TSA对克隆囊胚率的提高及同期发情的处理方法对胚胎移植的影响
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摘要
实验以牛胎儿成纤维细胞作为供体核,以牛卵母细胞作为受体胞质进行体细胞核移植,用80nmol／L曲古抑菌素A(Trichostatin A, TSA)处理牛胎儿成纤维细胞12 h,以TSA处理后的牛胎儿成纤维细胞为供体细胞进行胚胎重构,重构完成后继续处理克隆胚胎0、6、12和24 h。应用激光共聚焦显微镜检测供体细胞组蛋白H4K12乙酰化水平,并通过核移植检测不同时间处理的克隆胚胎发育率。
同时对不同同期发情方法对胚胎移植效果进行了比较,以寻找适合当地条件的同期发情方案。分别采用一次注射前列腺素(PG)、二次注射前列腺素(PG)、阴道埋植栓(CIDR)+前列腺素(PG)三种方法进行同期发情处理,并设自然发情组作胚胎移植效果比较。
1、TSA处理12 h的供体细胞组蛋白H4K12乙酰化水平显著增高；
2、80 nmol／LTSA处理12 h的克隆胚的囊胚发育率显著高于未处理组(21.9%vs 16.5%,p<0.05)；
3、TSA处理24h的重构胚的各期发育率均很低,与对照组差异显著；TSA处理6 h的重构胚的囊胚发育率与对照组无显著差异；TSA处理细胞对细胞融合率没有影响。
4、二次注射PG、CIDR+PG3、一次注射PG三种同期发情处理后的发情率分别为：74.70%、77.64%、71.19%,受体移植后的妊娠率鲜胚分达到52.12%、53.26%、50.44%,冻胚受胎率分别达到46.87%、41.44%、44.00%,这三组间差异不显著(P>0.05)。
5、自然发情利用率达到85.71%,显著高于二次PG法注射同期处理组,选取自然发情牛作为受体显著提高胚胎移植成功率。
The experiment lets bovine fetal fibroblasts become the ciliary cell,carries on the somatic cell nucleus transplant by the cow ovocyte as the acceptor butcher nature. Bovine fetal fibroblasts were seeded into DMEM containing 80nmol/L of TSA 12 h,then the nuclear transfer (NT) embryos that constructed with bovine fetal fibroblast cells were seeded into H-M199 (2-3h) and SOF containing 80nmol/Lof TSA for 0h、6h、12h、24h. Laser scanning confocal microscope for somatic cell histone H4K12 acetylation, and detected by nuclear transfer cloning of embryonic development rate at different time. At the same time,we compare the embryonic implantation effect in the method to the different time in order to find suitable local conditions estrus synchronization programs. Uses injection prostaglandin (PG), two injection prostaglandin (PG), the vagina to bury separately plants the hitch (CIDR)+the prostaglandin (PG) three methods to carry on in heat same time processing, and supposes the nature to be in heat group to make the embryonic implantation effect comparison.
1、TSA can increase the levels of histone acetylation of H4K12 after bovine fetal fibroblast Cells treated with 80 nmol/L TSA for 12 h(P<0.05).
2、80 nmol/L TSA treatment of nuclear transfer (NT) embryos for 12 h increased blastocyst development compared to controls (21.9%vs16.5%, P<0.05).
3、Marked difference existed in blastocyst development of nuclear transfer (NT) embryos which is treated with 80 nmol/L TSA for 24 h and control group (p<0.05); No marked difference existed in blastocyst development of nuclear transfer (NT) embryos treated with 80 nmol/L TSA for 6 h and control group (p<0.05).
4、The result of the three groups is similar,double PG injection, CIDR+PG treatment and the single injection of PG.The synchronization rates were 74.70%,77.64%,71.19%,correspondingly the pregnancy rate of fresh embryo was 52.12%,53.26%,50.44%, after embryo transfer, and that of frozen embryos is 46.87%,41.44%,44.00%.
5、The recipient utilization of spontaneous estrus was 85.71%, significantly higher than the double PG injection group, the success rate of embryo transfer can be remarkably improved by utilizing recipient of spontaneous estrtus.
同时对不同同期发情方法对胚胎移植效果进行了比较,以寻找适合当地条件的同期发情方案。分别采用一次注射前列腺素(PG)、二次注射前列腺素(PG)、阴道埋植栓(CIDR)+前列腺素(PG)三种方法进行同期发情处理,并设自然发情组作胚胎移植效果比较。
1、TSA处理12 h的供体细胞组蛋白H4K12乙酰化水平显著增高；
2、80 nmol／LTSA处理12 h的克隆胚的囊胚发育率显著高于未处理组(21.9%vs 16.5%,p<0.05)；
3、TSA处理24h的重构胚的各期发育率均很低,与对照组差异显著；TSA处理6 h的重构胚的囊胚发育率与对照组无显著差异；TSA处理细胞对细胞融合率没有影响。
4、二次注射PG、CIDR+PG3、一次注射PG三种同期发情处理后的发情率分别为：74.70%、77.64%、71.19%,受体移植后的妊娠率鲜胚分达到52.12%、53.26%、50.44%,冻胚受胎率分别达到46.87%、41.44%、44.00%,这三组间差异不显著(P>0.05)。
5、自然发情利用率达到85.71%,显著高于二次PG法注射同期处理组,选取自然发情牛作为受体显著提高胚胎移植成功率。
The experiment lets bovine fetal fibroblasts become the ciliary cell,carries on the somatic cell nucleus transplant by the cow ovocyte as the acceptor butcher nature. Bovine fetal fibroblasts were seeded into DMEM containing 80nmol/L of TSA 12 h,then the nuclear transfer (NT) embryos that constructed with bovine fetal fibroblast cells were seeded into H-M199 (2-3h) and SOF containing 80nmol/Lof TSA for 0h、6h、12h、24h. Laser scanning confocal microscope for somatic cell histone H4K12 acetylation, and detected by nuclear transfer cloning of embryonic development rate at different time. At the same time,we compare the embryonic implantation effect in the method to the different time in order to find suitable local conditions estrus synchronization programs. Uses injection prostaglandin (PG), two injection prostaglandin (PG), the vagina to bury separately plants the hitch (CIDR)+the prostaglandin (PG) three methods to carry on in heat same time processing, and supposes the nature to be in heat group to make the embryonic implantation effect comparison.
1、TSA can increase the levels of histone acetylation of H4K12 after bovine fetal fibroblast Cells treated with 80 nmol/L TSA for 12 h(P<0.05).
2、80 nmol/L TSA treatment of nuclear transfer (NT) embryos for 12 h increased blastocyst development compared to controls (21.9%vs16.5%, P<0.05).
3、Marked difference existed in blastocyst development of nuclear transfer (NT) embryos which is treated with 80 nmol/L TSA for 24 h and control group (p<0.05); No marked difference existed in blastocyst development of nuclear transfer (NT) embryos treated with 80 nmol/L TSA for 6 h and control group (p<0.05).
4、The result of the three groups is similar,double PG injection, CIDR+PG treatment and the single injection of PG.The synchronization rates were 74.70%,77.64%,71.19%,correspondingly the pregnancy rate of fresh embryo was 52.12%,53.26%,50.44%, after embryo transfer, and that of frozen embryos is 46.87%,41.44%,44.00%.
5、The recipient utilization of spontaneous estrus was 85.71%, significantly higher than the double PG injection group, the success rate of embryo transfer can be remarkably improved by utilizing recipient of spontaneous estrtus.
引文
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