5-Aza-CdR对德保黑猪手工克隆重构胚胎体外发育效果的影响

为了探讨5-氮杂-2'-脱氧胞苷（5-Aza-CdR）对德保黑猪手工克隆（HMC）重构胚胎体外发育效果的影响，本研究分别从供体细胞和重构胚入手，比较了5个不同处理浓度（0、5、10、20和40 nmol/L）5-Aza-CdR处理HMC重构胚的体外发育效果，筛选最佳处理浓度；在最佳浓度下比较5个不同处理时间（0、24、48、72和96 h）对HMC重构胚的体外发育效果，筛选最佳处理时间；用4个不同浓度（0、0.25、0.5和1 μmol/L）5-Aza-CdR结合最佳浓度和最佳时间处理供体和重构胚，比较其体外发育潜能。结果显示，与空白对照组相比，5、10、20和40 nmol/L 5-Aza-CdR处理72 h对重构胚卵裂率均无显著差异（P>0.05），20 nmol/L 5-Aza-CdR处理能显著提高重构胚的囊胚率（P<0.05），10和20 nmol/L 5-Aza-CdR处理均能显著提高囊胚细胞数（P<0.05），其中以20 nmol/L 5-Aza-CdR效果最佳；与空白对照组相比，利用20 nmol/L 5-Aza-CdR处理HMC重构胚72 h能显著提高重构胚的囊胚率和囊胚细胞数（P<0.05），其余处理时间对重构胚卵裂率、囊胚率和囊胚细胞数均无显著影响（P>0.05）；在囊胚的最佳处理浓度（20 nmol/L）和最佳处理时间（72 h）下，结合供体的4个处理浓度（0、0.25、0.5和1 μmol/L），同时处理重构胚和供体，各处理组HMC重构胚的发育潜能均有提高，但效果均不显著（P>0.05），其中0.25~0.5 μmol/L 5-Aza-CdR处理效果较佳。综上表明，适宜浓度（0.25~0.5 μmol/L）的DNA甲基化酶抑制剂5-Aza-CdR处理供体细胞72 h并结合20 nmol/L 5-Aza-CdR处理重构胚72 h均能有效提高德保黑猪HMC重构胚胎的体外发育潜能，该结果可为今后研究德保黑猪HMC胚胎DNA甲基化调控机制提供参考。

Effect of 5-Aza-CdR on the in vitro Developmental Potency of Reconstructed Embryos of Debao Black Pig Handmade Clone

To investigate the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR) on in vitro developmental potency of handmade clone (HMC) reconstructed embryos in Debao Black pigs,this study was performed from donor cells and reconstructed embryos,compared with developmental effects of 5-Aza-CdR treatment with HMC reconstructed embryos of five different concentrations (0,5,10,20 and 40 nmol/L) and five different treatment times (0,24,48,72,and 96 h),and screened for the optimal treatment concentration and time;Compared with in vitro developmental potency of four different concentrations (0,0.25,0.5 and 1 μmol/L) 5-Aza-CdR,combined with optimal concentration and optimal time to treatment with donor cells and reconstructed embryos.The results showed that there was no significant difference in the cleavage rate of reconstructed embryos between 5,10,20 and 40 nmol/L 5-Aza-CdR for 72 h compared with blank control group (P>0.05),20 nmol/L 5-Aza-CdR group significantly increased the blastocyst rate of reconstructed embryos (P<0.05).Both 10 and 20 nmol/L 5-Aza-CdR significantly increased the number of blastocysts (P<0.05),20 nmol/L 5-Aza-CdR was the best.Compared with blank control group,treatment with 20 nmol/L 5-Aza-CdR for 72 h significantly increased the blastocyst rate and blastocyst number of the reconstructed embryos (P<0.05),the remaining treatment time had no significant effect on the cleavage rate,blastocyst rate and blastocyst number of reconstructed embryos (P>0.05).Under optimal treatment concentration (20 nmol/L) and optimal treatment time(72 h),combined with four treatment concentrations (0,0.25,0.5 and 1 μmol/L) of the donor cells and treated with the reconstructed embryos and the donor cells simultaneously,the developmental potency of HMC reconstructed embryos in each treatment group was promoted,but the effect was not significant (P>0.05),which 0.25 to 0.5 μmol/L 5-Aza-CdR was better.In summary,the appropriate concentration (0.25 to 0.5 μmol/L) treatment of donor cells with DNA methylase inhibitor 5-Aza-CdR and treatment of reconstructed embryos with 20 nmol/L 5-Aza-CdR for 72 h could effectively improve in vitro developmental potency of HMC reconstructed embryos in Debao Black pig,which laid a foundation for the future study of DNA methylation regulation mechanism of HMC embryo in Debao Black pig.