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KMID : 0900919980220040425
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Korean journal of Animal Reproduction
1998 Volume.22 No. 4 p.425 ~ p.435
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Effects of Activation Regimens of Recipient Cytoplasm, Culture Condition of Donor Embryos and Size of Blastomeres on Development of Reconstituted Bovine Embryos
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Cho Sung-Gun
Lee Hyo-Jong
Choi Sang-Yong
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Abstract
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To improve the efficiency of nuclear transplantation in bovine, in this study the development in vitro of nuclear transferred (NT) embryos was compared by different activation regimens of the enucleated oocytes. The effect of developmental stage and culture system of donor nuclei on fusion and development in vitro of NT embryos were also evaluated. Oocytes were collected from Hanwoo ovaries obtained from slaughterhouse and matured in Ham′s F-10 supplemented with hormones. After 20~22 h maturation, the oocytes were vortexed to be free from cumulus cells and subsequently their nucleus and the first polar body were removed. Enucleated oocytes were divided into 3 groups for activation; the oocytes of group I were activated with ionomycin for 5 min and subsequently incubated in 6-dimetylarninopurine (DMAP) for 4 h, Those of group II were treated with DMAP for 4 h at 39 h after onset of in vitro maturation (IVM) and those of group III were kept in room temperature (25^{circ}C) for 3 h at 39 h after onset of IVM. After in vitro fertilization (IVF) the embryos for muclear donor were cultured either by group culture (20 embryos /50 {mu}ell drop) or individually (1 embryo /50 {mu}ell drop) for 4 day and 5 day. At day 4 and 5 after IVF, blastomeres were separated in calcium-magnesium free medium, and then classified into small (day 5: leq 38 {mu}{textrm}{m}, day 4: leq 46 {mu}{textrm}{m}) and large (day 5 : geq 38 {mu}{textrm}{m}, day 4 ; geq 46 {mu}{textrm}{m}). The separated blastomeres were replaced into enucleated and activated recipient cytoplasm. The blastomere-oocyte complexes were fused by electrically. The NT embryos were cultured in TCM-199 containing 10% FCS in 39^{circ}C, 5% CO_2 incubator for 7 day. The results obtained were summarized as follows; There were no differences in fusion and development to blastocyst between groups as group I (68%, 10%), group II (75%, 14%) and group III (73%, 9%), respectively. However, the cell number in blastocyst of NT embryos in group III were significantly fewer than in the other groups (P＜0.05). No differences in fusion and development to blastocyst were found between individual or group cultured and between small or large blastomeres of day 4 and day 5 donor embryos. From these results, it was concluded that the combination of ionomycin and DMAP, or treatment of DMAP at 39 h after onset of IVM were useful for the efficient of production of NT bovine embryos, and the individual cultured embryos could be simply used as donor nuclei for NT bovine embryo.
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KEYWORD
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Ionomycin, DMAP, Activation, Nuclear transplantation, Bovine
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