细胞松弛素B对猪孤雌胚胎和体外克隆胚胎发育能力的影响

Effects of Cytochalasin B on the Developmental Ability of Parthenogenetic Embryos and Cloned Embryos in vitro in Pigs

为探讨细胞松弛素B(cytochalasin B,CB)对猪孤雌胚胎和克隆胚胎发育能力的影响,本研究通过在猪体外胚胎培养基中添加不同浓度CB以及不同孵育时间的处理,筛选出CB对猪早期胚胎发育的最适浓度和最佳孵育时间,同时通过Hoechst33342染色检测猪体外囊胚孵化期的细胞数差异,进一步研究CB对孤雌胚胎和克隆胚胎发育的影响。结果显示,培养基中添加CB浓度为7.5μg/mL时孤雌胚胎和克隆胚胎的卵裂率分别为85.00%和90.23%,囊胚率为35.68%和42.58%,均显著高于其他各组(P<0.05);采用7.5μg/mL CB处理电激活后的孤雌胚胎和克隆胚胎,孤雌胚胎孵育4 h组的卵裂率(83.80%)和囊胚率最高(35.39%),与其他各组差异显著(P<0.05),而克隆胚胎孵育6 h组的卵裂率(83.98%)和囊胚率最高(55.62%),与其他各组差异显著(P<0.05)。此外,Hoechst33342染色结果显示,未添加CB处理的孤雌胚胎在囊胚孵化期的细胞平均数为28个,CB处理组的孤雌胚胎和克隆胚胎细胞平均数分别为36和52个,处理组和未处理组细胞数差异显著(P<0.05)。结果表明,猪体外孤雌胚胎用7.5μg/mL CB处理4 h可获得较高的卵裂率和囊胚率;体外克隆胚胎用7.5μg/mL CB处理6 h卵裂率及囊胚率最高,且囊胚期内细胞团细胞总数最多。CB处理有利于体外胚胎早期发育,提高克隆胚胎移植受孕率。

The aim of this research was to investigate the effect of cytochalasin B(CB)on the developmental ability of parthenogenetic embryos and cloned embryos in pigs.The optimal concentration and incubation time of CB on early porcine embryo development were screened out by testing different concentrations and treatment time of CB in porcine in-vitro embryo culture medium.At the same time,Hoechst33342 staining was used to observe the difference in cell number of porcine blastocysts during incubation,to study the effect of CB on the development of parthenogenetic embryos and cloned embryos.The results showed that the cleavage rates of parthenogenetic embryos and cloned embryos incubated with 7.5μg/mL CB in standard culture conditions were 85.00%and 90.23%,respectively,and the blastocyst rates were 35.68%and 42.58%,which were significantly higher than other groups(P<0.05);While the cleavage and blastocyst rates of the parthenogenetic embryos for 4 h group were 83.80%and 35.39%,and the cloned embryos form cleavage and blastocyst for 6 h group were 83.98%and 55.62%,there were significant differences by corresponding to other groups(P<0.05),respectively.In addition,embryos stained with Hoechst33342 showed that the average number of parthenogenetic embryos inner cells without CB were 28,and inner cells average numbers in the parthenogenetic and cloned embryos with CB were 36 and 52.There was a significant difference in the number of inner cells between the treated and untreated groups(P<0.05).The results showed that the parthenogenetic embryos in vitro were treated for 4 h with 7.5μg/mL CB,which resulted in higher cleavage and blastocyst rate;and the cloned embryos in vitro were treated for 6 h with 7.5μg/mL obtained the highest of the cleavage and blastocyst rate.The results showed that CB treatment was beneficial embryo early development in vitro and improved the pregnancy rate of cloned embryos.