Studies on bovine nuclear transfer(cloning)and related technologies

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Present studies were conducted to establish reliable bovine nuclear transfer(NT)procedure using embryonic blastomeres and somatic cells as nuclear donors, and to develop related technologies such as cryopreservation of oocytes/follicles and in vitro culture of preantral follicles.In experiments for NT using embryonic blastomeres, we have developed a new oocyte-enucleation technique with a high efficiency by using pre-activated oocytes. Clone calves were obtained after the transfer of NT embryos reconstituted with blastomeres, and we confirmed their consistent growth and fertility. In experiments for the production of clone calves using somatic cells as nuclear doner, we have investigated the several factors affecting the development of NT embryos. Results indicate that cell-cycle coordination between recipient cytoplast and nuclear donors is most critical, and that the combination of nuclear donor in the G0/G1 phase and recipient cytoplast in the M phase produces embryos with high deve … More lopmental capacity when somatic cells are used as nuclear donors. Clone calves were obtained after the transfer of NT embryos reconstructed with uterine epithelial cells, colostrum-derived mammary gland epithelial cells and ear-derived fibroblast cells ; however, abortion after the transfer of NT embryos and abnormalities in the clone calves were frequently observed.To prepare a large number of recipient oocytes, we have studied to develop a pre-antral follicle culture system and cryopreservation protocol for the cryopreservation of follicles/oocytes using cattle and mouse oocytes and preantral follicles. We confirmed that bovine oocytes derived from early preantral follicles could acquire developmental capacity if they were cultured for more than 8 days before maturation culture. A new vitrification solution(VS), a mixture of ethylene glycol and raffinose was developed after the several investigation using mouse oocytes. Using this new VS, we have succeeded to cryopreserve mouse preantral follicles, and confirmed that the oocytes derived from vitrified-cultured preantral follicles could develop to the blastocyst stage. Less