白藜芦醇处理对猪孤雌激活囊胚抗冻能力的影响

Effect of resveratrol on freezing tolerance of porcine parthenogenetic blastulas

为研究白藜芦醇(Resveratrol,RES)处理对猪孤雌激活囊胚抗冻能力的影响,利用2μmol/L RES在卵母细胞体外成熟、玻璃化冷冻与解冻和随后的胚胎培养过程中进行全程添加,观察其对猪孤雌激活囊胚冻后线粒体膜电位、TUNEL凋亡水平、多种Caspase活性、凋亡相关功能基因mRNA表达水平和体外发育能力的影响。结果显示:体外成熟和胚胎培养过程中添加RES能显著提高卵母细胞孤雌激活后的卵裂率和囊胚率(95.18%vs.85.79%,51.85%vs.41.46%,P<0.05)。RES全程添加后其孤雌激活囊胚的抗冻能力显著提高,表现为囊胚腔恢复率和囊胚细胞数增高(35.09%vs.31.25%,37.95 vs.31.8l,P<0.05),线粒体膜电位升高(0.92 vs.0.46,P<0.05),囊胚细胞凋亡比例下降(8.24%vs.10.13%,P<0.05),Pan-caspase、Caspase-8、Caspase-9和Caspase-3的荧光强度值和Caspase-8,Caspase-9,TNF-α基因表达水平下降,Bcl-2和SOD-1表达水平升高(P<0.05)。与冷冻囊胚相比,新鲜囊胚的线粒体膜电位水平、Bcl-2和SOD-1基因表达量更高;细胞凋亡比例、Pan^caspase、Caspase-8、Caspase-9、Caspase-3荧光强度值以及Caspase-8、Caspase-9、TNF-α基因表达水平则更低(P<0.05)。因此,RES全程添加可通过改善冻后猪孤雌激活囊胚的线粒体功能,降低囊胚细胞凋亡水平,从而提高其抗冻能力。

In order to evaluate the freezing tolerance of porcine parthenogenetic blastulas as influenced by resveratrol （RES）treatment,2 μmol/L RES was added in the whole courses of oocyte in vitro maturation ,freeze- thaw and embryo culture, and the mitochondrial membrane potentials of thawed porcine parthenogenetic blastulas,TUNEL apoptosis level, activities of several easpases, mRNA expression levels of genes related to apoptotic pathways and in vitro developmental capacity were investigated. The results showed that RES addition during in vitro maturation and embryo culture could significantly increase both cleavage and blastula rates （95.18% vs. 85.79% , 51.85% vs. 41.46% , P 〈 0.05 ）. RES addition significantly increased the freezing tolerance of porcine parthenogenetic blastulas, i. e. , the recovery rate of blastoeoeles and the cell number of blastulas increased （35.09% vs. 31.25 %, 37.95 vs. 31.81, P 〈 0.05 ） , the mitochondrial membrane potential improved （0.92 vs.O. 46.P〈O. O5）.the anontosis rate decreased （8.24% vs. 10. 13%, P 〈 O. 05 ）, the fluorescence intensities of Pan-caspase, Caspase-8, Caspase-9 and Caspase-3 and the expression levels of Caspase-8, Caspase-9 and TNF-α genes decreased （P 〈 0.05 ） , and the expression level of Bcl-2 and SOD-1 genes increased （P 〈 0. 05 ）. Compared with vitrified blastulas, the fresh blastulas were higher in the mitochondrial membrane potential and the expression levels of Bcl-2 and SOD-1 genes, and lower in the apoptosis rate and the fluorescence intensities of Pan-caspase, Caspase-3, Caspase-8, Caspase-9 as well as the expression levels of Caspase-8, Caspase-9, TNF-a genes （ P 〈 0.05 ）. It was concluded that RES addition could improve the mitochondrial function of vitrified porcine parthenogenetic blastulas, decrease their apoptosis level, and so enhance their freezing tolerance.