有无透明带猪卵母细胞电激活参数的优化及体外培养方式的建立

Optimization of Electro-activation Parameter for Zona-intact and Zona-free Porcine Oocytes Matured in vitro and Establishment of in vitro Culture Method

[目的]摸索无透明带猪体外成熟卵母细胞(ZFOs)电激活的条件和无透明带孤雌激活胚胎(ZFEs)的体外培养方式,为手工克隆法生产广西巴马小型猪体细胞核移植奠定基础。[方法]用不同场强、脉冲时程和脉冲次数激活ZIOs、ZFOs,用不同培养方式(微滴法和微穴法)培养ZIEs和ZFEs,检查第7天的囊胚率及囊胚的总细胞数。[结果]有带猪体外成熟的卵母细胞电激活最佳场强和脉冲时程为125 V/(mm.80μs),微滴法培养的囊胚发育率为(30.54±11.06)%;无带猪体外成熟的卵母细胞电激活最佳场强和脉冲时程为85V/(mm.80μs),微穴法培养,其囊胚发育率为(12.77±6.98)%;单次脉冲的激活效果好于多次脉冲。[结论]对有无透明带胚胎的培养方式,微穴法培养无透明带胚胎,微滴法培养有带胚胎较合适,一次脉冲可以有效激活卵母细胞。

[ Objective ]The aim was to explore the best conditions of electro-activation condition for zona- free porcine oocytes matured in vitro （ZFOs）and the in vitro culture method for Zona-free porcine embryos （ZFEs） ,in order to lay the foundation for GuangxiBama miniature porcine sowatic cell nuclear transfer embryo by hand-made cloning （HMC）.[Method] Under different electrical field strength and the pulse duratian,ZIEs and ZFEs were cultured by different methods（embryos in drops and wells） for 7 d in vitro. Blastocyst development rate of embryos and the total cell number of blastocysts were examined. [Result] The optimum electrical field strergth and the pulse duration were 125 V/（mm·80 μs）,with a blastocyst development rate being （30.54 ± 11.06） % for zona-intact activation embryo in drops. As for zona-free activation, embryos in wells under the conditions of electrical field strength and the pulse dttration at 85 V/（mm·80 μs）, the blastocyst development rate was （12.77±6.98）%. The activation effect of the multiple pulse was better than muhiple pulses. [Conclusion]As for zona-intact and zona-free porcine embtyos in vitro（ZIEs and ZEFs）, it was appropriate for ZIEs indmps and Zbgs in well. Oocytes was most effectively actived by a single pulse.