The sex ratio of bovine hatched blastocysts produced and cultured in vitro was examined by the polymerase chain reaction (PCR). A total of 190 hatched blastocysts was obtained between 188 and 248 h post insemination (hpi). The hatching rates expressed as a percentage of the total one-cell presumptive zygotes (0 hpi) and of cleaved embryos (72 hpi) were 31.8 and 46.7%, respectively. The sex of 189 embryos (99.5%) was successfully determined. The results of agarose gel electrophoresis clearly indicated that male embryos gave a double band with male-specific and gender-neutral primers, and females gave a single band with gender-neutral primers alone. The overall sex ratio was 51.9%(98/189), which did not significantly differ from the expected ratio of 1:1. Based on the observations at 12 h intervals, embryos were divided into three developmental groups according to the timing of hatching. The sex ratio of the fast group (188-200 hpi) was shifted to males (59.4%, 38/64), although with no significantly difference from the expected ratio of 1:1. On the other hand, the intermediate (212 hpi) and slow (224-248 hpi) groups had slightly more females than males (47.1%, 33/70) and the sex ratio close to the expected ratio of 1:1 (49.1%, 27/55), respectively.

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Three experiments were conducted to investigate the biological role played by cumulus cells in the early pregnancy of pig. In Exp 1, the proportions of oocytes with or without cumulus cells relating to follicular size were studied. The proportion of oocytes with cumulus cells increased by 63% in the oocytes collected from small follicles (1-4mm) compared with those from large follicles (≥5mm). In Exp 2, in vitro matured oocytes were fertilized with epididymal or ejaculated spermatozoa in vitro.
There was a significant difference in the rate of ovum development into 8-cell and 16-cell stage between epididymal and ejaculated spermatozoa. In Exp 3, the developmental ability of oocytes with or without cumulus cells was investigated. Oocytes nuded from cumulus complexes at in vitro fertilization could not develloped over the 4-cell stage.

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The aim of this study was to develop a simple and rapid method of cryopreservation of rat blastocysts by vitrification. Vitrification solution used here contained glycerol and polyethylene glycol as cryoprotective agents in a HEPES buffered saline (VS3). Two methods of vitrification, one-step and stepwise, were examined to explore a convenient and successful technique for vitrifying rat embryos. No obvious difference was found in post-thaw in vitro survival of vitrified embryos between the two methods of vitrification, suggesting that one-step method may be successful for the vitrification of rat blastocysts. Microscopical observations of the embryos revealed that prolonged exposure of the embryos to the VS3 induced the considerable swelling of embryonic cells, suggesting the intracellular influx of cryoprotective agents. Based on the morphological examinations, no serious damages were observed in the vitrified-thawed embryos after a short period of exposure time (5 min). The present results demonstrate that one-step method of vitrification would be successful for cryo-storage of rat blastocysts.

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Uncompacted 8-cell mouse embryos cultured in a Whittingham medium containing anti-Lewis X (Le^x) antibody underwent compaction, and developed into 16-cell embryos, though their blastomeres were all round and tended to be separated from each other. In such embryos no junctional complexes were seen of any type. These findings seem to suggest that Le^x epitope must play an important role in cell-to-cell recognitionin transformed embryos and in the subsequent formation of junctional complexes.

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We have studied on the relationships between substrate activity of MDH in TCA and energy substrates of bovine embryos from the 1-cell to blastocysts stages. MDH activity was calculated in single fresh embryos, using enzyme cycling method modified by us for single fresh embryos, and then each embryo estimated MDH activity was co-cultured with cumulus cells to check subsequent development.
We found out that the level of MDH activity was significantly higher (P<0.05) in AMLP buffer as compared with PBS (-) buffer as enzymatic reaction buffer during early stages of development; but the bovine embryos in AMLP buffer (pH 9.8) did not continue subsequent development after estimation of enzymatic activity. MDH activity of bovine embryos at unfertilized, 2, 4, 8-cell, morula and blastocysts stages was ranged from 5.20±1.06 to 12.18±1.86 (x10^-6 mol/embryo/hr). The activity was the highest at the blastocyst stages. MDH activity in the degenerated embryos was significantly lower (P<0.05) as compared with that in normal embryos. MDH activity increased markedly in the presence of glutamate, malate, α-ketoglutarate and DNP, indicating that via the TCA cycle that these substrates can be used as energy sources.

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In order to survey the relationships between metabolitic activity and develpmental ability of bovine embryo derived from in vitro fertilization, we modified an enzymatic cycling method in the present study. We measured lactate dehydrogenase (LDH) in preimplantation stages of single bovine embryo derived from IVF to examine the effect of LDH activity on a subsequent development in vitro. LDH activity of bovine embryos varied with the cleaving stages and embryos quality (Table 1, 2). LDH activity in the degenerated embryos was the highest as compared with that in normal embryos. It is suggested that the release of LDH by dying or degenerating embryos might offer an alternative for determining 2 the viability of embryos. Thus, the LDH activity proved to be a practical method fordistinguishing between nonviable and viable embryos.

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We have attempted an establishment of ES cells from bovine blastocysts fertilized in vitro co-cultured with fibroblast feeder cells of murine or bovine embryos. In this report, we have shown bovine ES-like cells from co-culture used murine fibroblasts as feeder layer. The ES-like cells were compared morphologically with trophoblast whose features are like ES cells. Furthermore, when the ES-like cells were exposed to 10-5M alltrans-retinoic acid, these cells differentiated into neuron-like cells. And, it was observed that the growth of ICMs of blastocysts used were slow, some of them hardly grew. These results may suggest that the growth of ICMs is influenced by differences of genome of each embryo, and also indicat that the property of ICMs is different between murine and bovin.

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