| 1. To study the effects of a few factors such as the state of COCs(cumulus oocytes complexes),the culture medium and the addictives of culture medium on the in vitro maturation(rVM) of oocytes in pigs,the ovaries of pigs are used as a source of oocytes for IVM research.First,COCs which are prepared from follicles in different diameters or different grades have significantly different maturation rate in IVM of porcine oocytes.The maturation rate of COCs from 2-6mm diamater follicles is 80.7%,and that of COCs from over 6mm diamater follicles is 81.2%,which is significangly higher than that of COCs from less than 2mm diamater follicles-which is 36.5%.Oocytes maturation of Grade A (80.7%)and B COCs(80.2%) is very significantly different from Grade C(21.2%).Second,culture medium of TCM199 and NCSU-23 are used for IVM of porcine oocytes,they show the same culture ability in IVM(The maturation rate in TCM199 is 79.8%,NCSU23 is 80.6%),but NCSU23 appears better culture quality in IVM maturation than TCM199.Third,addition of PMSG and hCG in the maturation medium had significangly effected on the maturantiong of IVM,the rate of maturation in group PMSG+hCG is 80.5% , 77.2%, 84.0%,but in group NO PMSG+hCG is 62.5% , 50.5% .respectively.When E2 is combined with PMSG and hCG in the culture,the rate of maturation is highest when these hormones are in effect in only first 24h(84.0%).The additon of rpGH of different content(10 ug/ml, 20ug/ml, 0) shows no significant difference to IVM,the maturation rate is 80.4% , 75.7% , 77.5% respectively.Addition of porcine follicular fluid(pFF) to mature medium promotes the in vitro maturation of oocytes.The maturation rate of Group pFF is 81.4%, 81.8%,which is significant higher than Group Without pFF(50.6%).2. Some key effect factors such as enucleation,activation and culture in the course of porcine somatic cells nuclear transfer(NT) using cumulus donor nulei were examined,in the method of microjection into cytoplasm of enucleated oocytes.The results were as follows:First,point-pressing method(PPM) is superior in enucleation rate and manipulation time to blind aspiration method(BAM).The rate of enucleation in PPM is 62.5%,which is significantly different from that in BAM,whose percentage is 50.7%.The manipulating time in PPM is lower than that in BAM(1.33/min vs 0.98/mim P<0.05).Enucleation efficiency is affected with chromosomes deviated from PB1. When oocytes were enuleated in 36-44h the rate of enucleation(when oocytes aged at 36-38h 60.9%;39-41,67.8%,41-44h,64.3%,respectively), significantly higher than in 44-48h(48.4%).Second,when somatic cells were pre-activated,the development rate of NT embryos increased significantly,increased from 20.1% to 28.0%.A23187can lead NT embryos to active and develop when respectively or combined with 6-DMAP. Third,when NT embryos were cultured in NCSU-23 the development rate(30.06%) is no significantly different from that in TCM199 contain cumulus single layer culture system(31.5%).However,the rate of development to 4-cell stage or above 4-cell cultured in NCSU-23 of NT embryos is increased(13.5%),which is significantly different in TCM199- cumulus system (3.9%).
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