猪植入前胚胎体外培养条件的优化

Optimization of in vitro Culture Conditions of Pig Preimplantation Embryos

探讨了更换胚胎培养液及添加FBS、高渗透压和不同浓度VE对猪卵母细胞体外受精(IVF)和孤雌激活(PA)胚胎体外发育的影响,进一步优化了猪植入前胚胎体外培养体系。试验一:在第2天、第4天更换新的培养液(换液组),在换液基础上第4天更换为添加10%FBS的培养液(FBS组)。试验二:胚胎分别在0.05 mol/L蔗糖(蔗糖组)和138 mmol/L氯化钠(氯化钠组)的PZM-3(300~320 mOsmol)中培养2 d后移至PZM-3(288 mOsmol)中培养5 d。试验三:在培养液中分别添加50、100和200μmol/L VE。对照组均在PZM-3(288 mOsmol)中培养7 d。结果表明:试验一,IVF和PA胚胎FBS组囊胚率显著高于对照组和换液组(P<0.05);试验二,IVF胚胎氯化钠组卵裂率、囊胚率均显著高于对照组与蔗糖组(P<0.05);试验三,IVF胚胎添加100μmol/L VE组囊胚率显著高于对照组(P<0.05)。结果提示,在换液的基础上添加FBS有利于猪IVF和PA胚胎的体外发育;氯化钠调节的高渗透压可以促进猪IVF胚胎的早期发育;添加100μmol/L VE可以改善猪IVF胚胎的体外发育体系。

This study examined the effect of medium change and supplemented with fetal bovine serum（FBS）,high osmolarity and the optimal concentrations of vitamin E on development of porcine embryos derived from in vitro fertilization（IVF） or parthenogenetic activation（PA）,in further to improve the in vitro culture conditions of pig preimplantation embryos.Experiment 1： The medium change group of embryos were cultured in PZM-3,which was changed to fresh medium on days 2 and 4.The FBS group of embryos were cultured in the change system up to day 4 and then cultured in PZM-3 supplemented with 10% FBS instead of BSA from Day 4 to 7. Experiment 2： Sucrose group and NaCl group embryos were cultured in a modified PZM-3（300 to 320 mOsmol） with 0.05 mol/L sucrose or increased NaCI to 138 mmol/L for the first 2 days, and then cultured in PZM-3（288 mOsmol） for the next 5 days. Experiment 3： Embryos were respectively cultured in a modified PZM 3 with 50, 100, 200 μmol/L vitamin E for the whole culture period. All the control group of embryos were cultured in PZM-3 （288 mOsmol） for 7 days. In the first experiment, the FBS group showed a significantly higher rate of blastocyst compared to the control and the medium change group both of IVF embryos and PA embryos （P^0.05）. In experiment 2, IVF embryos cultured in NaC1 group showed a significantly higher rate of cleavage and blastocyst compared to the control and sucrose group （P〈0. 05）. In the last experiment , IVF embryos cultured in a modified PZM-3 with 100 μmol/L vitamin E showed a significantly higher developmental rate to the hlastocyst stage compared to the control （P〈0.05）. These results indicate that a change to fresh medium and inclusion of FBS in the medium during the late stages of culture can promote the development on both of IVF and PA embryos. The higher osmolarity （increased NaCI to 138 mmol/L） at the early embryonic stage and the supplementing porcine embryo culture medium with 100μmol/L vitamin E can promote the in vitro development of porcine IVF embryo.