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 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.     

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.     

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.     

Influence of a bovine respiratory disease vaccine with a temperature‐sensitive modified live or killed infectious bovine rhinotracheitis component on oestrous cycle parameters and anti‐Müllerian hormone concentration in nulliparous heifers

The objective of this study was to examine the impact of a bovine respiratory disease complex (BRDC) vaccine with a temperature‐sensitive modified live vaccine (MLV) infectious bovine rhinotracheitis (IBR) component on oestrous cycle parameters and the follicular pool. Twenty‐four Holstein heifers (12.4 ± 0.5 months) previously calfhood vaccinated with an IBR MLV component were enrolled in two replicates (Spring; n = 10 and Fall; n = 14) and were blocked by pre‐vaccination bovine viral diarrhoea (BVD) serum neutralizing (SN) titres. Upon enrolment, heifers were oestrous synchronized with sampling beginning at detected oestrus. At their second heat, heifers were vaccinated with a BRDC calfhood vaccine with a MLV (MLV; n = 12) or killed (K; n = 12) IBR component and sampled for two additional cycles. Serum samples for oestrogen (E2) and progesterone (P4) as well as ultrasound data of ovarian structures were collected every other day. Serum samples for anti‐Müllerian hormone (AMH) were collected at oestrus and mid‐cycle for each cycle, and serum for titres was collected prior to and following vaccination. Data were analysed with the PROC MIXED and GLM procedures of SAS. There was no difference in pre‐ or post‐vaccination titres between MLV and K heifers (p > .5). Vaccination had no impact on P4 concentrations, P4 area under the curve, luteal tissue area, peak E2 production or oestrous cycle length (p > .05). Cycle number did impact AMH concentration (p < .05). In MLV heifers, AMH concentration was highest in cycle 1 (p < .05) while cycles 2 and 3 did not differ (p > .05). This was also true for the K heifers in the Fall replicate (p < .05). Within cycle 2, AMH concentrations were numerically lower between vaccine types (K = 308.22 ± 33.3 pg/ml, MLV = 181.13 ± 32.9 pg/ml; p > .05). Although no differences were seen in overall cycle parameters, differences in AMH concentrations may indicate a reduction of the follicular pool following vaccination and requires further investigation.     

Inhibitory effect of heat‐killed Lactobacillus strain on immunoglobulin E‐mediated degranulation and late‐phase immune reactions of mouse bone marrow‐derived mast cells

This study investigated the in vitro effect of Lactobacillus strains, a major group of probiotic lactic acid bacteria, on immunoglobulin E (IgE)‐ and antigen‐induced mast cell degranulation and subsequent gene expression. Bone marrow‐derived mast cells (BMMCs) from DBA/2 mice were cultured with heat‐killed Lactobacillus strains for 24 h. Some strains significantly inhibited IgE‐ and antigen‐induced β‐hexosaminidase release from BMMCs. Furthermore, Lactobacillus reuteri NBRC 15892, which exhibited the strongest inhibitory activity, significantly reduced the elevated interleukin (IL)‐4, IL‐13, tumor necrosis factor‐α, and cyclooxygenase‐2 expression levels that was induced by 1–2 h of stimulation with IgE and antigens. The suppressive effect of NBRC 15892 strain on BMMC degranulation was significantly reduced in the presence of a toll‐like receptor (TLR)2‐neutralizing antibody. In addition, downregulation of cell surface FcεRIα expression was observed after 6 h of NBRC 15892 treatment. These results suggest that some Lactobacillus strains inhibited IgE‐mediated mast cell degranulation and subsequent late‐phase reactions involving mast cells via a TLR2‐dependent mechanism with FcεRIα downregulation.     

The preliminary study on the effects of growth hormone and insulin‐like growth factor‐I on κ‐casein synthesis in bovine mammary epithelial cells in vitro

The effects of growth hormone (GH) and insulin‐like growth factor‐I (IGF‐I) on protein synthesis and gene expression of κ‐casein in bovine mammary epithelial cell in vitro were studied. The treatments were designed as follows: the growth medium without serum was set as the control group, while the treatments were medium supplemented with GH (100 ng/ml), IGF‐I (100 ng/ml), and GH (100 ng/ml) + IGF‐I (100 ng/ml). The quantity of κ‐casein protein was measured by ELISA, and the κ‐casein gene (CSN3) expression was examined by real‐time quantitative PCR (RT‐qPCR). Compared with the control group, all the experimental groups had greater (p < 0.05) expression of CSN3. The concentration of κ‐casein followed a similar response as CSN3, but the difference between the treatments and the control was not statistically significant (p > 0.05). Furthermore, no synergistic effect of GH and IGF‐I was observed for both the κ‐casein concentration and CSN3 expression. It is therefore concluded that GH or IGF‐I can independently promote the expression of CSN3 in bovine mammary epithelial cells in vitro.