体外受精生产性控犏牛胚胎条件的优化

Optimization of the Production Conditions for Sexed Embryos of Yak Cattle by in vitro Fertilization

为提高性控犏牛胚胎体外发育率,从性控精液的精子密度、受精时间、卵丘细胞的脱除程度、胚胎培养体系和胚胎培养环境各个环节进行优化。结果表明:(1)受精24h的卵裂率和囊胚率显著高于受精6h、18h(P<0.05);(2)性控精液的精子密度0.4×10~6~0.6×10~6个/mL时的卵裂率显著低于4×10~6~6×10~6个/mL组(P<0.05),但囊胚率差异不显著(P>0.05),均显著高于性控精液0.04×10~6~0.06×10~6个/mL的卵裂率和囊胚率(P<0.05);(3)卵丘细胞不脱除组的囊胚率(23.42%)显著高于半脱除组(13.77%)和完全脱除组的囊胚率(15.61%)(P<0.05);(4)输卵管上皮细胞共培养的囊胚率(24.63%)和卵丘细胞共培养的囊胚率(23.42%)差异不显著(P>0.05),但均显著高于FBS CR1aa培养液的囊胚率(P<0.05);(5)性控犏牛早期胚胎在低氧培养环境下囊胚率(23.42%)显著高于高氧培养环境(11.83%)(P<0.05),而孵化囊胚率差异不显著(P>0.05)。结果说明,生产性控犏牛胚胎最佳的条件是性控精液的浓度0.4×10~6~0.6×10~6个/mL,受精时间为24h,采取卵丘细胞不脱除的方法,胚胎培养方式采用输卵管上皮细胞共培养的方式,胚胎培养环境采用低氧环境。

In order to improve the productivity of sexed Yak cattle embryos in vitro,this experiment was conducted to optimize the links from sex-sorted sperm concentration,fertilization time,degree of removal cumuluscells,different embryo culture system and embryo culture environment.The results showed that：（1）the cleavage rate and blastocyst rate of fertilization 24 h was significantly higher than that of 6 h and 18 h（P〈0.05）;（2）The cleavage rate of sex-sorted sperm concentration of 4~6×10^6/mL was significantly higher than that of 0.4~0.6×10^6/mL（P〈0.05）,but blastocyst rate difference was insignificant（P〉0.05）,which were all significantly higher than the concentration of 0.04~0.06×10^6/mL（P〈0.05）;（3）The blastocyst rate of intact cumulus cells group was significantly higher than partial removal cumulus cells group and complete removal cumulus cells group（P〈0.05）;（4）The blastocyst rate of coculture with oviductal epithelial cells（24.63%）and cumulus cells（23.42%）were of no significant difference,but they were significantly higher than FBS CR1 aa medium（P〈0.05）;（5）The sex-control blastocyst cleavage rate in low oxygen group（23.42%）was significantly higher than high oxygen group（11.83%）（P〈0.05）,but the hatched blastocyst rates were not significantly different.The results indicated that sex sperm concentration of 0.4~0.6×10^6/mL,24 hfertilization time,intact cumulus cells method,co-culture with oviductal epithelial cells,embryo embryonic environment with low oxygen environment were the best condition of Yak cattle embryo production.