The effects of unilateral ovariectomy (ULO) before mating on maintenance of pregnancy, plasma progesterone levels and ovarian hypertrophy were examined using adult rabbits. The values related to reproductive efficiency, such as number of ovulations and implantation sites, implantation rates, size of uterine swellings, litter size, weight of young, birth rate, duration of pregnancy and peripheral plasma progesterone levels per doe, did not show any significant differences between ULO and intact does. The mean ovarian weight and the volume of ovaries after the second parturition in ULO and intact does became slightly larger than those of ovaries after the first parturition, though no significant differences were noticed between the first and the second. The ovarian weight and volume of ULO does after the second parturition was significantly larger than those of removed ones at ULO (p<0.05). These results suggest that ULO before mating did not affect the maintenance of pregnancy, fetal growth and litter size by the compensatory function of the remaining ovary in the ULO animal.

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The present studies were conducted to clarify the effective treatment for cows with unobserved estrus. In Exp. 1, 12 cows which had received 15 mg of PGF_2α on Day 12 after ovulation were divided into four groups of 3. They were injected with 200, 100, 50 and 25μg of LH-RH-A for Groups I, II, III and IV, respectively, 54 hrs after PGF_2α and inseminated at 24 hrs after LH-RH-A. In Exp. 2, 15 cows which had palpable corpus luteum (CL) were divided into three groups of 5. They were injected with 200, 100 and 50μg of LH-RH-A for Groups I, II and III, respectively, after PGF_2α and inseminated on the same time schedule as in Exp. 1. Ten control cows were in-seminated during spontaneous estrus. Ovulation was induced by 2 days after LH-RH-A and CL was formed in the cows of all groups. The LH levels at 2 hrs after LH-RH-A tended to increase in proportion to the dosage. The progesterone (P) levels in Exp. 1 in-creased gradually for 8-9 days after ovulation in the cows of Groups I and II as compared with that of Groups III and IV. The mean P level of Exp. 2 on Day 13 after ovulation was significantly (P<0.05) lower in Group I than in control. By the timed insemination, 1 cow of each group in Exp. 1, and 1 cow of Group I, 3 cows of Groups II and III, 6 cows of controls in Exp. 2 had conceived. These results indicate that the present treatments may be applicable to cows with unobserved estrus, and the effective dose of LH-RH-A is 25 or 50μg.

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This study was conducted to examine the effect of estrogen and/or pro-gesterone on intrauterine pressure during late stages of pregnancy in rats. On day 14 of pregnancy, rats were sham-ovariectomized or ovariectomized, and one of the con-ceptuses in an individual rat was replaced by a balloon. The volume of the balloon was adjusted daily to that of the conceptus in intact pregnant rats. Each ovariectomized pregnant rat with an intrauterine balloon was injected daily with either vehicle (oil), estradiol, progesterone or combined estradiol and progesterone from day 14. The intra-uterine pressure put on the balloon and the intraamniotic pressure in the conceptus with an alive fetus were measured on days 17 and 20. In some ovariectomized rats treated with either vehicle, estradiol or progesterone, the pressures could not be measured since these rats had the ruptured uterine wall over the balloon and/or no alive fetuses. Treatments with either estradiol or progesterone alone failed to lower the high intra-uterine and intraamniotic pressures induced by ovariectomy. Administrations of com-bined estradiol and progesterone, however, reduced the pressures to levels in sham-ovariectomized animals. These results suggested that estrogen and progesterone act synergistically to maintain a low intrauterine pressure during late pregnancy.

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The developmental changes and sex difference in activity of glucose-6-phosphate dehydrogenase (G-6-PDH) of bovine oocytes and early embryos derived from follicular oocytes fertilized in vitro were histochemically examined. Bovine embryos at two- to eight-cell stages were separated into two halves by pipetting after removal of zona pellucida by treating with 0.5% pronase. One half was assayed for G-6-PDH activity and the other was used to determine sex by chromosome analysis. There was no definite tendency in G-6-PDH activity from matured oocytes to the eight-cell embryos and no activity was observed at morulae stage. We found no sex difference in the G-6-PDH activity of bovine two- to eight-cell embryos derived from follicular oocytes. A high activity was observed in some embryos with four or six X-chromosomes.

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A commercial enzyme immunoassay kit (PREG-TEST, Teikoku Hormone Mfg. Co. Ltd., Tokyo) with polystyrene bead-solid phase for measuring progesterone in skim milk, blood plasma or serum of cows was applied to determine progesterone in mare serum. Water blank measured by the kit was 190 pg/ml. Recovery rates of pro-gesterone added to water and mare serum were 100-110% and 130-150%, respectively. Intra-assay coefficients of variation were 11.8-17.8% and inter-assay coefficients of vari-ation were 11.6-19.1%. Cross reactions of other steroids with the antibody of the kit were less than 5%, excepting 45.1% in 11α-hydroxyprogesterone and 10.5% in 5α-dihydroprogesterone. There was a significant correlation between progesterone concen-trations measured by the kit and radioimmunoassay (r=0.96, p<0.01). Progesterone levels in mare serum measured by the kit were 0.7ng/ml at the estrous stage and 7.0 ng/ml during the luteal phase. Progesterone levels in pregnant mares on 18-24 days after mating were higher than in non-pregnant mares. Progesterone levels decreased to less than 1 ng/ml 24 hours after administration of prostaglandin F_2α during the luteal phase.
The results indicate that the kit is the reliable and practical tool for determination of progesterone concentration in mare serum during the estrous cycle and early preg-nancy period.

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The present study was conducted to establish a suitable dilution method at room temperature for mouse 8-cell embryos (CD-1) vitrified by VS3 solution. Viability of the zygotes vitrified by VS1 and VS3 solutions was also examined. Superovulated 8-cell embryos and zygotes were equilibrated with VS3 or VS1 solutions and plunged into LN₂, . After warming, the vitrified-warmed embryos were diluted with various pro-cedures and cultured in vitro. The blastocysts obtained were transferred to pseudo-pregnant recipients. The high viability of vitrified-warmed mouse 8-cell embryos was obtained when the embryos were diluted with 0.5 M sucrose-HB1 solution for 5 min or with 1.5 M sucrose-HB1 and 0.5 M sucrose-HB1 solution for 5 min each at room tempera-ture; 90% and 87% of the embryos were recovered as morphologically normal after dilution, and 89% and 86% of these developed to blastocyst in vitro, respectively. After transfer to recipients, 60% and 59% of the blastocysts developed to full term. When the zygotes were vitrified by VS3 solution, the survived zygotes was few after dilution and culture in vitro. In contrast, 20% of vitrified and warmed zygotes by VS1 solution was developed to blastocysts, and live young were obtained after transfer. In conclusion, VS3 solution is effective for cryopreservation of mouse 8-cell embryos by vitrification, but not for the zygotes.

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To assess the potential of prepubertal gilts as a source of ova for swine embryo transfer research, (1) oocytes were aspirated from preovulatory follicles following treatment with exogenous gonadotropins and (2) the fertilization and developmental ability of these oocytes was examined by the gamete intrafallopian transfer (GIFT) method. Four-month-old prepubertal gilts (n=4) were treated with 2000IU of pregnant mare serum gonadotropin (PMSG) and 750 IU of human chorionic gonadotropin (hCG) to induce follicular growth. The numbers of follicles (5-7 mm) developed by this treat-ment were from 22 to 95 per gilt (52±15 SEM). The oocytes were transferred with ejaculated spermatozoa into three recipient gilts, two of which became pregnant and delivered three piglets. This demonstrates a potential method of embryo transfer using prepubertal gilts as donors of ova.

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Antisera to prostaglandins (PGs) E₂ and F_2α and indirect immunofluo-rescence were used to immunohistochemically demonstrate the presence of PGE₂ and PGF_2α in preimplantation embryos of hamsters, rats and rabbits. Fluorescence showing the presence of both PGF₂ and PGF_2α was observed in the cytoplasm of the eggs and embryos of all those species at the stages of unfertilized 1-cell to blastocyst. The intensity of the fluorescence was always weak in the eggs and embryos of hamsters, while strong at the 1-cell stage of rabbit eggs and 1-cell through 8-cell stages of rat eggs and embryos, but was weakened at the rest of the stages. In the 1-cell eggs of such species, no difference was observed in the in tensity of the fluorescence between the unfertilized and fertilized. These results, then, have clarified that the preimplantation embryos of hamsters, rats and rabbits constantly contain PGE₂ and PGF_2α in their early develop-mental stages.

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Development of the external genitalia in swine fetuses was studied macro-scopically, with respect to the formation of the labia and the sexual differentiation. The female urogenital fold, budding from a region on either side of the genital tubercle, gradually extended to the tip of the genital tubercle on day 82 of gestation and then began to ensheathe it with the corresponding fold on the other side, thus forming the labium pudendi which is homologous to the labium minus pudendi of the human. The genital swellings, which are known to become the labia majora in the human, did not take part in the formation of the labia. They became inconspicuous in the region cranial to the genital tubercle on day 45. The sex difference of the external genitalia was first observed on day 35; in the male, a small circular urogenital orifice and an anogenital raphe appeared in the region caudal to the genital tubercle; in the female, both structures were still not observed. The anogenital distance became clearly longer in male than in female on the day 40.

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Timed artificial inseminations (AI) were performed into the uterus of out-seasonally (Aug. to Sept., 1987) estrus induced ewes by a laparoscopy using frozen ram semen which is previously separated by bovine serum albumin (BSA) sedimentation method. An intravaginal sponge with 60 mg medoroxy-progesterone acetate (MAP) was remainded in the vagina of 137 mature Suffolk ewes for 9 days. PMSG (600IU/ewe) was injected i.m. on the day of sponge removal (Group A, n=72) or 2 days before sponge removal (Group B, n=65), and hCG (500IU/ewe) was administered i.m. 48 (Group A) or 24 hr (Group B) after the sponge removal. In groups A and B, AIs was carried out 48, 60 and 72 hr, and 24, 36 and 48 hr after sponge removal, respectively. The highest lambing rate (47.8%), significantly higher than any other groups (t<0.05) was achieved in ewes inseminated at 60 hr after the sponge removal. Sex ratios were 45.8% in 16 newborns produced by insemination of the top layered semen separated with the BSA column, and 43.5% in 13 newborns produced by insemination of the bottom layered one.

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