Go Kitahara

Plasma anti-Müllerian hormone as a biomarker for bovine granulosa-theca cell tumors: Comparison with immunoreactive inhibin and ovarian steroid concentrations

Granulosa-theca cell tumors (GTCTs) are the most frequently reported ovarian tumors in cattle. Clinically, GTCTs could be confused with other ovarian abnormalities; therefore, the only definitive diagnosis for such tumors is histopathology of a biopsy from the affected ovary. However, this is an invasive technique and unsuitable for farm conditions. As a result, the key aim of this study was to evaluate the diagnostic value of anti-Müllerian hormone (AMH), a glycoprotein hormone that is synthesized exclusively by ovarian granulosa cells, as a sensitive noninvasive biomarker for diagnosing GTCTs in cattle. To achieve this aim, we conducted two experiments. In experiment 1, four clinically healthy Japanese Black cows had blood samples taken daily over one estrous cycle to characterize their AMH profiles throughout the estrous cycle. Additionally, single blood samples were collected from 21 cyclic cows to clarify the physiological range of AMH. In experiment 2, cows with histologically confirmed GTCT (GTCT group, n = 9) and cows affected with cystic ovarian disease (COD group, n = 8) had one blood sample taken before extraction of the tumorous ovary or therapeutic treatment for the COD. Blood samples (n = 105) from cyclic cows (n = 25) in experiment 1 were assigned as a physiologically cyclic group (PC group). Plasma AMH, immunoreactive inhibin (ir-INH), estradiol-17β (E2), testosterone (T), and progesterone (P4) concentrations were assayed in all samples. In experiment 1, the mean plasma AMH concentration was 0.09 ± 0.003 ng/mL and did not show substantial fluctuation throughout the estrous cycle. In experiment 2, plasma AMH, ir-INH, and E2 concentrations were significantly elevated in the GTCT group in comparison with the PC group; among these parameters, only the AMH concentrations were significantly higher in the GTCT group than in the COD group. The area under the receiver operating characteristic curve of AMH for diagnosis of GTCT was 0.99 and was significantly higher than that of ir-INH (P < 0.001) and E2 (P < 0.01). Moreover, the AMH at a cutoff point of ≥0.36 ng/mL had the highest diagnostic accuracy (99.2%), sensitivity (100%), and specificity (99.1%) compared with the other tested parameters. In conclusion, plasma AMH concentration is probably a more reliable and sensitive biomarker for bovine GTCTs than the concentrations of ir-INH or ovarian steroids.

Risk factors for stillbirth and dystocia in Japanese Black cattle.

Stillbirth and dystocia are major factors reducing the productivity of beef cattle. The objective of this study was to determine the effect of season, parity and gestation length on the rates of stillbirth and dystocia in Japanese Black cattle. Calving records were obtained from 905 farms in Miyazaki Prefecture, Japan. Data were collected from 41,116 calvings in 15,378 (14.42% primiparous). There were 1013 stillbirths (2.46%) and 3514 dystocias (8.55%). The stillbirth rate in winter (December to February) (3.18%) was higher (odds ratio (OR) [95% confidence interval]: 1.008 [1.004-1.012]) than that in summer (June to August). Similarly, the dystocia rates in winter (OR: 1.011 [1.004-1.019]) and spring (March to May) (OR: 1.020 [1.013-1.027]) were significantly higher than in summer. For primiparous cows, the rates of stillbirth (OR: 1.010 [1.004-1.015]) and dystocia (OR: 1.053 [1.042-1.064]) were higher than in cows with fifth parity (reference parity). Stillbirth rates were higher in cows at ≥ 301 days of pregnancy (OR: 1.049 [1.035-1.062]) and those at ≤ 270 days of pregnancy (OR: 2.072 [2.044-2.101]) than those at between 281 and 290 days of pregnancy. Likewise, dystocia rates were higher in cows at ≥ 301 days of pregnancy (OR: 1.033 [1.008-1.059]) and those at ≤ 270 days of pregnancy (OR: 1.124 [1.095-1.154]) than those at between 281 and 290 days of pregnancy. Winter, primiparity, and long and short gestation lengths were risk factors for stillbirth and dystocia in this cohort of Japanese Black cattle.

Effects of stillbirth and dystocia on subsequent reproductive performance in Japanese Black cattle.

The objective of this study was to determine the effects of stillbirth and dystocia on subsequent reproductive performance in Japanese Black cattle. Data were collected for 34,763 calvings from 13,186 animals on 826 farms. Stillbirth was associated with a decreased first service conception rate compared to cows with a normal calving (44.4 versus 54.0%, respectively, P < 0.001). Cows with a normal calving required fewer artificial inseminations per conception and had a lower stillbirth rate at subsequent parity than those with stillbirth and dystocia (P < 0.001). Japanese Black cows with stillbirth and dystocia had inferior subsequent reproductive performance compared with those with a normal calving.

Multiple free-radical scavenging (MULTIS) capacity in cattle serum

Multiple free-radical scavenging (MULTIS) activity in cattle and human sera was evaluated with electron spin resonance spectroscopy. Scavenging rates against six active species, namely hydroxyl radical, superoxide anion, alkoxyl radical, alkylperoxyl radical, methyl radical, and singlet oxygen were quantified. The difference in the electron spin resonance signal intensity in the presence and absence of the serum was converted into the scavenging rates. Comparative MULTIS measurements were made in sera from eight beef cattle, three fetal calves and fifteen healthy human volunteers. Further, we determined the MULTIS value of albumin, the most abundant component in serum. MULTIS values in cattle sera indicated higher scavenging activity against most free radical species tested than human sera. In particular, cattle serum scavenging activities against superoxide and methyl radical were higher than human serum by 2.6 and 3.7 fold, respectively. In cattle serum, albumin appears to play a dominant role in MULTIS activity, but in human serum that is not the case. Previous data indicated that the abundance of uric acid in bovine blood is nearly 80% less than humans; however, this difference does not explain the deviation in MULTIS profile.

Serum selenium and liposoluble vitamins in Japanese Black cows that had stillborn calves.

Stillbirth and dystocia are major factors that negatively affect beef production. We sought to clarify serum selenium and liposoluble vitamin levels in Japanese Black cows that gave birth to stillborn calves (stillbirth cows). Blood samples were collected from 103 stillbirth cows and 95 cows that gave birth to healthy calves (control cows). Serum levels of selenium (45.8 ± 16.0 ng/ml) and vitamin A (73.0 ± 24.8 IU/dl) in stillbirth cows were lower (P<0.05) than those in control cows (52.2 ± 8.9 ng/ml and 93.3 ± 14.8 IU/dl, respectively). Our findings suggest that appropriate serum selenium and vitamin A levels are important for calving cows.

Presence of a temperature gradient among genital tract portions and the thermal changes within these portions over the estrous cycle in beef cows.

Abstract The aim of the present study was to describe the temperature of the different portions of the female genital tract and their relation to rectal temperature and to investigate the effect of steroid hormones profiles on these variables over the estrous cycle in cattle. Four nonpregnant Japanese Black cows were investigated daily over two successive estrous cycles using a digital thermometer with a long probe and rounded-end sensor to record the temperature of the rectum (RT), vagina (VT), cervix (CT), uterine body (UBT) and uterine horns (UHT). Blood samples were collected immediately before temperature recording to assay peripheral levels of progesterone (P4) and estradiol-17β (E2). Moreover, transrectal ultrasonography was carried out after temperature recording to monitor the ovulatory follicle and track ovulation. During the experiment, the ambient temperature and relative humidity were recorded for further calculation of the temperature humidity index (THI). The temperature within the genital tracts in these cows progressively increased towards the uterine horns from the vagina. The VT, CT, UBT and UHTs were significantly higher in association with peripheral P4 concentrations greater than 4 ng/ml (mid-luteal phase) when compared with lower peripheral P4 concentrations. The VT was more significantly (P<0.01) correlated to the CT, UBT and UHTs than RT. In conclusion, a temperature gradient was present among the vagina, cervix and uterus over the estrous cycle, and changes in peripheral P4 concentrations were associated with the thermal variations within these portions. The VT could be more beneficial than RT in monitoring temperature of deeper portions of the female genital tract in bovine.

Effects of intrauterine infusion of povidone-iodine on endometrial cytology and bacteriology in dairy cows with clinical endometritis

Endometritis is one of the major diseases causing infertility in the cow. Intrauterine infusion of povidone-iodine (PVP-I) is a common treatment. However, the optimal concentration of PVP-I for treating endometritis effectively remains unknown. We tested concentrations of 2.0% or 0.5% PVP-I for treating clinical endometritis in dairy cattle. In Experiment 1, bacteria isolated from the uterus were incubated with either 2.0% or 0.5% PVP-I, and the numbers of bacterial colonies were counted. In Experiment 2, 18 cows with clinical endometritis were treated with either 2.0% or 0.5% PVP-I (n=9 in each group). Cytology samples and bacteria were collected using a cytobrush on weeks 0 (W0), 1 (W1) and 2 (W2) after treatment. Subsequent reproductive performance was compared between the two groups. In Experiment 1, both concentrations had a similar antiseptic outcome. In Experiment 2, the percentage of polymorphonuclear neutrophils (PMN%) in the endometrial epithelium at W2 in the 2.0% group was significantly lower (P<0.05) than in the 0.5% group, although the PMN% decreased significantly from W0 to W2 (P<0.01) in both groups. Decreases in bacterial infection rates from W0 to W2 were similar in both groups. The first service conception rate was higher, numbers of services per conception were fewer, and time to conception was shorter in the 2.0% group than in the 0.5% group. Thus, an intrauterine infusion of 2.0% PVP-I was better than 0.5% in treating clinical endometritis in these dairy cattle.

Plasma anti-Müllerian hormone profile in heifers from birth through puberty and relationship with puberty onset

Abstract The current study aimed to define the plasma profile of anti-Müllerian hormone (AMH) and follicle stimulating hormone (FSH) in heifers during postnatal life until achieving puberty, as defined by plasma progesterone (P4) profile, to demonstrate a relationship between AMH and age of puberty onset. Blood samples collected from 11 Japanese Black female calves within 1 week after birth (W 0) and then biweekly until the sixth week after puberty (WP 6) were assayed for AMH, FSH, and P4. The heifers were classified into two groups based on age of puberty onset: ≤42 weeks (early puberty group; EP, n = 4) and ≥44 weeks (late puberty group; LP, n = 7). Minimal plasma AMH concentration occurring at W 0 gradually increased to its peak level by W 10 (fourfold higher than W 0; P < 0.01) before gradually declining to a steady plateau 6 weeks before puberty (WP -6). The AMH peak was preceded by a significant rise in plasma FSH at W 4, W 6, and W 8 compared with W0. Plasma AMH atW16 positively correlated withWP 4 andWP 6 (r = 0.69 and 0.71, respectively; P < 0.05). Overall plasma AMH and FSH was significantly higher and lower in EP compared with LP, respectively. In conclusion, heifers exhibit a characteristic plasma AMH profile during postnatal life, such that plasma AMH at an early prepubertal age could be a biomarker for precocious puberty and postpubertal AMH levels. Summary Sentence Plasma anti-Müllerian hormone (AMH) follows a characteristic profile in heifers during the postnatal period until puberty. Quantifying plasma AMH at early prepubertal age could be a useful biomarker for precocious puberty and postpubertal AMH levels.

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin-4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti-Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c-erbB-2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call-Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.

Associations of periparturient plasma biochemical parameters, endometrial leukocyte esterase and myeloperoxidase, and bacterial detection with clinical and subclinical endometritis in postpartum dairy cows

This study was aimed at demonstrating associations between peripheral biochemical parameters, endometrial leukocyte esterase (LE) and myeloperoxidase (MPO), and bacterial detection with the degree of endometrial inflammation, and determining the best time postpartum for diagnosing endometritis to predict subsequent fertility in dairy cows. Plasma albumin, blood urea nitrogen (BUN), total cholesterol (T-cho), NEFA, and BHBA concentrations were analyzed in 43 Holstein cows at 3, 5 and 7 weeks postpartum (W3, W5 and W7). Endometrial samples were collected at W3, W5 and W7 to examine LE and MPO activities, bacterial detection rates, and PMN% profiles. The 43 cows were divided into healthy (HE), subclinical endometritis (SE), and clinical endometritis (CE) groups, classified differently at W3, W5 and W7 based on the definitions of SE and CE for each of the three weeks pp. LE level had an association with PMN% in all weeks pp (P<0.05). Albumin and BUN levels had weak negative associations with endometrial PMN% at W3. Pathogenic bacterial detection rates were higher in the cows with endometritis at W3 and W5. Conception rate at first artificial insemination tended to be lower (P=0.057) in the cows diagnosed with endometritis at W3 than in the healthy cows. In conclusion, associations were found between endometrial LE and endometritis, but not for MPO and endometritis. Diagnosing endometritis in W3 may be the best moment to predict subsequent fertility.