NE2227: Contribution of Ovarian Function, Uterine Receptivity, and Embryo Quality to Pregnancy Success in Ruminants

(Multistate Research Project)

Status: Active

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SAES-422 Reports

Annual/Termination Reports:

[05/23/2023]

Date of Annual Report: 05/23/2023

Report Information

Annual Meeting Dates: 05/16/2023 - 05/17/2023
Period the Report Covers: 10/01/2022 - 09/30/2023

Participants

Dave Townson-URhode Island
Fransisco Diaz- Penn State
Cecily Bishop- Oregon State
Milo Wiltbank- UWisconsin
Yi Ren - Cornell
Amanda Patterson- UMissouri
Sarah Moorey-UTennessee
Dan Mathew-UTennessee
Soon Hun Cheong- Cornell
Jen Wood- UNebraska
Phil Bridges- UKentucky
Ron Butler- Cornell
Joy Pate- Penn State
Camilla Hughes- Penn State
Troy Ott- Penn State
Alvaro Garcia Guerra -Ohio State
Aileen Keating – Iowa State
Abby Maucieri – URhode Island
Kamilah Grant -USDA
Mark Mirando - USDA
Joanne Fortune -Cornell

Brief Summary of Minutes

Please see attached document

Minutes Attachment:

NE2227_Minutes_2023_PSU.docx

Accomplishments

Objective 1: Identify Mechanisms that Regulate Ovarian Function and Oocyte Quality during the Estrous Cycle   Identification of 22 metabolites that had positive relationship with serum estradiol concentration at the time of GnRH injection to induce ovulation. Determined a positive relationship between serum estradiol concentration at the time of GnRH injection to induce ovulation and oocyte ATP level ~18 hours later.   Generated new knowledge highlighting that heat-induced increases in body temperature in lactating dairy cows differentially impacts the cumulus and granulosa cell transcriptome of the periovulatory follicle.   Discovered that O-GlcNAcylation is expressed in KGN cells, an immortal granulosa cell tumor line that shares functional features of granulosa cells of mature, preovulatory follicles (e.g., FSH-responsiveness and estradiol production).   Demonstrated that manipulation of O-GlcNAcylation in KGN cells impairs cell proliferation and acutely perturbs oxidative phosphorylation, an effect observed previously with granulosa cells of bovine large antral follicles.   Preliminary experiments indicate inhibition of O-GlcNAc enhances estradiol production by primary cultures of bovine granulosa cells.   Determined the impact of altered insulin and glucose observed in dairy cattle experiencing heat stress (impact on the reproductive tract and elsewhere throughout the body. Recent work suggested that hypoglycemia during heat stress may be an important coping mechanism rather than a maladaptation to hyperthermia. Studies investigating the effect of hypoglycemia on follicle developmental dynamics and uterine function are currently underway.   Objective 2: Determine Factors Associated with Fertilization, Embryo Development, and Conceptus-Endometrial Interactions that Dictate Pregnancy Success   Using a day 16 conceptus-endometrial co-culture system, we are conducting studies to identify endometrial proteins and metabolites induced by IVP compared to IVD bovine embryos. Efforts are also underway to characterize the day 16 IVP and IVD bovine embryo transcriptome and secreted proteins/metabolites to better understand which conceptus secretory factors may be responsible for the altered endometrial transcriptome.    The gene codes for a KRAB-ZFP specifically expressed in bovine oocytes and early embryos and gene silencing experiments have demonstrated that ZNFO is required for early embryonic development in cattle. These results provide an essential step towards the identification of ZNFO regulated genes that play important roles during early embryonic development.   A study was completed to characterize three novel oocyte-specific lncRNAs, OOSNCR1, OOSNCR2 and OOSNCR3 using expression profiles generated from tissues, cells and embryos collected across the different stages of early embryonic development. Tissue expression data revealed detectable expression in only the fetal ovary for all three lncRNAs. Cell expression data revealed highest expression in oocytes with no significant difference seen between GV and MII oocytes. LncRNA expression was detectable but at lower levels in cumulus cells (CC) and even lower in theca (TC) and granulosa cells (GC). Expression data from the embryo panel suggests all three lncRNAs are maternally derived. Together, these data indicate a dynamic relationship among OOSNCR1, OOSNCR2 and OOSNCR3 and early reproductive events. Future studies aim to elucidate the functional roles of these lncRNAs during oocyte maturation and early embryonic development.   The role of ASIP in the bovine oocyte and early embryo was unknown. ATRN, MC3R, and MC4R were found to be expressed in both cumulus cells and oocytes. ATRN was also expressed in granulosa and theca cells and there was a significant effect of cell type as cumulus cells collected from MII COCs expressed ATRN at higher levels than granulosa and theca cells. Oocyte maturity level did not affect ATRN, MC3R, and MC4R expression levels.  There was a significant effect of embryo stage on ASIP expression. While ASIP was detected in each stage analyzed, ASIP levels were slightly reduced following oocyte maturation and remained at constant low levels until following completion of the embryonic genome activation at the 16-cell stage where it was observed ASIP levels increased in morula and blastocyst stage embryos.    Efforts are currently focused on exploring how the interleukin-6 (IL6) cytokine family acts as regulators of oocyte maturation, early embryo development, blastocyst formation, and early conceptus development in cattle. The work over the past year has provided strong evidence for implicating IL6 as a critical regulator of blastocyst development and inner cell mass formation. IL6 supplementation also improves conceptus elongation.   Exploration into how to modify our current bovine culture media formulations to improve embryo development and quality. Minor changes in formulation of SOF medium and the inclusion of an antioxidant cocktail are showing promise for improving the number and quality of transferable embryos.   Investigating the relationship between hypoglycemia during heat stress and pregnancy success on-farm.   Studies have determined how vitamin A and D supplementation in mid- to late gestation affects calving ease and birth weights of calves at parturition, as well as cow fertility at the following breeding. There was no effect of vitamin A and D treatment on calving ease or on birth weight, although calves had significantly elevated serum vitamin D in the treated group at time of birth, compared to controls, however these levels dropped to near control calves within one month of age.   Induction of pregnancy loss on day 35 of gestation in cattle results in luteolysis by gestation day ~45. During the second month of gestation basal prostaglandin F2α (PGF2α) is greater whereas pulse frequency and amplitude are similar to that at the expected time of luteolysis during the estrous cycle. It, however, is unclear whether PGF2α is responsible for luteolysis after induced conceptus demise. The aim, therefore, was to test the hypothesis that inhibition of PGF2α secretion (using flunixin meglumine) will delay luteolysis after induced conceptus demise. Luteolysis was identified in all animals, however, luteolysis tended to occur later in cows receiving flunixin meglumine (FM) than the untreated controls. In conclusion, inhibition of PGF2α secretion did not prevent but rather delayed luteolysis after induced conceptus demise, thus, providing partial support for the hypothesis that PGF2α is responsible for initiation of luteolysis after conceptus demise.   Objective 3: Develop and Evaluate Novel Reproductive Management Strategies and Technologies to Improve Reproductive Performance of Ruminants   Growth and nutrition during early development are major contributors to fertility and lifetime reproductive performance in cattle. Recent research findings demonstrate that changes in nutrition during gestation and early postnatal life result in functional and structural modifications in brain areas involved in the control of pubertal maturation in females. While these early modifications are likely to persist throughout life, their long-term effects remain unknown. Through a series of whole-animal, physiological, and molecular studies we have demonstrated that nutritional extremes during late gestation have only negligible effects on reproductive function in the heifer offspring. More specifically, maternal obesity or severe feed restriction during the last two trimesters of gestation did not alter age at puberty, pre-ovulatory follicle size, dominant follicle growth rate, CL size, endometrial thickness, antral follicle count, estrus expression, progesterone and estradiol concentrations during the estrous cycles, nor gonadotropin secretion in response to exogenous estradiol. These studies indicate that the adult reproductive phenotype in Bos indicus-influenced heifers is resilient to significant degrees of nutritional stress imposed during pre- and early postnatal periods, particularly if early gestation (first trimester) is avoided. These studies strongly suggest that adequate postnatal nutrition can overcome deficits programmed during late gestation in sexually-mature beef heifers. This information can guide management strategies for selection of replacement beef heifers.   A ruminant model of ovarian cryopreservation was investigated as part of an NIH-SIBR funded project in collaboration with 21st Century Medicine. The major goal of the study was to determine if a novel cryoprotectant along with a novel infusion and vitrification protocol would preserve most small follicle types on the ruminant ovary. This work found that perfusion techniques to vitrify whole ovaries are highly successful in preserving follicular function in ewes. Further studies are necessary to determine if these follicles produce fertilizable oocytes.   Presynchronization treatments administered before initiation of a CO-Synch type regimen are designed to improve ovulatory response and synchronization of follicular development. We conducted a study to determine the effect of various presynchronization strategies before initiation of a 6-day CO-Synch, on estrous expression and fertility to timed artificial insemination (TAI). Pregnancies per AI (P/AI) on D35 were greater for P4+PGF Presynch cows (66.7%) than for PGF Presynch (55.3%) and Control cows (50.9%). There were no differences in pregnancy loss between groups (5.9%). In conclusion, presynchronization with P4 and PGF prior to initiation of a 6-day CO-Synch treatment regimen improves fertility as a result of TAI in suckled beef cows.   Administration of FSH prior to ovum pick-up (OPU) in cattle improves in vitro embryo production (IVEP). Even though FSH is typically administered following a decreasing dose schedule, other dose schedules have not been evaluated. Thus, we evaluated the effect of FSH treatment pattern on ovarian response and IVEP. Pregnant Holstein heifers were randomly assigned to be administered p-FSH in a decreasing, constant, or increasing dose schedule, in a crossover design with a 14-day washout between sessions. Total number of follicles did not differ between decreasing, constant, and increasing groups. No differences were observed for number of viable COCs, number of COCs grade I, nor COC recovery rate. Cleaved oocytes, cleavage rate, and blastocyst rate did not differ between groups, resulting in a similar number of blastocysts per heifer. In conclusion, the pattern of administration for p-FSH prior to IVEP does not affect ovarian response, oocyte developmental competence nor embryo production.   Elucidated the underlying biology of differences in reproductive physiology of dairy cows with different genetic merit for fertility.   Generated evidence to support that cows of different genomic merit for fertility present different reproductive performance under common types of reproductive management.   Evaluated the response of dairy cows to different types of reproductive management strategies.   Identified associations between automated sensor monitoring systems data and the reproductive potential of dairy cattle.   Explored the value of combining genomic data with sensor and herd management data for predicting the future reproductive potential of cows.   Developed and tested reproductive management strategies that increase reproductive performance and improve herd management through use automated detection of estrus.   Described economic differences of cows of different genetic merit for fertility under different types of reproductive management programs.   Determined the economic value of days to pregnancy after calving (i.e., days open) on the profitability and survivability of dairy cows.   Developed data-analytic methods based on machine learning algorithms to predict the reproductive potential of dairy cows based on data collected by automated monitoring technologies and herd management and performance data.

Publications

Ault-Seay TB, Brandt KJ, Henniger MT, Payton RR, Mathew DJ, Moorey SE, Schrick FN, Pohler KG, Smith TPL, Rhinehart JD, Schneider LG, McLean KJ, Myer PR. Bacterial communities of the uterus and rumen during heifer development with protein supplementation. Front Anim Sci 2022. doi: 10.3389/fanim.2022.903909.   Ault-Seay TB, Moorey SE, Mathew DJ, Schrick FN, Pohler KG, McLean KJ, Myer PR. Importance of the female reproductive tract microbiome and its relationship with the uterine environment for health and productivity in cattle: A review. Front Anim Sci 2022.  doi: 10.3389/fanim.2023.1111636.   Ault-Seay TB, Payton RR, Moorey SE, Pohler KG, Schrick FN, Shepherd EA, Voy BH, Lamour KH, Mathew DJ, Myer PR, McLean KJ. Endometrial gene expression in response to lipopolysaccharide between estrous cycle phases and uterine horns in cattle. Front Anim Sci 2022. doi: 10.3389/fanim.2022.939876.   Bishop C, Selvaraj V, Townson DH, Pate JL, Wiltbank MC. History, insights, and future perspectives on studies into luteal function in cattle. J Anim Sci 2022  100: 1-15. doi.org/10.1093/jas/skac143   Bisinotto RS, Ribeiro ES, Greco LF, Taylor-Rodriguez D, Ealy AD, Ayres H, Lima FS, Martinez N, Thatcher WW, Santos JEP. Effects of progesterone concentrations and follicular wave during growth of the ovulatory follicle on conceptus and endometrial transcriptome in dairy cows. J Dairy Sci. 2022 Jan;105(1):889-903. doi: 10.3168/jds.2021-20193.   Byrd  MKH, Arneson AG, Soffa DR, Stewart JW, Rhoads ML. Human continuous glucose monitors for measurement of glucose in dairy cows. J Dairy Sci Comm. 2022. 3: 78-83.   Carr SN, Crites B, Pate JL, Hughes CHK, Matthews JC, Bridges PJ.  Form of supplemental selenium affects the expression of mRNA transcripts encoding selenoproteins, and proteins regulating cholesterol uptake, in the corpus luteum of grazing beef cows.  Animals. 2022. 12:313.    Chaney HL, Grose LF, LaBarbara JM, Sirk AW, Blancke AM, Sanchez JM, Passaro C, Lonergan P, Mathew DJ. Galectin-1 induces gene and protein expression related to maternal-conceptus immune tolerance in bovine endometrium. Biol Reprod. 2022. doi: 10.1093/biolre/ioab215.   Crites BR, Carr SN, Matthews JC, Bridges PJ. Form of dietary selenium affects mRNA encoding cholesterol biosynthesis and immune response elements in the early luteal phase bovine corpus luteum.  J Anim Sci. 2022. 100(7): doi:10.1093/jas/skac135.   Crites BR, Carr SN, Anderson LH, Matthews JC , Bridges PJ. Form of dietary selenium affects mRNA encoding interferon-stimulated and progesterone-induced genes in the bovine endometrium and conceptus length at maternal recognition of pregnancy. J Anim Sci.  2022. 100(7): doi:10.1093/jas/skac137.   Ealy AD, Pate JL, Ron Butler W. A synopsis of the NE1727 multistate project collection in the Journal of Animal Science. J Anim Sci. 2022 Jul 1;100(7):skac173. doi: 10.1093/jas/skac173.     García-Guerra A, Motta JCL, Hayden CB, Sala RV, Pereira DC, V. Absalon-Medina A, Moreno JF, Ross PJ. Advances in donor synchronization and superstimulation for OPU-IVEP:  the quest for enhanced oocyte quantity and quality. Proceedings of the Joint Convention of the American Embryo Transfer Association and the Canadian Embryo Transfer Association 2022, Pages 1-8.   Garza V, West SM, Cardoso RC. Review: Gestational and postnatal nutritional regulation of puberty and subsequent reproductive performance in heifers. Animal (accepted), 2022.   Hayden CB, Sala RV, Absalon-Medina VA, Motta JCL, Pereira D, Moreno JF, García-Guerra A. Synchronization of follicle wave emergence before ovarian superstimulation with FSH and ovum pick-up improves in vitro embryo production in pregnant heifers. Theriogenology 2022 188: 71-78.   Hardin, KN, dos Reis BR, Dias NW, Fiske DA, Mercadante VRG, Rhoads ML, Wilson TB, White RR. Growth and reproductive responses of heifers consuming endophyte-infected tall fescue seed with or without sodium bicarbonate supplementation. Appl Anim Sci. 2022 38(4):317-325. doi.org/10.15232/aas.2022-02273.   Horn, EJ, Read CC, Edwards JL, Schrick FN, Rhinehart JD, Payton RR, Campagna SR, Klabnik JL, Clark HM, Myer PR, McLean KJ, Moorey SE. Preovulatory follicular fluid and serum metabolome profiles in lactating beef cows with thin, moderate, and obese body condition. J Anim Sci. 2022. 1 (7): skac152. doi: 10.1093/jas/skac152.   Klabnik JL, Christenson LK, Pohler KG, Moorey SE, Rispoli LA, Gunewardena SSA, Payton RR, Schrick FN, Edwards JL. Heat-induced increases in body temperature in lactating dairy cows: impact on the cumulus and granulosa cell transcriptome of the periovulatory follicle. J Anim Sci. 2022. 1 (7): skac121. doi: 10.1093/jas/skac121   Lengi AJ, Stewart JW, Makris M, Rhoads ML, Corl BA. Heat stress increases mammary epithelial cells and reduces viable immune cells in milk of dairy cows. Animals (Basel). 2022. 12(20):2810. doi: 10.3390/ani12202810. PMID: 36290196; PMCID: PMC9597744.   Li Q, Chen KC, Bridges PJ, Matthews JC.  Pituitary and liver selenoproteins transcriptome profiles of grazing steers and their sensitivity to the form of selenium in vitamin-mineral mixes. Front Anim Sci.  2022. 3:911094. doi: 10.3389/fanim.2022.911094.   Lundberg AL, Jaskiewicz MN, Maucieri AM, Townson DH. Short Communication: Stimulatory Effects of TGFα in Granulosa Cells of Bovine Small Antral Follicles. J Anim Sci. 2022. 100:1-8. doi.org/10.1093/jas/skac105.   Maia TS, Guimarães HR, Garza V, Pohler KG, Cardoso RC, Williams GL. Early juvenile but not mid-to-late prenatal nutrition controls puberty in heifers but neither impact adult reproductive function. Biol Reprod. 2022 107(4):1035-45, 2022.   Mathew DJ, Peterson KD, Senn LK, Oliver MA, Ealy AD. Ruminant conceptus-maternal interactions: interferon-tau and beyond. J Anim Sci. 2022 Jul 1;100(7):skac123. doi: 10.1093/jas/skac123.   McCoski SR, Cockrum RR, Ealy AD. Short communication: Maternal obesity alters ovine endometrial gene expression during peri-implantation development. J Anim. Sci. 2022 Jul 1;100(7):skac090. doi: 10.1093/jas/skac090.   Moorey SE, Hessock EA, Edwards JL. Preovulatory follicle contributions to oocyte competence: Importance of the ever-evolving intrafollicular environment leading up to the LH surge. J Anim Sci. 2022. 1 (7): skac153. doi:10.1093/jas/skac153.   Moorey SE, Perry GA, Smith MF. Management Decisions to improve the reproductive performance of your herd: From calving to rebreeding. Applied Reproductive Strategies in Beef Cattle. 2022. San Antonio, TX.   Read CC, Edwards JL, Schrick FN, Rhinehart JD, Payton RR, Campagna SR, Castro HF, Klabnik JL, Moorey SE. Preovulatory serum estradiol concentration is positively associated with oocyte ATP and follicular fluid metabolite abundance in lactating beef cattle. J Anim Sci. 2022. 1 (7): skac136. doi: 10.1093/jas/skac136.   Soffa DR, Stewart JW, Arneson AG, Dias NW, Mercadante VRG, Rhoads RP, Rhoads ML. Reproductive and lactational responses of multiparous dairy cattle to short-term postpartum chromium supplementation during the summer months. J Dairy Sci Comm. 2022. doi:  https://doi.org/10.3168/jdsc.2022-0287.   Speckhart SL, Wooldridge LK, Ealy AD. An updated protocol for in vitro bovine embryo production. STAR Protoc. 2022 Dec 13;4(1):101924. doi: 10.1016/j.xpro.2022.101924.   Stewart JW, Arneson AG, Byrd MKH, Negron-Perez VM, Newberne HM, White RR, El-Kadi SW, Ealy AD, Rhoads RP, Rhoads ML. Comparison of production-related responses to hyperinsulinemia and hypoglycemia induced by clamp procedures or heat stress of lactating dairy cattle. J Dairy Sci. 2022 Oct;105(10):8439-8453. doi: 10.3168/jds.2022-21922.   Stoecklein KS, García-Guerra A, Duran BJ, Prather RS, Ortega M. Actions of FGF2, LIF, and IGF1 on Bovine Embryo Survival and Conceptus Elongation following Slow-rate Freezing. Front Anim Sci. 3. 2022. DOI: 10.3389/fanim.2022.1040064.   Walker BN, Nix J, Wilson C, Marrella MA, Speckhart SL, Wooldridge L, Yen CN, Bodmer JS, Kirkpatrick LT, Moorey SE, Gerrard DE, Ealy AD, Biase FH. Tight gene co-expression in BCB positive cattle oocytes and their surrounding cumulus cells. Reprod Biol Endocrinol. 2022 Aug 13;20(1):119. doi: 10.1186/s12958-022-00994-3.   Webb KL, Trotta RJ, Jia Y, Bridges PJ, Matthews JC. Influence of form of selenium supplementation and tall fescue toxicity on growth performance, serum parameters, and tissue mass of grazing beef steers.  Transl Anim Sci. 2022. 6(4):txac122. doi: 10.1093/tas/txac122.    Wooldridge LK, Keane JA, Rhoads ML, Ealy AD. Bioactive supplements influencing bovine in vitro embryo development. J Anim Sci. 2022 Jul 1;100(7):skac091. doi: 10.1093/jas/skac091.   Zhang M, Current JZ, Chaney HL, Yao J. Identification of the DNA binding element of ZNFO, an oocyte-specific zinc finger transcription factor in cattle. Gene. 2022 Aug 5;834:146655. doi: 10.1016/j.gene.2022.146655.   Rial C, Laplacette AL, Giordano JO. Effect of a targeted reproductive management program designed to prioritize insemination at detected estrus and optimize days to insemination on the reproductive performance of lactating dairy cows. J. Dairy Sci. 2022. 105:8411-8425. https://doi:10.3168/jds.2022-22082    Giordano JO, Sitko EM, Rial C, Perez MM, Granados GE. Use of multiple biological, management, and performance data for the design of targeted reproductive management strategies for dairy cows. J. Dairy Sci. 2022. 105:4669-4678. https://doi.org/10.3168/jds.2021-21476       Theses/Dissertations   Sarah N. Carr. Selenium form-induced changes in the early luteal phase corpus luteum, the blood, and the endometrium during early gestation in beef cows. PhD Dissertation. Kentucky.   Cassidy Ficker. Characteristics of Small Antral Follicles in Heifers Carrying the Bovine High Fecundity Allele Trio. Undergraduate Honors thesis. Ohio.   CB Hayden. Optimization of ovarian superstimulation before ovum pick-up and in vitro embryo production in pregnant cattle. MS thesis. Ohio.   Emma Hessock. Differential metabolome profiles in preovulatory follicular fluid of cattle with varying duration of proestrus. MS Thesis. Tennessee.   Emma Horn. Preovulatory follicular fluid and serum metabolome profiles in lactating beef cows with thin, moderate, and obese body condition. MS Thesis. Tennessee.   Ruben Lopez-Carrillo Jr. Ovarian protein hormones as biomarkers of fertility in dairy cows – Is it an acceptable model to predict infertility in dairy breeds? BS Thesis. Oregon.    Endya McKinley. Investigating the supplementation of IL-6, IL-11, & LIF at in vitro maturation to improve oocyte competency. MS Thesis. Virginia.   Katie Peterson. Identification of Endometrial Transcripts and Proteins Associated with the in vivo derived and in vitro produced bovine conceptus microenvironment. MS Thesis. Tennessee.   Casey Read. Effects of preovulatory follicle physiological status on oocyte metabolic capacity. PhD Dissertation. Tennessee.   Dallas Soffa  Effects of feed additives on uterine morphology and selected reproductive attributes. MS Thesis. Virginia.   Savannah L. Speckhart. Interleukin-6 and its Contribution to Embryogenesis in Cattle. PhD Dissertation. Virginia.   Emily Sitko. Genomically enhanced predictions of dairy cattle fertility: implementation in reproductive management and associations with cow reproductive biology and behavior. PhD Dissertation. Cornell University

Impact Statements

Specific impactful discoveries from the group include, but are not limited to: 1) describing the roles of pro-inflammatory cytokines and environmental stressors on follicle growth, steroidogenesis, luteal regression, and conceptus-uterine-immune interactions, 2) use of trace mineral supplementation to improve oocyte quality and increase systemic progesterone concentrations, 3) impact of beef cow body condition score and weight on the metabolome of follicular fluid and serum, 4) genetic determinants of ovulation and implantation rates, 5) endocrine and molecular mechanisms controlling ovulation rate in cows, 6) mechanisms associated with preimplantation embryo development and quality, 7) elucidate the underlying biology driving differences in dairy cow performance due to genetics, health, management, and environmental conditions, 8) developed novel reproductive management strategies that improve reproductive performance in dairy and beef herds, and 9) factors that cause pregnancy loss in cattle and development of methods to reduce losses. These 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. Members of the NE227 group (NY, IA, MS, OH, OR, PA, KY, MA, NE, TN, VT, WI, VA) 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. Greater than 50 students have enrolled in each of the last two offerings of the course. This accounted for a majority of the MS and PhD students across the experiment stations in this multistate project. 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.