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.

Anti-Müllerian hormone profiles as a novel biomarker to diagnose granulosa-theca cell tumors in cattle.

This study was carried out to evaluate the blood profile and tissue expression of Anti-Müllerian hormone (AMH) as a biomarker for granulosa-theca cell tumors (GTCTs) in cattle. Five cases with unilateral ovarian GTCTs (GTCT group) were investigated in comparison to other groups of Japanese Black cows, which had either cystic ovarian disease (COD group, n=5), a functional corpus luteum on Days 9 to 11 of the estrous cycle (Day 0=estrus; CL group, n=13) or received superovulation treatment (SOT group, n=13). We used transrectal ultrasonography and measured plasma AMH, estradiol-17ß (E(2)), progesterone (P(4)) and testosterone (T) levels. Moreover, GTCT tissues were collected and examined by immunohistochemical staining (IHC) for AMH. In the GTCT group, ultrasound images of GTCTs were variable and not definitive. However, the AMH level in the GTCT group (n=3, 58.1 ± 66.3 ng/ml) was significantly higher than in the COD, CL and SOT groups (0.1 ± 0.1 ng/ml for GTCT vs. COD, P<0.05; 0.2 ± 0.1 and 0.3 ± 0.2 ng/ml, respectively for GTCT vs. CL and SOT, P<0.01). The other hormonal levels in the GTCT group had no significant differences compared with the COD or SOT group. Neoplastic granulosa cells labeled with AMH antibody clearly demonstrated a variety of tissue patterns in all cases by IHC. To the best of our knowledge, this is the first study to investigate the blood profile and IHC of AMH in bovine GTCTs. Our findings indicate that AMH may be a novel biomarker to diagnose GTCTs in cattle.

Relationship between the peripheral concentrations of estradiol-17ß (E2) and preovulatory characteristics of cumulus-oocyte complexes (COCs) during superovulation treatment in Japanese Black cows.

The relationship between the peripheral concentrations of estradiol-17ß (E(2)) and the preovulatory characteristics of cumulus oocyte complexes (COCs) during superovulation treatment was investigated in Japanese Black cows. A superovulation regimen with FSH treatment in a descending manner was commenced on day 7 (n=3) or day 10 (n=2) of the estrous cycle (day 0=estrus). Peripheral blood was collected to measure E(2) concentrations twice a day throughout the treatment. Ovariectomies were performed at 100 h after the initial FSH treatment in five cows. Every follicle more than 8 mm in diameter was isolated from the ovaries, and cumulus-oocyte complexes (COCs) were gently aspirated. The COCs were then separated into three groups based on the characteristics of the cumulus (compact, expanded and denuded) and subgrouped based on the stage of the nucleus in the oocytes (GV, GVBD). Plasma E(2) concentrations tended to increase gradually and reached the peak level at around 84 h (E(2)-84: n=3) or 96 h (E(2)-96: n=2) after the initial FSH treatment. The ratio of COCs with expanded cumulus was significantly higher in E(2)-84 than in E(2)-96 (P<0.01). However, there was no difference in the ratio of oocytes showing GVBD between E(2)-84 and E(2)-96 (P=0.73), and the characteristics of the cumulus did not affect the stage of the nucleus in the oocytes in either groups (compact, expanded and nude; P=0.61, 0.81 and 1.00). It was possible that the time until the peak plasma E(2) concentrations after the FSH treatment could become an indicator for the maturation of follicles and oocytes in preovulatory follicles during superovulation treatment in Japanese Black cows.

Effect of sugar supplementation on rumen protozoa profile and papillae development in retarded growth calves.

The effect of sugar supplementation with 1 g/kg BW twice a week for eight weeks on rumen protozoa was determined in ten retarded growth calves. Rumen juice was sampled by abdominal paracentesis during the experiment. Papillae development of rumens excised by experimental laparotomy was macro- and micromorphologically determined before and after sugar supplementation in a selected calf. The numbers of Entodinium, Isotricha, Dasytricha and Epidinium protozoa increased by 3 to 12 folds after 1-3 wk of supplementation and subsequently decreased. The heights of the rumen papillae after sugar supplementation showed marked development compared with before supplementation (Post vs. Pre: 4.44 ± 0.43 vs. 1.36 ± 0.24 mm). Sugar supplementation accommodates the rumen protozoa profile and stimulates papillae development in retarded growth calves.

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces disruption of glands of the prostate and fibrosis in rhesus monkeys.

We investigated the effects that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure has on the prostate in rhesus monkey offspring. Dams received 0, 30 or 300 ng/kg TCDD subcutaneously on Day 20 of gestation, and then 5% of the initial dose was injected every 30 days until Day 90 after delivery. The offspring were maintained until reaching sexual maturity, and examined histopathologically. Dose-dependent decreases in glands of the prostate and widespread fibrosis were observed in offspring. It is noteworthy that 7 years from the final lactational TCDD exposure, inflammatory cell infiltration and disruption of glands of the prostate were still observed. Differential mRNA expression associated with fibrosis, inflammatory response and disruption of cell components were demonstrated by microarray analysis, with up-regulation of TGM4, TGFB1, COL1A1 and MMP2 confirmed. In conclusion, in utero and lactational exposure to TCDD induced dose-related prostatic fibrosis, indicating prostatic dysfunction and inducible semen quality reduction in second-generation rhesus monkeys.

Sugar supplementation stimulates growth performance in calves with growth retardation.

The growth effect of sugar supplementation was determined in 49 retarded growth calves. Calves were supplemented with sugar at 1 g/kg BW 2 times weekly for 8 weeks. Glucose tolerance tests prior to the experiment showed no difference between the retarded growth calves and normal growth controls. After sugar supplementation, the calves were classified into 4 groups characterized by high (H) or low (L) periodic changes in daily weight gain (DG) with a breakpoint of 0.8 kg/d in three periods, birth to sugar supplementation (Birth-Pre), the 8 weeks during supplementation (Pre-Post) and after feeding to delivery to market (Post-Market). The periodic DG showed a marked increase after supplementation in Pre-Post and Post-Market compared with before supplementation during Birth-Pre in 2 groups (0.93 and 1.11 vs. 0.51 kg/day for L-H-H [n=19], 0.66 and 1.19 vs. 0.42 kg/day for L-L-H [n=24]), but no difference was observed in L-H-L (n=3) and L-L-L (n=3). Peripheral blood was collected on the day before supplementation (Pre), 8 weeks after supplementation (Post) and eight weeks after cease of supplementation. The blood concentrations of both insulin-like growth factor-1 (IGF-1) and glucose showed significant increases in L-H-H and L-L-H, but decreases in non-esterified fatty acid were observed in L-H-H and L-L-L on day Post compared with day Pre, respectively (p<0.05). At delivery to market, the sugar-supplemented calves had body weights similar to the market average. The growth effect of sugar supplementation could be stimulated through rumen papillae development induce by sucrose, the main component of table sugar.

Effect of embryo transfer following artificial insemination (ETFAI) on reproductive performance in dairy cows in South-Western Japan.

In order to improve the reproductive performance during the summer period, embryo transfer (ET; Japanese black embryo) following artificial insemination (AI; Holstein semen: ETFAI) was conducted in dairy cows in south-western Japan (n=56). The conception rate was improved in cows with ETFAI compared with conventional AI, which served as the control (n=195; 30.4% vs. 13.8%, P<0.01). However, higher fetal loss was observed in ETFAI compared with the controls (38.1% vs. 7.4%, P<0.05). Four cases of twin pregnancy resulted in 2 singletons and a set of twins. There was no difference in the plasma progesterone level on d0 or d7 (d0=AI), but rather lower rectal temperature was observed on d7 or d8 (38.7 degrees C vs. 39.4 degrees C and 38.8 degrees C vs. 39.1 degrees C, P<0.05) in pregnant cows compared with those that were open. ETFAI could improve reproductive performance in dairy cows during the summer period in south-western Japan.

Two cases of bovine male pseudohermaphrodites with different endocrinological and pathological findings.

Two cases of bovine male pseudohermaphrodites (PH) were subjected for clinical investigation with transrectal ultrasonography, endocrinology with adoption of hCG-stimulation test, cytogenetics with analysis of sex chromosome and Y-specific DNA, and finally histological examination. Results were compared with normal calves. Case 1 was a 10-month-old calf with XX/XY chimeras, showing elevation of testosterone (T) levels, but no change in progesterone (P(4)) after hCG test, and possessed atrophied testes in the cavitas pelvis. Case 2 was an 18-month-old calf with SRY positive-XY chromosome, showing lower level of plasma T and P(4) after hCG test, and possessed atrophied testes and undifferentiated genital ducts. Both cases possessed female-like external genitalia with similar pathological findings, however endocrinological and cytogenetical aspects were different each other.

In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces a reduction in epididymal and ejaculated sperm number in rhesus monkeys.

A long-term developmental toxicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure was performed in rhesus monkeys and the effect on male reproductive organs was determined in the second generation. Dams received 0, 30 or 300 ng/kg TCDD subcutaneously on Day 20 of gestation, and then 5% of the initial dose was injected every 30 days until Day 90 after delivery. The offspring were maintained until reaching sexual maturity, and evaluated by semen analysis, and histopathology of the testes and epididymides. Ejaculated sperm concentration was severely reduced at 300 ng/kg, and sperm viability and activity were dose-proportionally reduced, although effects on spermatogenesis were slight. Histomorphometry revealed markedly reduced area of the ductus epididymis accompanying decreased reserved sperm in the 30 and 300 ng/kg groups. In conclusion, in utero and lactational exposure to TCDD induced a reduction of sperm quality in rhesus monkeys.

Efficacy of intravaginal progesterone administration combined with prostaglandin f(2alpha) for cystic ovarian disease in Japanese Black cows.

The objective of this study was to investigate cystic ovarian disease (COD) in commercial Japanese Black cows and to evaluate the efficacy of 7-day insertion of an intravaginal progesterone insert (CIDR) combined with prostaglandin F(2alpha) (PGF(2alpha)) at CIDR removal. Experiment 1 was conducted to group cysts into 4 patterns based on alteration of plasma progesterone (P(4)) concentrations on d -7 and d 0 (=CIDR insertion) with 1.0 ng/ml as the cut-off level by ultrasonographic examination of 28 cows with COD that were >or=40 days postpartum and anoestrous after calving. In Experiment 2, a total of 55 cows under the same conditions as in Experiment 1 were utilized, and the same regimen as in Experiment 1 was performed without 7 days of pre-observation before treatment. As a result, 92.9% of CLs on d 21 were highly formed in Experiment 1 and 83.6% were highly formed in Experiment 2. The conception rates within 60 days after CIDR removal were also satisfactory high and were 71.4% and 54.5%, respectively. There were no differences in any overall reproductive parameters between Experiments 1 and 2 (P>0.05). The average days between CIDR removal and conception were 24.4 +/- 5.3 and 24.0 +/- 6.5 days, respectively (P>0.05); therefore, the conception dates of the cows in Experiment 2 were at least 7 days earlier compared with Experiment 1. In conclusion, treatment with a CIDR and PGF(2alpha) against COD could minimize the risk of incorrect treatment and provide sufficient reproductive performance in Japanese Black cows.

Efficacy of intravaginal progesterone administration as an additional treatment on two types of timed AI protocols in a commercial herd of Holstein heifers.

Reproductive performance of two types of timed artificial insemination (TAI) protocols with or without intravaginal progesterone insert (CIDR) was investigated in a commercial herd of Holstein heifers. A total of seventy-four heifers with 14.4 months of age were allocated to two groups; Ovsynch (n=44) and estradiol benzoate (EB) used Heatsynch (EB-Heatsynch, n=30), and each group was additionally divided into two subgroups with CIDR insertion from day 0 to 7 (n=36) and without CIDR group (n=38). Blood was collected for progesterone (P4) analysis and ovarian finding was monitored with ultrasonography. Heifers in CIDR-treated group resulted in higher pregnancy rate as compared to No-CIDR-treated group (63.9% vs 21.1%, P<0.01). Heifers with functional corpus luteum (CL) on day 0 resulted in significantly higher pregnancy rate in CIDR-treated group than No-CIDR-treated group (day 0: 67.9% vs 13.0%, P<0.01). CIDR insertion suppressed the intermediate ovulation during the first 7 days and the period from the second GnRH or EB administration to TAI as compared to No-CIDR-treated group (first 7 days: 33.3% vs. 52.6%; P<0.05, before TAI: 11.1% vs. 37.0%; P<0.05). In conclusion, the selected TAI protocols with CIDR provided acceptable pregnancy rate and contributed to the economical improvement by shortening the average age of first calving approximately for 2.5 months as compared to the previous management without TAI protocols.

Uterine and ovarian blood flow in a Holstein Friesian cow with aplasia of one uterine horn.

The ovarian dynamics and uterine and ovarian blood flows of a 6-year-old (2 parturitions) Holstein Friesian cow with right uterine horn aplasia were observed during two estrous cycles. In one estrous cycle, a corpus luteum (CL) formed in the right ovary, but regression of the CL and subsequent ovulation were not observed. In the other estrous cycle, a CL formed in the left ovary and delayed regression of the CL and subsequent ovulation were observed. The blood velocity of the right uterine artery was lower than that of the left uterine artery throughout both estrous cycles when a CL formed in either the right and left ovary. The blood velocities of the right and left ovarian arteries were unaffected by right uterine horn aplasia and changed depending on the presence of a CL. These results indicated that the blood flow of the right uterine artery was very weak and that aplasia of one uterine horn affects the estrous cycle, especially CL regression.

GnRH treatment at CIDR insertion influences ovarian follicular dynamics in Japanese black cows.

Ovarian follicular dynamics and estrous synchronization after Gonadotropin-releasing hormone (GnRH) treatment at Controlled Internal Drug Releasing device (CIDR) insertion were investigated in Japanese Black cows. CIDR was inserted for eight cows at 7 days after estrus. Cows were allocated to either Group A: 8-day CIDR insertion with GnRH treatment on d 0 (n=4, d 0=CIDR insertion) or Group B: 8-day CIDR insertion (n=4). Both groups were injected with prostaglandin F2alpha (PGF2alpha) on d 7. Ultrasonography and blood sampling were performed twice daily. Intensive sampling was performed every 15 min for 8 hr to determine the pulsatile release of LH on d -1, d 5 and d 10. Three of four cows showed intermediate ovulation within 2 days after GnRH treatment during CIDR insertion in Group A, whereas no ovulation was found in Group B. Three of four cows in Group A and all four cows in Group B ovulated after CIDR removal. Plasma progesterone concentrations from d 3 to d 7 in three intermediate ovulatory cows in Group A (8.4 +/- 1.6 ng/ml) was significantly higher than those in Group B (4.1 +/- 1.2 ng/ml; 4 cows) during CIDR insertion (P<0.01). Interval to estrus and ovulation after CIDR removal was observed at 60.0 +/- 12.0 hr and 76.0 +/- 6.9 hr in three cows in Group A, and 75.0 +/- 15.1 hr and 93.0 +/- 20.5 hr in Group B, respectively. There was a significant increase in LH pulse frequency on d 10 compared on d -1 or d 5 in both groups (P<0.05), in addition those on d 10 in Group A tended to be higher than in Group B. As a result, GnRH treatment at CIDR insertion at 7 days after estrus induced intermediate ovulation with formation of corpus luteum (CL) and rather synchronized emergence of ovulatory follicle during CIDR insertion. These induced CL increased plasma progesterone concentrations and contributed to precise synchronization.

Distribution of estrogen receptor alpha in the dominant follicles and corpus luteum at the three stages of estrous cycle in Japanese black cows.

Distribution of estrogen receptor alpha (ERalpha) in the dominant follicle (DF) and corpus luteum (CL) at the three stages of estrous cycle in Japanese Black cows was evaluated by means of immunohistochemistry. Ovarian dynamics were observed twice daily using ultrasonography until the ovariectomy performed on Day 7 (First group, n=3), Day 10 (Second group, n=3) and Day 18 (Third group, n=3) (Day 0=estrus). Expression of ERalpha represented by immunohistological staining intensity in cells was determined using a light microscope equipped with a digital camera. A tendency toward higher expression were observed in theca interna (TI) of DF when compared with those in mural granulosa cells (mGC), antral granulosa cells (aGC) and theca externa (TE). ERalpha expression in the Third group was lower than that in the First Group in mGC, and it was also lower than that in the second group in TE (P<0.05). ERalpha expression in luteal cells was higher than those in the stromal cells in CL. No significant difference of ERalpha expression was observed within luteal or stromal cells, except in the Second group in the luteal cells, in which significantly higher expressions than that in the Third group (P<0.05) were observed. The results showed that, 1) ERalpha was present in developing DF on Day 7, early regressing DF on Day 10 and preovulatory DF on Day 18, especially in the TI, and a few were localized in the mGC, and 2) ERalpha was highly expressed in the luteal cells and the expression decreased in combination with regression of CL.

Dynamics and histological observation of first follicular wave in Japanese black cows.

AIMS: To determine the turnover of the first follicular wave in Japanese black cows and quantitative immunohistological characteristics of the previously in vivo identified dominant follicle (DF) and largest subordinate follicle (SF) derived from ovariectomy on Day 7 (3 cows) and Day 10 (3 cows) (Day 0=estrus). Six cases of first follicular wave in cows were observed twice daily by ultrasound scanning. The number of follicles, diameter of DF and SF, and prevalence of apoptotic granulosa cells (GC) and theca cells (TC) were studied by TUNEL methods. At follicular wave emergence, 13.5 +/- 9.5 Class I (2-5 mm in diameter follicles) were found 12 hr after ovulation, and increased its number until Day 1 pm. Future DF and SF observed retrospectively were 4.9 +/- 0.8 mm and 4.9 +/- 0.9 mm at wave emergence. Deviation of DF and SF occurred on Day 3 pm with mean diameters of 8.9 +/- 1.3 mm and 6.8 +/- 0.9 mm, respectively. DF developed until Day 8 am with a maximum diameter of 14.4 +/- 1.8 mm (n=3) and then regressed. The follicular wall of the DF had a characteristic image of a healthy follicle on Day 7 and slightly atretic DF on Day 10, whereas SF showed heavy atresia on both Day 7 and Day 10 under HE staining. In the prevalence of apoptotic cells, DF were 4.4 +/- 1.0% and 17.9 +/- 4.9% on Day 7 and on Day 10 in GC, respectively, and 2.4 +/- 0.7% and 8.0 +/- 1.4% on Day 7 and on Day 10 in TC, respectively. These results showed that, 1) the first follicular wave in cows is characterized by 24 hr recruitment of small follicles and a gradual divergence of growth rates in future DF and SF, and 2) early regression of DF on Day 10 was preceded by severe apoptosis.

Estrous synchronization using an intravaginal progesterone device in combination with gnrh or estradiol benzoate characterized by the initial ovarian conditions in Japanese black cows.

Estrous synchronization using a Controlled Internal Drug Releasing device (CIDR) in combination with GnRH or estradiol benzoate (EB) treatment was investigated in Japanese black cows characterized with initial ovarian conditions. A total of 142 cows were allocated to one of four treatments: insertion of CIDR for eight days (Group A: n=34), CIDR with 100 microg of GnRH on d 0 (Group B: n=54, d 0=CIDR insertion), CIDR with GnRH on d 0 and 1 mg of EB on d 10 (Group C: n=20) or CIDR with 2 mg of EB on d 0 and 1 mg of EB on d 9 (Group D: n=34). All cows received 25 mg of PGF(2alpha) on d 7 and blood was collected for progesterone (P4) analysis on d 0, 8, and 21. AI was performed at estrus, but in Group D timed AI was set following a day of EB treatment. Estrus was induced in 141/142 cows, and the majority of which occurred on d 10 and 11 (98 cows, 34 cows). GnRH treatment induced more intermediate ovulation than EB treatment in cows with CL on d 0 (19.0% vs. 0%). Ovulation after CIDR removal was significantly higher in cows with CL on d 0 compared to those without CL (87.0% vs. 71.4%). Group B showed higher conception rates than those combined with Groups C and D where EB was injected after CIDR removal (51.1% vs. 38.9%). Conception had no correlation with either CL existence on d 0 or intermediate ovulation on d 8. P4 concentrations on d 8 were significantly lower compared to those on d 0 or d 21. On d 21 in cows without intermediate ovulation, Group A showed significantly lower P4 concentrations than the other 3 groups. The data suggests that CIDR insertion with PGF(2alpha) treatment is an effective method for estrous synchronization irrespective of initial ovarian conditions, and GnRH treatment at CIDR insertion induces intermediate ovulation and improves the conception rate in Japanese black cows.