Regulation of Anti‐Müllerian Hormone and Its Receptor Expression around Follicle Deviation in Cattle

The anti‐Müllerian hormone (AMH) is an important marker of ovarian reserve and for predicting the response to superovulatory treatments in several species. The objective of this study was to investigate whether AMH and its receptor (AMHR2) are regulated in bovine granulosa cells during follicular development. In the first experiment, granulosa cells were retrieved from the two largest follicles on days 2 (before), 3 (at the expected time) or 4 (after deviation) of follicular wave. In the second experiment, four doses of FSH (30, 30, 20 and 20 mg) or saline were administered twice a day starting on Day 2 of the first follicular wave of the cycle. Granulosa cells and follicular fluid were collected from the two largest follicles 12 h after the last injection of FSH or saline. AMH mRNA abundance was similar in granulosa cells of the two largest follicles (F1 and F2) before deviation (Day 2), but greater in dominant (DF) than subordinate follicles (SF) at the expected time (Day 3) and after (Day 4) deviation (p < 0.05). In experiment 1, AMH mRNA levels declined in both DF and SF near the expected time and after deviation when compared to before deviation. There was no difference in AMHR2 mRNA levels before and during follicular deviation (p > 0.05), but they tended to be greater in DFs than SFs (p < 0.1) after deviation. Experiment 2 showed that AMH and AMHR2 mRNA in granulosa cells and AMH protein abundance in follicular fluid were similar (p > 0.05) between both co‐dominant follicles collected from the FSH‐treated cows. These findings indicate the followings: AMH mRNA levels decrease in both DFs and SFs during follicular deviation; granulosa cells from heathy follicles express more AMH mRNA compared to subordinate follicles undergoing atresia and FSH stimulates AMH and AMHR2 mRNA expression in granulosa cells of co‐dominant follicles.     

Effects of Dietary Contamination by Zearalenone and Its Metabolites on Serum Anti‐Müllerian Hormone: Impact on the Reproductive Performance of Breeding Cows

We investigated the effects of in vivo exposure to low zearalenone levels on the anti‐Müllerian hormone endocrine levels and the reproductive performance of cattle. Urine and blood samples and reproductive records were collected from two Japanese Black breeding female cattle herds with dietary zearalenone contamination below the threshold levels (<1 ppm) at 30 days after calving. Urinary zearalenone, α‐zearalenol and β‐zearalenol concentrations were measured by chromatography‐tandem mass spectrometry, and serum anti‐Müllerian hormone concentrations were determined along with serum biochemical parameters. Urinary concentrations of α‐zearalenol were significantly higher (p < 0.05) in cattle in Herd 1 than in cattle in Herd 2, reflecting the different amounts of zearalenone in the diet of the two herds. Although the number of 5‐mm and 10‐mm follicles of the herds and their fertility after artificial insemination were similar, the serum anti‐Müllerian hormone concentrations in herds 1 and 2 were 438.9 ± 48.6 pg/ml and 618.9 ± 80.0 pg/ml, respectively, with a trend towards a significant difference (p = 0.053), which may indicate differences in the antral follicle populations between herds. Thus, zearalenone intake from dietary feed, even when below the threshold zearalenone contamination level permitted in Japan, may affect the ovarian antral follicle populations, but not the fertility, of post‐partum cows.     

The Anti‐Müllerian Hormone Profile is Linked with the In Vitro Embryo Production Capacity and Embryo Viability after Transfer but Cannot Predict Pregnancy Outcome

The current study investigated the possibility of using the AMH concentration as a predictor of the ability of Korean Hanwoo cows to produce cumulus‐oocyte complexes, embryos that survive after transfer as well as the pregnancy outcome of surrogates. Eight sessions of ovum pick‐up (OPU) were performed with 19 donor cows at an interval of 3–4 days. Antral follicle count (AFC), oocyte quality and in vitro embryo development were recorded for each cow. Embryos produced from cows with different AMH profiles were transferred into recipients (n = 96). Cows in the high (≥0.25 ng/ml) and intermediate (0.1≥ to <0.25 ng/ml) AMH groups had a significantly higher AFC per OPU session (20.40 ± 1.36 and 16.91 ± 1.52, respectively; mean ± standard deviation) than cows in the low AMH group (<0.1 ng/ml; 12.19 ± 2.14). In addition, more cumulus‐oocyte complexes per donor were recovered in the high (11.46 ± 1.22) and intermediate (7.38 ± 0.83) AMH groups than in the low AMH group (4.77 ± 0.44). The percentage of oocytes reached blastocyst stage was significantly higher in the intermediate (47.0%) and high (38.5%) AMH groups than in the low AMH group (32.3%). The number of embryos produced per cow was higher in the high (3.9 ± 0.2) and intermediate (6.9 ± 0.6) AMH groups than in the low AMH group (2.2 ± 0.3). The percentage of embryos that gave birth to viable calves when transferred into recipients was higher for those derived from cows in the intermediate AMH group (50.7%) than for those derived from cows in the low (35.7%) and high (36.4%) AMH groups. In conclusion, a single measurement of AMH concentration predicted the in vitro embryo production potential of donor Korean native cows before OPU and is linked with embryo viability after transfer into recipients.     

Characterization of anti‐Müllerian hormone in a case of bovine male pseudohermaphroditism

The current report aimed to characterize plasma anti‐Müllerian hormone (AMH) in bovine male pseudohermaphroditism. The blood AMH concentration in a Japanese Black male pseudohermaphrodite calf was compared with pre‐ and post‐pubertal male and female calves and castrated calves. The concentration in the case was higher than in post‐pubertal males, castrated males, and pre‐ and post‐pubertal female calves (p < .05), but similar to that in pre‐pubertal male calves. After extraction of the testes, the concentration in the case dropped to a certain extent. The extracted testes expressed AMH, as detected by immunohistochemistry. This study is the first to show the characterization of AMH in a male pseudohermaphrodite calf. AMH levels in peripheral blood might be useful to diagnose male pseudohermaphroditism in cattle.     

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.     

Determination of the relationship between serum anti‐Müllerian hormone level and superovulatory response in Simmental cows

The most significant focal points of the embryo transfer technology are as follows: the selection of donors, the response of the selected donor to the superovulation protocol and the obtained number of the transferable embryos. For this purpose, it is suggested that donor selection can be done by anti‐Müllerian hormone (AMH) levels, and embryo production is evaluated. AMH is secreted by the granulosa cells of primordial, pre‐antral and antral follicles below 4 mm in the ovary, independent of FSH. Therefore, the aim of this study was to investigate the relationship between serum AMH levels and the number of corpus luteum (CL), total embryos and transferable embryos that were shaped after a uniform superovulation protocol. For this reason, 48 Simmental cows, which were located at General Directory of Agricultural Enterprises (region, province, etc. instead of the general directorate), were used as donors for the embryo transfer. Blood samples were taken at random, regardless of the stage of animal's sexual cycle. AMH levels were measured by enzyme‐linked fluorescent assay (ELFA) method of the miniVIDAS^® (bioMérieux SA) using AMH Bovine Test Kit. According to the statistical analyses of the obtained data, AMH levels were positively correlated with CL and total embryos (p < .05). No significant correlations between AMH and transferable embryos were approved (p > .05). It was also determined that each 200 pg/ml increase in serum AMH level resulted in one increase in CL number. Overall, considering the positive correlation between AMH level and the obtained number of CL and total embryos after a superovulation treatment, it was concluded that measuring blood AMH level prior to any further costly implementation may be an effective method in donor selection.     

A Cross‐Sectional Study Examining the Prevalence and Risk Factors for Anti‐Microbial‐Resistant Generic Escherichia coli in Domestic Dogs that Frequent Dog Parks in Three Cities in South‐Western Ontario,Canada

Anti‐microbial resistance can threaten health by limiting treatment options and increasing the risk of hospitalization and severity of infection. Companion animals can shed anti‐microbial‐resistant bacteria that may result in the exposure of other dogs and humans to anti‐microbial‐resistant genes. The prevalence of anti‐microbial‐resistant generic Escherichia coli in the faeces of dogs that visited dog parks in south‐western Ontario was examined and risk factors for shedding anti‐microbial‐resistant generic E. coli identified. From May to August 2009, canine faecal samples were collected at ten dog parks in three cities in south‐western Ontario, Canada. Owners completed a questionnaire related to pet characteristics and management factors including recent treatment with antibiotics. Faecal samples were collected from 251 dogs, and 189 surveys were completed. Generic E. coli was isolated from 237 of the faecal samples, and up to three isolates per sample were tested for anti‐microbial susceptibility. Eighty‐nine percent of isolates were pan‐susceptible; 82.3% of dogs shed isolates that were pan‐susceptible. Multiclass resistance was detected in 7.2% of the isolates from 10.1% of the dogs. Based on multilevel multivariable logistic regression, a risk factor for the shedding of generic E. coli resistant to ampicillin was attending dog day care. Risk factors for the shedding of E. coli resistant to at least one anti‐microbial included attending dog day care and being a large mixed breed dog, whereas consumption of commercial dry and home cooked diets was protective factor. In a multilevel multivariable model for the shedding of multiclass‐resistant E. coli, exposure to compost and being a large mixed breed dog were risk factors, while consumption of a commercial dry diet was a sparing factor. Pet dogs are a potential reservoir of anti‐microbial‐resistant generic E. coli; some dog characteristics and management factors are associated with the prevalence of anti‐microbial‐resistant generic E. coli in dogs.     

Use of Porcine Luteinizing Hormone at Oestrous Onset in a Protocol for Fixed‐Time Artificial Insemination in Gilts

The aim of this study was to evaluate the effect of porcine luteinizing hormone (pLH) given at oestrous onset in gilts, by different routes and doses, on the interval between onset of oestrus and ovulation (IOEO) and reproductive performance using a single fixed‐time artificial insemination (FTAI). A total of 153 gilts were submitted to oestrous detection at 8‐h intervals and assigned to three groups: control – without hormone application and inseminated at 0, 24 and 48 h after oestrous onset; VS2.5FTAI – 2.5 mg pLH by the vulvar submucosal route at oestrous onset and a single FTAI 16 h later; IM5FTAI – 5 mg pLH by the intramuscular route at oestrous onset and a single FTAI 16 h later. More VS2.5FTAI gilts (47.1%; p < 0.05) ovulated within 24 h after oestrous onset than control gilts (25.5%) whereas IM5FTAI gilts had an intermediate percentage (31.4%; p > 0.05). The IOEO tended to be shorter (p = 0.06) in VS2.5FTAI (30.2 ± 1.4 h) than in control (34.7 ± 1.4 h) gilts, but there was no difference (p > 0.05) between control and IM5FTAI (32.8 ± 1.4 h) gilts. Farrowing rate was not different (p > 0.05) among treatments. Total born piglets (TB) was lower (p < 0.05) in VS2.5FTAI (12.3 ± 0.4) than in control gilts (14.1 ± 0.4), whereas intermediate TB was observed in IM5FTAI gilts (13.3 ± 0.4). Due to the advancement of ovulation, reduction of the hormonal dose and the ease of application, the vulvar submucosal route would be the best option for FTAI protocols, but their negative impact on litter size remains to be elucidated. Taking into account the good fertility results obtained in IM5FTAI gilts whose ovulation was not advanced, the possibility of a single FTAI without any hormonal treatment should be further investigated, to establish reliable FTAI protocols for gilts.     

Changes of serum anti‐Müllerian hormone in a mare with granulosa cell tumour following surgery and reinitiation of follicular activity

Anti‐Müllerian hormone (AMH) has been reported to be elevated in mares with granulosa cell tumour (GCT). An 8‐year‐old Thoroughbred mare was presented for not being observed in oestrus after the beginning of the breeding season. Rectal palpation and ultrasonography revealed enlargement and cystic appearance of the left ovary while the right ovary was small with an anoestrous‐like appearance. The AMH concentration was 694.9 ng/ml. Presumptively diagnosed with GCT, the mare was subjected to tumour removal. Histopathology confirmed GCT. To evaluate changes of AMH concentration following surgery, blood samples were collected immediately prior to surgery, and on Days 1, 2, 4, 8, 16 and 32 after surgery. Thereafter, blood samples were collected monthly and also at the time the mare was observed in oestrus (148 days after tumour removal). The AMH concentrations decreased over the first 2 months after surgery (from 721.2 ng/ml to 0.1 ng/ml). Subsequently, AMH concentration increased and peaked at the time of oestrus expression (0.7 ng/ml). The mare then became anoestrous, and AMH concentration decreased and reached 0.2 ng/ml, which was not significantly different from the mean concentration of AMH in normal anoestrous mares (n = 5; 0.26 ± 0.07 ng/ml). In conclusion, the present report implies the potential use of AMH for determination of the time of follicular resumption in mares after GCT removal.     

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.     

Low Concentration of Anti-Müllerian Hormone in Mares with Delayed Uterine Clearance

Luteal dysfunction has been observed in mares with defective uterine clearance. Association of low ovarian reserve with luteal dysfunction and abnormal endometrial thickness has been reported in bovine. Anti-Müllerian hormone (AMH) has been indicated as a marker for ovarian reserve in bovine and originates primarily from the ovary in equine. The present study evaluated serum AMH concentration in mares with delayed uterine clearance versus that in mares without delayed uterine clearance. Of 49 mares assigned to the study, 12 and 37 mares were diagnosed with and without delayed uterine clearance, respectively. Delayed uterine clearance was determined based on history and the observation of intrauterine fluid in ultrasonographic examination 24 hours after natural breeding. Serum AMH concentration was measured during estrus. Concentration of AMH was lower in mares with delayed uterine clearance (0.4 ± 0.1 ng/mL) than in those without delayed uterine clearance (1.1 ± 0.1 ng/mL). In conclusion, the present study indicated possible associations between ovary-lined mechanisms and uterine clearance failure in mares.     

奶牛发情周期中生殖激素浓度与卵泡发育对比分析

选择从澳大利亚购进的2岁左右发情周期为21d育成母牛10头,用放射免疫测定法（RIA）测定发情周期血清中促卵泡素、促黄体素、孕酮、雌二醇17β的浓度,用B-型超声波诊断仪检测发情周期卵巢卵泡发育变化。结果显示：优势卵泡发育体积和FSH、LH、与E2浓度在奶牛发情周期中主要表现出两个波峰．对比分析显示奶牛在发情周期第12天（优势卵泡峰时）不发情与不排卵主要原因可能是体内前列腺素等激素量不足所致。     

Elevated Serum Anti-Müllerian Hormone in an Arabian Mare with Granulosa Cell Tumor

Anti-Müllerian hormone (AMH), produced by granulosa cells, has been reported to be elevated in mares with granulosa cell tumor. A 13-year-old Arabian mare was referred after 3 years of infertility. Rectal and ultrasonographic examination revealed enlargement and multicystic appearance of the left ovary. The mare had substantially higher concentration of AMH (21.6 ng/mL) compared with normal cyclic mares (n = 5; 0.36 ± 0.02 ng/mL). Testosterone (50 pg/mL) concentration was also elevated, but progesterone concentration (0.4 ng/mL) was lowered. Accordingly, the mare was presumptively diagnosed with granulosa-theca cell tumor and a left ovariectomy implemented using ventral midline approach under general anesthesia. Histopathological examination confirmed the diagnosis of granulosa-theca cell tumor. Serum concentrations of AMH decreased to 2 ng/mL and 0.5 ng/mL 2 and 4 weeks after tumor removal, respectively. In conclusion, the current report presents confirmatory evidence that evaluation of serum AMH could be useful for diagnosis of granulosa cell tumor in mare.     

Expression of Leptin and Long‐form Leptin‐receptor Proteins in Porcine Hypothalamus during Oestrous Cycle and Pregnancy

In this study, we examined the levels of leptin and OB‐Rb protein expression in the discrete areas of the porcine hypothalamus (mediobasal hypothalamus – MBH, pre‐optic area – POA, stalk median eminence – SME) during mid‐ and late‐luteal phases of the oestrous cycle (days 10–12 and 14–16) as well as two stages of pregnancy (days 14–16 and 30–32). The analysis showed that during the cycle, leptin protein expression in MBH was higher in the mid‐luteal phase than late‐luteal phase. In the case of OB‐Rb protein expression, a higher level was observed in MBH during the late‐luteal phase in comparison to the mid‐luteal phase, whereas in POA and SME the opposite dependence was noticed. In turn, during pregnancy, leptin protein expression in MBH and POA, and OB‐Rb protein expression in POA were more pronounced on days 14–16 than on days 30–32. In contrast, leptin protein content in SME as well as OB‐Rb protein in MBH and SME was higher on days 30–32 than during the earlier stage of pregnancy. Comparison of leptin and OB‐Rb protein expression between the cycle (days 10–12) and pregnancy showed a higher level of leptin and OB‐Rb protein contents in POA as well as in SME during pregnancy (on days 14–16 and 30–32, respectively). Yet, OB‐Rb protein expression in POA on days 30–32 of pregnancy was lower in comparison to days 10–12 of the cycle. Furthermore, during pregnancy, leptin protein expression in MBH was lower (days 14–16 and 30–32), whereas OB‐Rb protein expression in that area of hypothalamus was higher (days 30–32) in comparison to the mid‐luteal phase. Our results indicate that both leptin and OB‐Rb are synthesized in the porcine hypothalamus and suggest the participation of leptin in auto/paracrine regulation of these brain areas functions, including control of reproduction during the oestrous cycle and early pregnancy.     

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.