秋水仙胺对电融合方法体外制备牛四倍体胚胎的影响

Effect of demecocline treatment on producing bovine tetraploid embryos in vitro by electrofusion

通过添加秋水仙胺从而改善电融合方法体外制备牛四倍体胚胎的过程。首先检测牛早期胚胎的分裂时间,并由此确定于体外受精后26~30h时间段内挑选既同期化且优质的2细胞期胚胎用于电融合。电融合参数选取2次直流电压为0.75kV/Cm且脉冲时间60μs。并通过免疫荧光染色监测电融合后细胞核的重组过程。设立电融合对照组和电融合秋水仙胺处理组,处理组在电融合后随即处理0.05mg/L秋水仙胺6h,后彻底洗涤继续体外培养至囊胚期。处理组的融合率（84.4%vs 84.8%）、分裂率（74.3%vs 77.6%）和囊胚率（36.1%vs 39.4%）皆低于对照组,但差异均不显著。获取的囊胚期胚胎的核型分析结果显示,处理组的四倍体制备率显著高于对照组（59.4%vs 26.9%）。综上所述,对电融合后的胚胎处理0.05mg/L秋水仙胺6h显著提高了牛四倍体电融合方法体外制的备率。

The main objective was to improve the procedure for producing bovine tetraploid embryo in vitro by electrofusion of 2-cell stage embryos through demecolcine treatment.We first assessed early embryonic cleavage timing,and established the time quantum to collect synchronous and good quality 2-cell stage bovine embryos for electrofusion during 26-30hafter IVF.Double pulses of 0.75kV/Cm（DC）for 60μs were carried out as electrofusion protocol.Ploidy transition was monitored by immunofluorescence visualization of nuclear remodeling in a fused 2-cell stage embryos from cattle.Two groups were set up as fused control and fused demecolcine treated group with 0.05mg/L demecolcine for 6hafter fusion.We found that there was not significantly difference in fused rates（84.4%vs 84.8%）,cleavage rates（74.3%vs 77.6%）and blastocyst formation rate（36.1%vs 39.4%）between the treated group and control group.However,significantly higher tetraploid rate was detected in the treated group（59.4%vs 26.9%）compared to control by karyotype assessment.In conclusion,treatment with 0.05mg/L demecolcine for 6hafter fusion significantly improved bovine tetraploid rate.