| Summary: | 碩士 === 國立屏東科技大學 === 畜產系所 === 96 === The objective of this study was to investigate the effects of the enucleation timing on the enucleation accuracy, in vitro and in vivo development capability, and endogenous gene methylation status of the cloned bovine embryos. In experiment 1, the single clonal bovine ear fibroblast cells with transfected GFP gene was used for donor cells to examine the effect of enuclaetion timing on the enucleartion accuracy of bovine cloned embryos. The effect of oocyte maturity on the in vitro development of bovine cloned embryos was also investigated. The results showed that the in vitro development capability were not different between the cloned bovine embryos derived from single clonal and non-single clonal ear fibroblast cells (15.4 vs. 21.7％). The enucleation rate of the cloned embryos produced by the enucleation before activation (EBA) protocol was significantly higher than those embryos produced by the enucleation after activation (EAA) protocol (96.1 vs. 77.6％, P＜0.05). However, the enucleation accuracy were both 100％ in blastocyest stage of EAA -4h and EBA groups. The cloned embryos reconstructed with immature oocytes from 14 h IVM developed to 16-cell stage was significantly less than those embryos reconstructed with matured oocytes from 18 h IVM (11.7 vs. 37.5%, P＜0.05). The purpose of experiment 2 was to evaluate the effects of enucleation timing on the in vitro development and the methylation status of the endogenous gene in the cloned embryos. The results showed that the blastocyst rate of the cloned embryos produced by EAA-4h method was similar to those embryos produced by EBA method (23.3 vs. 13.0％), which were significantly higher than those embryos produced by EAA-8h method (15.5 vs. 5.5％, P＜0.05). One of the 6 recipients was become pregnant after transferring 10 cloned blastocysts produced by EAA-4h method, however the newbron was dead soon after birth. No pregnancy was found in 18 recipients after transferring 32 cloned blastocysts produced by EBA method. The methylation rate of Xist gene was not different between the cloned embryos produced by EBA and EAA-4h methods (81.6 vs. 76.6％, P＞0.05), which they were significantly higher than the IVF embryos (68.3％) (P＜0.05)and sperms (54.6％) (P＜0.05). However, these values were significantly lower than the donor cells (98.3％, P＜0.05). In conclusion, the enucleation accuracy was very high (76.1-100％) in cloned bovine embryos produced by EAA-4h method; however, their enucleation rate was lower than those embryos produced by EBA method. The development capability of cloned embryos might be decreased, when the recipient oocytes were derived from immatured oocytes and the enucleation time was delayed. The development ability and methylation rate of Xist gene of the cloned bovine embryos produced by EBA method were similar to those embryos produced by EAA-4h method. The cloned calf was generated by EAA-4h method suggesting that the EAA-4h protocol of somatic cell nuclear transfer technique was established successfully.
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