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KMID : 1023620040280010021
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Reproductive and Developmental Biology
2004 Volume.28 No. 1 p.21 ~ p.27
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Effects of Recipient Oocytes and Electric Stimulation Condition on In Vitro Development of Cloned Embryos after Interspecies Nuclear Transfer with Caprine Somatic Cell
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Lee M. Y.
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Abstract
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This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Recipient bovine and porcine oocytes were obtained from slaughterhouse and were matured in vitro according to established protocols. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS and primary fibroblast cultures were established in TCM-199 with 10% FBS. The matured oocytes were dipped in D-PBS plus 10% FBS ＋ 7.5 g/ml cytochalasin B and 0.05M sucrose. Enucleation were accomplished by aspirating the first polar body and partial cytoplasm which containing metaphase II chromosomes using a micropipette with an out diameter of 20∼30 m. A Single donor cell was individually transferred into the perivitelline space of each enucleated oocyte. The reconstructed oocytes were electric fusion with 0.3M mannitol fusion medium. After the electrofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. And porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6 ∼8 day at in air. Interspecies nuclear transfer by recipient bovine oocytes were fused with electric length 1.95 kv/cm and 2.10 kv/cm. There was no significant difference between two electric length in fusion rate(47.7 and 44.6%) and in cleavage rate(41.9 and 54.5%). Using electric length 1.95 kv/cm and 2.10 kv/cm in caprine-porcine NT oocytes, there was also no significant difference between two treatments in fusion rate(51.3 and 46.1%) and in cleavage rate(75.0 and 84.9%). The caprine-bovine NT oocytes fusion rate was lower(P＜0.05) in 1 pulse for 60 sec(19.3%), than those from 1 pulse for 30 sec(50.8%) and 2 pulse for 30 sec(31.0%). The cleavage rate was higher(P＜0.05) in 1 pulse for 30 sec(53.3%) and 2 pulse for 30 sec(50.0%), than in 1 pulse for 60 sec(18.2%). The caprine-porcine NT oocytes fusion rate was 48.1% in 1 pulse for 30 sec, 45.2% in 2 pulse for 30 sec and 48.6% in 1 pulse for 60 sec. The cleavage rate was higher(P＜0.05) in 1 pulse for 30 sec(78.4%) and 1 pulse for 60 sec(79.4%), than in 2 pulse for 30 sec(53.6%). In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer and 30.6% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than parthenotes(37.4%).
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KEYWORD
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Somatic cell, Nuclear transfer, Fusion, Blastocyst, Caprine
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