Influence of time after the removal of nocodazole from nuclear donors on the development of reconstituted embryos in bovine nuclear transplantation.

The present study examined the influence of post-cleavage time of nuclear donors on the development of reconstituted embryos in bovine nuclear transfer. Blastomeres of 16-cell stage embryos derived from in vitro-maturation, fertilization and culture were used as nuclear donor source. They were treated with 10 microM nocodazole for 12 hr. Blastomeres that cleaved within 3 hr after the removal of nocodazole were used for the study. Metaphase II (M-II) oocytes were used as recipient cytoplasm. In experiment 1, donor blastomeres at 6, 11 and 15 hr after the removal of nocodazole and donor blastomeres not treated with nocodazole were transferred into ethanol-exposed and enucleated oocytes. The reconstituted embryos produced by donor blastomeres at 6 hr after the removal of nocodazole had a significantly higher developmental rate to the blastocyst stage than those at 15 hr and the untreated groups (P < 0.01). In experiment 2, blastomeres at 6 hr after the removal of nocodazole used as nuclear donors were transferred into ethanol-exposed and enucleated M-II oocytes. The reconstituted embryos with ethanol-exposed and enucleated oocytes as recipient cytoplasm had a significantly higher rate of initial-cleavage (P < 0.05) and development to the blastocyst stage (P < 0.01) than non ethanol-exposed and enucleated M-II oocytes. These results demonstrate that the development of reconstituted embryos was improved when cleaved donor blastomeres after the removal of nocodazole were immediately transferred (at 3-6 hr post-cleavage) into activated enucleated oocytes by exposure to ethanol.