Overview of Anti-Müllerian hormone (AMH) and association with fertility in female cattle

Anti-Müllerian hormone (AMH) is produced by granulosa cells of early-antral follicles found on the ovary. After production, it enters circulation and can be detected from a blood sample with an ELISA. Multiple works have found that circulating AMH is a reliable marker of the antral follicle population (AFP) of an animal as well as directly correlated to an animal's response to a superovulation protocol. Research has also found high repeatability within an animal's oestrous cycle. Further use of AMH may be valuable as a reproductive management tool, based on previous research linking productive life with circulating AMH in heifers and success to various breeding protocols by AMH concentration. The aim of this review was to summarize previous works describing basic function of AMH as well as explore recent research examining AMH as a reproductive tool and measurement of fertility in dairy animals.     

Immunohistochemical and qPCR determination of the expression and serum level of anti‐Müllerian hormone in pre‐pubertal,intact and ovarian remnant syndrome detected bitches

The aim of this study was to determine the serum concentrations, ovarian presence and expression of anti‐Müllerian hormone (AMH) in pre‐pubertal, bitches with signs of ovarian remnant syndrome (ORS) and intact bitches. In addition, we aimed to verify the suitability of serum AMH concentrations for diagnostic purposes in sterilized bitches and/or in suspected cases of ORS in the field of veterinary medicine. For this purpose, 36 healthy female dogs divided into six groups: proestrus, oestrus, dioestrus, anoestrus, pre‐pubertal and ORS. Serum AMH concentrations were determined by electrochemiluminescence immunoassay, and ovarian presence and distribution of AMH was confirmed by immunohistochemical and qPCR techniques. According to the results of qPCR, while the expression values of AMH were at the highest concentrations in the proestrus and oestrus, there was a statistically significant decrease in these values at the later stages of the cycle (p < 0.05). According to hormone analysis, the serum AMH values of the ORS group had decreased significantly compared with the proestrus and oestrus (p < 0.05). Although serum AMH levels of ORS group were increased compared with anestrus and pre‐pubertal groups, this increase was statistically non‐significant (p > 0.05). Immunohistochemically, AMH expression was first observed in the granulosa cells of primordial follicles in folliculogenesis. Expression values were the highest in the proestrous and oestrus groups, but values from bitches in later stages of the cycle were statistically significant decrease in comparison with these groups (p < 0.05). As a result, AMH concentration and expression were found to be higher in proestrus and oestrus than in other periods (p < 0.05). In addition, the measurable level of AMH concentration in bitches with ORS is an indication that it can be used in the diagnosis of ORS.     

Ovarian Follicular Dynamics. A review with Emphasis on the Bovine Species. Part II: Antral Development,Exogenous Influence and Future Prospects

During an oestrous cycle, a cohort of antral follicles develops into – depending on the species – one or more ovulatory follicles. The bovine oestrous cycle is characterized by two to three such cohorts or growth waves, only the last of which will result in an ovulation. In every growth wave, several antral follicles are recruited for development. Recruited follicles are subjected to a selection process, whereby ever decreasing levels of follicle stimulating hormone (FSH) are available to the FSH dependent follicles. In the cow, a single follicle from the cohort will acquire dominance. The ability of the dominant follicle to prosper under basic FSH levels is ascribed to a transition in hormone dependency from FSH to luteinizing hormone. The exact follicle selection mechanism remains, however, to be elucidated. The beginning of this article focuses on the recruitment, selection and dominance phases in antral follicle development. Subsequently, the conditions leading to successful maturation and ovulation are discussed. The next section expounds upon the mechanisms for exogenous modulation of follicular dynamics with the aim of superovulation/superstimulation, and finally prospective future research directions are sketched.     

Ovarian follicular growth, function and turnover in cattle: a review

Studies in cattle assessing changes in number and size of antral follicles, concentrations of estradiol, androgens and progesterone in serum and follicular fluid, and numbers of gonadotropin receptors per follicle during repetitive estrous cycles and postpartum anestrus are reviewed. The rate of growth of small follicles (1 to 3 mm) into larger follicles increases as the estrous cycle progresses from d 1 to 18 (d 0 = estrus). Size of the largest antral follicle present on the ovary also increases with advancement of the estrous cycle. Most large follicles (greater than 10 mm) persist on the ovarian surface for 5 d or more between d 3 and 13 of the bovine estrous cycle. After d 13, most of these large follicles are replaced more frequently by new growing follicles (turnover) with an increased probability for recruitment of the ovulatory follicle after d 18. More research is needed to determine the time required for growth of bovine follicles from small to large antral size and evoke recruitment of the ovulatory follicle. Factors that regulate selection of the ovulatory follicle are unknown but may involve increased frequency of LH pulses in blood, altered blood flow and(or) changes in intrafollicular steroids and proteins. Quantitative evaluation of ovarian follicles indicated occurrence of consistent short-term changes in fluid estradiol and numbers of luteinizing hormone receptors in cells of large follicles only during the pre-ovulatory period. Presumably, low concentrations of follicular estradiol found during most of the estrous cycle are not due to a lack of aromatizable precursor or follicle-stimulating hormone receptors. Follicular fluid concentrations of progesterone increase only near the time of ovulation. Little is known about changes in follicular growth, turnover and function during postpartum anestrus in cattle. However, preliminary data suggest that the steroidogenic capacity of large follicles changes markedly during the postpartum period.     

Practical aspects of the fertility of dairy cattle

To establish sustainability in the dairy industry, it is important that cows become pregnant at a biologically optimal time and at an economically profitable interval after calving. In this review, the results obtained from Holstein cattle in an experimental herd for dairy research are summarized. First, the effect of age at first calving of heifers on productive and reproductive performance was examined. A reduction in calving age from 25.1 to 21.5 months with the same growth rate during the first 12 months after birth had no negative effects on the heifers' performance. Second, the postpartum follicular dynamics of lactating cows were traced in relation to their fertility, and the emergence and fate of cystic ovarian follicles were examined. The premature initiation of ovarian activity does not always improve the fertility of cows as indicated by the number of days open. Third, the occurrences of anestrous ovulation during the early postpartum period were analyzed with reference to the frequency of reversion to anestrus. The premature onset of estrous activity also did not improve fertility, and relapse back into anestrus after the onset of the estrous cycle often occurred during the breeding period. Fourth, some indices for the occurrence of postpartum reproductive events were evaluated as an indicator of the reproductive performance of lactating cows. The milk yield and percentage of body weight loss could be indicators for reproductive events. Finally, the potency of a pedometry system for the detection of typical and atypical estrous behaviors of heifers and lactating cows was evaluated in terms of efficiency and accuracy. The location of the pedometers and housing conditions for the animals affected the estrus detection of the system. These results represent the reproductive potential of modern high-yielding dairy cattle and provide a baseline to evaluate their reproduction.     

Anti-Mullerian hormone and its relationship to ovulation response and fertility in timed AI Bos indicus heifers

This study evaluated the association between plasma anti-Mullerian hormone (AMH) concentration and fertility in Nelore (Bos indicus) heifers submitted to timed artificial insemination (TAI). At the onset of the synchronization protocol, heifers (n = 289) received a subcutaneous P4 ear implant (3 mg) and 2 mg of oestradiol benzoate. Eight days later, the P4 implant was removed and 0.5 mg of oestradiol cypionate, prostaglandin (0.265 mg, i.m.) and equine chorionic gonadotropin (300 UI, i.m.) was administered, and TAI was performed 48 hr after ear implant removal. Ovarian ultrasound evaluations were performed to measure number of ovarian follicles, dominant follicle size and ovulation response. Pregnancy diagnosis was performed by ultrasound 30 days after AI. Heifers with greater circulating AMH had more antral follicles, a smaller dominant follicle near timed ovulation and lower ovulation response to the timed AI protocol compared to heifers with lower circulating AMH. Although AMH and pregnancy outcome had a quadratic-shaped pattern, AMH was not significantly associated with fertility. In conclusion, heifers with lower AMH had larger follicles towards the end of the synchronization protocol and greater ovulation responses, whereas greater circulating AMH was unrelated to conception success.     

Non Cat-Like Ovarian Cycle in the Eurasian and the Iberian Lynx – Ultrasonographical and Endocrinological Analysis

The Iberian lynx is considered the most endangered felid species. Therefore, an ex situ conservation program was initiated to protect this species from extinction. Additional knowledge on lynx reproduction biology and reliable methods for reproductive monitoring are important for developing a captive breeding program. The aim of this study in lynx was to implement transrectal ultrasonography to visualize ovarian structures (follicles, corpora lutea) and to assess ovarian activity in addition to analysis of serum progesterone and oestradiol. Because of limited access to Iberian lynxes, the less-endangered Eurasian lynx and bobcat were also studied in this comparative study. Recent endocrinological studies based on faecal and urinary progesterone and oestrogen metabolites revealed that steroid profiles in both these species were alike and did not follow the typical pattern of other felids. Pregnancy diagnosis was not possible, since progesterone concentrations did not differ between pregnant and pseudopregnant animals. Progesterone was also detected after parturition as well as after weaning until the onset of a new oestrous cycle. In the present study, the presence of corpora lutea during the non-breeding season was confirmed by ultrasonography and by elevated serum levels of progesterone averaging 3.56 ± 1.3 ng/ml in Eurasian and 6.1 ± 0.26 ng/ml in Iberian lynx, respectively. The ultrasonographical findings on the ovarian structures suggest strongly that corpora lutea developed after ovulation stay active until November and regress before the onset of the next oestrus.     

Relationship among ovarian follicular status,developmental competence of oocytes,and anti‐Müllerian hormone levels: A comparative study in Japanese wild boar crossbred gilts and Large White gilts

The aim of this study was to investigate the ovarian follicular development, developmental competence of oocytes, and plasma anti‐Müllerian hormone (AMH) levels of Japanese wild boar crossbred (wild hybrid) gilts, whose litter size is inferior to that of European breeds. Ovary and plasma samples were collected from two different breeds of gilts (wild hybrid and Large White breeds). The ovaries from the wild hybrid gilts had a lower average numbers of secondary follicles and vesicular follicles in ovarian cross‐sections and of good quality oocytes collected from ovarian follicles as compared with those from Large White gilts (p < 0.05). The development rate to the blastocyst stage of good quality oocytes after in vitro maturation, fertilization and culture was also lower (p < 0.05) in wild hybrid gilts than in Large White gilts. Plasma AMH levels with >0.16 ng/ml were detected in 8.3% of the examined wild hybrid gilts and 33% of the Large White gilts. These results indicate that the low reproductive performance of wild hybrid breed may result in part from low numbers of vesicular follicles and good quality oocytes, and low developmental competence of oocytes. Moreover, plasma AMH levels may support low number of vesicular follicles in ovaries of wild hybrid gilts.     

Effect of growth differentiation factor‐9 (GDF‐9) on the progression of buffalo follicles in vitrified–warmed ovarian tissues

To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor‐9 (GDF‐9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF‐9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen–thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the presence or absence of GDF‐9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF‐9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF‐9 was detected in mural granulosa cells and theca cells of pre‐antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF‐9. In corpora lutea, GDF‐9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF‐9 and cultured control that indicated the GDF‐9 treatment has no effect on the primordial to primary follicle transition. GDF‐9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre‐antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF‐9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14 days, on the expression of GDF‐9. In conclusion, treatment with GDF‐9 was found to promote progression of primary follicle that could provide an alternative approach to stimulate early follicle development and to improve therapies for the most common infertility problem in buffaloes (ovarian inactivity).     

Distribution and Heterogeneity of Mast Cells in Female Reproductive Tract and Ovary on Different Days of the Oestrus Cycle in Angora Goats

The physiological distribution of mast cells (MCs) in the reproductive tract and ovary of 12 Angora goats was determined using light microscopic histochemical techniques. Uterus (corpus uteri and cornu uteri), uterine cervix, uterine tubes (isthmus and ampulla) and ovary samples were obtained by laparatomy from groups of animals during metoestrus, dioestrus and proestrus (days 5, 10 and 16 of the oestrous cycle). Tissues were fixed in Mota's fixative (basic lead acetate) for 48 h and embedded in paraffin. Six-micrometre-thick sections were stained with toluidine blue in 1% aqueous solution at pH 1.0 for 5 min and alcian blue-Safranin at pH 1.0 for 30 min. MCs were generally associated with blood vessels in all reproductive organs. In the uterus, they were concentrated mainly in the close of the uterine gland and deep stroma in the endometrium. Higher MC numbers were observed by toluidine blue staining in the uterus, uterine cervix and uterine tubes on days 10 (corpus uterine: 4.7 ± 3.8 and cornu uterine: 4.9 ± 3.5) and 16 (corpus uterine: 5.9 ± 4.5 and cornu uterine: 5.4 ± 2.4) of the oestrous cycle compared with day 5 (p < 0.05). Mast cells were not observed in the follicles, the corpus luteum and the underside of the surface epithelium of the ovarian cortex, but were observed in the interstitial cortical stroma and the ovarian medulla. In the ovary, MC numbers were significantly higher on day 16 of the oestrous cycle (cortex: 3.4 ± 2.4 and medulla: 5.7 ± 4.5, p < 0.05). Safranin-positive connective tissue MCs were not observed in the uterine tube on any occasion. These results indicate oestrous cycle-related changes in the number and location of MCs in goat reproductive organs.     

Monitoring follicular development in cattle by real-time ultrasonography: a review.

The application of real-time ultrasonography to monitoring ovarian function in mammals has advanced the understanding of follicular dynamics and its regulation. Follicular development is a wave-like sequence of organised events. The waves consist of the synchronous growth of small (4 to 5 mm) antral follicles, followed by the selection and growth of one dominant follicle which achieves the largest diameter and suppresses the growth of the subordinate follicles. In the absence of luteal regression, the dominant follicle eventually regresses (becomes atretic) and a new follicular wave begins. The dominant follicle regulates the growth of the subordinate follicles, because the appearance of the next wave is accelerated if the dominant follicle is ablated, and delayed if the lifespan of the dominant follicle is prolonged. During bovine oestrous cycles, two or three successive waves emerge, on average, on the day of ovulation (day 0) and day 10 for two-wave cycles, and on days 0, 9 and 16 for three-wave cycles. During the oestrous cycle there are thus two or three successive dominant follicles, and the last of these ovulates. Ovarian folliculogenesis is a complex process involving interactions between pituitary gonadotrophins, ovarian steroids and non-steroidal factors. Subtle changes in the hormonal milieu regulate folliculogenesis and the emergence of a follicular wave is preceded by a small increase in the concentration of plasma follicle-stimulating hormone. The mechanisms that promote the selection of a dominant follicle have not been elucidated, but considerable progress has been made in understanding follicular development and its regulation. Most treatments designed to control the development of follicular waves have been based on the physical or hormonal removal of the suppressive effect of the dominant follicle, and the consequent controlled induction of the emergence of a new follicular wave. The studies reviewed here describe current methods for regulating the bovine ovarian cycle, interesting models for future studies, and information that may be used for improving reproductive efficiency.     

Seasonal reproduction in the mare: possible role of plasma leptin, body weight and immune status

The primary objective of this study was to evaluate the possible role of leptin, body weight and immune status on reproductive activity throughout the transition period from cyclicity to seasonal anestrus, during anestrus and resumption of ovarian activity in Lusitano mares. Mares in good body condition were monthly monitored throughout 2 years (10 mares in each year) for evaluation of their reproductive status by sequential ultrasonography and plasma progesterone determinations. On the second year, all mares were weighed. Progesterone and leptin were assayed by radioimmunoassay (RIA). Parameters of the immune status (phagocytosis and oxidative burst of neutrophils, characterisation of circulating lymphocyte subsets) were also evaluated. Phagocytosis and oxidative burst in blood neutrophils were measured by flow cytometry using commercially available kits. Lymphocyte subsets were assessed by indirect immunofluorescence staining after incubation with monoclonal antibodies specific for CD2, CD19, CD4, CD8 cells markers by flow cytometry. Natural killer cells and B cells were estimated mathematically. No significant difference was found in phagocytosis, oxidative burst and circulating lymphocyte subsets at anestrus and at either phase of the estrous cycle (p>0.05), suggesting that the immune status of the mare was not influenced by the seasonal changes in ovarian activity. This study also suggests that body weight has a direct relationship with plasma leptin levels. Increased concentrations of this hormone in circulation might be associated with the restart or maintenance of ovarian cyclicity in Lusitano mares.     

Influence of follicular ablation during lactation on postweaning interval to estrus, ovulation rate, and endocrine function in sows

Duroc sows farrowed second litters in March and lactated 35 +/- 2 days. At 36 hr before weaning, electrocautery of follicles greater than or equal to 3 mm in diameter (n = 8) or sham surgery (n = 5) was performed to test the hypothesis that ablation of medium-sized follicles would prolong the duration of postweaning anestrus. Number of follicles and diameters at surgery were: 1.3 +/- .6 (greater than 5 mm diameter), 26 +/- 1 (3 to 5 mm) and greater than 20 (less than 3 mm). Blood samples were collected at 15 min intervals for 3 hr beginning at -12, 0, 12, 60 and 96 hr from weaning. Interval to estrus was 3.4 +/- .2 days in seven of eight cauterized sows and 3.6 +/- .6 days for sham-surgery sows. The remaining cauterized sow was anestrus at slaughter, 40 days after weaning. Number of corpora lutea and pregnancy rate were 15.8 +/- .6 and 92%, respectively, and were similar between sham-surgery and cauterized sows. Concentration of follicle stimulating hormone (FSH) at 12 hr before weaning was greater in sows subjected to electrocautery than for sham-surgery sows, but FSH values were similar at other sampling times. Concentrations of estradiol were similar at all times for both treatment groups. Luteinizing hormone (LH) was higher (P less than .05) at 60 hr in cauterized sows because of the onset of the preovulatory LH surge in one sow. We conclude that destruction of medium-sized ovarian follicles before weaning did not influence postweaning reproductive performance.     

Plasma Antimullerian Hormone as a Predictor of Ovarian Antral Follicular Population in Bos indicus (Nelore) and Bos taurus (Holstein) Heifers

In Bos taurus cattle, antimullerian hormone (AMH) has been demonstrated to have a high degree of correlation with ovarian antral follicle count and the number of healthy follicles and oocytes. To document the correlation between the plasma concentration of AMH and follicular number in Bos indicus and Bos taurus heifers, Nelore (Bos indicus, n = 16) and Holstein heifers (Bos taurus, n = 16) had their ovarian follicular waves synchronized. After synchronization, ovarian antral follicular population (AFP) was evaluated three times at 60‐day (d) intervals (T‐120 d, 120 days before plasma AMH determination; T‐60 d, 60 days before; and T0, at the time of plasma AMH determination). The plasma AMH concentration was positively correlated with the number of ovarian follicles on the day of the follicular wave emergence in Bos indicus (Nelore) and Bos taurus (Holstein) heifers at each evaluation time (p < 0.05). The AFP was higher in Bos indicus (Nelore) than in Bos taurus (Holstein) heifers (p < 0.05). Similarly, the AMH concentration was higher in Bos indicus (Nelore) than in Bos taurus (Holstein) heifers (p < 0.0001). When heifers were classified as to present high or low AFP according to the mean of the AFP within each genetic group, high‐AFP heifers presented a greater (p < 0.0001) AMH concentration than low‐AFP heifers, regardless of the genetic group. In conclusion, the AFP is positively correlated with plasma AMH concentration in both Bos indicus (Nelore) and Bos taurus (Holstein) heifers. Furthermore, Bos indicus (Nelore) heifers presented both greater plasma AMH concentrations and AFP than Bos taurus (Holstein) heifers.     

Follicle Wave Growth in Cattle

Ovarian follicle growth in cattle culminates in the selection of a single dominant follicle which attains the ability for final maturation and ovulation once or twice during the luteal phase and at the end of the oestrous cycle, as well as during other reproductive states. This review will describe in detail the first follicle wave of the cycle leading to selection of the first wave dominant follicle, indicating the specific gonadotrophin dependencies of cohort and dominant follicles, and relating follicle fate to steroidogenesis. As a differential gonadotrophin response of growing antral follicles during the follies‐stimulating hormone (FSH) decline may determine which follicle becomes selected, first wave follicles are also characterized in relation to intrafollicular growth factors, which may modify the gonadotrophin response, such as inhibins and members of the insulin‐like growth factor (IGF) family. Subsequently, the follicular control of the transient FSH rise and decline so crucial to dominant follicle selection will be discussed. It is concluded that successful hormonal manipulation of follicle wave growth and dominant follicle selection will depend on our detailed understanding of the gonadotrophin requirements of differentiating wave follicles.     

Follicular development in cattle; changes in their count and size during the ovarian cycle

Follicular development was examined by transrectal ultrasound scanning in 12 heifers during 51 oestrous cycles. Internal diameters of largest and second largest follicles and the number of smaller ovarian vesicles were determined. Diameters of dominant follicles showed inverse growth pattern to the second largest follicles and numbers of smaller follicles (greater than or equal to 5 mm). There was an increase in diameters of the largest follicles from beginning of dioestrous to day 9 and from time of luteolysis to ovulation, which was coincident which a decrease in diameters of the second largest follicles and numbers of smaller ovarian vesicles. Smaller follicles increased in count and diameter, when the dominant follicle achieved its largest dimension and started to regress. The cyclic corpus luteum had no local influence on diameters of the largest and second largest follicles in the ovary bearing the corpus luteum versus the contralateral ovary. Internal diameters of oestrous follicles measured 14.7 +/- 2.6 mm in heifers and 15.3 +/- 2.9 mm in cows at the day of oestrous (p greater than 0.05; t-test). Dioestrous follicles with similar size were detected during various stages of the oestrous cycle. The diameter of the dominant follicle is not an accurate criterion for determining the stage of the oestrous cycle.     

Pre‐Selection Test to Identify High Responder Donor Goats

The aim of the study was to evaluate the feasibility of pre‐selection of high or low responder does prior to the superovulatory protocols. Twenty Saanen does received 800 IU of equine chorionic gonadotropin (eCG) at the end of long‐term progestogen treatment. Fourteen days later, a second progestogen protocol associated with a multiple‐dose follicle stimulation hormone (FSH) treatment (5 IU/kg of FSH, in six decreasing doses between days 4 to 6 of the protocol) was administered. Transrectal ultrasound was used to assess the follicular status at the beginning of superovulatory treatments, at the oestrous onset and on the seventh day of the oestrous cycle for counting corpora lutea (CL). A significant lower number of CL was obtained in eCG‐treated in comparision with FSH‐treated does (p < 0.05). A quartic regression was able to explain the relationship between the number of CL in response to both treatments (r²=0.50; p < 0.05). Seventy per cent (14 of 20) of does maintained the same ovulatory response (high or low) after treatments. The Kappa (κ = 0.40; p < 0.05) and Spearman (rs = 0.39; p = 0.08) coefficients were able to show a relationship between treatments. Regarding the follicular status, there is a significant relationship between the number of small follicles (r = 0.71; r²=0.47; p < 0.01) and total follicles (r = 0.60; p < 0.01) at eCG and first FSH dose with the number of CL. Moreover, it was found a negative relationship between the presence of large follicles and the number of CL in response to eCG treatment (r = ?0.44; p < 0.05), but not from FSH (p > 0.05). In conclusion, the screening test with eCG has the potential to identify Saanen does that will better respond to the superovulatory protocol with FSH. In addition, it highlighted the importance of an ultrasound evaluation prior to the beginning of superovulatory treatments with FSH to characterize the follicular status and identify the potential donors of high ovulatory response in MOET programmes in goats.     

Association of Fertility with Numbers of Antral Follicles within a Follicular Wave During the Oestrous Cycle in Beef Cattle

The association between conception rate at first service and numbers of follicles developed during a follicular wave was examined in 102 suckled beef cows and 14 heifers. Follicular development was monitored using ultrasonography for either two (trial 1) or three (trial 2) consecutive oestrous cycles (pre-breeding, breeding and post-breeding equivalent). Animals were examined on alternate days from day 6 after first oestrus (day 0) until ovulation and from day 6 after insemination until next ovulation or day 24 of pregnancy and were observed for oestrus twice daily and inseminated artificially at either the second (trial 1) or third oestrus (trial 2). Cows were classified as having two or three waves of follicular development for each oestrous cycle. Numbers of follicles >or=4 mm per wave were determined, and based on the maximum diameter they attained, were classified as small (4-6 mm), medium (7-10 mm) or large (>or=11 mm) follicles. Total numbers of follicles, and primarily numbers of small and medium follicles, were affected by trial and within trial by cow, oestrous cycle and follicular wave. Heifers had more small and total numbers of follicles, but fewer large follicles than cows in trial 1 (p < 0.05). The average number of antral follicles per wave in the breeding cycle or post-breeding period did not affect conception rates, which averaged 84%. Repeatability of the total numbers of antral follicles between and among oestrous cycles and follicular waves ranged from 0.01 to 0.97. In conclusion, fertility was not affected by the numbers of antral follicles >or=4 mm in diameter in a single follicular wave.     

Anti‐Mullerian Hormone Concentration and Antral Ovarian Follicle Population in Murrah Heifers Compared to Holstein and Gyr Kept Under the Same Management

This study was performed to evaluate plasma concentrations of anti‐Mullerian hormone (AMH) and the ovarian antral follicle population (AFP) in different genetic groups. Cyclic heifers (13 Bubalus bubalis [Murrah]; 15 Bos taurus [Holstein] and 10 Bos indicus [Gyr]) were maintained under the same management and were synchronized with two doses of 150 μg IM d‐cloprostenol administered 14 days apart. After the second d‐cloprostenol treatment, heifers had their ovaries scanned daily by ultrasound to define the day of ovulation. On the same day, the AFP was determined and a plasma sample was collected to measure AMH. Murrah heifers had less AFP (25.6 ± 2.1 follicles; p = 0.01) and plasma AMH concentration (0.18 ± 0.03 ng/ml; p < 0.001) than Gyr (60.0 ± 12.2 follicles and 0.60 ± 0.12 ng/ml of AMH); however, data were similar when compared to Holstein (35.9 ± 6.8 follicles and 0.24 ± 0.06 ng/ml of AMH) heifers. Regardless of genetic background, there was a positive relationship between the AFP and plasmatic AMH concentration (Murrah [r = 0.62; p < 0.01], Holstein [r = 0.66; p < 0.001] and Gyr [r = 0.88; p < 0.001]). Also, when heifers were classified according to high‐ or low‐AMH concentration based on the average within each genetic group, high‐AMH heifers had greater (p < 0.0001) AFP than low‐AMH heifers. In conclusion, both Murrah and Holstein heifers presented lower plasma AMH concentration and AFP when compared to Gyr.     

Determination of serum anti‐Müllerian hormone concentrations for the diagnosis of granulosa‐cell tumours in mares

Reasons for performing study: Endocrinological assays are important for evaluation of mares with granulosa‐cell tumours (GCTs), and our research in mares indicates that anti‐Müllerian hormone (AMH) may be a good biomarker for this type of ovarian tumour. Objectives: To evaluate the use of serum AMH concentrations for endocrine diagnosis of GCTs in mares. Methods: Archived serum samples (n = 403) previously assayed for determination of serum inhibin, testosterone and progesterone concentrations (GCT panel) were assayed for serum AMH concentrations using a heterologous enzyme‐linked immunosorbent assay previously validated by our laboratory. For a subset (n = 44) of these samples, a clinical diagnosis of GCT was confirmed by histopathology. Results: Overall, the sensitivity of AMH (98%) for detection of histologically confirmed GCTs was significantly (P<0.05) greater than that of either inhibin (80%) or testosterone (48%) or the combination of inhibin and testosterone (84%). Conclusions: Determination of serum AMH concentrations is a useful biomarker for detection of GCTs in the mare. Potential relevance: Measurement of serum AMH concentrations can be used for diagnosis of GCTs in the mare. As serum AMH concentrations do not vary significantly during the oestrous cycle or pregnancy, interpretation of these results is not confounded by these physiological states.