| The research is performed to study the effect of oocytes with different paternal genetic background on development of cloned and parthenogenetic embryos. The conclusion is demonstrated by several facts:1. Preparation of donor cellsThe purpose of the research is to reveal how different cell isolation methods (enzymatic digestion and tissue fragment) and serum starvation (Cell density>90%) influence the rate of cell viability respectively. The results show that enzymatic digestion method required 3 d and tissue fragment method require 7 d, and with no difference in cell viability (P>0.05).2. Preparation of oocyteTo study the number of litter, ratio of female and male and efficiency of hyperovulation, we take hybriding between three different strains male (129/Sv, C3H, ICR ) and KM female as experimental group and mating of between KM male and KM female as control group. The result shows that there is no significant difference between experimental group (129/Sv×KM F1,C3H×KM F1,ICR×KM F1) and control group (KM) ( 9±1.60, 9±2.40, 8±1.60 vs 8±1.97,P>0.05; 49.5%, 54.3%, 52.5% vs 49.4%, P>0.05 ,respectedly). There is also no significant difference between experimental group and control group (34.0±4.5, 31.9±8.4,34.9±7.5 vs 36.7±9.1,P>0.05;27.9±10.4 , 26.7±7.4 vs 28.9±10.7,P>0.05, respectedly) after hyperovulting F1 of experimental and control group. This demonstrats that it does not affect the number of litter and ratio of F1 female and male, as well as the quality of F1 hyperovulation.3. Effect of oocytes with different paternal genetic backgrounds on the successful rate of somatic nuclear transfer①to improve the efficiency of injected cytoplasm, the study compared the injecting needles made of two different capillaries (100mm×0.1mm and 100mm×0.2mm ) . 100mm×0.2mm can significantly save the injection time(<30s vs >1min)and successful rate(86% vs 5%, P<0.05) than 100mm×0.1mm.②the research evaluated the effect of paternal genetic backgrounds on somatic cell nuclear transfer in mice. Taking hybrid F1 female of 129/Sv×KM F1, C3H×KM F1 and ICR×KM F1 as experimental groups respectively, and female KM mice as control group, viability of oocytes after micromanipulation, the cleavage rates and blastocytes rates of reconstruction embryos in vitro between experiemental groups and control are compared. The results indicate that the rates of successful enucleation in 129/Sv×KM F1, C3H×KM F1 and KM are significantly higher than that of ICR×KM (78.0%, 82.9%, 81.0% vs 63.9%; P<0.05); however, the successful injection rate of 129/Sv×KM is significantly higher than that of C3H×KM F1, ICR×KM F1and KM(83.0% vs 59.6%, 55.5%, 71.4%; P<0.05). The efficiency of artificial activation of 129/Sv/Sv×KM is significantly higher than that of C3H×KM F1, ICR×KM F1 and KM(97.3% vs 85.2%, 81.7%, 78.3%; P<0.05);and the cleavage rate and blastocyte rate of C3H×KM are significantly higher than that of ICR×KM F1 and KM×KM F1(84.5%,28.2% vs 63.2%,11.4%,64.5%,16.5%; P<0.05).③taking the cloned prenuclear and bolstocyst derived from 129/Sv×KM F1, C3H×KM F1 and KM oocyte as experimental group, natural fertilized prenuclear and blastocyst as control, the study is performed to compare the successful rate of embryo transfer in oviduct and uterus. The result shows that control group is highly developed to full-term than treated group with both embryo transfer in oviduct and uterus (0%,0%,0% vs 41.6%, P<0.05; 0%,0%,0% vs 60.3%, P<0.05); in the meanwhile, the treated group has no inserted dot in uterus. This demonstrated that the capillary of 100mm×0.2mm can improve the efficiency of injection; different the paternal genetic backgrounds can influence the efficiency of somatic cell nuclear transfer in mice, in which the oocytes derived from C3H paternal genetic backgrounds can enhance the efficiency of somatic cell nuclear transfer in mice; cloned embryo from129/Sv×KM F1, C3H×KM F1 and KM oocyte can not continue to develop beyond the blastocyst after embryonic transfer.4. Effect of oocytes with two paternal genetic backgrounds on in vitro development of parthenogenetic embryo.The research evaluated the effect of two artificial activation methods on development of two paternal genetic backgrounds parthenogenetic embryo. Taking hybrid F1 female of 129/Sv×KM F1, C3H×KM F1 as experimental groups, and female KM mice as control group, the result shows that there is no significant difference between experimental group and control group, ratio of one pronuclear and rate of fragment at 2 cell stage for ion+6-DMAP is higher than Sr2++CB, rate of blastocyst for Sr2++CB is higher than ion+6-DMAP for each group. Meanwhile, rate of blastocyst for experimental group is higher than control group in both activation stimuli. In conclusion, the Sr2+ +CB activation is optimal comparing with ion+6-DMAP. Experimental groups can improve the in vitro development of parthenogenetic embryo by introducing paternal genetic background of 129/Sv and C3H.5. Effect of oocytes with two paternal genetic backgrounds on rate of isolation of ntES and pES ntES and pES are isolated from cloned and parthenogenetic blastocyst of treated group (129/Sv×KM F1, C3H×KM F1) and control group (KM). The result shows that the attach rate of experimental group is higher than control group (13.0%,10.0% vs 0%, P<0.05) after cultivating the cloned blastocyst on MEF feeder. ntES is not isolated from the experimental group because no obvious ICM outgrowth is observed. The attach rates of treated and control group are not significantly different (67.8%, 66.7% vs 55.0%, P>0.05) after cultivating the cloned blastocyst on MEF feeder. 5, 6, 6 attached ICM outgrowth are isolated from KM, C3H×KM F1 and 129/Sv×KM F1, respectedly. A ICM outgrowths from KM is only cultivated to 4 passages because the cell cluster became black. A ICM outgrowths from C3H×KM is cultivated to 7 passages because the cell clusters are infected by bacteria. A ICM outgrowths from 129/Sv×KM F1 is successfully cultivated to 50 passages. The result revealed that ntES is difficult to isolate from the cloned blastocysts, but pES is easy to isolated from the parthenogenetic blastocysts.
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