Hideo Kamomae

Improvement of pregnancy rate in Japanese Black cows by administration of hCG to recipients of transferred frozen-thawed embryos.

Japanese Black primiparous and multiparous beef cows (n = 120) were selected as recipients and randomly divided into three groups (A, B, and C) of 40 recipients each. Group A received an intramuscular (i.m.) treatment of 1500 IU human chorionic gonadotropin (hCG) on day 1 (day 0 = onset of estrus), while Group B received an i.m. treatment of hCG on day 6. Group C received an i.m. treatment of 5 ml saline on day 6 as a control. On day 7, frozen-thawed embryo transfer was conducted in all groups, and pregnancy was diagnosed by palpated per rectum 40-50 days after the transfer. Twelve recipients were randomly selected from each group. Plasma progesterone (P) and estradiol-17beta (E2) concentrations were determined in these recipients on days 6, 7 and 14, and at the time of pregnancy diagnosis, and their ovaries were examined for a corpus luteum and follicles by palpated per rectum. The pregnancy rate in Group B was higher (67.5%. P < 0.05) than the rate in Group C (45.0%) and in Group A (42.5%). The plasma P concentration on day 14 tended to be higher although not significantly in Group B than in Groups C and A. At the time of pregnancy diagnosis, the blood P concentration of pregnant recipients in Group B was higher (P < 0.05) than that of those in Groups C and A. The plasma E2 concentrations on days 7 and 14 were lower (P < 0.05) in Group B than in Groups C and A. These results showed that administration of hCG 6 days after estrus improved the pregnancy rate for non-surgical frozen embryo transfer 7 days after estrus by enhancing luteal function and depressing E2 secretion.

Ultrasonic observations on the turnover of ovarian follicular cysts and associated changes of plasma LH, FSH, progesterone and oestradiol-17β in cows

Changes in the diameters of individual follicular structures on ovaries were measured by transrectal ultrasonography for 29 to 40 days and the plasma concentrations of luteinising hormone (LH) follicle-stimulating hormone (FSH), progesterone and oestradiol-17 beta were determined in four cows with ovarian cysts. When these structures decreased in size, new follicular structures appeared and developed into cysts. Progesterone concentrations in plasma were below 1.0 ng ml-1 during the experimental periods. Plasma concentrations of oestradiol-17 beta fluctuated. The mean concentration of oestradiol-17 beta in plasma differed (P < 0.01) depending on the stage of the cyst. No preovulatory surges of LH were detected during the developmental stage of the cysts.

Fasting-induced suppression of pulsatile luteinizing hormone secretion is related to body energy status in ovariectomized goats

The effect of energy status on the response of luteinizing hormone (LH) pulse frequency to acute short-term energy deficiency created by fasting in estradiol-treated ovariectomized Shiba goats was studied in two experiments. In experiment 1, eight goats whose mean body weight (BW) was 25.6 +/- 5.8 (mean +/- S.D.)kg were fed 500 g hay cubes daily for 1 week. Then they were fasted for 3 days. Blood samples were collected for 4 h at 6 min intervals on the last day of feeding, first, second and third day of fasting for LH analysis. The goats were divided into light (<24 kg, n = 4) and heavy (> or =24 kg, n = 4) groups for data analysis. There was no difference in LH pulse frequency between the last day of feeding and each day of fasting in the heavy group. LH pulse frequency was significantly (P < 0.05) suppressed on the second day (3.3 +/- 1.3 pulses/4 h) and on the third day (2.3 +/- 1.9 pulses/4 h) relative to the day prior to fasting (4.8 +/- 1.5 pulses/4 h) in the light group. In experiment 2, BW plus a body mass index (BMI (body weight (kg)/withers height (m)/body length (m))/10 [corrected]) were measured to define energy status. Nine goats (BW, 25.6 +/- 5.8 kg) were fed 500 g hay cubes daily for a week and then fasted for 3 days. Then they were divided into two groups offered either a maintenance (n = 4) or a restricted (n = 5) level of feeding for 4 weeks. The restricted level of feeding was 30% of maintenance requirement based on the BW recorded weekly. The feeding level was then adjusted to maintain BW for a further week followed by 3 day fasting for restricted animals. Blood samples were collected for 6 h at 10 min intervals on the day prior to fasting and on third day of fasting before and after the dietary manipulation. BW (26.6 +/- 2.2 to 26.8 +/- 3.8 kg) and gBMI (8.4 +/- 0.4 to 7.8 +/- 0.3) remained constant over the period prior to fasting for the maintenance animals but were significantly lower (P < 0.05) after 4 weeks for the restricted goats (BW, 26.3 +/- 2.1 to 21.5 +/- 2.4 kg; gBMI, 8.4 +/- 0.9 to 6.9 +/- 0.7). There was no significant difference in the LH pulse frequency between feeding and fasting day in both sampling periods in the maintenance group. In the restricted group, LH pulse frequency was not suppressed by fasting in the first sampling period (6.8 +/- 2.9 to 5.2 +/- 2.5 pulses/6 h), whereas it tended to be suppressed (4.8 +/- 3.1 to 1.6 +/- 2.3 pulses/6 h; P < 0.06) and was significantly (P < 0.05) correlated to body weight (r = 0.70) and gBMI (r = 0.81) after the dietary manipulation. These results suggest that the suppressive effect of short-term energy restriction (fasting) on pulsatile LH secretion is related to body energy status.

Intermittent nutritional stimulus by short-term treatment of high-energy diet promotes ovarian performance together with increases in blood levels of glucose and insulin in cycling goats

The aim of this study was to determine if short-term intermittent treatments of high-energy diet have any stimulatory effects on ovarian function and metabolic status in goat. Cycling Shiba goats were divided into treatment (TG; n=6) and control (CG; n=6) groups. After the detection of ovulation (1st ovulation, Day 0) by ultrasonography, a high-energy diet (250% of maintenance) was fed to the TG from Day 12 to Day 15 (4 days) and from Day 18 to Day 21 (4 days). The high-energy diet comprised 1000 g hay-cubes and 300 g of concentrated feed/head/day (approximately 15 MJ of digestible energy/day). The CG was offered maintenance diet throughout the experiment. Transrectal ultrasound examinations were conducted every other day during the luteal phase and were conducted daily during the follicular phase. Blood samples were collected daily from Day -2 to 7 days after ovulation (2nd ovulation) following the nutritional treatment for analysis of follicle stimulating hormone (FSH), progesterone, oestradiol, glucose and insulin in plasma. Two wave-like rises in the concentrations of glucose and insulin appeared in response to the intermittent nutritional stimulus. Mean plasma concentrations of glucose and insulin were significantly (P<0.05) higher in the TG than in the CG on almost all days during each treatment period. For ovarian performance, the total number of follicles that developed on the day prior to the 2nd ovulation in the TG (7.5 ± 2.7) was significantly higher than that of the CG (4.5 ± 2.6). Ovulation rate of the 2nd ovulation (4.5 ± 1.0) in the TG was significantly higher than that of the 1st ovulation (3.0 ± 0.6) of the TG and than both 1st (2.5 ± 0.5) and 2nd (2.7 ± 0.5) ovulations of the CG. No significant difference was detected in the concentrations of FSH, progesterone and oestradiol throughout the experiment between TG and CG. The present experiment demonstrated that short-term intermittent nutritional stimulus from the luteal phase increased the total number of ovulatory follicles and the ovulation rate in association with increasing plasma concentrations of glucose and insulin in goats.

Recombinant human activin A stimulates development of bovine one-cell embryos matured and fertilized in vitro

The effects of recombinant human activin A on the development of bovine one‐cell embryos matured and fertilized in vitro were investigated. In experiment 1, one‐cell embryos were cultured in a chemically‐defined medium, of modified synthetic oviduct fluid supplemented with 1 mg/ml polyvinyl alcohol (mSOF‐PVA), containing different concentrations of activin (0, 0.1, 1, 10, and 100 ng/ml) until 240 hr after in vitro fertilization. The addition of ‐1 ng/m activin to mSOF‐PVA improved development to the blastocyst stage (14.5–17.1%), compared with no addition of activin (5.6%). However, there was no significant difference in hatching rate of embryos among treatments. In experiments 2 and 3, the embryos were also cultured in MSOF‐PVA at various periods of exposure to 10 ng/ml activin to evaluate (development to the morula and blastocyst stages, respectively. The proportion of morulae was significantly higher in culture with activin at 20–120 hr postinsemination (37.2%) than with control (25.7%). Total number of cells in morulae at 120 hr postinsemination significantly increased by the addition of activin at 20–72 hr (26.1 cells) and 20–120 hr (24.2 cells) postinsemination, compared with control (20.1 cells). When activin was added to the medium during 20–120 hr and 20–192 hr postinsemination, the percentages of blastocysts (18.0% and 18.7%, respectively) were significantly higher than in the control (9.6%). However, the total number of cells in blastocysts was not significantly different. These results demonstrate that activin stimulates the development of bovine one‐cell embryos to the morula and blastocyst stages in vitro.

Comparison between lactating and non-lactating dairy cows on follicular growth and corpus luteum development, and endocrine patterns of ovarian steroids and luteinizing hormone in the estrous cycles

The dynamics of ovarian follicle, corpus luteum (CL), and peripheral plasma ovarian steroids were compared between lactating and non-lactating cows, and a possible association of pulsatile luteinizing hormone (LH) secretion with the dynamics was examined. Lactating (n=5) and non-lactating (n=5) cows were monitored daily for follicle and CL throughout two consecutive estrous cycles (Day 0: day of ovulation). Blood samples were collected daily and at 15 min intervals for 8h on Days 2, 4, 6, 8, and 14 of the second cycle. Lactating cows had larger CL (25.4 ± 1.8mm vs. 23.5 ± 1.5mm, P<0.01) and greater progesterone concentrations (4.6 ± 1.0ng/ml vs. 3.9 ± 0.9 ng/ml, P<0.01) during mid-luteal phase compared with non-lactating cows. Maximal diameters of the first wave dominant follicle (17.2 ± 1.8mm vs. 15.5 ± 0.8mm) and the ovulatory follicle (17.9 ± 1.2mm vs. 15.2 ± 0.8mm) were greater (P<0.05) in lactating cows than in non-lactating cows during the estrous cycles with two follicular waves, but no significant differences were detected between the groups during the estrous cycles with three follicular waves. Plasma estradiol concentrations did not differ between the groups throughout the experiment. Lactating cows had more LH pulses from Days 2 to 14 than non-lactating cows. These results imply that differences in ovarian dynamics may exist between lactating and non-lactating cows, for which the increased number of LH pulses observed in lactating cows may have responsibility.

Responses of gonadotropin secretion to short-term dietary supplementation in ovariectomized goats with different body weights

The responses of luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion to acute dietary supplementation were studied in goats with different body weights. Ovariectomized Shiba goats (n=11) were used and were maintained with a feed of 100% of their energy requirement. They were implanted subcutaneously with an oestradiol capsule and were divided into light (LBW; <or=24kg, n=6, mean+/-S.D., 21.8+/-2.7kg) and heavy body weight (HBW; >24kg, n=5, mean+/-S.D., 32.0+/-6.3kg) groups on the basis of their body weights at 8 days before the start of treatment. At the start of treatment (Day 1), the level of a feed changed to 250% of their energy requirement and this level was maintained for 7 days in both groups. Blood samples were collected daily from Day -7 to Day 7 for the analysis of FSH, glucose, and insulin profiles in plasma. Frequent blood samples were also collected at 10min intervals for 6h on Day 0, Day 3, and Day 7 for analysis of LH pulses. LH pulse frequency increased significantly on Day 3 as compared with that on Day 0 in both the HBW (7.4+/-0.5pulses/6h vs. 6.2+/-0.8pulses/6h, p<0.05) and LBW (6.5+/-0.8pulses/6h vs. 5.5+/-0.5pulses/6h, p<0.05) groups, whereas it decreased on Day 7 (HBW, 6.4+/-0.9pulses/6h; LBW, 6.3+/-1.6pulses/6h, p>0.05 vs. Day 0). Plasma glucose and insulin concentrations increased temporarily from Day 2 to Day 4 and then decreased to the level before the start of dietary supplementation in both groups. There was no significant difference in the LH pulse frequency or daily concentrations of FSH, glucose, or insulin between the HBW and LBW groups throughout the experimental period. The present study indicated that acute dietary supplementation stimulates pulsatile LH secretion in parallel with a rise of blood glucose and insulin levels. However, the influence of body weight on these responses between light and heavy animals was not observed.

Different effects of subnormal levels of progesterone on the pulsatile and surge mode secretion of luteinizing hormone in ovariectomized goats.

Abstract This study tested the hypothesis that endocrinological threshold levels of progesterone that induce negative feedback effects on the pulsatile and surge modes of LH secretion are different. Our approach was to examine the effects of subnormal progesterone concentrations on LH secretion. Long-term ovariectomized Shiba goats that had received implants of silastic capsules containing estradiol were divided into three groups. The high progesterone (high P) group received a subcutaneous implant of a silastic packet (50 × 70 mm) containing progesterone, and the low progesterone (low P) group received a similar implant of a small packet (25 × 40 mm) containing progesterone. The control (non-P) group received no treatment with exogenous progesterone. Blood samples were collected daily throughout the experiment for the analysis of gonadal steroid hormone levels and at 10-min intervals for 8 h on Days 0, 3, and 7 (Day 0: just before progesterone treatment) for analysis of the pulsatile frequency of LH secretion. Then estradiol was infused into the jugular vein of all animals at a rate of 3 μg/h for 16 h on Day 8 to determine whether an LH surge was induced. Blood samples were collected every 2 h from 4 h before the start of the estradiol infusion until 48 h after the start of the infusion. In each group, the mean ± SEM concentration after progesterone implant treatment was 3.3 ± 0.1 ng/ml for the high P group, 1.1 ± 0.1 ng/ml for the low P group, and <0.1 ng/ml for the non-P group, concentrations similar to the luteal levels, subluteal levels, and follicular phase levels of the normal estrous cycle, respectively. The estradiol concentration ranged from 4 to 8 pg/ml after estradiol capsule implants in all groups. The LH pulse frequency was significantly (P < 0.05) suppressed on Day 3 (6.2 ± 0.5 pulses/8 h) and on Day 7 (2.6 ± 0.9 pulses/8 h) relative to Day 0 (9.0 ± 0.5 pulses/8 h) in the high P group. In both the low P and non-P groups, however, the changes of pulsatile frequency of LH were not significantly different, and high pulses (7–9 pulses/8 h) were maintained on each of the 3 days they were tested. An LH surge (peak concentration, 100.3 ± 11.0 ng/ml) occurred in all goats in the non-P group, whereas there was no surge mode secretion of LH in either the high P or the low P group. The results of this study support our hypothesis that the threshold levels of progesterone that regulate negative feedback action on the LH pulse and the LH surge are different. Low levels of progesterone, around 1 ng/ml, completely suppressed the LH surge but did not affect the pulsatile frequency of LH secretion.

Relationships Between the Appearances and Changes of Estrous Signs and the Estradiol-17β Peak, Luteinizing Hormone Surge and Ovulation During the Periovulatory Period in Lactating Dairy Cows Kept in Tie-stalls

Lactating Holstein-Friesian cows kept in tie-stall barn were used as subjects in this study. Rectal examination, ultrasonography and blood sampling were conducted every other day and then daily after the day on which diameter of the corpus luteum decreased. After the luteal diameter decreased for 2 consecutive days, rectal and ultrasound examinations, blood sampling, and observation of estrous signs were conducted at 6-h intervals. Most of the estrous signs became obvious with the increase in estradiol-17β (E2) and became most remarkable 24 to 30 hours before ovulation, at which point the E2 peak and luteinizing hormone (LH) surge were achieved, and then weakened which progression to ovulation. The correlation between the intensity of four estrous signs (hyperemia and swelling of the intravaginal part of the uterus, opening of the external uterine orifice and viscosity of the cervical mucus) and the plasma E2 concentration was higher than that of three estrous signs (swelling of the vulva, contraction of the uterus, diameter of uterine horn) and the plasma E2 concentration. The relaxation of the intravaginal part of the uterus showed a unique change compared with the other estrous signs, and it became most obvious 6, 12 and 18 h before ovulation; this obviously relaxed period was consistent with the generally accepted theoretical optimal time for artificial insemination (AI), i.e., 6 to 24 h after initiation of estrus. These results suggest that observation of estrous signs by vaginoscopic examination gave useful information for detection of the optimal timing of AI in the periovulatory period in lactating dairy cows kept in a tie-stall barn.

Effect of Intrauterine Infusion with Liquid Paraffin on Phagocytes Migrating to Mucus of External os of the Cervix in Cows

The objective of this study was to investigate the effect of intrauterine infusion with liquid paraffin (LP) on phagocytic migration into the uterus of cows. Smears of swab samples of the external os of the cervix and discharges collected inside the vagina were obtained in multiparous dairy cows (n = 10) that had been infused with 50 ml of LP (LP group: n = 5) or physiological saline (PS group: n = 5) on day 10 or 11 after ovulation (day 0: ovulation). The samples were collected for cytological examination 0 (just before), 0.25, 1, 2, 3, 4, 6, 8, 12 and 24 h after LP or PS infusion and then at daily intervals until subsequent ovulation. The number of neutrophils increased significantly (p < 0.05) for 8 days compared with the pre-infusion level in the LP group and for 2 days in the PS group. The average numbers of neutrophils in the LP group were significantly (p < 0.05) greater than those in the PS group on 3, 4, 5 and 8 days after infusion. The number of monocytes from 6 h to 8 days after LP infusion was significantly higher than that before infusion (p < 0.05). The average numbers of monocytes at 4 and 6 h and 1 day after infusion in the LP group were significantly higher than those in the PS group. These findings indicate that LP stimulates phagocytic migration into the uterine lumen in cows and that LP infusion into the uterus might enhance uterine defence mechanisms during uterine infection.