| Nuclear transfer (NT) is a complex embryo biotechnology involving in many manipulation steps, and it is an important factor influencing the total efficiency of NT. Simplification and standardization of the proceeds are critical to improve the efficiency of NT. This study was conducted using cumulus cells (CCs) as donor cells to investigate the effects of enucleation, reconstruction and delayed activation on NT, some other related issues such as the in vitro maturation (IVM) of pig oocytes were also studied. The study aimed to provide simpleer and more feasible methods of acquirement of CC, enucleation, reconstruction and delayed activation which were applicable in pig NT. Three conclusions were drawed based on the analysis of experimental data.1, The study on the IVM of oocytes used as recipients in pig somatic cell nuclear transfer was conducted. Using modified TCM199 as the main culture medium and the eduction of first polar body as the symbol of maturation, the effects of different grades of cumulus oocyte complexs (COCs) and the addictives of culture medium on the IVM of porcine oocytes were explored. The results indicated that:(1), When the oocytes were cultured by different grades, Grade A (253/324,78.09%)and B porcine COCs (520/689, 75.47%) had significantly higher IVM rates than that of ungraded (794/1240, 27.01%) and Grande C COCs (37/137, 64.03%) (P<0.05).(2), The addition of 10% porcine follicular fluid (PFF) (490/1139, 43.02%) or 10% (OPS) (599/1426, 42.00%) to modified TCM199 did not improve the IVM rate as compare to control (modified TCM199,607/1264, 48.02%) (P>0.05), and there was no difference between the effects of PFF and those of OPS on IVM rate.(3), The addition of 10% oestrus calf serum (OCS) (1027/1397, 73.51%) or 10ng/ml EGF (1040/1368, 76.02%) significantly improved the IVM rate as compared to control, the effect of OCS and EGF on IVM was not different (P>0.05).The results showed that the grading of oocytes before culture and the additions of 10% OCS and 10ng/ml EGF were advantageous to the IVM of oocytes used for NT.2, Study on the pig cumulus cell nuclear transfer was conducted. Using CC as donor cell and electrical stimulus to activate the oocytes, the critical steps in porcine cumulus cell nuclear transfer such as enucleation, reconstruction, acquirement of CC and delayed activation were studied. The results showed:(1), Effects of different acquirement of CC on NT: The CC which was directly divided from COC or passaged for 4 generations was used as donor cell in NT. These two kind of CC had no different effct on the early development of porcine reconstructed oocyte (P>0.05); the cleavage rate, 4-8 cell formation rate,and morula formation rates of reconstructed oocyte using these two kinds of CC as donor cell were 37.76% (37/98) /38.93% (51/131), 12.24% (12/98) /12.21% (16/131), 2.04% (2/98) /1.53% (2/131), respectively.(2), Among different enucleation methods such as blind aspiration (BA), bisbenzimide (Hoechst 33342) stain method (BS), blind aspiration combined with bisbenzimide stain method (BABS). BS (136/156, 87.18%) had the highest enucleation rate (P<0.05), the enucleation rate of BABS (133/185, 71.89%) is higher than that of BA (164/274, 55.85%) (P<0.05) while lower than that of BS (P<0.05). BA cost the least time (1.2min/oocyte (274)) (P<0.05). The results showed that BS is superior to BA in pig cumulus cell nuclear transfer.(3), The NT oocytes using perivitelline microinjection (PM) which placing a donor cell in the perivitelline space of an enucleated recipient oocyte had higher cleavage rate and 4-8 cell formation rate than those of intracytoplasmic microinjection (IM) which injecting an donor cell nucleus into an enucleated oocyte. The morula formation rate of PM and IM was not different (P>0.05), but after 4-8 cell was formed, the morula formation rate of PM was significantly lower than that of IM (P<0.05). The cleavage rate, 4-8 cell formation rate, morula formation rate and morula formation rate in 4-8 cell formed oocytes of PM and IM was 40.11 %(71/111)129.15% (83/279), 11.86% (21 /177) /6.81 % (19/279), 1.75% (3/177) /1.79% (5/279) ,14.29% (3/21) /26.32% (5/19) .respectively.(3) The donor cell nucleus was settled in the cytoplasm of enucleated recipient oocyte for Oh, 1.5h, 3.0h, 4.5h, 6.0h, 7.5h or 9.0h before activation. The results showed that the cleavage rate was the highest when the reconstructed was equilibrated for 7.5h (51/114, 44.74%) and 9.0h (56/124, 45.16%) (P<0.05), and there is no difference between them. However, when activation was delayed for 1.5h( 14/123,11.38%), 3.0h (13/113, 11.50%) ,4.5h (14/110, 12.73%) or 6.0h (13/120, 10.83%) , higher 4-8 cell formation rate was observed (P<0.05), and there's no difference among them (P>0.05). The cleavage rate(13/113, 14.69%), 4-8 cell formation rate(l/l 13, 0.85%), and morula formation rate(0%) was the lowest when the oocyte was immediately activated after NT (P<0.05). The results indicated that the delayed activation of reconstructed oocytes is necessary in porcine cumulus cell nuclear transfer, and 4.5h for interaction of nucleus and cytoplasm is advantageous to the morula formation of reconstructed oocytes.In conclusion, the grading of oocytes and the additions of 10% OCS and 10ng/ml EGF to modified TCM199 were advantageous to the IVM of porcine oocytes. As far as the acquirement method of CC was concerned, the CCs directly divided from COC and these passaged for 4 generations had no significant difference in effect on NT. When it came to enucleation, bisbenzimide (Hoechst 33342) stain method had the highest enucleation rate, while blind aspiration cost the least of time. The NT oocytes using perivitelline microinjection and intracytoplasmic microinjection had no significant difference in developing rate, but the former microinjection method was more advantageous for the reconstructed...
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