牛卵母细胞及胚胎的玻璃化冷冻研究
摘要
本实验对牛卵母细胞在成熟培养时间、玻璃化液处理时间、玻璃化载体、所带颗粒细胞层数、玻璃化液及浓度5个方面进行了比较选择,优化了牛卵母细胞的玻璃化冷冻保存方法。本实验还对牛胚胎在发育阶段、玻璃化冷冻载体的玻璃化冷冻效果进行了研究。(1)体外成熟培养22h后的牛母细胞冷冻效果较好,其冷冻解冻体外受精后的卵裂率和囊胚率分别为41.80%和15.23%,与成熟培养12h后的卵母细胞差异显著(p <0.05),与成熟16h、24h的卵母细胞差异不显著(p >0.05)。(2)卵母细胞在冷冻保护液中处理30S的玻璃化冷冻效果较好,其冷冻解冻体外受精后的卵裂率及囊胚率分别为41.18%和14.70%,囊胚率方面:30S组与15S、60S组差异极显著(p <0.01);与45S组差异显著(p <0.05)。(3)不同载体的对比:对玻璃OPS管、塑料OPS管和0.25ml细管作为卵母细胞的载体进行玻璃化冷冻效果比较后发现:玻璃OPS管和塑料OPS管冷冻效果较好。三种载体的卵母细胞解冻后无论体外受精的卵裂率还是囊胚率均无显著差异(p>0.05)。0.25ml细管玻璃化冷冻效果稍低于OPS玻璃化冷冻法,但差异不显著(p>0.05)(4)保留2~3层颗粒细胞的卵母细胞玻璃化冷冻的冷冻效果较好,其冷冻解冻体外受精后的卵裂率和囊胚率分别为40.13%和15.13%。囊胚率方面:2～3层颗粒细胞组与不含颗粒细胞组差异显著(p<0.05),2～3层颗粒细胞组与完整颗粒层组差异均不显著(p > 0.05)。完整颗粒层卵母细胞的卵裂率与不含颗粒层卵母细胞的卵裂率21.21%差异显著(p<0.05),含2～3层颗粒细胞的卵母细胞的卵裂率与不含颗粒层的卵母细胞差异极显著(p<0.01)。(5)通过两种冷冻保护剂及不同配比浓度玻璃化冷冻牛卵母细胞解冻后体外受精的卵裂率,体外培养的囊胚率的对比得出EDFS30这种组合及配比浓度的牛卵母细胞玻璃化冷冻效果较好。(6)不同发育时期的牛体外培养胚胎玻璃化冷冻效果得出冷冻牛囊胚效果比冷冻牛桑椹胚效果好。牛囊胚玻璃化冷冻解冻后形态正常率为85.71%,继续体外培养,孵育率为34.13%。桑椹胚冻后孵育率与囊胚组差异不显著(p>0.05),但结果低于后者。(7)玻璃化冷冻牛囊胚时使用玻璃细管、塑料OPS管效果较好,但与0.25ml细管的玻璃化冷冻效果差异不显著(p >0.05)。可以用0.25ml细管作为牛囊胚玻璃化冷冻的承载工具。通过上述实验为本实验室初步建立了牛卵母细胞及胚胎的玻璃化冷冻体系。
The study on bovine oocyte vitrification frozen were carried out from 5 aspects: the vitricationfreezen on different maturation time of bovine oocyte; the bovine oocyte vitrified by different vitrificationfluid and the same vitrification fluid with different concentration; the different carrier used in the bovineoocytevitrificationfrozen;theoocytestayatthesamevitrificationfluidwithdifferenttime;theoocytetakedifferent layer number of cumulus cell when it vitrified frozen. I also take study on the bovine embryovitrificationfrozenfrom2aspects:thevitrificationfrozenonthedifferentdeveloptimeofbovineembryo;thedifferentcarrierusedinthebovineembryovitrificationfrozen.Theconclusionare:
(1)The companany on the impact of the vitrication frozen on different develop time of bovine oocytefindthat:theimpactofvitrificationfrozenonbovineoocytewhichafter22hoursinvitro maturecultureisbetter. The oocyte morphologically normal rates after vitrification frozen is 94.14%.The bovine oocytecleavagerateis41.80%andtheblastocystrateis15.23%.
(2) The companany on the impact of the vitrication frozen on the oocyte stay at the same vitrificationfluid with different time find that:the impact of vitrification frozen on bovine oocyte which stay in thevitrification fluid for30second isbetter.Theoocyte morphologicallynormalrates aftervitrification frozenis93.53%.Thebovineoocytecleavagerateis41.18%andtheblastocystrateis14.70%.
(3)Through the contrast the impact of the different carrier used in the bovine oocyte vitrificationfrozen found that: the impact of glass straw carrier and plastic open pulled straw carrier which used inbovineoocytevitrificationfrozenisbetter.Whenuseglassstrawcarriertheoocyte morphologicallynormalrates after vitrification frozen is 91.20%.The bovine oocyte cleavage rate is 41.30%and the blastocyst rateis14.52%.
(4) Through the contrast the impact of vitrification frozen on oocyte which took the different numberof cumulus cell layer I found that: the impact of vitrification frozen on oocyte which take 2~3 layers ofcumulus cell is better than other number of cumulus cell layer. After vitrification frozen the oocytemorphologicallynormalrates is90.79%.The bovine oocyte cleavage rate is40.13%and the blastocyst rateis15.13%.
(5) Through the contrast the impact of vitrification frozen on oocyte which vitrified by differentvitrificationfluidandthesamevitrificationfluidwithdifferentconcentrationfoundthat: thecombinationandconcentrationofEDFS30hasbetterimpactofbovineoocytevitrificationfrozen.
(6) Through the contrast the impact of vitrification frozen on the different develop time of bovineembryo I found that: The impact of vitrification frozen on bovine blastocysts stage is better than theimpact of vitrification frozen on bovine morula stage. After vitrification frozen the bovine blastocystsmorphologically normal rates is 85.71%. After invitro culture the developing rates of bovine blastocystsis34.13%.
(7)The impact of glass straw carrier and plastic open pulled straw carrier which used in bovineblastocysts vitrification frozen is better. But the impact of 0.25ml tubule carrier used in bovine blastocystsvitrification frozen is not prominence difference with them. So we can use the 0.25ml tubule carrier inbovineblastocystsvitrificationfrozen.
The study on bovine oocyte vitrification frozen were carried out from 5 aspects: the vitricationfreezen on different maturation time of bovine oocyte; the bovine oocyte vitrified by different vitrificationfluid and the same vitrification fluid with different concentration; the different carrier used in the bovineoocytevitrificationfrozen;theoocytestayatthesamevitrificationfluidwithdifferenttime;theoocytetakedifferent layer number of cumulus cell when it vitrified frozen. I also take study on the bovine embryovitrificationfrozenfrom2aspects:thevitrificationfrozenonthedifferentdeveloptimeofbovineembryo;thedifferentcarrierusedinthebovineembryovitrificationfrozen.Theconclusionare:
(1)The companany on the impact of the vitrication frozen on different develop time of bovine oocytefindthat:theimpactofvitrificationfrozenonbovineoocytewhichafter22hoursinvitro maturecultureisbetter. The oocyte morphologically normal rates after vitrification frozen is 94.14%.The bovine oocytecleavagerateis41.80%andtheblastocystrateis15.23%.
(2) The companany on the impact of the vitrication frozen on the oocyte stay at the same vitrificationfluid with different time find that:the impact of vitrification frozen on bovine oocyte which stay in thevitrification fluid for30second isbetter.Theoocyte morphologicallynormalrates aftervitrification frozenis93.53%.Thebovineoocytecleavagerateis41.18%andtheblastocystrateis14.70%.
(3)Through the contrast the impact of the different carrier used in the bovine oocyte vitrificationfrozen found that: the impact of glass straw carrier and plastic open pulled straw carrier which used inbovineoocytevitrificationfrozenisbetter.Whenuseglassstrawcarriertheoocyte morphologicallynormalrates after vitrification frozen is 91.20%.The bovine oocyte cleavage rate is 41.30%and the blastocyst rateis14.52%.
(4) Through the contrast the impact of vitrification frozen on oocyte which took the different numberof cumulus cell layer I found that: the impact of vitrification frozen on oocyte which take 2~3 layers ofcumulus cell is better than other number of cumulus cell layer. After vitrification frozen the oocytemorphologicallynormalrates is90.79%.The bovine oocyte cleavage rate is40.13%and the blastocyst rateis15.13%.
(5) Through the contrast the impact of vitrification frozen on oocyte which vitrified by differentvitrificationfluidandthesamevitrificationfluidwithdifferentconcentrationfoundthat: thecombinationandconcentrationofEDFS30hasbetterimpactofbovineoocytevitrificationfrozen.
(6) Through the contrast the impact of vitrification frozen on the different develop time of bovineembryo I found that: The impact of vitrification frozen on bovine blastocysts stage is better than theimpact of vitrification frozen on bovine morula stage. After vitrification frozen the bovine blastocystsmorphologically normal rates is 85.71%. After invitro culture the developing rates of bovine blastocystsis34.13%.
(7)The impact of glass straw carrier and plastic open pulled straw carrier which used in bovineblastocysts vitrification frozen is better. But the impact of 0.25ml tubule carrier used in bovine blastocystsvitrification frozen is not prominence difference with them. So we can use the 0.25ml tubule carrier inbovineblastocystsvitrificationfrozen.
引文
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