| Somatic cell nuclear transfer in porcine is significant, especially as models for the study of human -disease, or the pig is generally accepted as the specie of choice for xenotransplantation.The paper investigated In vitro maturation of porcine oocytes, fusion and activation and in vitro culture. All of these will be providing effective technology parameter for production of cloned pigs. The results are as follows:The hormone requirements in different stages of porcine oocytes:Porcine oocytes were cultured in NCSU23+10%pFF+10IU/mLPMSG+10IU/mLhCG+10ng/mLEGF for 44h in vitro, or from mature at 22h after culture, culture for 22h in the medium without hormone supplement subsequent for another 22h. Results showed: different stages, hormone supplement was not significantly different in the rate of maturation; but in the later free- hormone-test cleavage rate and blastocyst formation were higher than the fronter(P<0.05).The effects of different activation methods:⑴The effects of different electric field strength on parthenogenetic activation:for 2.0kv/cm, the rates of cleavage and blastocysts formation were significantly higher than others (1.6kv/cm,2.4kv/cm) (P<0.05); The effects of cycloheximide exposure before or after electrical activation of in vitro-matured porcine oocytes on the subsequent PAs(Parthenogenic activation)development: The MⅡoocytes exposured to PZM3 medium containing cycloheximide 10μg before electrical activation showed higher cleavage rate(P<0.05)and blastocyst formation than the control; The MⅡoocytes exposured to PZM3 medium containing cycloheximide 10μg for 0,10,60min before electrical activation, The MⅡoocytes exposured to cycloheximide showed better development both in 10min and 60min.Compared to control, oocytes treated with 60min exposured to cycloheximide had a higher cleavage rate, and a significantly higher blastocyst formation (P<0.05); The MⅡoocytes exposured to PZM3 medium containing cycloheximide 10μg after electrical activation showed higher cleavage rate and blastocyst formation(P<0.05); The MⅡoocytes exposured to PZM3 medium containing cycloheximide 10μg for 3h,4h,5h after electrical activation, no difference was found in both cleavage rate and blastocyst formation; The MⅡoocytes were exposured to PZM3 medium containing cycloheximide 10μg immediately or exposured to that from 1h later or exposured to PZM3 alone after electrical activation, cleavage rate and blastocyst formation showed the highest in the second treatment(P<0.05).The effects of different embryo density conditions on PAs:25, 45, 65 embryos per 50μl medium drop, cleavage rate and blastocyst formation were higher in 45:50 group, but there were no significant differences(P>0.05).The effect of trichostatin A (TSA), an inhibitor of histone deacetylase, on the maturation of porcine oocytes and development of parthenogenetic embryos in vitro, the different concentrations and treatment duration of TSA in vitro maturation and culture embryo investigated:⑴The maturation oocytes to the metaphase II (M II) stage cultured with 5nM trichostatin A was not significantly different from the control treatment, meanwhile 5nM TSA treatment supported a higher cleavage and blastocyst development rate (P<0.05);⑵The parthenogenetic embryo exposured in 50nM TSA supported a higher cleavage and blastocyst formation (P<0.05);⑶The parthenogenetic embryo exposured in 50nM TSA for up to 24h supported a higher cleavage and blastocyst formation than the others (P<0.05).⑴The SCNTs were fusioned after injection (20～40min,1～2h), 1～2h is too long for the development, it improved the fusion rate (P<0.05), but the fragetion rate was significantly higher than the other (P<0.05), 20～40min recovery was suitable for the blastocyst formation;⑵The SCNTs were activated in different time (0h,0.5～1h,1.5～2h)after fusion, The results showed we should activate SCNTs 1.5～2h later.The effect donor cell transfected or not to the SCNTs, there was no significantly different between them(P<0.05).In conclusion, we had established a stable platform for the production of porcine embryos by somatic cell nuclear transfer.
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