Genomic heritability and genome-wide association analysis of anti-Müllerian hormone in Holstein dairy heifers.

Anti-Müllerian hormone (AMH) is an ovarian growth factor that plays an important role in regulation of ovarian follicle growth. The objectives of this study were to estimate the genomic heritability of AMH and identify genomic regions associated with AMH production in a genome-wide association (GWA) analysis. Concentrations of AMH were determined in 2,905 dairy Holstein heifers genotyped using the Zoetis medium density panel (Zoetis Inclusions, Kalamazoo, MI) with 54,519 single nucleotide polymorphism (SNP) markers remaining after standard genotype quality control edits. A linear mixed model was used to model the random effects of sampling day and genomics on the logarithm of AMH. The genomic heritability (± standard error of the mean) of AMH was estimated to be 0.36 ± 0.03. Our GWA analysis inferred significant associations between AMH and 11 SNP markers on chromosome 11 and 1 SNP marker on chromosome 20. Annotated genes with significant associations were identified using the Ensembl genome database (version 88) of the cow genome (version UMD 3.1; https://www.ensembl.org/biomart). Gene set enrichment analysis revealed that 2 gene ontology (GO) terms were significantly enriched in the list of candidate genes: G-protein coupled receptor signaling pathway (GO:0007186) and the detection of chemical stimulus involved in sensory perception (GO:0050907). The estimated high heritability and previously established associations between AMH and ovarian follicular reserve, fertility, longevity, and superovulatory response in cattle implies that AMH could be used as a biomarker for genetic improvement of reproductive potential.

Development of the "waveless" bovine model.

Development of a bovine model without ovarian follicular waves (waveless) and transient increases in gonadotropin secretion during estrous cycles may lead to new methods to more consistently regulate ovulatory follicle growth thereby improving efficiency of embryo transfer. We hypothesized that the GnRH antagonist acyline would inhibit gonadotropin secretion thereby blocking follicular waves, ovarian function and ovulation during estrous cycles of cattle. To test this hypothesis, beef heifers (n = 5 per group) were treated twice daily with vehicle (control) or 25 or 50 µg/kg acyline beginning 12 h after GnRH-induced ovulation and ending 21 days later. Each animal was subjected to ovarian ultrasonography for 25 days to monitor number and growth of follicles ≥3 mm in diameter and growth of the corpus luteum (CL). Blood samples were taken at various intervals to determine circulating concentrations of FSH, LH, anti-Müllerian hormone (AMH, marker of small follicle growth) and progesterone. Results show that acyline treatment decreased or blocked: circulating concentrations of LH, transient FSH spikes associated with emergence of follicular waves, preovulatory FSH/LH surges, follicular waves, dominant follicle development, CL growth, and progesterone production. In contrast, the largest acyline dose increased AMH concentrations. In conclusion, long-term acyline treatment blocks follicular waves but not growth of preantral and small antral follicles (≤ 3 mm). Future studies will determine if the waveless bovine model, which has enhanced development of preantral and small antral follicles, can be used to develop new methods to improve predictability of response of cattle to superovulation.

A single ultrasound determination of ≥25 follicles ≥3 mm in diameter in dairy heifers is predictive of a reduced productive herd life.

Fertility and productive herd life (time in herd after birth of first calf) are inferior in dairy cows with relatively low compared with intermediate but not high numbers of follicles growing during ovarian follicular waves. The present study, therefore, tested the hypothesis that fertility and productive herd life are lower in dairy heifers with high follicle numbers compared with age-matched herdmates with fewer follicles. To test this hypothesis, 11 to 15 mo old Holstein heifers were subjected to a single ultrasound measurement of the number of follicles ≥3 mm in diameter. Heifers were classified into a high- (≥25 follicles), mid- (16-24), or low-range (≤15) follicle number group (FNG). All heifers not removed from the herd before first calving (n = 408) had the opportunity to start their fifth or sixth lactation after birth of their first calf. During this time, performance and health parameters for each individual were recorded daily by herd managers. Results showed that heifers in the high-range FNG had a 180-d shorter productive herd life, reduced survival rate, and greater probability of being culled after birth of the first calf, as well as fewer lactations compared with heifers in the low-range FNG. Cows in the high-compared with the mid- or low-range FNG also had greater involuntary culling rates, days open, and services per conception, and lower pregnancy rates during the first, second, or third lactations. We concluded that dairy heifers with ≥25 follicles ≥3 mm in diameter have suboptimal fertility and a shorter productive herd life compared with herdmates with fewer follicles.

Anti-Müllerian Hormone (AMH) and fertility management in agricultural species.

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.

Concentration of anti-Müllerian hormone in dairy heifers is positively associated with productive herd life.

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.

Heritability and impact of environmental effects during pregnancy on antral follicle count in cattle.

Previous studies have documented that ovarian antral follicle count (AFC) is positively correlated with number of healthy follicles and oocytes in ovaries (ovarian reserve), as well as ovarian function and fertility in cattle. However, environmental factors (e.g., nutrition, steroids) during pregnancy in cattle and sheep can reduce AFC in offspring. The role that genetic and environmental factors play in influencing the variability in AFC and, correspondingly, the size of the ovarian reserve, ovarian function, and fertility, are, however, poorly understood. The present study tests the hypothesis that variability in AFC in offspring is influenced not only by genetic merit but also by the dam age and lactation status (lactating cows vs. nonlactating heifers) and milk production during pregnancy. Antral follicle count was assessed by ultrasonography in 445 Irish Holstein-Friesian dairy cows and 522 US Holstein-Friesian dairy heifers. Heritability estimates for AFC (± standard error) were 0.31 ± 0.14 and 0.25 ± 0.13 in dairy cows and heifers, respectively. Association analysis between both genotypic sire data and phenotypic dam data with AFC in their daughters was performed using regression and generalized linear models. Antral follicle count was negatively associated with genetic merit for milk fat concentration. Also, AFC was greater in offspring of dams that were lactating (n=255) compared with nonlactating dams (n=89) during pregnancy and was positively associated with dam milk fat concentration and milk fat-to-protein ratio. In conclusion, AFC in dairy cattle is a moderately heritable genetic trait affected by age or lactation status and milk quality but not by level of dam's milk production during pregnancy.

Effects of maternal environment during gestation on ovarian folliculogenesis and consequences for fertility in bovine offspring.

Mammals such as cattle, swine, sheep and humans are born with a highly variable number of ovarian follicles and oocytes in the ovaries that dwindle during ageing and are never replenished. This variation in the ovarian reserve is reflected in the numbers of antral follicles in the ovaries at all ages after birth. As numbers of follicles in ovaries are determined during gestation, the role of maternal nutrition and health during gestation (at time of ovarian development in their foetuses) has been investigated as factors that may impact oogonia proliferation and thus follicle numbers post-natally. These studies have found that both nutrition and health impact numbers of follicles in their offspring. The idea that numbers of follicles and oocytes in ovaries impact fertility is a long-held belief in reproductive biology. This has recently been tested in cattle, and it has been shown that cows with a relatively high number of antral follicles in ovaries have higher pregnancy rates, shorter calving to conception intervals and fewer artificial inseminations during the breeding season compared with cows with a lower number of follicles, and similarly, heifers with many follicles had higher pregnancy rates than those with fewer follicles. Studies summarized in this review highlight the importance of the maternal environment during gestation in determining the size of the ovarian reserve in their offspring and also the contribution of the ovarian reserve to subsequent fertility in cattle.

Does size matter in females? An overview of the impact of the high variation in the ovarian reserve on ovarian function and fertility, utility of anti-Müllerian hormone as a diagnostic marker for fertility and causes of variation in the ovarian reserve in cattle.

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.

Evidence that high variation in antral follicle count during follicular waves is linked to alterations in ovarian androgen production in cattle.

Androgens have an important role in ovarian follicular growth and function, but circulating androgen concentrations are also associated with ovarian dysfunction, cardiovascular disease, and metabolic disorders in women. The extent and causes of the variation in androgen production in individuals, however, are unknown. Because thecal cells of follicles synthesize androstenedione and testosterone, variation in production of these androgens is hypothesized to be directly related to the inherently high variation in number of healthy growing follicles in ovaries of individuals. To test this hypothesis, we determined whether thecal CYP17A1 mRNA (codes for a cytochrome P450 enzyme involved in androgen synthesis), LH-induced thecal androstenedione production, androstenedione concentrations in follicular fluid, and circulating testosterone concentrations were lower in cattle with relatively low versus high number of follicles growing during follicular waves and whether ovariectomy reduced serum testosterone concentrations. Results demonstrated that cattle with a low follicle number had lower (P<0.05) abundance of CYP17A1 mRNA in thecal cells, reduced (P<0.01) capacity of thecal cells to produce androstenedione in response to LH, lower (P<0.01) androstenedione concentrations in ovulatory follicles, and lower (P<0.02) circulating testosterone concentrations during estrous cycles compared with animals with high follicle number. Also, serum testosterone in cattle with low or high follicle number was reduced by 63 and 70%, respectively, following ovariectomy. In conclusion, circulating androgen concentrations are lower in cattle with low versus high number of follicles growing during follicular waves, possibly because of a reduced responsiveness of thecal cells to LH.

Inherent capacity of the pituitary gland to produce gonadotropins is not influenced by the number of ovarian follicles > or = 3 mm in diameter in cattle.

We hypothesised that higher serum FSH concentrations in cattle with low v. high follicle numbers during follicular waves are caused by a different capacity of the pituitary gland to produce gonadotropins. Dairy cows with high (> or = 30; n = 5) and low (< or = 15; n = 5) follicle numbers were selected and serum concentrations of oestradiol and FSH during an oestrous cycle were measured. Cows were ovariectomised at oestrus and bled frequently up to 8 days after ovariectomy. After 33 days, cows were injected with gonadotropin-releasing hormone (GnRH) and bled intensively up to 8 h after GnRH injection. One day later, animals were injected with follicular fluid (FF) from bovine follicles and were bled intensively up to 2 days after the first injection. Serum concentrations of FSH and LH were measured. After 2 days, cows were killed and their pituitary glands collected. Prior to ovariectomy, serum oestradiol concentrations were similar between groups, whereas FSH concentrations were higher in cattle with low v. high numbers of follicles. No differences were detected in serum gonadotropin concentrations after ovariectomy, GnRH injection or FF challenge between groups. The results indicate that the inherent capacity of the pituitary gland to secrete gonadotropins does not differ between cattle with high v. low numbers of follicles during follicular waves.

Causes and consequences of the variation in the number of ovarian follicles in cattle.

In cattle we have noted that the antral follicle count (AFC, follicles > or = 3 mm in diameter) varies greatly among animals (from 5 to 50), is repeatable within animals, and is highly correlated with the total number of healthy follicles in ovaries. Also, animals with low AFC have higher serum concentrations of FSH and LH, but lower concentrations of Anti-Mullerian Hormone, progesterone and androgens than animals with high AFC. We have investigated the effect of maternal environment during gestation on their offspring AFC by restricting maternal nutrition to 60% of maintenance requirements (compared with 100% in controls) during the first third of gestation. Calves born to nutritionally restricted mothers had 60% lower AFC compared with calves born to mothers fed control diets. In other studies we have evidence to indicate that fertility may be compromised in animals with low AFC due to effects on oocytes, progesterone and the endometrium compared with animals with high AFC. To examine this directly we assessed AFC in post-partum dairy cows and found that cows with a high AFC had higher pregnancy rates, shorter calving to conception intervals and received fewer services during the breeding season compared with cows with a low AFC. In addition, the high variation in follicle numbers in adults may not only be reflective of reproductive disorders and suboptimal fertility, but there is evidence to indicate that it may be associated with alterations in the function of other non-reproductive systems (e.g. cardiovascular) that may have profound effects on the animal's health and welfare.

Evidence that high variation in ovarian reserves of healthy young adults has a negative impact on the corpus luteum and endometrium during estrous cycles in cattle.

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.

Variation in the ovarian reserve is linked to alterations in intrafollicular estradiol production and ovarian biomarkers of follicular differentiation and oocyte quality in cattle.

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).

Antral follicle count reliably predicts number of morphologically healthy oocytes and follicles in ovaries of young adult cattle.

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.

Follicle numbers are highly repeatable within individual animals but are inversely correlated with FSH concentrations and the proportion of good-quality embryos after ovarian stimulation in 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 (> or =25) or low (< or =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.

Evidence for autocrine or paracrine roles of alpha2-macroglobulin in regulation of estradiol production by granulosa cells and development of dominant follicles.

alpha(2)-Macroglobulin (alpha(2)-M) inhibits proteinases and modulates the actions of growth factors and cytokines. Despite the key roles proteinases, growth factors, and cytokines have in folliculogenesis, the role of alpha(2)-M in follicular development is unknown. Our objectives were to: 1) determine whether granulosa cells produce alpha(2)-M and have alpha(2)-M receptors, 2) examine the effect of alpha(2)-M on estradiol production by granulosa cells, 3) establish whether amounts of alpha(2)-M and alpha(2)-M receptors were altered during dominant nonovulatory follicle development, and 4) examine alpha(2)-M's mechanism of action. The results demonstrated that bovine granulosa cells contain 5.2- and 15-kb mRNAs and 720- and 500-kDa proteins that correspond, respectively, to sizes of mRNAs and proteins for alpha(2)-M and the alpha(2)-M receptor. Treatment of granulosa cells with alpha(2)-M resulted in a specific dose-responsive increase in estradiol production. Cell viability, cell number, and the amount of aromatase in granulosa cells were not altered by alpha(2)-M. Treatment of granulosa cells with factors that bind alpha(2)-M or its receptor did not mimic alpha(2)-M action. Although intrafollicular amounts of alpha(2)-M remained unchanged, amounts of alpha(2)-M receptor in granulosa cells were strongly inversely associated with concentrations of estradiol in dominant and subordinate follicles. Based on these results, we concluded that alpha(2)-M may have autocrine or paracrine roles in granulosa cells potentially important for regulation of estradiol production and development of dominant follicles.

Evidence for a negative intrafollicular role for inhibin in regulation of estradiol production by granulosa cells.

Intrafollicular concentrations of inhibin A and estradiol vary inversely during development of dominant follicles in cattle. Thus, we hypothesized that inhibin has a negative autocrine or paracrine effect on estradiol production by granulosa cells. To examine this hypothesis, a homologous model system was used to test the effects of bovine antibovine inhibin antibodies, bovine inhibin, and a peptide fragment of bovine inhibin (bINH) on capacity of granulosa cells isolated from individual estrogen-active or -inactive dominant or subordinate follicles to produce estradiol during short-term (18 h) serum-free culture. Immunoblot analysis of media demonstrated that granulosa cells basally produce different molecular weight forms of inhibin, similar to those in bovine follicular fluid. Immunoneutralization of endogenous inhibin in culture with different doses (12.5-1000 microg) of highly purified bovine antibovine inhibin antibodies increased estradiol production 2- to 15-fold, compared with controls. Preadsorption of the anti-inhibin antibodies with bINH precursors or bovine pro-alpha(C) suppressed the capacity of anti-inhibin antibodies to enhance estradiol production by granulosa cells, compared with controls. Treatment of granulosa cells with an immunoaffinity-purified preparation of bINH suppressed basal estradiol production 60%, compared with controls. In contrast, treatment of granulosa cells with the bINH peptide increased estradiol production 14-fold, compared with controls. Based on these results, we concluded that both anti-inhibin antibodies and bINH blocked the suppressive local effects of basally produced inhibin on estradiol production during culture of granulosa cells and that inhibin has a negative autocrine or paracrine effect on the in vitro capacity of granulosa cells isolated from dominant or subordinate follicles to produce estradiol.

Development and validation of a short-term, serum-free culture system for bovine granulosa cells: evaluation of the effects of somatotropin and growth hormone-releasing factor on estradiol production.

The objective of this study was to develop and validate a short-term, serum-free culture system to determine whether recombinant bovine somatotropin (rbST) or recombinant bovine growth hormone-releasing factor (rbGRF) altered the estradiol-producing capacity of bovine granulosa cells isolated from dominant or subordinate follicles of the first follicular wave. Thus, ovaries were obtained at an abattoir from cows that were between d 2 to 5 or 6 to 10 of the estrous cycle. Three size classes of follicles were isolated from each cow's ovaries: small (2 to 5 mm in diameter), medium (6 to 14 mm), or the largest (6 to 19 mm). In vivo steroid-producing capacity of follicles was assessed by measuring concentration of estradiol, progesterone, androstenedione and 5alpha-dihydrotestosterone in each follicle. In vitro steroid-producing capacity was assessed by culturing granulosa cells from the different follicle sizes for 48 h in serum-free media with 19-OH androstenedione and measuring the estradiol and progesterone concentrations in media at the end of culture. The effect of different doses of FSH, rbST, or rbGRF on estradiol and progesterone production by granulosa cells from each follicle size class during d 2 to 5 or 6 to 10 was also evaluated. A high percentage (91.7%) of the largest follicles obtained on d 2 to 5 was estrogen-active (estradiol > progesterone) compared with other follicle classifications (d 2 to 5, small = 0%, medium = 13.8%; d 6 to 10, small = 0%, medium = 3.3%, largest = 33.3%). Estradiol was highest (P < 0.05) in the largest follicle on d 2 to 5 and correlated positively with follicle diameter. The pattern of in vitro production of estradiol by granulosa cells from the different follicle size classes reflected the original in vivo capacity of follicles to produce estradiol. However, only granulosa cells from the largest estrogen-active follicle on d 2 to 5 produced more estradiol than progesterone in vitro. Progesterone production by granulosa cells from all follicle classifications was increased by FSH, but FSH only enhanced estradiol production by granulosa cells from the largest estrogen-active follicles on d 2 to 5. Recombinant bST blocked the FSH-induced increase in estradiol by granulosa cells from the largest estrogen-active follicles on d 2 to 5, whereas rbGRF had no effect on steroid production. Based on these results, we concluded that short-term, serum-free culture of bovine granulosal cells obtained from first-wave follicles at an abattoir could be used to reflect reliably the original in vivo estradiol-producing capacity of granulosal cells, and that neither rbST nor rbGRF enhance basal or FSH-induced estradiol production by bovine granulosa cells from first-wave follicles.

Effect of long-term infusion with recombinant growth hormone-releasing factor and recombinant bovine somatotropin on development and function of dominant follicles and corpora lutea in Holstein cows.

The objective of this study was to evaluate the effects of recombinant bovine growth hormone-releasing factor (rGRF) or recombinant bovine somatotropin (rbST) on growth and function of the first-wave dominant follicle and corpus luteum. Primiparous Holstein cows (117 d postpartum) were infused with 12 mg/d of rGRF (n = 10) or 29 mg/d of rbST (n = 10) for 63 d, and non-infused cows (n = 10) were controls. At slaughter on d 5 of an estrous cycle, blood and ovaries were collected and data from cows with a corpus luteum were analyzed (control, n = 8; rGRF, n = 5; rbST, n = 6). Treatment with rGRF or rbST increased somatotropin (ST) and IGF-I in serum similarly compared with controls. In contrast, rbST-treated cows had higher concentrations of ST in follicular fluid (FF) compared with rGRF-treated and control cows. In addition, rbST, but not rGRF, increased the number and decreased the size of estrogen-active follicles (EA; estradiol > progesterone concentrations in FF), increased the abundance of IGF binding proteins-2, -3, and -4 in FF from EA follicles, and increased the number but decreased the size of corpora lutea and decreased concentration of progesterone in serum compared with controls. Based on these results, we concluded that long-term infusion of rbST alters growth and function of the first-wave dominant follicle and the corpus luteum in cattle.

Effect of long-term immunization against inhibin on sperm output in bulls.

To determine the effect of neutralization of inhibin on sperm output, 12 Holstein bulls were paired by birth date and weight on Day 1 of age. Each bull was actively immunized against bovine inhibin alpha1-26 gly-tyr (bINH) conjugated to human alpha globulin (HAG, n = 6 bulls) or HAG alone (controls, n = 6) at 60 days of age; booster immunizations were administered at 90, 104, 124, 270, and 395 days of age. Body weights and scrotal circumferences were measured at the time of primary immunization and at 10 days after each booster. In addition, jugular blood was obtained at 60, 70, 100, 114, 134, 280, and 405 days of age, during the 3-wk sperm collection period, and during a 6-h blood-sampling period after sperm collection to determine bINH antibody titer and concentrations of FSH, LH, testosterone, and estradiol. Beginning at 405 days of age, sperm output was measured 3 days/wk for 3 wk with two successive ejaculates collected each day for a total of 18 ejaculates per bull. During Days 60-405 of age, the increase in titer of bINH antibodies, scrotal circumference, and serum concentration of FSH was greater (p < 0.01) for the bINH-immunized compared with control bulls. There were significant (p < 0.01) pair x treatment interactions for sperm output and serum FSH and LH concentrations. Specifically, bINH-immunized bulls for four of the six pairs had nearly 50% greater serum FSH concentrations and sperm output. For the remaining two pairs, sperm output was lower and FSH was either lower or only marginally higher in the bINH-immunized bulls compared with controls. Also, the control bulls for the two remaining pairs produced more sperm than all but one bINH-immunized bull, and had markedly higher serum LH concentrations than all other bulls. To summarize, enhancement of sperm output after immunization against inhibin depends on the subsequent increment in FSH concentrations. We conclude that inhibin suppresses spermatogenesis. Thus, methods to immunoneutralize inhibin may have merit as a therapeutic route to enhance sperm production in reproductively maturing bulls.