THE IMPACT OF THE OVARIAN RESERVE ON FERTILITY AND RESPONSE TO SUPEROVULATION IN CATTLE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1019586
• Project Start Date: Oct 1, 2019
• Project End Date: Sep 30, 2024
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
ANIMAL SCIENCE

Non Technical Summary
Diagnostic or genetic procedures to predict which heifers in herds have suboptimal fertility, poor health and(or) low milk production are not available. It is well established thatpoor health, low milk production and suboptimal fertility cause life span of dairy cattle inherdsto be relatively short (~2.5 to 3 lactations). Thus, one of the major annual costs to dairy cattle operations is herd replacements. Consequently, development of new, more consistent diagnostic, therapeutic or genetic methods to predict or enhance fertility and(or) to select for high fertility cattle with improved herd healthy and longevity, which is this investigator's long-term research goal, would clearly have a major economic impact on the cattle industry. Results from our laboratory show that approximately 25% of young adult cattle in a herd have very low numbers of eggs and follicles in ovaries, decreased ovarian function, poor responsiveness to superovulation and poor fertility,and that use of ovarian ultrasonography to determine number of follicles on ovaries or measurement of a blood marker known as anti-Mulllerian hormone (AMH) can be used to identify these cattle. The purpose of our studies is threefold: First, we will remove blood and analyze AMH concentration in heifers to determine if AMH concentration can be used to predict future fertility, health and level of milk production of these individuals after calving. Second, high concentrations of FSH used to superovulate cattle for embryo transfer are detrimental to embryo survival especially in cattle with low AMH and low follicle numbers. Thus, we will determine the mechanisms whereby FSH impairs embryo survival and test if use of relatively low FSH doses during superovulation of dairy heifers with low AMH and low follicle numbers improves their response to superovulation. Third, we will determine if the high amounts of follicle stimulating hormone (FSH) secreted during estrous cycles of multiparous dairy cows with low AMH and low follicle numbers impairs function of ovulatory follicles, which may cause infertility, and if therapies to lower FSH secretion improve ovulatory follicle function in these individuals.Outcomes of these studies will provide new knowledge relevant to a better understanding ot the impact of FSH and number of follicles (or concentration of AMH) on ovarian function, which provides a foundation for new methods to improve fertility in cattle. In addtion, we will directly establish if potential new diagnostics (AMH) or therapies (reduction in FSH secretion) can potentially be used to enhance fertility in cattle. Collectively, this new information provides reproductive scientists with a new understanding of the importance of AMH concentrations and follicle numbers to ovulatory follicle function and the cattle industry with new therapeutic approaches to improve efficiency of reproductive management.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3410	1020	100%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1020 - Physiology;

Keywords

dairy cows, ovarian reserve, superovulation, anti-mullerian hormone, fertility

reproduction

amh

fsh

follicle stimulating hormone

Goals / Objectives
Goal I: To determine if AMH can be used to predict fertility and longevity in the herd in dairy cows. Goal II: To determine the mechanisms whereby FSH impairs oocyte/embryo quality during superovulation and to develop new methods to improve responsiveness of cattle with a low antral follicle count (AFC) and small ovarian reserve (low egg numbers) to superovulation. Goal III: To determine if the chronically elevated secretion of follicle stimulating hormone (FSH) during follicular waves has a negative impact on ovulatory follicle function, oocyte quality, and embryo survival in multiparous dairy cows with a low AFC and small ovarian reserve.

Project Methods
RESEARCH METHODS/EXPERIMENTAL PROCEDURES:Objective I: To determine if AMH can be used to predict fertility and longevity in dairy cattle. The overarching hypotheses for this study is that young adult heifers with a relatively small ovarian reserve, and correspondingly low AMH concentrations and low AFC also have suboptimal fertility and reduced herd longevity compared with their age-matched herdmates with larger ovarian reserves. To test this hypothesis, a total of 3000, 12-month-old heifers at Green Meadow Farms Inc were used in our study to complete the following Aims:Aim 1 determined if differences existed in conception rates and incidence of diseases/disorders between heifers with low vs higher AMH and if circulating AMH concentrations were correlated with any genotype. Results showed that AMH concentration was a genetically heritable trait (32%) but fertility in heifersand incidence of disease were not correlated with AMH. The potential negative impact of a diminished ovarian reserve on fertility may be age-dependent however and productive herd life (from birth of first calf to removal from the herd) cannot be examined in Aim 1. Thus, Aim 2 is independent of Aim 1 because it will determine if conception rates, culling rate and reasons for culling, and herd longevity differ after calving between the heifers identified in Aim 1 as having small (low AMH) vs larger (high AMH) ovarian reserves. Data recorded by farm employees on DC305 records is currently being analyzed.Objective II: To determine the mechanisms whereby FSH impairs oocyte/embryo quality during superovulation and to develop new methods to improve responsiveness of cattle with a low AFC (reduced ovarian reserve) to superovulation. Our central hypothesis is that FSH doses in excess of the SOVMAX during ART impair ovulatory follicle number/function, oocyte and embryo quality and embryo survival irrespective of donor's age or AFC. The following Aims test this hypothesis:Aim I: Establish the relationship between FSH dose, SOVMAX and AFC. Hypothesis: The SOVMAX does not exceed the individual's AFC and FSH doses in excess of those that achieve the SOVMAX decrease ovulatory follicle numbers.Aim II: Determine if FSH doses exceeding SOVMAX increase indices of ovulatory follicle function linked to high oocyte/embryo wastage during ART. Hypothesis: FSH doses exceeding SOVMAX disrupt the FSH signaling system in granulosal cells thereby triggering a cascade of intrafollicular events that impair ovulatory follicle function, oocyte and embryo quality, and blastocyst development in vitro.Aim III: Determine if FSH doses that cause high oocyte/embryo wastage are also detrimental to embryo survival. Hypothesis: FSH doses impairing oocyte quality and embryo development/quality in vitro also decrease pregnancy rate following transfer of morphologically high quality embryos.Aim IV: Test potential therapies to increase responsiveness to superovulation and embryo yield in individuals with a small ovarian reserve. Hypothesis: Suppression of endogenous FSH and LH secretion and associated number of antral follicles previously exposed to high endogenous FSH/LH concentrations enhances responsiveness of individuals with a small ovarian reserve and chronically high FSH to superovulation.Objective III: To determine if the chronically elevated FSH secretion during follicular waves has a negative impact on ovulatory follicle function, oocyte quality, and embryo survival in multiparous dairy cows with a low AFC. The strong linkage between high FSH secretion, aberrant gonadotropin signaling cascades, ovarian dysfunction and poor fertility, coupled with our findings that cattle with naturally chronically heightened FSH secretion have diminished ovarian function and are refractory (not fully responsive) to gonadotropin stimulation, support the following overarching hypothesis for this study: Chronically elevated FSH secretion during follicular waves in multiparous cows with low follicle numbers has a negative impact on the gonadotropin signaling cascades thereby impairing differentiation, function, and capacity of granulosal cells to respond to gonadotropins during ovulatory follicle development.To test our hypothesis, we will use multiparous dairy cows. Aim I will conduct a series of functional assays to determine if granulosal cells in ovulatory follicles have a reduced capacity to produce estradiol and undergo luteinization because one or more components of the FSH or LH protein kinase A (pKA) signaling cascade, which is the predominant signaling pathway for gonadotropins, are defective in cattle with naturally chronically elevated vs lower FSH secretion during follicular waves. Results will for the first time establish if high FSH secretion during follicular waves is linked both to an aberrant pKA signaling cascade in granulosal cells and reduced capacity of these cells in ovulatory follicles to respond to the preovulatory LH surge, which would explain why luteal progesterone production is low during estrous cycles of cattle with naturally elevated FSH secretion. While Aim I establishes the impact of elevated FSH secretion during follicular waves on ovulatory follicle function, the mechanisms whereby the high FSH secretion initially enhances function of small antral follicles but later in the same wave impairs ovulatory follicle function, as implied by our previous studies, are unknown. Moreover, pKA independent pathwaysand a variety of regulators of FSH action could also be modified by chronically high FSH secretion at unknown stages of dominant follicle development and thus explain the reduction in responsiveness of granulosal cells to FSH and LH action we observe in cattle with chronically elevated FSH secretion during follicular waves. Thus, in Aim II we will conduct transcriptome analysis to identify the genes, gene families, pathways and regulatory elements in granulosal cells that may differ at key points in dominant follicle development (pre-deviation, deviation, preovulatory) between cattle with chronically elevated (low AFC, small ovarian reserve) vs lower FSH concentrations (high AFC, large ovarian reserve) during follicular waves. Whether the diminished function we observe as small antral follicles develop into ovulatory follicles is caused by the chronically elevated FSH secretion per se during the ovulatory follicular wave and thus independent of differences in follicle numbers during follicular waves is unknown. Aim III, therefore, takes a physiological approach to determine if a reduction in peak FSH secretion during ovulatory follicular waves in cattle with naturally chronically elevated FSH secretion enhances ovulatory follicle function.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Target audience will be scientists interested in improving reproductive efficiency and embryo transfer in cattle, cattle producers and veterinarians, and clinicians interested in improvement of assisted reproductive technologies (ART) in women. Changes/Problems:Progress has been slowed due to Covid because this investigator is not allowing his research team to go to the local abattoir to collect bovine ovaries. This source of ovaries typically provides the tissues necessary to develop and validate various techniques including histological analyses of cumulus oocyte complexes (COCs) and blastocysts, and rates of IVF, cleavage and blastocyst development. Consequently, this investigator has to use live animals to provide COCs to validate techniques which slows progress. In addition, progress has been slowed due to loss of key personnel that were hired in other positions. Thus, new investigators had to be hired and trained.. What opportunities for training and professional development has the project provided?Opportunities for training and professional developent were provided to a PhD student,two visiting scientists and undergraduates during completion of several studies in my laboratory. All individuals were involved in superovulation andblood sampling of cattle,hormone assays, ovarian ultrasonography to monitor growth of ovulato;ry follicles during superovulation, ultrasound-guided needle biopsy and recovery of cumulus-oocyte complexes (COCs) from conscious cattle, ovary recovery atautopsy of cattle,isolation of ovarian follicles and contents including granulosal, thecal and cumulus cells and oocytes which were processed for RNAseq analyses, isolation of follicular fluid from excised follicles which was processed for hormone assays, and recovery, grading and staining and histological analyses of COCs. The PhD student and visiting scientistswere involved in record-keeping, developed abstracts and posters, attended conferences and presented orally their results. In addition, the PhD student and visitingscientistwere responsible for training undergraduates to assist in completion of the aforementioned projects and procedures andhave been involved in preparation of manuscripts for publication. How have the results been disseminated to communities of interest?Results of studies have been presented in international and local scientific meetings which have been attended primarily by reproductive scientistsand animal technicians and practioners interested in embryo transfer. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: We are currently evaluatingif maternal environment including somatic cell count, number of lactations and fertility of dams impacts AMH concentration in their daughtersand if AMH concentration in heifers is predictive of level of milk production. Analyses of these results including publication should be completed during the next reporting period. Goal II: RNAseq analysis has been completed on granulosa, theca, cumulus and oocytes of ovulatory follicles (12 hours after the last Folltropin-V injection and prior to ovulatory dose of hCG) isolated from Holstein heifers superovulated with 2 (industrydose) vs 6 ml (excessive dose) Folltropin-V per injection (total = 8 injections). The excessive FSH doses during superovulation resulted numerous significant differences in abundance of RNAs for a variety of pathways.Bioinformatic analyses are ongoing to understand which molecular pathways are disrupted by the excessive FSH doses during superovulation. Anew study has been designed to determine the impact of the industry vs excessive FSH dose on germinal vesicle breakdown and parthenogenetic activation of oocytes is planned and should be completed in the next reporting period. Goal III: No studies planned.

Impacts
What was accomplished under these goals? IMPACT: Fertility in dairy heifers (60% to 70% conception rate following first artificial insemination) drops about 40% to 50% or more after calving. This well-established, rapid decline in fertility makes reproductive efficiency poor and costs associated with suboptimal fertility high in dairy cows compared with heifers. The reason for the rapid decline in fertility in heifers post-calving is unknown. Therefore, development of new methods to identify the heifers that will exhibit suboptimal fertility post-calving is this investigator's first research goal. Such methods could be used to either remove (cull) the low fertility heifers from the herd or to identify the low fertility heifers to subject to new fertiliity enhancing therapies to improve overall herd fertility and thus reduce costs linked to suboptimal fertility. Also, the reason for the high variation of responsiveness of cattle to superovulation, which may be related to their fertility, is unkown. This longstanding problem of extreme differences among cattle in responsiveness to superovulation is further compounded by the general lack of knowledge of whether the dose of hormone used to stimulate superovulation causes this high variability. Taken together, these unresolved problems greatly hinder widespread use of assisted reproductive technologies in donor cattle to propagate genetically superior offspring in a herd. Thus, this investigator's second research goal is to develop new methods to optimize hormone doses used for ovarian stimulation to enhance ovulatory follicle function and egg quality in individual cows and thus improve predictability of outcomes of in vitro fertilization and embryo transfer. This investigator's research team established that total number of high-quality eggs in ovaries of cattle varies remarkably from 2000 to 36,000 in 12-month-old heifers. Because eggs are rapidly depleted and never replenished as heifers (and all mammals) age, this investigator tested the hypothesis that a relatively low number of eggs in ovaries of heifers negatively impacts ovarian function and fertility thereby reducing herd longevity and response to superovulation including IVF and embryo transfer outcomes. The investigator's research team established that concentration of anti-Mullerian hormone (AMH) in blood is positively correlated with ovarian function, egg and follicle numbers, and response to superovulation, and that 20 to 25% of cattle in a typical herd have a relatively low AMH concentration and reduced ovarian function and egg numbers. Next, my research team measured AMH in over 3000 Holstein heifers and established that although AMH concentrations in blood (and presumably egg numbers) is very different among individuals, their subsequent fertility (e.g., conception rates), health, and herd longevity over the next nearly five lactations were similar. These results implied that a single AMH measurement in Holstein heifers is not predictive of their future fertility, health or herd longevity. However, the caveat in our interpretation of these results is the following: A single determination of AMH concentration in 11- to 12-month old heifers does not indicate the age-related rate of decline in AMH, egg numbers and ovarian function that may vary greatly among heifers after breeding, calving and completion of lactations. Consequently, determination of AMH concentration in 11- to 12-month old heifers and again in the same individuals after completion of their first lactation may be a much better test of whether AMH concentrations is useful to predict future fertility, health and longevity of dairy cows. My research team established that AMH concentration in blood is not only a reliable biomarker for egg numbers, ovarian function and response to superovulation, but also is a moderately genetically heritable trait (33%, highest for any reproductive trait). Thus, new breeding schemes could be used to genetically enhance egg numbers, ovarian function and response to superovulation in dairy cattle. These genetically superior cows would have several potential economically important benefits compared with cattle with fewer egg numbers, including for example consistently higher quantity of eggs in ovaries to recover surgically for IVF and commercial embryo production, reduced requirement for hormone per injection and perhaps fewer injections during superovulation to enhance number of embryos recovered and outcomes during traditional embryo transfer procedures, and sustained fertility during stressful environments such as inadequate nutrition or high ambient temperatures, or following new genetic breeding schemes to enhance wellness (health, production and longevity) that may produce unintended negative consequences on egg and follicle numbers and ovarian function. My research team has also established that very high doses of hormone used to stimulate superovulation in cattle with low egg numbers usually does not enhance the superovulation response compared with lower doses. In addition, high hormone doses have negative effects on ovulatory follicle function which may impair egg quality and hinder embryo transfer outcomes. Thus, use of high hormone doses to enhance the superovulation response in heifers with low egg numbers is economically wasteful needlessly driving up costs of embryo transfer. Results of these studies also show that AMH concentration for heifers with low egg numbers is useful to predict how well these heifers will respond to superovulation. For example, even though all heifers in our study had relatively low AMH and egg numbers, the heifers within this group with the highest AMH and greatest egg numbers responded best to superovulation. This finding may enable practitioners to use AMH concentration in blood to select cattle in the herd with the highest egg numbers that may require much lower hormone doses during superovulation to optimize embryo transfer or IVF outcomes than their herd mates with lower AMH and egg numbers. Goal 1: After determination of circulatingAMH concentrations in nearly 3000, 11- to 12-month old Holstein heifers, results of analyses of DC305 herd records maintained for nearly five lactations indicate that AMH cconcentration is not predictive of the future fertility, culling rates, productive herd life or longevity of these heifers. Goal II: Results of analysis of alterations in ovulatory follicle development and function have been completed and submitted for publication. Results show that high FSH doses are detrimental to ovulatory follicle function and ovulation rate and thus do not enhance response to superovulation in small ovarian reserve heifers. Analysis of steroid environment,morphology of cumulus cells,as well as RNAseq analysis has been completed on granulosa, theca, cumulus and oocytes of ovulatory follicles (12 hours after the last Folltropin-V injection and prior to ovulatory dose of hCG) isolated from Holstein heifers superovulated with 2 (industrydose) vs 6 ml (excessive dose) Folltropin-V per injection (total = 8 injections). The excessive FSH doses during superovulation resulted premature luteinization of ovulatory follicles and numerous significant differences in abundance of RNAs for a variety of pathways.Bioinformatic analyses are ongoing to understand which molecular pathways are disrupted by the excessive FSH doses during superovulation.In addition, a preliminary study has been completed to learn and validate oocyte pick up technique, expected efficiency of egg recovery,and staining procedures to evaluate maturation of cumulus-oocyte complexes. Goal III: No progress.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Succu S, Sale S, Ghirello G, Ireland JJ, Evans ACO, Atzori AS, Mossa F. 2020. Exposure of dairy cows to high environmental temperatures and their lactation status impairs establishment of the ovarian reserve in their offspring. J Dairy Sci Accepted for publication.
• Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Karl K, Jimenez F, Gibbings, Ireland JLH, Clark Z, Tempelman, R, Latham KE, Ireland JJ. 2020. Negative impact of excessive doses of FSH during superovulation on ovulatory follicle function in small ovarian reserve heifers. Submitted Biol Reprod
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Clark Z, Thakur M, Leach RE, Ireland JJ. 2020. FSH dose is negatively correlated with oocyte retrieval: analysis of ~650,000 assisted reproductive technology cycles. Submitted Fert Steril
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Mossa F, Latham KE, Ireland JJ, Veiga-Lopez A. 2019. Undernutrition and hyperandrogenism during pregnancy: role in programming of cardiovascular disease and infertility. Mol Reprod Dev 2019:1-10.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Mossa F., Ireland J. 2019. PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Anti-Mullerian hormone (AMH): a biomarker for the ovarian reserve, ovarian function and fertility in dairy cows. J Anim Sci. 97: 1446-1455.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Clark ZL, Karl KR, Ruebel ML, Chen O, Herzog RL, Huffman SM, Gibbings E, Latham KE, Ireland JJ. 2019. Evidence that excessive FSH during superovulation of cattle induces premature luteinization of ovulatory follicles. Abstract presented to Michigan Alliance for Reproductive Technologies and Sciences (MARTS) Conference, Heritage Room, University Club, Lansing, MI, May 31. (received 2nd place poster award)
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Karl KR, Jimenez-Krassel F, Gibbings E, Ireland JLH, Tempelman R, Latham KE, Ireland JJ. 2019. The effect of different doses of Follicle-Stimulating Hormone during superovulation on ovarian function in dairy cattle. Abstract presented to 10th Annual Michigan Alliance for Reproductive Technologies and Sciences (MARTS) Conference, Heritage Room, University Club, Lansing, MI, May 31. (received 1st place poster award)
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: . Jimenez-Krassel F, Ireland JLH, Berger L, Neuder L, Pursley JR, Ireland JJ. Impact of maternal health and parity of dairy cows on size of the ovarian reserve, fertility and health of female offspring. Abstract presented to the 10th Annual Michigan Alliance for Reproductive Technologies and Science (MARTS) Conference, The University Club, Heritage Room, Lansing, MI, May 31.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Succu S, Sale S, Ghirello G, Ireland JJ, Evans ACO, Atzori AS, Mossa F. 2020. High environmental temperatures during early fetal life may impair the ovarian reserve in cattle. International Embryo Technology Society 46th Annual Conference, New York, New York, January 16-19.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Clark ZL, Karl KR, Ruebel ML, Jimenez-Krassel FJ, Ireland JLH, Tempelman RJ, Thakur M, Leach RE, Gibbings E, Chen O, Herzog RL, Huffman SM, Latham KE, Ireland JJ. 2020. Excessive FSH doses during superovulation decrease oocyte recovery in women and adversely impact ovarian function in cattle. Virtual poster presented at the 53rd Annual Meeting of the Society for Study of Reproduction (SSR), July 9-12, Shaw Center, Ottawa, Ontario, Canada
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: . Karl KR, F Jimenez-Krassel, E Gibbings, K Latham, JJ Ireland. 2019. The effect of different doses of follicle-stimulating hormone during superovulation on ovarian function in dairy cattle. Abstract submitted to IETS 45th Annual Conference, New Orleans, La, Sheraton New Orleans Hotel, January 20-23.