钙离子及山梨醇对猪卵母细胞孤雌激活的影响

探讨电激液中不同Ca2+浓度与不同电脉冲强度相互关系对猪体外成熟卵母细胞孤雌激活的影响;以及山梨醇电激活液在猪孤雌激活中的应用。结果表明,当Ca2+浓度不高时,同时增加电激液Ca2+浓度和电脉冲强度能协同促进孤雌胚胎的卵裂率和囊胚率的提高,Ca2+浓度为0.05和0.1mmol/L时,分别以1.6kV/cm和1.2kV/cm激活,得到的卵裂率为73.75%、74.70%;囊胚率为37.50%、36.83%,显著高于其余各组(P0.05);当Ca2+过高,增加脉冲强度卵母细胞孤雌发育能力反而下降,退化率升高;以山梨醇液进行电激活,在1.6kV/cm时,卵裂率和囊胚率最高,分别为77.23%和34.15%;与甘露醇电激液不同,施加交流电,山梨醇液不能对孤雌胚胎发育起到促进作用;将山梨醇、甘露醇电激液等体积混合,交流脉冲后进行电激活,1.2、1.6kV/cm组卵裂率分别为72.33%和70.03%,囊胚率分别为31.03%和29.60%,虽然略低于甘露醇组(77.07%、36.03%),但优于山梨醇组(69.63%、26.93%),差异不显著(P0.05)。以上结果说明,猪卵母细胞激活所需内流Ca2+浓度存在临界值,临界值内,提高Ca2+浓度或电参数,都能提高卵母细胞的激活效果;超过临界值,效果相反;山梨醇液能够取代甘露醇液用于猪卵细胞的电激活;同时以山梨醇和甘露醇作为电激液的基本成份,完全可以用于猪卵母细胞的电激活或融合。

Effect of calcium concentrations and activation media on parthenogenetic activation in pig

The relationship between Ca~(2+) concentration in activation medium and electrical pulse strength on parthe-nogenetic activation of porcine ooctyes was examined in this paper,and the application of sorbitol was also discussed.It was showed that : with low Ca~(2+) concentration, the increase of electrical pulse strength had positive effect on the development of parthenogenetic embryos. The cleavage rates (73.75%, 74.70%) and blastocyst rates (37.50%,36.83%) obtained under 0.05 mmol/L Ca~(2+) and 1.6 kV/cm pulse strength,and 0.1 mmol/L Ca~(2+) and 1.2 kV/cm,respectively,were significantly higher than those of other conditions (P<0.05). Under excessive level of Ca~(2+) con-centration,however,increase electrical pulse strength led to high degradation and low development rate,using sorbi-tol as electro-activation medium, the highest cleavage (77.23%) and blastocyst rates (34.15%) could be obtained under 1.6kV/cm DC;with the AC pulse before DC pulse, the sorbitol electroporation medium could not promoting parthenogenetic development; using equally mixed sorbitol and mannitol as electro-activation medium, the cleavage rates were 72.33% and 70.03% under 1.2 and 1.6 kV/cm electrical pulse,and blastocysts rates were 31.03% and 29.60% ,respectively. The development of mixture group was a little lower than that of mannitol group (77.07%,36.03 %), and higher than sorbitol group(69.63%, 26.93%), but no significantly difference(P>0.05). The results demonstrated that parthenogenetic activation of porcine oocytes needed a threshold levelof Ca~(2+) concentration. In-crease Ca~(2+) concentration and electrical pulse strength could improve the development within this level,otherwise not. The mannitol could be replaced by sorbitol as fundamental component in electro-activation of porcine oocytes.Mixture of sorbitol and mannitol also got a satisfied activation results.