小鼠卵母细胞去核方案的优化

为了优化小鼠卵母细胞去核方案，本研究对比了透明带磨口刺入和透明带直接刺入两种去核方法（分别简称磨口法和刺入法）、蔗糖处理与否、CB的浓度及去核针内径对小鼠卵母细胞的去核效率的影响，及CB浓度对去除部分胞质（不去核）的孤雌胚早期发育的影响。结果：（1）无论是磨口法还是刺入法，添加3％蔗糖和对照组之间的去核成功率及去核操作时间差异均不显著（P>0.05）；（2）CB浓度为5μg/mL、10μg/mL和20μg/mL时，各试验组的操作时间差异不显著（P>0.05），但10μg/mL的CB操作速度最快，磨口法三个试验组间去核成功率差异不显著（P>0.05），刺入法在CB浓度为5μg/mL、10μg/mL时去核成功率显著高于20μg/mL组（P<0.05），磨口法去核成功率显著高于刺入法（P<0.05）；（3）去除部分胞质的孤雌胚卵裂率各试验组之间差异不显著（P>0.05），20μg/mLCB处理组囊胚率显著低于其它处理组（P<0.05）；（4）在CB浓度为10μg/mL的操作液中，分别用内径为10μm、15μm和20μm内径的去核针，各试验组间操作时间差异不显著（P>0.05），15μm内径的去核针操作速度较快，磨口法去核成功率差异不显著（P>0.05），15μm去核针去核成功率较高，刺入法应用20μm去核针的去核成功率显著低于其它组（P<0.05）。结果表明，磨口法在10μg/mL CB和15μm内径去核针条件下提高了去核成功率(90.8%)及去核速度（50sec/个），且不影响孤雌胚的体外发育率。

Optimization of the Enucleation Protocols for Mouse Oocytes

To optimize the enucleation protocols of mouse oocytes, the study examined the effects of the enucleation methods of abradeding the zona pellucida(AZP) or sticking the zona pellucida (SZP)straightly、sucrose treatment or not、concentration of CB and inner diameter of pipettes on enucleation efficiency，and the effects of the concentration of CB on parthenogenetic embryos removed partial cytoplasm from 1/8 to 1/12. The results showed (1) there were no significant differences between supplemented 3% sucrose group and control group on manipulating time and enucleation rate achieved by AZP or SZP（P＞0.05）; (2) 5μg/mL、10μg/mL and 20μg/mL CB treatments had no significant effects on manipulating time（P＞0.05）. AZP had no significantly different enucleation rates among 5μg/mL、10μg/mL and 20μg/mL CB groups（P＞0.05）；however, in SZP, the enucleation rates of 5μg/mL、10μg/mL CB treatments were significant higher than that of 20μg/mL（P<0.05）; (3) the cleavage rate of parthenogenetic embryos had no significant difference between various groups（P＞0.05）. Blastocyst rate of 20μg/mL CB group was significant lower than that of other groups（P<0.05）; (4) the enucleation rates of 10μm、15μm and 20μm inner diameter of pipettes were no significantly different in AZP（P＞0.05）; the enucleation rates of 10μm and 15μm inner diameter of pipettes were higher than that of 20μm in SZP（P<0.05）. The enucleation rates achieved by AZP was significant higher than that achieved by SZP （P<0.05） . The results indicate that the optimized protocol combining AZP, 10μg/mL CB with 15μm inner diameter of pipettes significantly improves the efficiency of enucleation, and can be used for nuclear transfer on mouse.