【作者】 徐贵江；

【导师】 吴信生； 李碧春；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2010， 硕士

【摘要】 本实验以44只新西兰母兔为实验素材,主要研究兔卵巢表面卵母细胞体外成熟、体外受精以及受精卵的不同体外培养条件,以优化兔卵母细胞体外成熟、体外受精以及受精卵培养的体系,实验结果如下:1.采用切割法、针刺挤压法收集卵巢表面卵母细胞,检验不同采集方法对收集卵巢卵母细胞数量的影响。结果表明:针刺挤压法的所获得的卵母细胞数(23.63/只)显著高于切割法(16.50/只)(p<0.05)。2.比较添加不同浓度的FSH、LH对兔卵母细胞体外成熟的影响,结果表明: 1 U /mL FSH+2 U/mL LH的成熟率最高,达到46. 62 %,极显著高于对照组(TCM199+10 %FCS)的成熟率(13. 71 %,p<0.01),显著高于高浓度组(10 U /mL FSH+20 U/mL LH)的成熟率(26.83 %,p<0.05)。3.比较了不同的成熟培养时间对卵母细胞成熟率的影响,以成熟培养液培养卵母细胞,分别在成熟培养24 h、30 h、36 h、40 h对部分卵母细胞用透明质酸酶处理后观察第一极体排出率,其余部分加精子观察受精率。培养24 h、30 h、36 h、40 h的成熟率分别为21.92 %、46.62 %、52.77 %、30.71 %,这就表明:成熟时间为30、36 h的成熟率显著高于成熟时间为24、40 h的成熟率(p<0.05)。4.以TCM199+FCS+FSH+LH+E2+丙酮酸钠为基础成熟培养液,分别添加50 ng/mL及100 ng/mL的EGF、10 ng/mL及20 ng/mL的SCF、10 ng/mL及20 ng/mL的LIF,然后比较了EGF、SCF及LIF对卵母细胞成熟率的影响。结果表明:50 ng/mL及100 ng/mL的EGF能显著地提高了卵母细胞成熟率(基础成熟培养液、添加50 ng/mL及100 ng/mL的EGF组的成熟率分别为49.04 %、61.39 %和65.79 %)。SCF对卵母细胞成熟率无明显影响(成熟率分别为54.17 %、50.00 %和45.83 %),而LIF对卵母细胞成熟有明显的抑制作用(成熟率分别为54.19 %、12.50 %和4.17 %)。5.比较了上游法、直接洗涤法和Percoll密度梯度离心法三种精子处理方法对兔精子回收率及卵裂率的影响,三种方法的回收率分别为7.8 %、93.9 %、86.9 %,受精后的卵裂率分别为0 %、39.05 %、54.29 %,结果表明:Percoll密度梯度离心法是一种较理想的精子处理方法。6.比较了常规体外受精与显微注射卵裂率,结果显示,常规体外受精卵裂率(63.97 %)极显著高于显微注射卵裂率(25.65 %)(p<0.01)。7.比较了TCM199+10% FCS和DMEM+10 %FCS对胚胎(取自常规体外受精的2-细胞)发育的影响,结果表明两者之间无显著差异(p>0.05)。8.以TCM199+10 %FCS为基础的受精卵培养液,分别添加一定浓度的维生素C(100μmol/L)及β-巯基乙醇(50μmol/L),然后比较了它们对胚胎(取自常规体外受精的2-细胞)发育的影响,表明结果,100μmol/L的维生素C及50μmol/Lβ-巯基乙醇对胚胎发育无显著影响(p>0.05)。更多还原

【Abstract】 In this paper , we took 44 female New Zealand Rabbits as object, mainly researched in vitro maturation,fertilization of oocyte on ovarian surface and in vitro culture of zygote,our purposes were optimizing the system of in vitro maturation,fertilization of rabbit oocyte and in vitro culture of zygote,results were as follows.1. Cumulus and oocyte complex (COCs) on the surface of ovaries were gotten by the method of cutting with Blade or stab-extruding with needle,the number of COCs gotten by the method of stab-extruding with needle(23.63 per rabbit )were significant larger than by the method of cutting (16.50 per rabbit )(P <0.05).2. Supplemented with diffirent concentration of FSH+LH in the maturation medium,then COCs were IVM under the condition of 38.5°C,5 % CO2 and saturated humidity,the time of IVM was 30 h,the results showed that the rate of maturation by supplementing 1 U/mL FSH+2 U/mL LH was significent higher than 10 U/mL FSH+20 U/mL LH,and more significent higher than contrast group(TCM199+10 %FCS).The rate of maturation were respectively 46. 62 %,26.83 %,13. 71 %.3. This experiment compared the rate of maturation of oocytes by the time of 24 h,30 h,36 h or 40 h,the results indicated that culture time of 30 h or 36 h were better than 24 h or 40 h(P <0.05).The rate of maturation were 21.92 %,46.62 %,52.77 %, 30.71 % respectively.4. Basic maturation medium was TCM199+FCS+FSH+LH+E2+pyruvic sodium,added epidermal growth factor (EGF)50 ng/mL,EGF 100ng/mL,stem cell factor (SCF)10 ng/mL,SCF 20 ng/mL,leukemia inhibitory factor(LIF)10 ng/mL,LIF 20 ng/mL,then counted rate of first polar body emission by the time of 30 h .Results manifested the first polar body emission rate in group of Adding 50 ng / mL and 100 ng / mL EGF were significantly higher(P <0.05),first polar body emission rate of contrast group,50 ng / mL group and 100 ng / mL was respectively 49.04 %,61.39 % and 65.79 %,SCF could not enhance the first polar body emission rate,first polar body emission rate was respectively 54.17 %,50.00 % and 45.83 %,LIF obviously restrained the maturation of oocyte,first polar body emission rate was respectively 54.19 %,12.50 % and 4.17 %.5. Test compared three methods of sperm handlling on sperm recovery rate and cleavage rate , they were swimming-up , direct washing and density gradient centrfugation,results showed that the method of density gradient centrfugation was a ideal method(sperm recovery rate of the three ways were 7.8 %,93.9 % and 86.9 %,cleavage rate after the fertilization were 0 %,39.05 % and 54.29 %).6. Cleavage rate of in vitro fertilization and cleavage rate of intracytoplasmic sperm injection was compared,result was that the cleavage rate of former(63.97 %)was more significant higher than the later(25.65 %)(P <0. 01).7. The experiment compared embryo developmental potential capacity between TCM199+10 %FCS group and DMEM+10 %FCS group,the result indicated that they have no significant difference(P >0. 05).8. Supplemented with vitmin C(100μmol/L)and mercaptoethanol (50μmol/L)in the zygote culture medium,zygote of 2-cell (from in vitro fertilization)were cultured under the condition of 38.5℃,5% CO2 and saturated humidity,then statisticed the rate of 4-cell and of 8-cell or later,the results were that this three groups have no significat difference,Whether the rate of 4-cell zygote or rate of 8-cell zygote or later.更多还原