To survey the relationship between metabolic activity and developmental ability of bovine embryos derived from in vitro fertilization, we modified an enzymatic cycling method inthe present study. We measured 6-phosphogluconate dehydrogenase activity (6PGD) in singlebovine embryos derived from in vitro fertilization in preimplantation stages and checked theirdevelopmental ability. When measured by adding 6-phosphogluconate as a substrate, 6PGD activi-ties were 62.8±9.2, 77.4±3.3, 103.4±6.8, 96.2±0.5 and 120.6±3.4 (×10^-9 mol/embryo/h) in the 2-cell, 4-cell, 8-cell, morula and blastocyst stage embryos, respectively, indicating significant differ-ences between groups at the 2-or 4-cell stages and blastocyst stage (P<0.05). In all cell stageembryos, the level of 6PGD activity increased by adding the substrate. When subsequent develop-ment was examined after measurement of 2-cell, 4-cell, 8-cell and morula stage embryos, thedevelopmental rate to blastocyst stage ranged from 18.8 to 61.0% and did not vary among the different stages.

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This study was undertaken to confirm whether early pregnancy factor (EPF)-like substance(s) was secreted by fertilized bovine ova. Using the rosette inhibition test, EPF-like substance(s) was detected in the culture media of fertilized bovine ova, while this substance was not found in the culture media of unfertilized bovine ova. EPF-like activity, analyzed by HPLC-gel permeation chromatography, was present between molecular weights of 16 and 37 KD. In conclu-sion, EPF-like substance(s) is secreted by fertilized bovine ovum and has a molecular weight of16-37 KD.

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The influence of sera in culture medium on hatching of equine blastocysts was examined. Non-surgically recovered early blastocysts were cultured for 10 days in TCM199 supplemented with 10% (v/v) of: (1) fetal bovine serum (FBS, n=5), (2) fetal equine serum (FES, n=4), (3) mare serum collected at the day of ovulation (MS-O, n=6), or (4) mare serum collected at day 8 of pregnancy (MS-P, n=5). Mean embryonic diameters at the beginning of culture ranged from 165.3 to 177.3 μm. Embryos in MS-P started to hatch when their mean diameter reached 286.2 μm at day 8.6 of embryonic age. Embryos in MS-O, FES and FBS started to hatch at 322.5μm (day 10.0), 379.3μm (day 8.1) and 405.4μm (day 10.0), respectively. Judged from the maxi-mum diameter of the embryos during the culture period, fetal serum (FES: 1, 556.5μm; FBS: 942.2μm) supported the in vitro development of the embryos better than mare serum (MS-O; 677.5 μm, MS-P; 489.0μm). The hatching manner observed most frequently in FBS and MS-P supplemented media was a prolonged process characterized by either squeezing through a small opening in the zona pellucida or tearing open the zona pellucida. In contrast, a sudden escape from the zona pellucida was found in MS-O (3/5) and FES (2/4). The concentrations of E₂ and P₄ in FBS, FES, MS-O and MS-P were determined by EIA (E₂: 0.074, 9.150, 0.010, and 0.013 ng/ml; P₄: 0.800, 8.350, 2.390, and 6.340 ng/ml, respectively). In conclusion, the source of sera for supple-mentation of culture media influenced in vitro development of equine early blastocysts, especially the process of hatching.

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The GnRH neuron was investigated immunohistochemically in the hypothalamus and other diencephalic areas of mature male Shiba goats. Morphological characteristics and the distri-bution pattern of cell bodies immunoreactive to GnRH (GnRH-ir) as well as the projection of GnRH-ir fibers were examined in serial coronal sections (40 μm thick) extending from the diagonal band of Broca to the mammillary complex in the rostro-caudal direction using a specific mono-clonal antibody (LRH 13). The GnRH-ir cell bodies were classified morphologically into two sub-types, i.e. bipolar and multipolar, based on the number of processes. The distribution pattern of GnRH-ir cell bodies was strikingly similar in all the animals examined, and 51% of total immunore-active neurons were found in a restricted region including the preoptic area, supraoptic nuclei and the vicinity of the organum vasculosum of the lamina terminalis. The major fiber projections were seen in the neurohemal organs, namely the median eminence and the organum vasculosum of the lamina terminalis. The GnRH fibers were also found in the mammillary complex and the habenular system but not in the amygdaloid complex.

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A study was conducted to examine if gonadotropin-releasing hormone (GnRH) neurons show sex-related differences in the hypothalamus of the mature goat. The localization of GnRH neurons were made evident immunohistochemically using a monoclonal antibody against GnRH in coronal sections (40μm thick) of the hypothalamic area of male and female goats. The localization of immunoreactive GnRH (GnRH-ir) cell bodies was assessed according to the stereotaxic coordinates of the brain atlas for the Shiba goat, and was compared between the male and the female brains. The morphological characteristics and distribution pattern of GnRH-ir neurons, and the projection pattern of GnRH-ir fibers were similar between the sexes. On the other hand, the number of GnRH-ir neurons showed sexual dimorphism; the estimated total number of GnRH cell bodies in the hypothalamus of the male goat (4, 020 ± 572, Mean ± SEM) was greater (P<0.01) than that for the female (2, 223 ± 284). These results indicate that the goats of both sexes have a similar GnRH neurosecretory system, although the male goat has a greater population of GnRH neurons in the rostral hypothalamus.

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The pattern of cell proliferation of early rat embryos in vivo was examined. Naturally mated female rats were sacrificed every 2 to 3 h between 0000 h on Day 1 (day of estrus) and 2200 h on Day 5 of pregnancy. Embryos were obtained by flushing the oviducts and uterus with M2 medium. Embryonic stages were morphologically categorized into 12 stages, i.e. 1-cell, 2-cell, 3-cell, 4-cell, 5-7 cell, 8-cell, 8-cell-morula, morula, early blastocyst, blastocyst, large expanded blastocyst, and hatched blastocyst. The 2-cell stage lasted 31 h and was longer than any other stage. Duration of the 1-, 4-, 5-7-and 8-cell stage was 23, 11, 5, and 4 h, respectively. Embryonic shape at the 4-cell stage was either square or rhomboidal, and embryos at the 8-cell stage were either circular or rectangular. After the 8-cell stage, an increase in cell numbers was more dependent on time than the progression of developmental stages; thus, the number of blastomeres in each embryonic stage after the 8-cell stage varied considerably. Understanding the chronology of rat embryonic development in vivo will provide the basic knowledge that is required for manipulating the rat embryo.

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It has been accepted that the majority of XX⇔XY chimeric mice develop into adult males. The present study was designed to examine the gonads of XX⇔XY chimeric mice to determine whether the co-existence of chromosomally female (XX) and male (XY) cells affects sex differentiation. Southern blot analysis using a Y chromosome specific DNA fragment as a probe revealed the sex chromosomal constitution of C57BL/6N⇔DDD/1 chimeric mice. The histological features of the gonads in XX⇔XY chimeric mice were observed at both fetal and adult stages. The gonads in XX⇔XY chimeras were ovotestes at the fetal stage on 15 days post coitum (d.p.c.), had predominantly testicular components after 16 d.p.c., and were testes at adult stage. This observation indicates that the fetal ovotestes in XX⇔XY chimeras develop into testes in adults. In addition, testes of some adult chimeras which were not dominantly composed of XY cells showed co-existence of gametic and agametic seminiferous tubules.

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Long-term effects of progesterone implants on the advancement of puberty were investigated in immature female guinea pigs. A subcutaneous implant of progesterone-filled tubing was given to immature females at 1 day of age and removed at 5, 10, 15, 20, or 25 days of age. Continuous treatment with progesterone implants until 15 or 20 days of age significantly advanced the mean age at first vaginal opening (25.5 ± 0.6, n=6 and 28.3 ± 0.5 days of age, n=6, respectively) as compared with controls (35.3 ± 1.6 days of age, n=6). The mean age at first ovulation (29.8 ± 1.7 days, n=6) in females implanted until 15 days of age was also significantly earlier than controls (37.7 ± 1.9 days, n=7). Ovaries at 24 days of age taken from the animals treated with a progesterone implant until 15 days of age had a significantly higher number of preovulatory follicles as compared with controls. These results indicate that long-term treatment with progesterone during the early infant period induces precocious puberty in female guinea pigs.

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Mouse haploid and diploid parthenogenetic embryos were cultured in 25 μl of Whitten's medium using 1, 5, 10 or 20 embryos. Improvement in development was observed at higher embryo densities. Development of haploid parthenogenetic embryos was improved in terms of both the percentage of blastocysts and the number of cells per blastocyst. In diploid parthenoge-netic embryos, an increase in the number of cells per blastocyst was observed. These results demonstrate that the development of parthenogenetic embryos is affected by embryo density. In vitro culture of 10-20 parthenogenetic embryos together can enhance their preimplantation development.

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The efficacy of the use of controlled internal drug releasing (CIDR^R) device containing 1.9g progesterone in inducing estrus synchronization of heifers in Japan was first attested. Eighty-eight Holstein heifers were randomly designated for the 7, 12, and 14 days of CIDR^R insertion periods. The effects of CIDR^R with and without estradiol-benzoate (E₂) and prostaglandin F₂α (PGF₂α) at 7, 12 and 14 days of insertion periods were evaluated and compared in terms of progesterone concentrations during the 1st and 2nd day of insertion periods, at CIDR^R removal and at the AI time; retention rates, estrus incidence, and pregnancy rates.
An overall CIDR^R device retention rate of 85.2% (75/88), estrus incidence of 90.7% (68/75) and pregnancy rate of 63.3% (38/60) were recorded. Specifically, estrus incidence and pregnancy rates in both 7 days (86.8 and 60.7%) and 12 days (93.3 and 61.5%) insertion groups showed encouraging results. No significant difference was found between treatment groups. However, the 14 days insertion period without the use of additional hormones showed the highest pregnancy rate of 83.3% (5/6). An E₂ capsule implanted at insertion and PGF₂α injected at CIDR^R removal showed no effect on pregnancy rate, but the incidence of 'standing' estrus increased significantly significantly (p<0.05) with PGF₂α treatment.

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