猪卵母细胞体外成熟和孤雌激活参数研究

Studies on Porcine Oocyte in Vitro Maturation and Parthenogenetic Activation

本研究比较了按不同方法贮藏的培养液对猪卵母细胞体外成熟及成熟后孤雌激活对胚胎发育的影响。此外,还探索了针对初情期前母猪体外成熟卵母细胞和孤雌激活方案。结果如下:①1.4 kv/cm、100μs、1DC电激活后,卵母细胞死亡率明显高于2.0 kv/cm、30μs、1DC和2.0 kv/cm、60μs、1DC处理组,但激活后胚胎卵裂率和囊胚率相似;②使用钙离子载体和6-二甲基氨基嘌呤(Ionomycin+6-DMAP)处理后,胚胎卵裂及囊胚率都明显不如电激活处理;③6次独立试验结果证明:经4℃冷藏和-20℃冷冻保存的培养液用于猪卵母细胞培养,其体外成熟率、孤雌激活后的卵裂率及囊胚发育率无显著差异(P>0.05)。说明成熟卵在2.0 kv/cm、30μs、1DC或者2.0 kv/cm、60μs、1DC电击参数激活下可以降低死卵率;按本试验设计的电激活方案处理初情期前母猪成熟卵优于化学激活;在-20℃冷冻保存猪卵母细胞成熟液是可行的。

The purposes of the present study were to evaluate cold storage of maturation medium on nuclear maturation, subsequent development in vitro of these oocytes after parthenogenetic activation, and to determine an optimal protocol for porcine oocyte activation. The results were as following： （1）Death rate of oocytes after electrical stimulation was significantly higher in group of 1.4 kv/cm,100μs,1DC than that in group of 2.0 kv/cm,30μs,1DC or in group of 2.0 kv/cm,60μs,1DC. The rates of cleavage and blastocyst formation, however, were similar among these goups. （2）When chemicals inomycin and 6- DMAP were used as activation agents, the cleavage and blastocyst rates were both poorer than when electrical stimulation was used. （3）After 6 replicates, we found that the rates of nuclear maturation, cleavage, blastocyst formation were all similar between oocytes matured in culture media prepared from cold storage in -20℃ and 4℃. In conclusion, optimal activation protocol was determined and cold storage of maturation medium under -20℃ is feasible without compromising developmental competence of porcine oocytes.