牛卵泡卵母细胞体外成熟、体外受精及受精卵体外培养的研究
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摘要
摘要
本研究利用屠宰黄牛卵巢，对卵巢表面2-8mm卵泡卵母细胞的体外成熟（IVM）--体外受精（IVF）--受精卵的体外发育（IVD）进行了系列研究，并探讨了其影响因素，结果如下：
1.采用400μl并且不覆盖石蜡油的U形培养皿培养卵母细胞及受精卵，卵母细胞的体外成熟率、卵裂率及受精卵的桑椹胚、囊胚发育率与经典的覆盖石蜡油的微滴（50μl）培养卵母细胞的相应指标间差异不显著，表明在培养环境相对稳定的情况下，可以采用U形皿（不覆盖石蜡油）培养卵母细胞和胚胎，以简化胚胎体外生产的程序。
2..采用M-199+10%发情牛血清（ECS）不添加激素培养牛卵泡卵母细胞，获得了较高的卵母细胞体外成熟率（80.67%），卵裂率（57.07%）和桑囊率（30%），进一步证明在成熟培养液中单独添加10%ECS是可行的，适宜于在生产中推广应用。
3.对冻精体外获能的精子处理方法上游法、Percoll法及直接离心洗涤法进行了比较实验。虽然三组受精卵卵裂率无明显差异（P>0.05），上游法处理组与Percoll法处理组，Percoll法处理组与洗涤法处理组桑囊率差异均不显著（分别为29.10%，28.47%和28.47%，22.41%%；P>0.05），但上游法处理组与洗涤法处理组桑囊率差异显著（分别为29.10%和22.41%，P<0.05），表明在考虑成本的商业化胚胎生产中，上游法更适于应用。
4.比较了改良台氏液（FERT-TALP）作为受精液时，6-8小时精卵分开与18-20小时精卵分开对受精卵体外发育的影响效果，结果两个处理组间卵裂率、8-细胞发育率及桑囊率差异均不显著（P>0.05），表明6-8小时精卵分开不影响受精卵体外发育，更有利于胚胎体外生产的实际。
5用人胚胎培养基IVC-ONETM（0-8细胞阶段）和Quinn's D3+HTF（8-细胞后发育阶段）+10%ECS培养牛胚胎获得了与M-199培养基相似的效果（牛培养基处理组与人培养基处理组8-细胞发育率和桑囊率分别为60.63%，64.83%和26.60%，32.76%）。提示可以通过研究人胚胎培养基的组成及对胚胎的作用机理优化牛胚胎的体外培养体系。
6.建立了相对比较实用而且有效的牛卵泡卵母细胞IVM-IVF-IVC程序：利用U形培养皿和M-199+25mMHepes+10%D0ECS高密度（50-60枚/400μl）培养牛卵泡卵母细胞23h后，用改良台氏液加6mg/mlBSA和5mM咖啡因及上游法洗涤精液、分离活精子及获能（另加10μg/ml 肝素）；精卵在受精液中共同孵育6-8小时后转移至M-199+10%ECS+颗粒细胞单层或输卵管上皮细胞单层中培养，直至发育到桑椹胚或囊胚。
Abstract
In this paper, cattle ovaries collected from Xi'an slaughterhouse were used, ability of bovine 2-8mm follicular oocytes for in vitro maturation(IVM), in vitro fertilization(IVF), in vitro culture(IVC) of fertilized eggs were studied, and factors affecting IVM、IVF and IVC were also investigated. The results were as follows:
⒈ The corresponding maturation rate ,cleavage rate of oocytes and in vitro development rate of morula or blastocyst had no significant differences(P>0.05) between in U-shaped glass service,which volume is 400μl and no mineral oil overlaided and in traditional droplet,which was overlaided by mineral oil and volume is 50μl. The results indicated that U-shaped glass service of which volume is larger than conventional culture service can simplify the procedure of in vitro production of bovine embryos and may be used as a kind of medium for culturing oocytes and embryos under steady cultural circumstance.
⒉ Single addition of 10%D0ECS(V/V) in maturation medium obtained higher maturation rate (80.67%),cleavage rate(57.07%) and development rate of morula or blastocyst(30.00%) .The results indicated further that single addition of 10% D0ECS is practicable,and and is fit for applying spreadly.
⒊ Comparative experiments were carried out among swimming-up method, Percoll method and Washing method of treatment methods of frozen--semen in order to observe respective effect of these 3 methods on the in vitro development ability of fertilized eggs. The results shows that there was not any significant differences among cleavage rate (P>0.05) of fertilized eggs treatedby frozen-semen from swimming-up method, Percoll method and Washing method respectively, there was not any significant differences of in vitro development rate of morula or blastocyst between treatment of swimming-up method and of Percoll method; between treatment of Percoll method and of Washing method(29.10%和28.47% :28.47%和22.41%；respectively, P>0.05),but there was significant differences of in vitro development rate of morula or blastocyst between treatment of swimming-up method and of Washing method(29.10%and 22.41%，respectively,P<0.05).The result indicated that swimming-up method is more suitable for applying in IVP of bovine embryos than Percoll method and Washing method under circumstance of considering cost.
⒋ Compared with 18-20 h' oocyte-sperm interaction when m--Tyrode's was used as in vitro fertilization medium ,6-8h' oocyte-sperm interaction has not significant effect on the cleavage rate of zygote,in vitro development rate of 8-cell and morula or blastocyst(P>0.05).This result indicated that conventional 18-20h'oocyte-sperm interaction can be substituted by 6-8h'oocyte-sperm interaction so as to accord further practicality of IVP of bovine embryo.
⒌ Embryonic culture medium of human,that is IVC-ONETM（0-8cell stage）and Quinn's D3+HTF（8cell-morula or blastocyst developmental stage）were used tentatively for culturing bovine embryo, and gained the same effect as bovine culture medium --TCM-199 (IVP rate of 8-cell and morula or blastocyst were 60.63%，64.83%and 26.60%，32.76% respectively between treatment of human's embryonic culture medium and of bovine culture medium.).This result implied that research of both composition of human's embryonic culture medium and its mechanism to embryo can contribute to optimizing in vitro culture system of bovine embryo.
⒍ Comparatively feasible and effective IVM-IVF-IVC procedure of bovine follicular oocytes was established as follows: Bovine cumulus-enclosed oocytes were matured in tissue culture medium-199(with Earle's salts)buffered with 25mM Hepes and supplemented with 10%(v/v)ECS, which solution filled in U-shaped glass service and volume is 400μl and no mineral oil overlaided. After 23h of IVM, matured oocytes and sperms which come from frozen-semen treated by swimming-up method were put in IVF medium contained 6mg/ml bovine serum albumin(BSA) , 10μg/ml heparin and 5mM caffeine. Oocytes and sperms were incubated all together for 6-8h, then presumed
本研究利用屠宰黄牛卵巢，对卵巢表面2-8mm卵泡卵母细胞的体外成熟（IVM）--体外受精（IVF）--受精卵的体外发育（IVD）进行了系列研究，并探讨了其影响因素，结果如下：
1.采用400μl并且不覆盖石蜡油的U形培养皿培养卵母细胞及受精卵，卵母细胞的体外成熟率、卵裂率及受精卵的桑椹胚、囊胚发育率与经典的覆盖石蜡油的微滴（50μl）培养卵母细胞的相应指标间差异不显著，表明在培养环境相对稳定的情况下，可以采用U形皿（不覆盖石蜡油）培养卵母细胞和胚胎，以简化胚胎体外生产的程序。
2..采用M-199+10%发情牛血清（ECS）不添加激素培养牛卵泡卵母细胞，获得了较高的卵母细胞体外成熟率（80.67%），卵裂率（57.07%）和桑囊率（30%），进一步证明在成熟培养液中单独添加10%ECS是可行的，适宜于在生产中推广应用。
3.对冻精体外获能的精子处理方法上游法、Percoll法及直接离心洗涤法进行了比较实验。虽然三组受精卵卵裂率无明显差异（P>0.05），上游法处理组与Percoll法处理组，Percoll法处理组与洗涤法处理组桑囊率差异均不显著（分别为29.10%，28.47%和28.47%，22.41%%；P>0.05），但上游法处理组与洗涤法处理组桑囊率差异显著（分别为29.10%和22.41%，P<0.05），表明在考虑成本的商业化胚胎生产中，上游法更适于应用。
4.比较了改良台氏液（FERT-TALP）作为受精液时，6-8小时精卵分开与18-20小时精卵分开对受精卵体外发育的影响效果，结果两个处理组间卵裂率、8-细胞发育率及桑囊率差异均不显著（P>0.05），表明6-8小时精卵分开不影响受精卵体外发育，更有利于胚胎体外生产的实际。
5用人胚胎培养基IVC-ONETM（0-8细胞阶段）和Quinn's D3+HTF（8-细胞后发育阶段）+10%ECS培养牛胚胎获得了与M-199培养基相似的效果（牛培养基处理组与人培养基处理组8-细胞发育率和桑囊率分别为60.63%，64.83%和26.60%，32.76%）。提示可以通过研究人胚胎培养基的组成及对胚胎的作用机理优化牛胚胎的体外培养体系。
6.建立了相对比较实用而且有效的牛卵泡卵母细胞IVM-IVF-IVC程序：利用U形培养皿和M-199+25mMHepes+10%D0ECS高密度（50-60枚/400μl）培养牛卵泡卵母细胞23h后，用改良台氏液加6mg/mlBSA和5mM咖啡因及上游法洗涤精液、分离活精子及获能（另加10μg/ml 肝素）；精卵在受精液中共同孵育6-8小时后转移至M-199+10%ECS+颗粒细胞单层或输卵管上皮细胞单层中培养，直至发育到桑椹胚或囊胚。
Abstract
In this paper, cattle ovaries collected from Xi'an slaughterhouse were used, ability of bovine 2-8mm follicular oocytes for in vitro maturation(IVM), in vitro fertilization(IVF), in vitro culture(IVC) of fertilized eggs were studied, and factors affecting IVM、IVF and IVC were also investigated. The results were as follows:
⒈ The corresponding maturation rate ,cleavage rate of oocytes and in vitro development rate of morula or blastocyst had no significant differences(P>0.05) between in U-shaped glass service,which volume is 400μl and no mineral oil overlaided and in traditional droplet,which was overlaided by mineral oil and volume is 50μl. The results indicated that U-shaped glass service of which volume is larger than conventional culture service can simplify the procedure of in vitro production of bovine embryos and may be used as a kind of medium for culturing oocytes and embryos under steady cultural circumstance.
⒉ Single addition of 10%D0ECS(V/V) in maturation medium obtained higher maturation rate (80.67%),cleavage rate(57.07%) and development rate of morula or blastocyst(30.00%) .The results indicated further that single addition of 10% D0ECS is practicable,and and is fit for applying spreadly.
⒊ Comparative experiments were carried out among swimming-up method, Percoll method and Washing method of treatment methods of frozen--semen in order to observe respective effect of these 3 methods on the in vitro development ability of fertilized eggs. The results shows that there was not any significant differences among cleavage rate (P>0.05) of fertilized eggs treated
⒋ Compared with 18-20 h' oocyte-sperm interaction when m--Tyrode's was used as in vitro fertilization medium ,6-8h' oocyte-sperm interaction has not significant effect on the cleavage rate of zygote,in vitro development rate of 8-cell and morula or blastocyst(P>0.05).This result indicated that conventional 18-20h'oocyte-sperm interaction can be substituted by 6-8h'oocyte-sperm interaction so as to accord further practicality of IVP of bovine embryo.
⒌ Embryonic culture medium of human,that is IVC-ONETM（0-8cell stage）and Quinn's D3+HTF（8cell-morula or blastocyst developmental stage）were used tentatively for culturing bovine embryo, and gained the same effect as bovine culture medium --TCM-199 (IVP rate of 8-cell and morula or blastocyst were 60.63%，64.83%and 26.60%，32.76% respectively between treatment of human's embryonic culture medium and of bovine culture medium.).This result implied that research of both composition of human's embryonic culture medium and its mechanism to embryo can contribute to optimizing in vitro culture system of bovine embryo.
⒍ Comparatively feasible and effective IVM-IVF-IVC procedure of bovine follicular oocytes was established as follows: Bovine cumulus-enclosed oocytes were matured in tissue culture medium-199(with Earle's salts)buffered with 25mM Hepes and supplemented with 10%(v/v)ECS, which solution filled in U-shaped glass service and volume is 400μl and no mineral oil overlaided. After 23h of IVM, matured oocytes and sperms which come from frozen-semen treated by swimming-up method were put in IVF medium contained 6mg/ml bovine serum albumin(BSA) , 10μg/ml heparin and 5mM caffeine. Oocytes and sperms were incubated all together for 6-8h, then presumed
引文
参考文献
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