Semen samples were concentrated by centrifugation and then frozen. Effects of this pre-freezing centrifugation on the quality (motility, live sperm rate, and abnormal sperm rate) of thawed semen were examined afterwards.
1. The concentrating procedure before freezing semen improved the motility and the live sperm rate, while lowered the abnormmal sperm rate.
2. Similar results were obtained between the samles centrifuged immediately after the dilution and after the cooling equilibration.
Changes in centrifugation speed did not affect the live sperm rate and abnormal sperm rate. But the motility was improved and the sperm concentration was increased by accelerating the speed of centrifugation.
3. Increase in the proportion of the supernatant removed resulted in decrease in the motility and live sperm rate and increased in the abnormal sperm rate. Addition of the thawing solution improved the semen quatity. These effects were expressed more remarkably as sperm concentration increased. Optimum dilution rate at the thawing was not changed by different proportions of supernatant removed. The motility and live sperm rate were improved by increasing the proportion of supernatant removed when the semem was optimum diluted at thawing.

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Beef cows were raised in different raising system, and we estimated levels of progesterone, glucose and lipids in blood, the number of days for the occurrence of postpartum estrus, and insemination efficiency. The results obtained were as follows:
1) Body weight after calving decreased slightly until either 6 weeks in grazing cows or 7 weeks in non-grazing cows. After these periods, no significant changes were observed.
2) Levels of total cholesterol, phospholipids and triglyceride in serum decreased on the day of calving, and then showed the tendency to increase. While NEFA level increased on the day of calving, and then slowly decreased. NEFA and triglyceride levels in general were higher in grazing cows than those of non-grazing cows. 3) Plasma progesterone levels were between 2.610.1 ng/ml 1 week before calving in grazing cows and 3.04.3 ng/ml in non-grazing cows and the levels fell sharply below 0.5 ng/ml on the day of calving. A small peak was observed before the first postpartum estrus in grazing cows but no such a peak was observed in non-grazing cows. After artificial insemination, plasma progesterone levels more than 2.0 ng/ml continued for 6 weeks for both grazing and non-grazing cows. 4) The first estrus appeared 42.2 ± 13.3 days after partrition in grazing cows and 72.0 days for non-grazing cows. No significant difference in number of services before conception was observed between grazing and non-grazing cows

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A solid-phase competitive enzyme immunoassay of gonadotropin releasing hormone (GnRH) was examined. Enzyme-labeled antigens were prepared by coupling synthetic GnRH with peroxidase using bis-diazotized benzidine and purified by Sephadex G-25 column chromatography. Antiserum raised against [Glu¹]-GnRH-rabbit serum albumin conjugate in a male horse was used. Immunoglobulin G (IgG) fraction of the antiserum used for a solid-phase was prepared by an (NH₄)₂SO₄ precipitation method, purified by Sephacryl S-300 column chromatography, and adsorbed to a polystyrene ball.
Three-tenths ml of 0.05 M phosphate buffer (pH 7.5), 0.1 ml of standard or sample solution, 0.1 ml of peroxidase-labeled GnRH solution and a solid-phase polystyrene ball were added in a test tube. After incubation at 4°C for 24 hours, a polystyrene ball was washed three times with 2 ml of saline, and transferred to a new tube. The enzyme activity was measured with a spectrophotometer (absorbance at 492 nm).
A dose-response curve was obtained between 1 to 1000 ng/ml of GnRH when a solid-phase coated with 50 μg/ml IgG solution and peroxidase-labeled GnRH solution (×100) were used. Acid extract of bovine pituitary stalk produced dose-response curve indistinguishable from synthetic GnRH, but those of anterior lobe, liver and muscle didn't produced.
These results show that GnRH is assayed by this method.

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Sperm rich portions of fresh boar ejaculates were washed twice and preincubated in a modified Krebs-Ringer bicarbonate solution (Toyoda et al., 1971) at different concentrations for various periods at 37°C under 5% CO₂ in air. The preincubated spermatozoa were then added to pig eggs matured either in vivo or in vitro. Final sperm concentration at insemination was adjusted to 5 × 10⁵ cells/ml in each experiment.
The rate of penetrated eggs, as examined at 20 h after insemination, was markedly increased with elevated sperm concentrations during preincubation and with the increased preincubation periods, reaching 83-100% with spermatozoa preincubated at a concentration of 40 × 10⁸ cells/ml for 4 h. These spermatozoa showed vigorous, hyperactivated movement when diluted in fertilization medium.
Penetrated eggs at stages of anaphase to telophase of second meiosis were frequently observed at 4 h and female pronucleus was observed at 6 h after insemination. While in vitro matured eggs did not develop further due to failure in male pronucleus formation, in vivo matured eggs reached the 2-cell to 8-cell stage after 48 h in culture.
These results demonstrated the possibility of in vitro capacitation of boar ejaculated spermatozoa in a defined medium and suggested that sperm concentration during preincubation is important to capaci-tate spermatozoa.

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Sexual behavior of 1 male and 3 female Hokkaido brown bears in captivity was observed 62 days from May 1 to July 1, 1984 at Maruyama Zoo. Copulation took place in the following sequence: mounting, pelvic thrusting, quivering and dismounting. The average copulation time was 23 minutes. Mating was observed from early May to late June. All of three females were not mounted consecutively during a 20-day mating period.

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The present investigation was undertaken to examine the incidence of pregnan-cy and the number of implantation sites in adult rats treated with varying doses of gonad-otropins, and to explore the factor(s) causing a diminished incidence of implantation. Adult female rats were primed with 10-40 IU of PMSG at 1100-1300 h on the day of early diestrus. The half of PMSG-primed rats were further injected with the same doses of hCG 52-54 h after PMSG injection. These gonadotropin-treated rats were mated with males and examined for the number of implantation sites 8 days after mating (day 8). The incidence of pregnancy decreased, in a dose-dependent manner, from 95.0 to 17.5% in PMSG (10-40 IU)-primed rats and from 90.0 to 27.5% in PMSG-hCG-primed rats. The number of implantation sites in pregnant rats treated with 10 and 20 IU of either PMSG or combined PMSG and hCG was comparable to the value observed for the untreated control rats. However, the number of implantation sites per mating rat was significantly less in rats treated with 30 and 40 IU of either PMSG or combined PMSG and hCG than in control rats (P< 0.001). Administrations of prolactin (10 or 15 IU/day) from days 1 through 7 significantly increased both the incidence of pregnancy and the number of implantation sites in rats treated with 40 IU of combined PMSG and hCG (P<0.05-0.001). Injection of prolactin (15 IU/day) were found to maintain serum progesterone at high levels (152.7-237.2 ng/ml) during days 2-8 of pregnancy. These results suggest that the diminished number of implantation sites in adult rats treated with large doses of gonadotropins may be at least partially due to an insufficient progesterone secretion from the corpora lutea.

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Mating behavior of four Suffolk rams and its effect on fertility in 47 hormonally-treated ewes during the non-breeding season were investigated. Each ram was allowed to mate 11 or 12 ewes in a 5 × 8 m breedig pen (single-sire mating). The observation was continuous for 48 hs after hormonal treatment and joining of rams. The items of observa-tion were numbers of mounts and services per ewe, ratio of mounts/services per ram, intervals and distribution order of services in each ram. Mating behavior varied markedly between rams. The mean numbers of mounts and services per ewe were 12.6 and 3.3, 35.7 and 6.6, 8.4 and 1.8, and 36.0 and 2.5 for the four rams (A, B, C, and D, respectively). The ratio of mounts/services (14.6) in the youngest ram (D) was significantly (P< 0.01) higher than the ratio of the other three rams. The distribution order of services to estrous ewes by each ram was also variable. Ram mating preference was observed, especially in the youngest ram. However, these differences in ram mating behavior did not relate to detection of estrous ewes. Although the proportions of lambed ewes against the number of marked ewes were not significantly different among the rams used, it was revealed that there was a significant difference (P< 0.05) in lambing rates between rams A and D. Maiden ewes had lower (P< 0.025) lambing rate (27.8%) than parous ewes (71.4%). It was considered from the present results that mating behavior of rams and status of ewes (maiden or parous) could be the important factors affecting on fertility during the non-breeding season.

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