Inflammatory Changes and Composition of Collagen during Cervical Ripening in Cows.

Dystocia and stillbirths in cows pose a high risk of loss of both dams and fetuses, thereby resulting in high economic losses. One of the causes of these problems is birth canal abnormalities. Thus, to prevent these occurrences, it is necessary to understand the mechanisms underlying cervical ripening. Although physiological inflammatory responses and changes in collagen composition have been reported in humans and mice, related information is scarce for cows. We observed inflammatory changes and changes in the collagen composition in the cervix from late pregnancy to parturition to clarify some of the physiological changes associated with cervical ripening during normal calving in cows. Cervical mucus and tissue samples were collected from 41 Japanese Black cows at 200, 230, and 260 days of gestation and at 7-day intervals thereafter until parturition. The percentage of polymorphonuclear neutrophils (PMN%) in the mucus was calculated, and interleukin (IL)-8 concentration was determined by enzyme-linked immunosorbent assay. Blood samples were collected from the jugular vein, and leukocyte counts were determined. Picrosirius red-stained cervical tissue specimens were observed under a polarizing microscope, and the percentage of type I and type III collagen areas in the cervical tissue were calculated. The PMN% in cervical mucus was lowest at 200 days gestation (12−13 weeks before delivery), significantly increased 5 weeks before (21.7 ± 0.04), and was highest 1 week before calving (50.9 ± 0.04). IL-8 levels were increased at 295 days compared with those at 200 days of pregnancy (p < 0.05). No significant changes were observed in the white blood cell counts. The percentage of type I collagen in the cervical tissue reached a maximum (91.4 ± 0.02%) on day 200, significantly decreased after 274 days (3 weeks before calving), and continued to decrease thereafter until the week of parturition. There was no significant change in type III collagen levels. The results suggest that cervical ripening progresses when PMNs begin to infiltrate the cervix at around 260 days of gestation (5−4 weeks before parturition), IL-8, which increases at the end of pregnancy, mobilizes PMNs, and enhances inflammation, and that type I collagen changes are useful as an indicator of cervical ripening.

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

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 W 0. Plasma AMH at W 16 positively correlated with WP 4 and WP 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.

The effects of extracellular ATP and its receptor antagonists on pig oocytes during in vitro maturation.

We measured the ATP concentrations in the porcine follicular fluid derived from three sizes of follicles (small: 6 mm in diameter). Then, the effects of pre-treatment (100 µM each for 30 min before maturation) with antagonists for extracellular ATP receptor P2X or P2Y on the nuclear maturation rate of cumulus-cell-enclosed (COs) or -denuded oocytes (DOs) up to the preovulatory stage in the presence or absence of 20 nM ATP (a similar concentration to that of medium-sized follicle fluid) were investigated. The antagonists used were pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) or reactive blue 2 (RB2), for extracellular ATP receptor P2X and P2Y, respectively. In addition, the embryonic development rates of COs pre-treated with RB2 were also evaluated. It was found that when the follicular sizes increased, the ATP concentrations significantly decreased (P < 0.05). No differences were observed in the nuclear maturation rates among all COs, regardless of pre-treatment with (+) or without (-) PPADS and in the presence (+) or absence (-) of ATP during maturation. In contrast, the nuclear maturation rate of the COs, but not DOs, in the ATP(-) RB2(+) group was significantly lower (P < 0.05) than that of the ATP(-) RB2(-) and ATP(+)RB2(-) groups. The pronuclear formation and blastocyst formation rates by parthenogenetic activation in the ATP(-) RB2(+) and ATP(+) RB2(+) groups were significantly lower (P < 0.05) than those in the ATP(-) RB2(-) group. In conclusion, it is suggested that the nuclear maturation of porcine oocytes may be influenced by the ATP receptor P2Y present in the cumulus cells.

Effects of AI protocol and time interval from onset of estrus to AI on conception rate in Japanese Black cows.

Two different artificial insemination (AI) protocols were investigated to determine the effect of the time interval from the onset of estrus (as detected by a pedometer) to the AI on the conception rate in Japanese Black cows. Seventy-three cows were divided into two groups that received AI either after the induction of synchronized ovulation (synchronized AI group; n = 26) or at the time of observed estrus (control AI group; n = 47). Each group was further divided into two subgroups, which were artificially inseminated either 0-12 h (early AI group; n = 21) or 12-24 h (late AI group; n = 52) after the onset of estrus. There was no significant difference in the conception rate between the synchronized AI and control AI groups. The AI protocol and the detection of estrus using a threshold of counted steps (as measured by a pedometer) in this study could be effective for planned reproduction management without the observation of standing estrus in Japanese Black cows.

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

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 (P(4)) and estradiol-17ß (E(2)). 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 P(4) concentrations greater than 4 ng/ml (mid-luteal phase) when compared with lower peripheral P(4) 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 P(4) 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.

Anti-Müllerian hormone (AMH) profiles as a novel biomarker to evaluate the existence of a functional cryptorchid testis in Japanese Black calves.

Anti-Müllerian hormone (AMH) and testosterone (T) profiles in blood were investigated before and after an hCG stimulation test to assess their sensitivity and specificity for the existence of a functional cryptorchid testis in Japanese Black calves. The hCG (3,000 IU) was administered on Day 0, and peripheral blood was collected on Days 0 (just before hCG injection), 5 and 7 in intact male calves (Intact; n=19), bilateral castrated calves (Castrated; n=17), unilateral cryptorchid calves, which abdominal testis could been extracted (Uni-crypto; n=9). Castration of a descended testis was carried in the Castrated and Uni-Crypto groups on Day -14. The AMH detectability and the optimum cut-off point for T levels using the receiver operating characteristic curve were verified to characterize the cryptorchid testis. AMH values on Day 0 were 21.1 ± 5.1 and 29.0 ± 7.5 ng/ml in the Intact and Uni-crypto groups, respectively (Mean ± SEM). AMH levels were under the detection limit in the Castrated group (i.e., < 0.006 ng/ml). T showed its peak levels on Day 5 in the Intact group (26.8 ± 4.2 ng/ml), while it remained low in the Castrated group (< 0.9 ng/ml) and did not show a significant difference in the Uni-crypto group. The detectable levels for AMH was 0.006 ng/ml, and the optimum cut-off point for T was 0.9 ng/ml; the sensitivity and specificity for evaluation of testicular descent into the scrotum were 1.0 for both the AMH and T levels. The detection rates in the Uni-crypto group using them were 1.0 and 0.57 for AMH on Day 0 and T on Days 5 or 7, respectively. In conclusion, plasma AMH profiles could be used as a novel biomarker to evaluate the existence of a functional cryptorchid testis in Japanese Black calves.