| Since the first mammalian cloning, nuclear transfer methods have not been changed appreciably. Most laboratories still rely mainly on micromanipulation-assisted enucleation by aspiration, followed by injection of the donor cell. The method still requires sophisticated and considerable skills from the operator, putting cloning beyond the reach of many laboratories. In this research the effects of different activation and activators dose on parthenogenetic development, and maturation culture time, parameter of electric fusion, activation and culture method for nuclear transfer were compared so that to establish a simply nuclear transfer procedure.I. Parthenogenetic activation of ovine oocytes1. Zona-free oocytes were treated with 0μM, 2.5μM, 5μM Ionomycin and 2mM 6-DMAP for the parthenogenetic activation, the rates of cleavage were 18.8%, 78.9% and 73.9%, the development rates to blastocysts were 0%, 13.7%, 11.3%, respectively. Zona-intact oocytes were treated with same methods, the rates of cleavage were 14.5%, 72.5% and 88.5%, the development rates to blastocysts were 0%, 13.8% and 14.7%, respectively. The developmental rates of treatment groups for zona-intact and zona-free oocytes were significant high than that of control. There were no significant differences between different treament groups.2. Zona-intact and zona-free oocytes cultured in WOWs (pressed with heatedrod) were treated with 5uM Ionomycin and 6-DMAP for parthenogenetic activation, the rates of cleavage were 70.4% and 69.2% respectively. Zona-intact and zona-free oocytes were treated with 7% ethanol and 6-DMAP for parthenogenetic activation, the rates of cleavage were 33.3% and 23.0% respectively. The cleavage rates of oocytes treated with Ionomycin groups were significant high than that with 7% ethanol groups. The results indicated that cleavage rates were not been affected by zona-intact or zona-free of oocytes, but the cleavge oocytes cultured in WOWs did not develop to blastocyst.II. Somatic cell nuclear transfer of zona-free ovine oocytes1. Effects of oocytes maturation culture time on the expelling and attaching rates of first poly body were observed. The expelling rates of first poly body were 69.9%, 76.2%, 82.3% and 83.8% when oocytes were cultured 18-19h, 20-2lh, 23-24h and 26-27h, and the attaching rates of the first poly body were 77.7%, 62.6%, 41.3% and 25.5%, respectively. The results indicated that the appropriate oocyte maturation timewas 18-19h.2. Electric fusions were performed in 1.3 kv/cm, 1.5 kv/cm and 1.9 kv/cm under the condition of 20us, 2pulse, 1 second, and the fusion rates were 30.6%, 48.2% and 88.2% respectively. The results indicated that the optimum parameter of electricfusion was 1.9kv/cm.3. Enucleated zona-free oocytes and somatic cells were fused to reconstruct embryos, and the embryos were activated by Ionomycin and 6-DMAP, the cleavage and blastocyst rates were 82.1% and 10.4% after cultured in four-well dish.Reconstructed embryos were activated with 7% ethanol and 6-DMAP, and the cleavage and blastocyst rates were 40.3% and 0%, respectively. The cleavage and blastocyst rates of oocytes treated with Ionomycin groups were significant high than that with 7% ethanol groups. The results indicated Ionomycin and 6-DMAP have good result. Reconstructed embryos were activated by the same methods, the cleavage and blastocyst rate were 62.1% and 6.1% after cultured in the WOWs (only pressed) , there was no significant difference compared with that in four-well dish. When zona-free ovine oocytes was irradiated by UV(l/4) beyond 10s , most of ovine reconstruct embryos can not cleave to blastocyst after activation.The results indicated the zona-free cloning method might be easier to adopt than conventional cloning procedure for users, and was a easy procedure.
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