Researches On Optimization And Simplification Of Cumulus Cell Nuclear Transfer Procedures In Porcine

In order to simplify nuclear transfer (NT) procedure and improve NT efficiency, this experiment studied the optimal culture conditions and duration of porcine oocytes used for NT. In-vitro maturated oocytes were adopted as recipients and cumulus cells derived directly from COCs as donor cells; the effects of enucleation, injection, CB and activation on NT efficiency and the following development of reconstructed oocytes were researched.The results showed that when TCM199 was used as basic medium for in-vitro maturation (IVM) of porcine oocytes, supplement with AA or EGF to TCM199 can significantly improved IVM rates comparing to AA+EGF group or control(P<0.01). The IVM rates of these four groups were 80.6%, 81.2%, 64.4%, 66.2%, respectively, AA and EGF groups as well as AA+EGF group and control had no difference in IVM rates(P>0.05). The mature oocytes were parthenogenetically activated, as a result, the cleavage rate between AA and EGF group was similar (74.8%vs74.2%, P>0.05), while they were all significantly higher than those in AA+EGF group and control (61.5%, 63.4%, P<0.01); the rates of development to 8-cell stage on cultured oocytes in the AA, EGF, AA+EGF groups and control were 32.6%, 35.5%, 26.9%, 28.1%, respectively; among them, the rates of AA and EGF groups were both extremely significantly higher than that of AA+EGF group and control (P<0.01), the rate of EGF group was significantly higher than that of AA group (P<0.05), there's no difference between AA+EGF group and control (P>0.05). Therefore, supplement of TCM199 with certain AA or EGF can improve the IVM rate and quality of porcine oocytes.When the porcine oocytes were maturated for 39~48h in vitro, as the oocyte age increased, the IVM rate was enhanced, while the enucleation rate declined; during the 39~41h, 42~44h and 45~48h, the porcine oocyte IVM rates were 70.8%, 80.1 %, 81.2%, respectively; the enucleation rates were 82.4%, 82.2%, 79.6%, respectively. After the following culture of these reconstructed oocytes, the cleavage rate in 39~41h group was significantly lower than that in 42~44h group (37.2%vs41.8%) and 45~48h group (37.2%vs43.0%) (P<0.01), the rate of development to 8-cell stage on cultured oocytes in 39~41h group was significantly lower than that in 42~44h group (15.0%vsl7.5%, P<0.05) and 45~48h group (15.0%vs20.9%, P<0.01); 45~48h group had both higher cleavage rate and proportion of embryos with more than 8-cell than 42~44h group, but the difference was not significant (P>0.05). Consequently, adoption of IVM oocytes aged 45~48h is advantageous to improve the overall enucleation rate and developmental competence of reconstructed oocytes.