奶牛卵母细胞玻璃化冷冻方法及胚胎程序化冷冻研究
摘要
卵母细胞和胚胎冷冻技术在家畜遗传资源保存方面具有广泛和潜在的应用价值。本试验用OPS管或GMP管对牛卵母细胞玻璃化冷冻和用麦管对胚胎程序化冷冻进行研究。在冷冻时,对基础液、不同发育阶段的牛卵母细胞、保护剂、冷冻载体、解冻步骤进行比较试验。同时研究三步平衡法和一步细管法冷冻牛胚胎和移植受胎率效果。
1.基础液中添加牛血清白蛋白或胎牛血清对GV期牛卵母细胞的冷冻效果。冻后对照组和两个试验组形态正常率分别为45%,65.1%,65.6%,成熟率分别为28.%,44.4%,44.2%,卵裂率分别为11.7%,23.8%,21.3%,试验组与对照组差异显著,但添加BAS和FCS两组之间差异不显著。
2.不同发育阶段的牛卵母细胞玻璃化冷冻效果。冷冻GV期和MⅡ期,结果形态正常率分别为71.3%和68.8%,差异不显著(p>0.05);成熟率分别为45%和47.6%,差异显著(p<0.05);卵裂率分别为23.3%和31.1%,差异显著(p<0.05)。
3.不同颗粒细胞层数的牛卵母细胞玻璃化冷冻效果。裸卵,含2～4层颗粒细胞,颗粒细胞完整的卵母细胞冻后形态正常率分别为21.7%,60.3%,60.7%,成熟率分别为6.7%,46%, 44.2%,卵裂率分别为1.6%,30.2%,24.6%。结果含2～4层颗粒细胞的卵母细胞冷冻效果最好。
4.不同玻璃化冷冻溶液对OPS法冷冻GV期牛卵母细胞体外发育的影响。在EFS30和EFS40液中,冻后形态正常率分别为67.2%和66.7%,差异不显著(p>0.05);成熟率分别为36.2%和37.6%,差异不显著(p>0.05)。在EDFS30和EDFS40中,冻后形态正常率分别为73.1%和75.4%,差异不显著(p>0.05);成熟率分别为42.9%和43.7%,差异不显著(p>0.05)。而在EFS30液和EDFS30液中,冻后形态正常率分别为67.2%和73.1%,差异显著(p<0.05);成熟率分别为36.2%和42.9%,差异显著(p<0.05),EFS40与EDFS40在影响成熟率上差异也显著。
5.不同玻璃化冷冻溶液对GMP法冷冻GV期牛卵母细胞体外发育的影响,在EFS30和EFS40液中,冻后形态正常率分别为63.5%和63.9%,差异不显著(p>0.05);成熟率分别为33.7%和36.1%,差异不显著(p>0.05);在EDFS30液和EDFS40液中,冻后形态正常率分别为78.6%和75%,差异不显著(p>0.05);成熟率分别为43.8%和47.6%,差异不显著(p>0.05)。EDFS30和EDFS40均是牛GV期卵母细胞良好的玻璃化冷冷冻液。
6.采用OPS法和GMP法冷冻牛GV期卵母细胞,在EFS40中冻后形态正常率分别为68.4%和74.1%,差异不显著(p>0.05);成熟率分别为47.3%和48.4%,差异不显著(p>0.05);在EDFS40液中冷冻后形态正常率分别为74.6%和75.4%,差异不显著(p>0.05);成熟率分别为42.4%和49.1%,差异不显著(p>0.05);OPS管和GMP管都是比较理想的冷冻载体。
7.不同解冻程序对玻璃化冷冻的牛卵母细胞发育影响。采用EFS30玻璃化冷冻液,OPS法冷冻GV期牛卵细胞,通过一步法,三步法,五步法解冻,形态正常率分别为32.3%,51.7%,49.1%,成熟率分别为25.4%,43.4%,36.7%,卵裂率分别为18.7%,30%,29.8%,三步解冻法效果较好。
8.程序化冷冻牛胚胎移植效果。采用三步平衡法和一步细管法,结果显示,用10%甘油作抗冻剂,三步平衡冷冻,三步解冻的胚胎移植后受胎率为38.3%和用15%乙二醇作抗冻剂一步细管法冷冻胚胎移植后受胎率为36.9%,两者差异不显著(p>0.05)。
Cryopreservation of oocytes and embryos is a potentially valuable technique for conservation of farm animal genetic resource and population. The bovine oocytes were vitrified by different vitrification fluid and the same vitrification fluid with open pulled straw(OPS) or glass micro-pipe(GMP).The influence of different priming solution,cryoprotectants,carrier,freezing and thawing methods on developmental capability of bovine oocytes was examined and frozen embryos of bovine also was transferred.
1. The GV-oocytes were vitrified by the same vitrification fluid with different concentration adding BAS or FCS. Supplementing concentration of BSA or FCS can influence rates of morphologically normal and matured oocytes.
2.The bovine oocytes in different stage(GV and MⅡ) were vitrified. There were no statistical significant differences in rates of morphologically normal (71.3%,68.8%,p>0.05)and rates of matured oocytes(45%,47.6%,p>0.05).There was significant difference in rate of cleavge(23.3%,31.1%,p<0.05).
3. Through the contrast impact of vitrification frozen on oocytes with the different number of cumulus cell layer .It was found that,the impact of vitrification frozen on oocyte with 2~4 layers of cumulus cell is better than other number of cumulus cell layer.After vitrification of the oocytes, rates of morphologically normal oocytes were 21.7%,60.3%,60.7%, cleavage rates were 1.6%,30.2%,24.6%.
4.In GV-stage oocytes, Through the contrast impact of vitrification on GV-stage oocytes vitrified by the same vitrification fluid with different concentration and different vitrification fluid ,There was no significant difference in rate of morphologically normal oocytes (67.2%,66.7%,p>0.05) and rate of maturation (42.9%,43.7%,p>0.05) with EFS30 or EFS40. But there was significant difference in rate of morphologically normal oocytes and the maturation rate of the oocytes vitrified between EFS and EDFS.
5. Through the contrast impact of vitrification frozen on oocyte vitrified by different vitrification fluid and the same vitrification fluid with different concentration, EDFS has better impact of bovine oocyte vitrification frozen.
6.In GV-stage oocytes, there was no difference in rates of morphologically normal oocytes(68.4%,74.1%,p>0.05),the maturation rate(47.3%,48.4%,p>0.05)by OPS or GMP with EFS40, while there was difference between EFS40 and EDFS40 vitrification in the maturation rates(48.4%, 49.1%,p<0.05)by GMP .
7.The rates of morphologically normal and matured of oocytes with three-step or five-step thawing method were higher than one-step,while there was difference in rates of morphologically normal oocytes(32.3%,51.7%,49.1%,p<0.05),rates of maturation(25.4%,43.4%,36.7%,p<0.05)and rates of cleavage(18.7%,30%,29.8%,p<0.05).
8.Effect of transferring bovine ftozen embryos showed that there was no significant differences in pregnancy between one and three-step two methods.Pregnancy rate of bovine embryos frozen by three-step programmed freezing with supplementing concentration of 10% GLY was 38.3%,and pregnantcy rate of bovine embryos frozen by one-step programmed freezing with supplementing concentration of 15% EG was 39.6%.
1.基础液中添加牛血清白蛋白或胎牛血清对GV期牛卵母细胞的冷冻效果。冻后对照组和两个试验组形态正常率分别为45%,65.1%,65.6%,成熟率分别为28.%,44.4%,44.2%,卵裂率分别为11.7%,23.8%,21.3%,试验组与对照组差异显著,但添加BAS和FCS两组之间差异不显著。
2.不同发育阶段的牛卵母细胞玻璃化冷冻效果。冷冻GV期和MⅡ期,结果形态正常率分别为71.3%和68.8%,差异不显著(p>0.05);成熟率分别为45%和47.6%,差异显著(p<0.05);卵裂率分别为23.3%和31.1%,差异显著(p<0.05)。
3.不同颗粒细胞层数的牛卵母细胞玻璃化冷冻效果。裸卵,含2～4层颗粒细胞,颗粒细胞完整的卵母细胞冻后形态正常率分别为21.7%,60.3%,60.7%,成熟率分别为6.7%,46%, 44.2%,卵裂率分别为1.6%,30.2%,24.6%。结果含2～4层颗粒细胞的卵母细胞冷冻效果最好。
4.不同玻璃化冷冻溶液对OPS法冷冻GV期牛卵母细胞体外发育的影响。在EFS30和EFS40液中,冻后形态正常率分别为67.2%和66.7%,差异不显著(p>0.05);成熟率分别为36.2%和37.6%,差异不显著(p>0.05)。在EDFS30和EDFS40中,冻后形态正常率分别为73.1%和75.4%,差异不显著(p>0.05);成熟率分别为42.9%和43.7%,差异不显著(p>0.05)。而在EFS30液和EDFS30液中,冻后形态正常率分别为67.2%和73.1%,差异显著(p<0.05);成熟率分别为36.2%和42.9%,差异显著(p<0.05),EFS40与EDFS40在影响成熟率上差异也显著。
5.不同玻璃化冷冻溶液对GMP法冷冻GV期牛卵母细胞体外发育的影响,在EFS30和EFS40液中,冻后形态正常率分别为63.5%和63.9%,差异不显著(p>0.05);成熟率分别为33.7%和36.1%,差异不显著(p>0.05);在EDFS30液和EDFS40液中,冻后形态正常率分别为78.6%和75%,差异不显著(p>0.05);成熟率分别为43.8%和47.6%,差异不显著(p>0.05)。EDFS30和EDFS40均是牛GV期卵母细胞良好的玻璃化冷冷冻液。
6.采用OPS法和GMP法冷冻牛GV期卵母细胞,在EFS40中冻后形态正常率分别为68.4%和74.1%,差异不显著(p>0.05);成熟率分别为47.3%和48.4%,差异不显著(p>0.05);在EDFS40液中冷冻后形态正常率分别为74.6%和75.4%,差异不显著(p>0.05);成熟率分别为42.4%和49.1%,差异不显著(p>0.05);OPS管和GMP管都是比较理想的冷冻载体。
7.不同解冻程序对玻璃化冷冻的牛卵母细胞发育影响。采用EFS30玻璃化冷冻液,OPS法冷冻GV期牛卵细胞,通过一步法,三步法,五步法解冻,形态正常率分别为32.3%,51.7%,49.1%,成熟率分别为25.4%,43.4%,36.7%,卵裂率分别为18.7%,30%,29.8%,三步解冻法效果较好。
8.程序化冷冻牛胚胎移植效果。采用三步平衡法和一步细管法,结果显示,用10%甘油作抗冻剂,三步平衡冷冻,三步解冻的胚胎移植后受胎率为38.3%和用15%乙二醇作抗冻剂一步细管法冷冻胚胎移植后受胎率为36.9%,两者差异不显著(p>0.05)。
Cryopreservation of oocytes and embryos is a potentially valuable technique for conservation of farm animal genetic resource and population. The bovine oocytes were vitrified by different vitrification fluid and the same vitrification fluid with open pulled straw(OPS) or glass micro-pipe(GMP).The influence of different priming solution,cryoprotectants,carrier,freezing and thawing methods on developmental capability of bovine oocytes was examined and frozen embryos of bovine also was transferred.
1. The GV-oocytes were vitrified by the same vitrification fluid with different concentration adding BAS or FCS. Supplementing concentration of BSA or FCS can influence rates of morphologically normal and matured oocytes.
2.The bovine oocytes in different stage(GV and MⅡ) were vitrified. There were no statistical significant differences in rates of morphologically normal (71.3%,68.8%,p>0.05)and rates of matured oocytes(45%,47.6%,p>0.05).There was significant difference in rate of cleavge(23.3%,31.1%,p<0.05).
3. Through the contrast impact of vitrification frozen on oocytes with the different number of cumulus cell layer .It was found that,the impact of vitrification frozen on oocyte with 2~4 layers of cumulus cell is better than other number of cumulus cell layer.After vitrification of the oocytes, rates of morphologically normal oocytes were 21.7%,60.3%,60.7%, cleavage rates were 1.6%,30.2%,24.6%.
4.In GV-stage oocytes, Through the contrast impact of vitrification on GV-stage oocytes vitrified by the same vitrification fluid with different concentration and different vitrification fluid ,There was no significant difference in rate of morphologically normal oocytes (67.2%,66.7%,p>0.05) and rate of maturation (42.9%,43.7%,p>0.05) with EFS30 or EFS40. But there was significant difference in rate of morphologically normal oocytes and the maturation rate of the oocytes vitrified between EFS and EDFS.
5. Through the contrast impact of vitrification frozen on oocyte vitrified by different vitrification fluid and the same vitrification fluid with different concentration, EDFS has better impact of bovine oocyte vitrification frozen.
6.In GV-stage oocytes, there was no difference in rates of morphologically normal oocytes(68.4%,74.1%,p>0.05),the maturation rate(47.3%,48.4%,p>0.05)by OPS or GMP with EFS40, while there was difference between EFS40 and EDFS40 vitrification in the maturation rates(48.4%, 49.1%,p<0.05)by GMP .
7.The rates of morphologically normal and matured of oocytes with three-step or five-step thawing method were higher than one-step,while there was difference in rates of morphologically normal oocytes(32.3%,51.7%,49.1%,p<0.05),rates of maturation(25.4%,43.4%,36.7%,p<0.05)and rates of cleavage(18.7%,30%,29.8%,p<0.05).
8.Effect of transferring bovine ftozen embryos showed that there was no significant differences in pregnancy between one and three-step two methods.Pregnancy rate of bovine embryos frozen by three-step programmed freezing with supplementing concentration of 10% GLY was 38.3%,and pregnantcy rate of bovine embryos frozen by one-step programmed freezing with supplementing concentration of 15% EG was 39.6%.
引文
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