【作者】 蔡元；

【导师】 张兆旺； 田见晖；

【作者基本信息】 甘肃农业大学 ， 动物遗传育种与繁殖， 2004， 硕士

【摘要】 本文以屠宰场废弃的猪卵巢为材料,进行了体外成熟培养、孤雌激活、孤雌胚的核型分析等一系列试验，旨在探寻适于猪卵母细胞ICSI的激活方法。研究内容有三方面：①观察了有极体卵母细胞电激活后7h内极体排出的时间分布情况，以及有极体卵母细胞电激活后间隔不同时间再用化学物质激活对其孤雌发育能力的影响，并对其孤雌胚的核型进行了分析；②比较了猪卵母细胞成熟44h后，有极体卵母细胞和无极体卵母细胞电激活后排出极体的差异，以及两类卵母细胞电激活后间隔不同时间再用化学物质激活之后其孤雌发育能力的差异；③研究了在37℃-38.5℃生理盐水中保存猪卵巢，运回实验室生理盐水温度的下降对猪卵母细胞体外成熟的影响。结果如下：①电激活后4h时有78.43%的卵排出第二极体，此后，每小时内的第二极体排出率明显减少；②电激活后间隔0h（对照）、3h、4h、5h、6h再分别用6-二甲基氨基嘌呤（6-Dimethylaminopurine,6-DMAP）、 放线菌酮(cycloheximide, CHX)处理6h，各组卵裂率、囊胚细胞数差异不显著(p>0.05)，但是，在6-DMAP处理中对照组囊胚率极显著高于其余各组（p<0.01），在CHX处理中对照组囊胚率极显著高于间隔5h、6h两组(p<0.01)，而其余各组之间差异不显著(p>0.05)；③电激活后间隔4h再分别用6-DMAP、CHX和CB（细胞松弛素Ｂ，Cytochalasin B）+6-DMAP处理6h，各组卵裂率差异不显著（p>0.05），对照组（对照同②中6-DMAP的对照）囊胚率极显著高于其余各组(p<0.01)，其余各组囊胚率显著性均无差异(p>0.05)。对照组中无单倍体囊胚，二倍体囊胚占88.24%，但6-DMAP、CHX、CB+6-DMAP中产生的囊胚60-70%是单倍体，且相互之间无显著性差异（p>0.05）。以上结果说明，猪卵母细胞电脉冲处理后间隔不同时间用DMAP、CHX激活，激活作用随时间的延长而减弱；猪卵母细胞电激活后4h再用6-DMAP、CHX、CB+6-DMAP激活，产生<WP=6>的囊胚大部分为单倍体，该方法适于猪卵母细胞ICSI的激活。猪卵母细胞成熟44h后①有极体卵母细胞排出第二极体的比例很高，而无极体的卵母细胞排出极体(第一极体或第一、第二极体)的比率很低。电激活的第4h，78.43%的有第一极体的卵能排出第二极体，此后，每h内的第二极体排出率明显减少；无极体卵每h排出极体的比率低且很缓慢，电激活后4h时仅有37.74%的卵排出极体②电激活后间隔0h（对照）、3h、4h、5h、6h再分别用6-DMAP、CHX处理6h时，6-DMAP组中，卵裂率a组极显著高于f、g、h、i、k组(p<0.01)；b、c、d、e组极显著高于f、g组(p<0.01)，显著高于h、i、k组（p<0.05），其余各组差异均不显著(p>0.05)。囊胚率a组极显著高于e、f、g、h、i、k组（p<0.01），显著高于b、c、d组p<0.05)，其余各组差异均不显著（p〈0.05）；在CHX处理中，各组的卵裂差异不显著（p>0.05），囊胚率a组极显著高于d、e、h、k组（p<0.01），显著高于f、i组p<0.05），其余各组差异均不显著（p>0.05）③无极体卵母细电激活后间隔4h再分别用6-DMAP、CHX和CB+6-DMAP处理6h，各组卵裂率、囊胚率均差异不显著（p>0.05）。以上结果说明，无极体卵母细胞发育能力比有极体卵母细胞差；无极体卵母细胞电激活处理后，部分卵母细胞虽然也可以排出第一极体，但此后再用化学激活也不能提高其孤雌发育的能力；第一极体的存在是卵母细胞成熟 的一个很重要的特征。在37－38.5℃的生理盐水下保存猪卵巢，运回实验室后，当温度下降为31℃、32℃、33℃、34℃、36℃时卵巢上卵母细胞的成熟率和淘汰率分别为27.86±7.31％、21.34±5.12％、28.32±7.49％、21.14±4.82％、17.70±2.27％和53.42±12.10％、48.05±21.04％、43.93±12.77％、58.19±6.82％、62.06±12.03％，各组间差异不显著（p>0.05）。结果表明，在37－38.5℃的生理盐水下保存猪卵巢，运回实验室后，当温度下降到31－36℃时对卵母细胞的成熟没有影响。更多还原

【Abstract】 IVM,parthenogenetic activation and karyotype analysis were performed in porcine oocytes in this thesis.Development and karyotype of parthenogenetic embryo were mainly investigated. meanwhile,The situation of extrusion about the polar bodies from the porcine oocytes activated by electricity were observed in 7h ; Then development of oocytes that had the first or no polar bodies were compared; Effect of decreased saline temperature of preserving ovaries on development in vitro maturation were studied. The result of the experiment is described as following: 78.54% of oocytes extrude the second polar bodies after they were activated by electricity, and subsequently the rate of SPB in an hour dramatically decreased; Porcine oocytes were cultured in vitro for 0h (control), 3h, 4h, 5h and 6h after electrical activation , then they were treated in 6-DMAP/CHX ,there were no statistical different in the mean number of nuclei per blastocysts and cleavage rate (p>0.05).But blastocysts rate of the control group in 6-DMAP group was significantly higher than other groups (p<0.01). Blastocysts rate of the control group in CHX group was significantly higher than interval 5h, 6h groups (p<0.01); In the experiment about chemical treatments for 6h after electric activation in 4h intervals , there was no significant different for cleavage rate (p>0.05), but blastocysts rate of the control group was significantly higher than other groups (p<0.01). Blastocysts cultured in control almost were diploids, blastocysts in other groups mostly were haploids , and there were no significant different (p>0.05) . The result showed that the activation effect gradually declined when interval time become long; Blastocystes activated by electricity and 6-DMAP,CHX,CB+6-DMAP (with 4h interval between them) mostly were haploid. <WP=8>Rate of oocytes that extruded the SPB were very high for oocytes which had the first poplar bodies, conversely rate of oocytes extruded the first poplar bodies or SPB were very low for oocytes which had not the polar bidies after oocytes were maturated in vitro for 44h. Porcine oocytes were cultured in vitro for 0h (control), 3h, 4h, 5h and 6h after electrical activation , then they were treated in 6-DMAP/CHX . cleavage percentage of the a,b.c,d,e group in 6-DMAP group was higher than other groups (p<0.05)..Blastocysts percentage of the a group was significantly higher than other groups (p<0.05). there were no statistical different for cleavage rate in CHX group (p>0.05)Blastocysts percentage of the a group was higher than d,e,f,i,k,h groups (p<0.05); In the second study, there was no significant different for cleavage rate and blastocysts rate (p>0.05), The result showed that development of oocytes which had no poplar bodies were worse than that of oocytes which had the first polar bodies ;Oocytes which had not the polar bidies could extrude the bodies ,but development couldn`t be enhanced even though they were activated by chemical activated ;The first polar bodies are an important character of oocytes maturation. There were no significant different for IVM when saline temperature decreased from 37℃-38.5℃ to 31℃,32℃,33℃,34℃,36℃。更多还原