| Xenogeneic somatic cell cloning technology to protect and save rare and endangered animals, human organ transplantation has opened up a new way, at present, although the kind of animals born from the somatic cell nuclear transfer, but the success rate lower limits of the technology in practical application. Interspecies nuclear transfer is still facing great difficulties, need to find the appropriate combination of donor and recipient cells and early embryonic development environment. In order to study animal species nuclear transfer knowledge and experience, we study of Yanbian Goat-Sheep reconstructed embryo fusion and activationIn this research, we use Yanbian milk goat ear skin fibroblast cells as donor cells, we use MⅡsheep oocytes of enucleated nuclear for transfer receptor, constructed goat-sheep heterogeneous reconstructed embryo. We designed a series of experiments to optimize the Yanbian milk goats-sheep fusion and activation effect of reconstructed embryos, study of different electric field strength, pulse frequency, pulse duration, equilibrium time, blending concentration of fusion and activation method on the fusion rate of reconstructed embryos.The results are as follows:1) mannitol concentration of 0.25mol/L,0.28 mol/L when the. fusion rate of reconstructed embryos (50.12% vs52.74%) and cleavage rate (53.50% vs55.52%) not significantly different (P> 0.05), but with 0.30mol/L group difference was significant (P<0.05),0.28 mol/L group morula development rate (17.36%) was significantly higher than the other two groups, significant difference (P<0.05);2) when the electric field strength for the 1000v/cm fusion rate of reconstructed embryos with the other significant difference between groups (P<0.05), the electric field strength 1200v/cm,1400v/cm and 1600v/cm reconstructed embryos when Fusion was no significant difference between the rate (P> 0.05); 1400v/cm the cleavage rate (60.84%) and blastocyst rate (8.14%) the highest, and 1200 v/cm was not significant when compared (P> 0.05), but the difference compared with the other groups were significantly (P<0.05); 3) two times the fusion pulse rate and pulse one significant difference (P<0.05), but the electric pulses, compared with three times the difference was not significant (P> 0.05); two pulses of the cleavage rate (69.84%) and blastocyst rate (6.14%) was significantly higher than one and three pulses, the difference was significant (P<0.05); but the one and three no significant difference between pulses (P> 0.05).4) when the pulse duration is 40μs fusion rate of reconstructed embryos (65.56%) than other groups, but the difference was not significant (P> 0.05), but the cleavage rate (58.90%) and the other three groups (10μs, 20μs,80 u s) significant difference (P<0.05). Pulse duration is 40μs morula development rate 18.16%, and group 10μs,80 u s group difference was significant (P<0.05), compared 20μs but the difference was not significant (P> 0.05)5) 30 min,60min,90min,120min different equilibrium time of the fusion rate was not significant (p> 0.05), but the 90min group, the cleavage rate (70.62%) was significantly higher than the other three groups, significant difference (p< 0.05).90min group, blastocyst rate was 8.10%, significantly higher than 30min and 120min group, but compared with the 60min was no significant difference (p> 0.05).6) EH+CHX, EH+DMAP, Ion+CHX, Ion+DMAP activation cleavage rate of four was not significant, but the growth rate in the morula, Ion +6-DMAP group (21.30%) was significantly higher than the other three groups (18.90%,14.12%,15.34%), EH+CHX group and EH+6-DMAP group and Ion+6-DMAP group were significantly different. EH+6-DMAP group and Ion+6-DMAP was no significant difference between groups. |
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