卵丘细胞和培养条件对牛体外胚胎生产效率的影响
摘要
体外受精(in vitro fertilization,IVF)技术是牛体外胚胎生产(in vi(?)ro embryoproduction,IVEP)的关键环节之一,该技术应用于奶牛性控胚胎的生产和优良肉牛品种的快速繁育有着巨大的商业前景。目前牛胚胎的体外生产技术已得到很大提高,但是还存在着生产效率较低、培养体系不稳定、体外胚胎质量不高、移植妊娠率较低等问题。本研究利用屠宰场大量的废弃黄牛卵巢,对影响牛IVF胚胎生产效率的主要环节进行了系统的研究,旨在优化牛IVEP程序,建立起一套高效的牛体外胚胎生产体系。研究结果如下:(1)体外成熟(in vitromaturatiom,IVM)22-24h后,对卵母细胞外包裹的卵丘细胞分别进行全脱除(裸卵)、部分脱除和全保留的处理,受精后发现三组间卵裂率分别为46.8%、77.7%和78.9%;囊胚率分别为7.7%、24.2%和25.6%;囊胚孵化率分别为12.5%、62.5%和68.9%。裸卵受精卵裂率、囊胚率和囊胚孵化率均极显著低于有卵丘细胞包裹的卵母细胞(P＜0.01)。(2)采用不同大小的微滴(20μl、50μl和80μl)培养牛IVF胚胎,各组卵裂率分别为86.4%、80.5%和76.0%,差异不显著(P＞0.05);囊胚率分别为35.0%、36.4%和28.2%,差异不显著(P＞0.05);囊胚孵化率分别为63.3%、62.5%和51.8%,在20μl组和50μl组之间差异不显著(P＞0.05),但均显著高于80μl组(P＜0.05)。(3)分别以CR1和SOF为牛IVF胚胎培养基础液,两组卵裂率分别为82.9%vs.79.1%;囊胚率分别为35.2%vs.30.9%;囊胚孵化率分别为71.9%vs.69.6%,差异均不显著(P＞0.05),CR1组稍高于SOF组。结果发现,CR1和SOF两种基础液都能支持牛体外受精胚胎获得很好的发育能力,但CR1基础液的效果略好于SOF。(4)牛IVF胚胎培养液为CR1,2天后添加5%FBS,与CR1+5%FBS全程培养相比,前者卵裂率显著低于后者(71.8%vs.81.9%,P＜0.05),两组囊胚率和囊胚孵化率差异均不显著(P＞0.05);若前3天培养液为CR1+3mg/ml BSA,后换成CR1+5%FBS,与CR1+5%FBS全程培养相比卵裂率无显著差异(84.5%vs.79.9%,P＞0.05),囊胚率显著提高(30.9%vs.23.8%,P＜0.05)。结果表明,培养前期添加3mg/mlBSA,后期添加5%FBS的二步培养法更有利于牛IVF胚胎的发育。(5)从培养的第0、2、3和5天开始培养液中添加5%FBS,牛IVF胚胎囊胚发育率分别为23.4%、27.7%、34.2%和35.5%。第3天和第5天添加FBS对囊胚率无显著影响,第2天添加FBS组的囊胚率高于第0天添加组,但差异不显著(P＞0.05)。第3天或第5天添加FBS与第0天或第2天添加组相比,显著提高了囊胚发育率(P＜0.05)。结果表明,从培养的第3天或第5天添加FBS牛IVF胚胎的发育潜力更好。(6)在胚胎培养的第3天,培养液中添加不同浓度的FBS(5%、10%和20%),囊胚率分别为35.6%、34.6%和27.0%,5%和10%血清组囊胚率高于(?)0%血清组,但差异不显著(P＞0.05);囊胚孵化率分别为70.4%、68.3%和55.4%,20%血清组囊胚孵化率显著降低(P＜0.05)。结果显示,在胚胎培养的第3天添加5%FBS效果最佳。(7)培养液中分别添加1mg/mlPVA、3mg/mlBSA和5%FBS,囊胚率分别为18.3%、24.2%和31.9%,囊胚孵化率分别为21.7%、44.3%和62.1%,FBS组显著高于BSA组(P＜0.05),极显著高于PVA组(P＜0.01);BSA组显著高于PVA组(P＜0.05)。结果表明FBS作为蛋白质来源的培养效果优于BSA,不添加蛋白质(PVA组)不利于胚胎的发育。(8)胚胎培养液中分别添加0、1、10和100 ng/ml的leptin,各组卵裂率无显著差异(P＞0.05);囊胚率分别为30.7%、31.5%、36.8%和26.3%,leptin浓度为10ng/ml时囊胚率显著高于100ng/ml组(P＜0.05),其余各组间无显著差异;各组囊胚孵化率无显著差异(P＞0.05)。结果表明胚胎培养液中添加10ng/ml的leptin为宜。
综上所述,牛卵母细胞体外受精前全保留卵丘细胞,体外受精胚胎的基础培养液选择CR1aa,从培养的第3或5天添加5%的FBS和添加10ng/ml的瘦素对牛体外胚胎生产效率最高。
Bovine IVF technology is one of the most important procedures for bovine in vitro embryo production(IVEP).Yhe objective of this study was to investigate effects of many factors such as cumulus cells,type of medium,size of drops,BSA,FBS and leptin on development of bovine IVF embryos,and to establish an reliable in vitro system in which bovine oocytes can be matured,fertilized and cultured up to the blastocyst stage.Bovine ovaries were collected from slaughterhouse and transported to the laboratory in physiological saline at 20℃to 30℃within 10-12h.Cumulus oocyte complexes were aspirated from follicles 2 to 8 mm in diameter and were in vitro matured for 22-24h.In experiment 1,the expanding Cumulus cells enwrapped oocytes were treated as follows 1)all removal,2)partial removal,and 3)intact.After in vitro fertilization,the zygotes were in vitro cultured.The cleavage rate was 46.8% vs.77.7%and 78.9%;the blastocyst rate was 7.7%vs.24.2%and 25.6%;the hatched blastocyst rate was 12.5%vs.62.5%and 68.9%respectively.There were significant differences among the three groups(P＜0.01).In experiment 2,after in vitro fertilization,the zygotes were cultured in different sizes of culture drops:1)20μl, 2)50μl,and 3)80μl.The cleavage rate and the blastcyst rate were 86.4%vs.80.5% vs.76.0%and 35.0%vs.36.4%vs.28.2%respectively.There were no significant differences between(P＞0.05).But the the hatched blastcyst rate of in 80μl group was significantly lower than that in 20μl and 50μl groups(63.3%,62.5%and 51.8%, P＜0.05).In experiment 3,the zygotes were cultured in CR1 or in SOF medium supplemented 5%FBS.The cleavage rate was 82.9%vs.79.1%;the blastcyst rate was 35.2%vs.30.9%and the hatched blastcyst rate was 71.9%vs.69.6%.There were no significant differences between the two media.In experiment 4,the zygotes of treatment group were cultured in CR1 for 3 days and then were transferred to CR1 supplemented with 5%FBS.The cleavage rate was significantly lower than that of the zygotes were cultured in CR1 supplemented with 5%FBS for all days(71.8% vs.81.9%,P＜0.05).When the zygotes of treated group were cultured in CR1+3mg/ml BSA for 3 days and then were transferred to CR1 supplemented with 5%FBS.The cleavage rate was not different from that of control group.But the blastocyst rate of embryos in treatment group was significantly higher than that of control group(30.9 %vs.23.8%).In experiment 5,the blastocyst rate was 23.4%,27.7%,34.2%and 35.5%respectively when the culture medium was supplemented with 5%FBS at 0 (control group),2nd,3rd or 5th day.The blastocyst rate of treated group was higher than that of control group.But the blastocyst rate in the medium supplemented with FBS at the 3rd or the 5th day was significantly higher than that in the conrol group(P＜0.05). The hatched blastcyst rate was not different among all groups.in experiment 6,the olastocyst rate was 35.6%,34.6%and 27.0%respectively when the culture media were supplemented with 5%,10%or 20%FBS at 3rd day.The blastocyst rate was not significantly different among 3 groups.The hatched blastocyst rates of 5%and 10%FBS groups were higher than that of 20%FBS group(70.4%,68.3%and 55.4 %respectively;P＜0.05).In experiment 7,bovine zygotes were cultured in three type of media:CRlaa supplemented with 0.1mg/ml PVA,0.3mg/ml BSA or 5%FBS.The cleavage rate of embryos cultured in these media was not significant different(77.5% vs.79.9%and 78.6%;P＞0.05).The blastocyst rate of embryos cultured in medium CRlaa supplemented with 0.1 mg/ml PVA was lower than that of embryos cultured in medium CR1aa supplemented with 0.3mg/ml BSA(18.3%vs.24.2%;P＜0.05)and significantly lower than that of embryos cultured in medium CR1aa supplemented with 5%FBS(18.3%vs.31.9%;P＜0.01).The hatched blastocyst rate of embryos cultured in medium CR1 aa supplemented with 0.1 mg/ml PVA was lower than that of embryos cultured in medium CRlaa supplemented with 0.3mg/ml BSA(21.7%vs. 44.3%;P＜0.05)and significantly lower than that of embryos cultured in medium CRlaa supplemented with 5%FBS(21.7 vs.62.1%;P＜0.01).In experiment 8,the zygotes were cultured in CR1aa supplemented with 0,1,10 or 100 ng/ml leptin.The cleavage rate and the hatched blastocyst rate were not significantly differet among the tour groups(P＞0.05).But the blastocyst rate of embryos cultured in medium supplemented with 10 ng/ml leptin was siginificantly higher than that of embryos cultured in medium supplemented with 100 ng/ml leptin(36.8%vs.26.3%;P＜0.05). In conclusion,the present study indicates that the fertilization competence and the developmental competence will be depressed when the expanding cumulus cells enwrapped were removed.Two-step culture system,which applies serum-free medium for early bovine embryos till cleavage stage,and utilized 5%serum containing media on the 3rd or 5th day of in vitro culture may be effective fore in vitro production systems.The embryos cultured in medium supplemented with 10 ng/ml leptin support well developmental competence.
综上所述,牛卵母细胞体外受精前全保留卵丘细胞,体外受精胚胎的基础培养液选择CR1aa,从培养的第3或5天添加5%的FBS和添加10ng/ml的瘦素对牛体外胚胎生产效率最高。
Bovine IVF technology is one of the most important procedures for bovine in vitro embryo production(IVEP).Yhe objective of this study was to investigate effects of many factors such as cumulus cells,type of medium,size of drops,BSA,FBS and leptin on development of bovine IVF embryos,and to establish an reliable in vitro system in which bovine oocytes can be matured,fertilized and cultured up to the blastocyst stage.Bovine ovaries were collected from slaughterhouse and transported to the laboratory in physiological saline at 20℃to 30℃within 10-12h.Cumulus oocyte complexes were aspirated from follicles 2 to 8 mm in diameter and were in vitro matured for 22-24h.In experiment 1,the expanding Cumulus cells enwrapped oocytes were treated as follows 1)all removal,2)partial removal,and 3)intact.After in vitro fertilization,the zygotes were in vitro cultured.The cleavage rate was 46.8% vs.77.7%and 78.9%;the blastocyst rate was 7.7%vs.24.2%and 25.6%;the hatched blastocyst rate was 12.5%vs.62.5%and 68.9%respectively.There were significant differences among the three groups(P＜0.01).In experiment 2,after in vitro fertilization,the zygotes were cultured in different sizes of culture drops:1)20μl, 2)50μl,and 3)80μl.The cleavage rate and the blastcyst rate were 86.4%vs.80.5% vs.76.0%and 35.0%vs.36.4%vs.28.2%respectively.There were no significant differences between(P＞0.05).But the the hatched blastcyst rate of in 80μl group was significantly lower than that in 20μl and 50μl groups(63.3%,62.5%and 51.8%, P＜0.05).In experiment 3,the zygotes were cultured in CR1 or in SOF medium supplemented 5%FBS.The cleavage rate was 82.9%vs.79.1%;the blastcyst rate was 35.2%vs.30.9%and the hatched blastcyst rate was 71.9%vs.69.6%.There were no significant differences between the two media.In experiment 4,the zygotes of treatment group were cultured in CR1 for 3 days and then were transferred to CR1 supplemented with 5%FBS.The cleavage rate was significantly lower than that of the zygotes were cultured in CR1 supplemented with 5%FBS for all days(71.8% vs.81.9%,P＜0.05).When the zygotes of treated group were cultured in CR1+3mg/ml BSA for 3 days and then were transferred to CR1 supplemented with 5%FBS.The cleavage rate was not different from that of control group.But the blastocyst rate of embryos in treatment group was significantly higher than that of control group(30.9 %vs.23.8%).In experiment 5,the blastocyst rate was 23.4%,27.7%,34.2%and 35.5%respectively when the culture medium was supplemented with 5%FBS at 0 (control group),2nd,3rd or 5th day.The blastocyst rate of treated group was higher than that of control group.But the blastocyst rate in the medium supplemented with FBS at the 3rd or the 5th day was significantly higher than that in the conrol group(P＜0.05). The hatched blastcyst rate was not different among all groups.in experiment 6,the olastocyst rate was 35.6%,34.6%and 27.0%respectively when the culture media were supplemented with 5%,10%or 20%FBS at 3rd day.The blastocyst rate was not significantly different among 3 groups.The hatched blastocyst rates of 5%and 10%FBS groups were higher than that of 20%FBS group(70.4%,68.3%and 55.4 %respectively;P＜0.05).In experiment 7,bovine zygotes were cultured in three type of media:CRlaa supplemented with 0.1mg/ml PVA,0.3mg/ml BSA or 5%FBS.The cleavage rate of embryos cultured in these media was not significant different(77.5% vs.79.9%and 78.6%;P＞0.05).The blastocyst rate of embryos cultured in medium CRlaa supplemented with 0.1 mg/ml PVA was lower than that of embryos cultured in medium CR1aa supplemented with 0.3mg/ml BSA(18.3%vs.24.2%;P＜0.05)and significantly lower than that of embryos cultured in medium CR1aa supplemented with 5%FBS(18.3%vs.31.9%;P＜0.01).The hatched blastocyst rate of embryos cultured in medium CR1 aa supplemented with 0.1 mg/ml PVA was lower than that of embryos cultured in medium CRlaa supplemented with 0.3mg/ml BSA(21.7%vs. 44.3%;P＜0.05)and significantly lower than that of embryos cultured in medium CRlaa supplemented with 5%FBS(21.7 vs.62.1%;P＜0.01).In experiment 8,the zygotes were cultured in CR1aa supplemented with 0,1,10 or 100 ng/ml leptin.The cleavage rate and the hatched blastocyst rate were not significantly differet among the tour groups(P＞0.05).But the blastocyst rate of embryos cultured in medium supplemented with 10 ng/ml leptin was siginificantly higher than that of embryos cultured in medium supplemented with 100 ng/ml leptin(36.8%vs.26.3%;P＜0.05). In conclusion,the present study indicates that the fertilization competence and the developmental competence will be depressed when the expanding cumulus cells enwrapped were removed.Two-step culture system,which applies serum-free medium for early bovine embryos till cleavage stage,and utilized 5%serum containing media on the 3rd or 5th day of in vitro culture may be effective fore in vitro production systems.The embryos cultured in medium supplemented with 10 ng/ml leptin support well developmental competence.
引文
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