影响绵羊卵母细胞玻璃化冷冻效果的因素研究
摘要
卵母细胞玻璃化冷冻保存对于珍稀和濒危种质资源保存、“卵子库”的建立、胚胎生物技术的发展具有重要意义。本实验以绵羊卵母细胞为研究模型,通过比较卵母细胞不同发育阶段、卵丘细胞有无及冷冻前用紫杉醇及细胞松弛素B预处理对绵羊卵母细胞玻璃化冷冻保存效果的影响,并对解冻后卵母细胞进行体外受精、孤雌激活,结果表明:(1)体外成熟24h绵羊卵母细胞(MⅡstage)与未成熟卵母细胞(GV stage)相比,玻璃化冷冻-解冻后FAD染色存活率为(70.63% vs. 58.51%);体外受精卵裂率(38.42%vs.22.68%),囊胚率(5.89%vs.0)具有显著性差异(P<0.05)。表明:成熟的绵羊卵母细胞更适宜冷冻保存。(2)卵丘细胞的有无对玻璃化冷冻MⅡ期卵母细胞解冻后存活率、卵裂率、囊胚率均无显著性差异(P>0.05)。(3)采用浓度为0.5μmol/L紫杉醇预处理绵羊卵母细胞冷冻保存效果最佳,解冻后卵母细胞的存活率(82.38%)与其他三个实验组相比有显著差异性(P<0.05);体外受精卵裂率(51.17%)和桑囊胚率(20.30%)亦显著高于其他实验组(P<0.05),但低于对照组(69.42%,34.77%)(P<0.05);0.5μmol/L紫杉醇处理组孤雌激活卵裂率(53.61%)和桑囊胚率(22.64%),显著高于其他实验组,但于对照组(73.40%,33.79)(P<0.05)。(4)7.5μg/mL的细胞松弛素B预处理绵羊卵母细胞冷冻-解冻后的存活率(64.50% vs.68.87%)无显著性差异,体外受精卵裂率及桑囊胚率和孤雌激活卵裂率及桑囊胚率两组之间亦无显著性差异(P>0.05),但均显著低于对照组(P<0.05)。
综上所述,与GV期绵羊卵母细胞相比,MⅡ期卵母细胞更适宜冷冻保存; 0.5μmol/L紫杉醇有利于提高绵羊体外成熟卵母细胞玻璃化冷冻保存效果。
Oocyte vitrification is of great significance for rare and endangered animal germplasm conservation , "egg bank" established, and the development of embryonic biotechnology. In this study, we take ovine oocytes as the research model to study the effects of different developmental stages of oocytes, with or without cumulus cells and pre-treatment with Taxol and Cytochalasin B on the cryopreservation efficiency of ovine oocytes, and the frozen-thawed oocytes are used for in vitro fertilization and parthenogenetic activation,status the cleavage rate and blastocyst rate for evaluation.The results show that: (1) in vitro maturation 24 h ovine oocytes(MⅡstage) and immature oocytes(GV stage) vitrification are compared, and the thawed viability for FAD staining viability rate (70.63%vs.58.51%), after IVF, cleavage rate(38.42%vs.22.68%), blastocyst rate (5.89%vs.0)are significant differences (P<0.05). It indicated that: mature ovine oocytes is more suitable for cryopreservation. (2) with or without culumus cells has no significant difference for MII oocytes vitrification in survival rate and cleavage rate or blastocyst rate (P>0.05).(3)Pre-treatment the ovine oocytes with the concentration(0.5μmol/L) of taxol cryopreservation best effect, after thawed, the efficience was verified by parthenogenetic activation and in vitro fertilization.The results showed that: the survival rate(82.38%)in 0.5μmol/L taxol group was significantly higher (P < 0.05) than those in three groups; there was significant diferences between 0.5μmol/L taxol group and those three treatment groups in cleavage rate ( 51.37% ) and morula-blastocyst rate(20.30%) of in vitro fertilization of frozen-thaw oocytes(P < 0.05), but was significantly lower (P < 0.05) than the control(69.42%,34.77%)(P<0.05). And pre-treatment with 0.5μmol/L taxol mature in vitro ovine oocytes, the cleavage rate(53.61%)and morula-blastocyst rate(22.64%) of parthenogenetic activation of frozen-thaw oocytes was higher than those three treatment groups(P < 0.05), but was significantly lower than the control(73.40%,33.79)(P<0.05). (4)The survive rate of frozen-thawed oocytes between in vitrification group without cytochalansin B treatment and with cytochalansin B treatment (64.50% vs.68.87%) was no significant difference(P>0.05). After in vitro fertilization (IVF) and parthenogenetic activation, the cleavage and morula-blastocyst rates of the frozen-thawed oocytes in both the vitrification (without cytochalansin B treatment) and cytochalansin B treatment group were also no significant difference(P>0.05),but also lower than those in the control(P<0.05).
In conclusion,compare with GV stage ovine oocytes, MⅡstage oocytes are more suitable for cryopreservation; 0.5μmol/L taxol is benificial to improve the cryopreservation effect of in vitro maturation sheep oocytes .
综上所述,与GV期绵羊卵母细胞相比,MⅡ期卵母细胞更适宜冷冻保存; 0.5μmol/L紫杉醇有利于提高绵羊体外成熟卵母细胞玻璃化冷冻保存效果。
Oocyte vitrification is of great significance for rare and endangered animal germplasm conservation , "egg bank" established, and the development of embryonic biotechnology. In this study, we take ovine oocytes as the research model to study the effects of different developmental stages of oocytes, with or without cumulus cells and pre-treatment with Taxol and Cytochalasin B on the cryopreservation efficiency of ovine oocytes, and the frozen-thawed oocytes are used for in vitro fertilization and parthenogenetic activation,status the cleavage rate and blastocyst rate for evaluation.The results show that: (1) in vitro maturation 24 h ovine oocytes(MⅡstage) and immature oocytes(GV stage) vitrification are compared, and the thawed viability for FAD staining viability rate (70.63%vs.58.51%), after IVF, cleavage rate(38.42%vs.22.68%), blastocyst rate (5.89%vs.0)are significant differences (P<0.05). It indicated that: mature ovine oocytes is more suitable for cryopreservation. (2) with or without culumus cells has no significant difference for MII oocytes vitrification in survival rate and cleavage rate or blastocyst rate (P>0.05).(3)Pre-treatment the ovine oocytes with the concentration(0.5μmol/L) of taxol cryopreservation best effect, after thawed, the efficience was verified by parthenogenetic activation and in vitro fertilization.The results showed that: the survival rate(82.38%)in 0.5μmol/L taxol group was significantly higher (P < 0.05) than those in three groups; there was significant diferences between 0.5μmol/L taxol group and those three treatment groups in cleavage rate ( 51.37% ) and morula-blastocyst rate(20.30%) of in vitro fertilization of frozen-thaw oocytes(P < 0.05), but was significantly lower (P < 0.05) than the control(69.42%,34.77%)(P<0.05). And pre-treatment with 0.5μmol/L taxol mature in vitro ovine oocytes, the cleavage rate(53.61%)and morula-blastocyst rate(22.64%) of parthenogenetic activation of frozen-thaw oocytes was higher than those three treatment groups(P < 0.05), but was significantly lower than the control(73.40%,33.79)(P<0.05). (4)The survive rate of frozen-thawed oocytes between in vitrification group without cytochalansin B treatment and with cytochalansin B treatment (64.50% vs.68.87%) was no significant difference(P>0.05). After in vitro fertilization (IVF) and parthenogenetic activation, the cleavage and morula-blastocyst rates of the frozen-thawed oocytes in both the vitrification (without cytochalansin B treatment) and cytochalansin B treatment group were also no significant difference(P>0.05),but also lower than those in the control(P<0.05).
In conclusion,compare with GV stage ovine oocytes, MⅡstage oocytes are more suitable for cryopreservation; 0.5μmol/L taxol is benificial to improve the cryopreservation effect of in vitro maturation sheep oocytes .
引文
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