The size distribution of ovarian follicles was examined histologically duringpregnancy in the Wistar rats. The number and size distribution of healthy and atretic follicles larger than 350 μm in diameter were determined for each day of pregnancy. Throughout pregnancy and day of parturition, the mean number of healthy follicles larger than 350 μm per ovary ranged from 17.6 to 32.6 and the number was maximum 32.6 on day 2 (day 1 of pregnancy = day sperm positive). Higher number (6.8-8.1) of healthy follicles larger than 550 μm per ovary were observed on days 7-9 and 22, while the number decreased markedly on days 14-21. There was an inverse relation between the number distribution of healthy follicles of 350-549 μm and that of healthy follicles larger than 550 μm. The mean number of atretic follicles larger than 350 μm per ovary ranged from 5.0 to 13.7. These results indicate that the development of small follicles to large follicles occurs mainly on about days 7-9 and 22 of pregnancy.

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The object of this study was to adopt a triple-stain technique for identifying human acrosome-reacted spermatozoa and its simplified technique to boar spermatozoa. Spermatozoa were incubated in the uteri isolated from hamsters and mice and stained by these techniques. Stained smears disclosed spermatozoa with the following four patterns: a) light brown postacrosomal regions with red acrosomes (live sperm with normal acrosomes); b) light brown postacrosomal regions with unstained acrosomal regions or damaged acrosomes (live sperm without normal acrosomes); c) dark brown postacrosomal regions with dark red acrosomes (dead sperm with normal acrosomes); d) dark brown postacrosomal regions with unstained or dark blue acrosomal regions or damaged acrosomes (dead sperm without normal acrosomes). There was a significant positive correlation between the percentage of live sperm without normal acrosomes (category b) and that of zona-free hamster eggs penetrated (triple-stain technqiue: r=0.836; P<0.01; n=7, simplified technique: r=0.603; P<0.01; n=19). These results indicate that the technique can be used toidentify boar acrosome-reacted spermatozoa. However, staining by the simplified technique produced misleading results, i.e., a higher than "real" percentage oflive spermatozoa without normal acrosomes. This suggests that the triple-stain technique is more suitable for boar spermatozoa than the simplified technique.

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Activities of Δ⁵-3β-hydroxysteroid dehydrogenase (Δ⁵-3β-HSD), 20β-HSD and 17β-HSD in Sertoli and Leydig cells of 0-50 days old infant rats born of busulphan-treated dams were histochemically quantitated using dehydroepiandrosterone (DHA), pregnenolone, 17α-hydroxypregnenolone, 20β-hydroxyprogesterone, testosterone and estra-diol-17β as the substrates. All the HSD activities were detectable in Sertoli cells of 20-50 days old infant animals born of both the control and the busulphan-treated dams. At the age of 40 days, activities of individual HSD in the cells were higher in infants born of the treated dams than in animals born of the control ones. When DHA and pregnenolone were used as the substrates, Leydig cells of infant rats born of the treated dams exhibited a higher Δ⁵-3β-HSD activity as did the cells of control infant animals. Compared to control infants, however, the other HSD activities were consistently lower in Leydig cells of infant rats born of the treated dams.

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A microtitre plate enzyme immunoassay (EIA) kit for the determination of the concentration of plasma progesterone was evaluated.
The procedure of the EIA kit is very simple and brief; the assay completes in approximately 1.5 hours. Progesterone concentrations in bovine blood plasma can be measured without extraction.
The intra-assay coefficients of variation (CV, N=10) of the absorbances and the concentrations were 4 to 5% and 8 to 16%, respectively. The inter-assay CV (N=8) were 9 to 15%. The inter-assay CV (N=6) of the absorbances of the provided standards in the same batch were 6 to 10%. The detection limit of progesterone was 2.5 pg/well.
The correlation between progesterone concentrations in bovine blood plasma meas-ured by EIA and RIA was quite high (r=0.97, p<0.001).
The results of the present examination suggest that the kit can be used to measure plasma progesterone with sufficient sensitivity and precision, and will be useful for laboratory and veterinary practices.

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An attempt was made to identify the fluorescent body (F-body), with Quina-crine mustard (Q-M) staining, in the spermatozoa from 7 mammalian species (human, bull, boar, dog, rabbit, rat and mouse). Washed sperm suspension in PBS was either stained with Q-M solution for 40 min or 24 hr, or treated with Protease and then stained with Q-M for 40 or 100 min. The final concentration of Q-M in sperm suspension was 0.025 mg/ml. The microscopic examination, using a reflected fluorescence microscope (B-exciter filter; 390490 nm, Optical suppression filter; ?? 510 nm), revealed that the same F-body as that in human sperms was present in all of these species. After 24 hr staining, F-body was found in 28.5, 28.4, 36.2, 3.0, 9.7 and 9.3% of sperms of bull, boar, rabbit, dog, rat and mouse, respectively. Enzyme treated specimens showed higher inci-dences (43.8, 44.0, 47.6 and 41.0% for bull, boar, rabbit and dog, respectively, and 20.4 and 20.7% for mouse and rat, and 32.5% for human) of the F-body than those in the specimens stained 24 hr without enzymatic digestion. These findings led to the conclusion that the F-body is present in common with spermatozoa of many mammalian species including man and gorila.
The successful detection of F-bodies in the sperms from various mammalian species is attributed to; (1) the long-term submersion of specimens in the Q-M solution for staining and (2) the treatment of the membrane by protein digestion which yielded more favorable condition for staining in many animal species.

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Ultrastructure of the fibrous strands and crystalloids in the mouse blastocysts developed from the 2-cell stage in vitro was observed in a medium containing bovine serum albumin (BSA) and in a medium containing polyvinylpyrrolidone (PVP).
Many fibrous strands composed of a bundle of fibrils, 150 Å in diameter, were distributed evenly in the cytoplasm of the 2-cell embryos. A few crystalloids composed of densely packed fibrous strands, in a loosely shaped lattice structure, were also observed in the cytoplasm of the 2-cell embryos.
Similar number of fibrous strands was observed in the cytoplasm of trophoblast cells and inner-cell-mass cells of the blastocysts developed in either medium. However, the number of fibrous strands greatly decreased as compared with the 2-cell embryos. The crystalloids showing clear lattice structure were observed in the cytoplasm of tropho-blast cells and inner-cell-mass cells of blastocysts developed in either medium. The number of crystalloids greatly increased as compared with the 2-cell embryos. The in-creasing ratio in the number of crystalloids observed in the blastocysts developed in a medium containing BSA was greater than in the blastocysts developed in a medium containing PVP.

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A microsurgical method which is available for sexing of pig embryo was developed. A total of 22 pig expanding blastocysts was subject to being bisected into two hemispheres by a glass needle: one was "inner cell mass (ICM)-half" which contained the whole ICM and approximately half of the trophoblast, and the other was "the trophoblast (Tr)-half" which had the remaining half of the trophoblast. After the microsurgery, the pairs of ICM and Tr halves were cultured for 20 hr, and the ICM halves were transplanted to recipient gilts and Tr halves were used for chromosome analysis. The rate of development of the transplanted ICM halves were 63.2% (12/19). This is approximately equal to the usual results obtained in transplantation of pig embryos. Sixteen (80%) of 20 chromosome specimens from the Tr halves showed one or more metaphase plates. The average of cells per specimens was 65.8. This result indicated that the micromanipulation method presented here is a suitable one for determining the sex of blastocysts of pigs. Enough cells for specimen preparation can be collected without descreasing embryo survivability.

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Melatonin in Silastic capsules was implanted subcutaneously to examine the effect on reproductive activities of anestrous ewes under natural photoperiods. The constant supply of melatonin from the capsules increased plasma melatonin levels in a dose dependent manner, and high concentrations exceeding the endogenous night peaks were maintained throughout the day in the ewes carrying two capsules. On May 8 (Day 0) non-lactating ewes (n=23) were divided into 3 groups, and were either implanted with one (MEL 1, n=7) or two (MEL 2, n=6) capsules, or left untreated (control, n=10). Plasma progesterone concentrations in the MEL 2 group rose and became significantly higher than those in the control group from Day 56 onwards. This suggests a stimulatory effect of melatonin on quiescent ovarian function. In the MEL 1 group, however, no apparent effect was observed, probably due to an insufficiency in basal melatonin ele-vation. These results suggest that the continuous presence of high melatonin levels in the circulation nullified the anti-gonadal effect of long days, presumably by blunting the endogenous rhythm of melatonin levels responsible for conveying information of the environmental photoperiod.

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A newly deviced cervical mucus remover was tested to improve a method for removing the cervical mucus before flushing the embryos. The embryos were collected in a newly deviced collective dish.
The recovery rate of embryos was significantly higher with the new procedure than that with the cervical dilator. Namely, the number of embryos recovered were 12.05±7.86 for the former vs 8.52±6.82 for the latter (P<0.05). Five of 43 donors had flushing trouble when the cervical dilator was used for removing the cervical mucus. However, there was no trouble when the newly deviced cervical mucus remover was used for re-moving the mucus.

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