Methods to predict numbers of healthy oocytes in the ovaries of young adults could have important diagnostic relevance in family planning and animal agriculture. We have observed that peak antral follicle count (AFC) determined by serial ovarian ultrasonography during follicular waves is very highly reproducible within individual young adult cattle, despite 7-fold variation among animals. Herein, we tested the hypothesis that AFC is positively associated with the number of morphologically healthy oocytes and follicles in ovaries and with serum concentrations of anti-Müllerian hormone (AMH), an indirect marker for number of healthy follicles and oocytes in ovaries. In the present study, age-matched young adult cattle (12-18 mo old) were subjected to serial ultrasonography to identify animals with a consistently high (> or =25 follicles that were > or =3 mm in diameter) or low (< or =15 follicles) AFC during follicular waves. Differences in serum AMH concentrations, ovary weight, and number of morphologically healthy and atretic follicles and oocytes were determined. The phenotypic classifications of cattle based on AFC during follicular waves or AMH concentrations both predict reliably the relative number of morphologically healthy follicles and oocytes in ovaries of age-matched young adult cattle.

BACKGROUND: The significance of the high variation in numbers of follicles produced during reproductive cycles in humans and cattle is unknown. METHODS: We selected beef heifers with high (25) or low (15) numbers of ovarian follicles and determined the association with alterations in FSH and estradiol concentrations, as well as responsiveness to superstimulation and embryo quality. The variation in follicle numbers was also compared with oocyte quality in natural cycles using IVF and abattoir sourced bovine ovaries. RESULTS: Results show that: (i) FSH was lower (P < 0.03) in animals with high compared with low follicle numbers per follicle wave; (ii) after superovulation, in the high versus low follicle number group, the number of oocytes/embryos recovered after insemination (10.6 + + + + + 2.7 versus 4.7 + + + + + 0.7) and the number of transferable embryos (5.4 + + + + + 1.3 versus 3.8 + + + + + 0.8) per animal were greater (P < 0.05), whereas the proportion of transferable embryos (50.7% versus 79.8%) was lower (P < 0.05); (iii) in unstimulated animals, the numbers of high-quality oocytes harvested and in-vitro fertilized oocytes developing into blastocysts were up to 4-fold greater (P < 0.05) for ovaries with high versus low numbers of follicles, but the proportions of oocytes developing into blastocysts were similar in the two groups. CONCLUSION: Phenotypic classification based on numbers of follicles may be useful to improve superovulation procedures. The lower proportion of transferable embryos following superovulation of ovaries with high numbers of follicles is probably not the result of differences in the quality of oocytes before superovulation.

The extent, causes, and physiological significance of the variation in number of follicles growing during ovarian follicular waves in human beings and cattle are unknown. Therefore, the present study examined the variability and repeatability in numbers of follicles 3 mm or greater in diameter during the follicular waves in bovine estrous cycles, and we determined if the variation in number of follicles during waves was associated with alterations in secretion of FSH, estradiol, inhibin, and insulin-like growth factor I (IGF-I). Dairy cattle were subjected to twice-daily ultrasound analysis to count total number of antral follicles 3 mm or greater in diameter throughout 138 different follicular waves. In another study, blood samples were taken at frequent intervals from cows that consistently had low or very high numbers of follicles during waves and were subjected to immunoassays. Results indicate the following: First, despite an approximately sevenfold variation in number of follicles during waves among animals and marked differences in age, stage of lactation, and season of the year, a very highly repeatable (0.95) number of follicles 3 mm or greater in diameter is maintained during the ovulatory and nonovulatory follicular waves of individuals. Second, variation in number of follicles 3 mm or greater in diameter during waves and the inverse association of number of follicles during waves with FSH are not directly explained by alterations in the patterns of secretion of estradiol, inhibin, or IGF-I. Third, ovarian ultrasound analysis can be used reliably by investigators to identify cattle that consistently have low or high numbers of follicles during waves, thus providing a novel experimental model to determine the causes and physiological significance of the high variation in antral follicle number during follicular waves among single-ovulating species, such as cattle or humans.

The mechanism whereby the inherently high variation in ovary size and the total number of high-quality oocytes in ovaries (ovarian reserve) impact on ovarian function and fertility, diagnostics to measure the size of the ovarian reserve and the factors that cause variation in the ovarian reserve are unknown. Our results show that cattle can be phenotyped reliably based on the number of antral follicles growing during follicular waves (antral follicle count, AFC). Young adult cattle with a consistently low v. a high AFC have smaller gonads, a markedly diminished ovarian reserve and many other phenotypic characteristics usually associated with ovarian aging and infertility. A powerful new approach based on a single measurement of serum concentration of anti-Müllerian hormone (AMH) is described to test the longstanding hypothesis that the size of the ovarian reserve is positively associated with fertility. Also, new evidence shows that maternal environment has a critical role in regulation of the high variation in the ovarian reserve and perhaps fertility in offspring. These results support the conclusion that the inherently high variation in the ovarian reserve, potentially caused by alterations in the maternal environment, has a negative impact on ovarian function that may result in suboptimal fertility in young adult cattle, and a single AMH measurement can be used reliably in future studies to determine if fertility is suboptimal in young adult cattle with low circulating AMH concentrations and a correspondingly diminished ovarian reserve.

Low progesterone concentrations and diminished ovarian reserves (total number of healthy oocytes) during reproductive cycles are linked to infertility in single-ovulating species like cattle. However, the extent and mechanisms whereby the inherently high variation in ovarian reserves may negatively affect progesterone production are unknown. Cattle were chosen to address these questions because the size of their ovarian reserves can be predicted based on an antral follicle count (AFC) during follicular waves. The present study determined if progesterone concentrations, differentiation and function of the corpus luteum (CL), and endometrial thickness differed during reproductive cycles of age-matched healthy young adult cattle with low versus high AFC during follicular waves. The results showed that, despite enhanced LH secretion, progesterone concentrations were lower during estrous cycles for animals with low versus high AFC. Animals with low versus high AFC also had a decreased basal, LH-, and 25-hydroxycholesterol-induced capacity of luteal and granulosal cells to produce progesterone, reduced amounts of STAR and mRNAs for STAR and LH receptor in the CL, and no change in endometrial thickness during estrous cycles. Taken together, these results 1) supported the conclusion that high variation in ovarian reserves of young adults is associated with alterations in differentiation and function of the CL and 2) provided insight into the potential factors that may cause suboptimal luteal function (e.g., heightened LH secretion and desensitization of the LH receptor, diminished LH responsiveness, diminished STAR, inherent deficiency in capacity of granulosal cells to undergo luteinization) and infertility (e.g., low progesterone, poor endometrial growth) in individuals with diminished ovarian reserves.

Reliable biomarkers predictive of productive herd life (time in herd after birth of first calf) have heretofore not been discovered in dairy cattle. However, circulating concentrations of anti-Müllerian hormone (AMH) are positively associated with number of follicles or antral follicle count (AFC), ovarian function, and fertility, and approximately 25% of cows have a relatively low AFC and low AMH concentrations. The present study tested the hypothesis that heifers with the lowest AMH concentrations have suboptimal fertility and are removed from a herd for poor reproductive performance at a greater rate, and therefore have a shorter productive herd life compared with age-matched herdmates with higher AMH. To test this hypothesis, 11- to 15-mo-old Holstein heifers (n=281) were subjected to a single measurement of AMH. All heifers not removed from the herd had the opportunity to complete 2 lactations and start their third lactation after calving. During this time, performance and health parameters for each individual were recorded daily by herd managers. Results showed that the quartile of heifers with the lowest AMH concentration also had, on average, a shorter productive herd life (by 196 d), a reduced survival rate after birth of the first calf, the lowest level of milk production (first lactation), the lowest total percentage of cows pregnant (across all lactations), the highest culling rates (first and second lactations and overall), and the highest culling rate for poor reproduction (first lactation) compared with age-matched herdmates with higher AMH. We concluded that a single determination of AMH concentration in young adult dairy heifers may be a simple diagnostic method to predict herd longevity, and AMH may be a useful phenotypic marker to improve longevity of dairy cows.

The mechanisms whereby the high variation in numbers of morphologically healthy oocytes and follicles in ovaries (ovarian reserve) may have an impact onovarian function, oocyte quality, and fertility are poorly understood. The objective was to determine whether previously validated biomarkers for follicular differentiation and function, as well as oocyte quality differed between cattle with low versus a high antral follicle count (AFC). Ovaries were removed (n = 5 per group) near the beginning of the nonovulatory follicular wave, before follicles could be identified via ultrasonography as being dominant, from heifers with high versus a low AFC. The F1, F2, and F3 follicles were dissected and diameters determined. Follicular fluid and thecal, granulosal, and cumulus cells and the oocyte were isolated and subjected to biomarker analyses. Although the size and numerous biomarkers of differentiation, such as mRNAs for the gonadotropin receptors, were similar, intrafollicular concentrations of estradiol and the abundance of mRNAs for CYP19A1 in granulosal cells and ESR1, ESR2, and CTSB in cumulus cells were greater, whereas mRNAs for AMH in granulosal cells and TBC1D1 in thecal cells were lower for animals with low versus a high AFC during follicle waves. Hence, variation in the ovarian reserve may have an impact on follicular function and oocyte quality via alterations in intrafollicular estradiol production and expression of key genes involved in follicle-stimulating hormone action (AMH) and estradiol (CYP19A1) production by granulosal cells, function and survival of thecal cells (TBC1D1), responsiveness of cumulus cells to estradiol (ESR1, ESR2), and cumulus cell determinants of oocyte quality (CTSB).

A reliable, easy to assess marker for fertility in agricultural species would be highly desirable and Anti-Müllerian Hormone (AMH) is a promising candidate. This review summarizes recent findings concerning AMH and its role in fertility management, mainly in cattle. It focuses on (1) alterations in circulating AMH concentrations from birth to puberty and during estrous cycles; (2) correlation of circulating AMH concentrations with ovarian follicle numbers and ovarian reserve; (3) factors that impact circulating AMH concentrations; (4) use of AMH as a predictor of fertility. Circulating AMH concentrations can be easily and reliably measured with a single blood sample in adult cattle because AMH varies minimally during the estrous cycle and is repeatable across multiple cycles. Circulating AMH concentrations are positively associated with several measures of fertility. Dairy heifers with low compared with higher AMH concentrations subsequently had lower pregnancy rates, higher probability of being culled after birth of their first calf and shorter herd longevity. Also, AMH is predictive of response to superovulation in cattle and sheep. Several factors contribute to the variability in AMH concentrations among individuals; for example, beef cattle have higher AMH than dairy cattle. Nutritional imbalances, disease and endocrine disruptors during fetal life may negatively program the size of the ovarian reserve and consequently serum AMH concentrations and potential fertility in adulthood. We conclude that AMH may be a predictor of fertility and herd longevity in cattle, whereas in sheep and other farm species, the potential association between AMH and reproductive performance remains largely unexplored. Free Italian abstract: An Italian translation of this abstract is freely available at http://www.reproduction-online.org/content/154/1/ R1/suppl/DC1. Reproduction (2017) 154 R1-R11