| The oocyte activation is one of the key step of nuclear transfer (NT) in mammal, and the efficiency of oocytes activation affects the efficiency of NT directly. Nowadays the efficiency of NT in mammal is very low, one reason is that the oocytes are not activated enough. By now, many mammalian oocytes activation have been researched, but the study on pig oocytes activation is not enough.This study was undertaken to explore the methods , such as electric-activation, CHX, cytochalasin B (CB) for activation of the in-vitro matured porcine oocytes, and the effect on development after activation, in order to obtain an optimal method for NT of porcine. Five conclusions were drawed based on the analysis of experimental data.1,The influence of the electricity activation parameter to the activation effect of heavyconstruction embryo in the nucleus transplants.The influence of electric-field intensity to nucleus transplant : when the fixed electricity pulse width is 80μs and the electricity pulse frequency is 1, we choice 1.3 KV/cm electric-field intensity , the embryo rate of above 8 cells is 5.00% (6/120), which is Remarkably higher than the electric-field intensity is 1.0 KV·cm-1 experimental group (2.50%, 3/120) ,and the diversity is remarkable(P < 0.05).when the electric-field intensity continues to elevate, The oocyte cleavage rate and the embryo rate of above 8 cells drops insteadly. When the electric-field intensity is 1.6 KV·cm-1and 1.9KV·cm-1, the embryo rate of above 8 cells respectively is 2.50% (3/120) and 0.83 % (3/120) ,it is lower remarkably than the 1.3 KV/cm experiment group (P<0.01) .the effect of electricity pulse frequency to nucleus transplant :When the electric-field intensity is 1.3 KV·cm-1,the electricity pulse width is 80μs and electricity pulse frequency is 1, The oocytes cleavage rate and the embryo rate of above 8 cells is 32.50% (39/120) and5.00% (6/120) , it is remarkably higher than 2 times of pulses (26.67%,32/120;1.67%,2/120) and 3 times of pulses (22.50%,27/120; 1.67%,2/120).and the diversity is heteropolarly remarkable(P < 0.01). Explained that 1 times of pulse may cause the pig oocyte full activation.the result above indicated :using 1.3 KV·cm-1 electric-field intensity,80μs arries 1 times of pulses electricity activation condition to the pig heavy construction embryo can obtain a better embryonic development rate.2,The effect of CHX density to nucleus transplant: when the heavy construction embryo after electricity activates were processed for 4 hours with 7μg/ml and 10μg/mlCHX, the embryo rate Obtained of above 8 cells arrives respectively is 6.67% (8/120) ,7.50% (9/120) , it is remarkably higher than 4μg/ml group (0.83%,1/120) and 13μg/ml group (0.83%,1/120) , and the diversity is heteropolarly remarkable (P < 0.01). But the difference between 7μg/ml group and 10μg/ml group is not remarkable (P > 0.05) . The test result indicated that: when the reconstruction embryo were processedafter electricity activation ,then Put them in NCSU-23 including CHX whose density is between 7μg/ml～10μg/ml for 4 hours can obtain the best embryonic development rate.3,The effect of 6-DMAP density to nucleus transplant influence: when the heavy construction embryo after electricity activates were processed for 4 hours with 0.5mM/L,1mM/L和2 mM/L 6-DMAP, Along with above density increase the 8 - cells embryo rate(3.33 %, 4/120; 5.83%, 7/120; 9.17%, 11/120) has the increase thetendency, But egg cleavage rate (38.33%, 46/,120; 40.00%, 48/, 120; 40.83%, 49/,120)thechange is not obvious, When 6 - DMAP density up to 4mM/L, we may obtain a higher cleavage rate and above 8 cells embryo rate (45.83%, 55/120; 13.33%, 16/120) , Misses heteropolarly remarkably is higher than other groups (p<0.01) ; This time again increases 6 - DMAP density like 6mM/L group, its cleavage rate and above 8 - cells embryo rate (38.33%, 46/120; 7.50, 9/120) instead drops.4,The influence of different activation methods to nucleus transplant: the result demonstration that The electricity activates + CHX group and the electricity activates + 6-DMAP group's cleavage rate (40.83%, 49/120; 45.83%, 55/120) and above 8 cells embryo rate (7.50%, 9/120; 13.33 %, 16/120) all is higher than the electricity activation group's the cleavage rate(32.50%, 39/120)and above 8 cellsthe embryo rate(5.00%, 6/120), It showed that it can enhance the embryo the cleavage rate and the embryo growth rate to use CHX or 6-DMAP processing after electricity. The electricity + 6-DMAP group's cleavage rate and above 8 cellsthe embryo rate all is higher than the electricity +CHX group's cleavage rate and above 8 cells embryo rate,it showed that 6-DMAP is better than CHX, we use the electricity + 6-DMAP is an ideal activation method.5,The effect of preactivation of enucleated oocytes on NT: the enucleated oocytes underwent preactivation or not has received intracytoplasmic nucleus injection. Take heavy construction embryo's cleavage rate and above 8 cellsembryo rate as target (similarly hereinafter), Compared with two methods to nucleus transplant effect influence. The test results show: The activation group and the comparison group's cleavage rate respectively is 25.83% (31/120),32.50% (39/120) ,and the difference is remarkable (p<0.01) ; the embryo rate of above 8～cell respectively is 2.50%,5.00 %, the difference is remarkable too (p<0.05) . The result indicated that we do not need the pre-activation on enucleated oocytes during porcine NT.In summary, Under our laboratory condition, Using the Ningbo xin zhi company's CRY-3 cytomixis meter, We may not carry on the pre- activation to the enucleated oocytes on NT ; Using the electricity activation and the actidione jointly is a better activation method; ofter 1.3KVcm-1 80μs and one pulse's electricity activation condition, Then Changing into NCSU-23that contains 4mM/L 6 - DMAP may obtain an ideal activation effect.
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