猪卵泡卵母细胞体外成熟与冷冻保存

研究了激素、猪卵泡液、不同类型血清对猪卵泡卵母细胞体外成熟的影响 ;比较了卵泡直径小于 2 mm、2～ 5 mm和大于 5 mm的卵母细胞体外成熟能力的差异 ,并对不同发育阶段猪卵母细胞的冷冻保存进行了研究。结果表明 :猪卵母细胞体外培养 4 8h时 ,培养的前 2 4 h培养液中加入激素 ,后 2 4 h去掉激素 ,卵母细胞的 A级成熟率 (5 1.73% )和总成熟率 (83.2 5 % )最高 ,极显著高于前 2 4 h不加激素 ,后 2 4 h添加激素培养的成熟率 (P<0 .0 1) ;也显著高于不含激素的培养液连续培养 4 8h的成熟率 (P<0 .0 5 ) ;但与添加激素连续培养 4 8h的成熟率差异不显著 (P>0 .0 5 )。在体外成熟培养液中 ,添加 10 % (体积分数 ) ECS的成熟率 (72 .86 % )显著高于添加 10 % (体积分数 ) NCS的成熟率(6 2 .2 1% ) (P<0 .0 5 ) ,而添加 10 % p FF则抑制卵母细胞的体外成熟。随着卵泡直径的增大 ,卵母细胞体外成熟能力逐渐增强。采用程序冷冻保存方法 ,成熟卵母细胞的成活率 (35 .5 9% )显著高于培养前 (2 4 .6 4 % )和培养 2 4 h(2 3.36 % ) (P<0 .0 5 )。培养 2 4 h(77.2 2 % )和培养成熟 (72 .81% )的卵母细胞解冻后的形态完整率均显著高于培养前(5 3.2 4 % ) (P<0 .0 5 )     

肝素、咖啡因、丙酮酸钠、BSA对延边黄牛卵母细胞体外受精效果的影响

试验为延边黄牛体外胚胎产业化生产奠定基础,进行了肝素、咖啡因、丙酮酸钠、BSA对延边黄牛体外受精效果的影响研究。结果表明,以改良BO液为基础液,添加20 μg/mL肝素时,精子顶体反应率和受精率分别为82.52%和75.34%,高于添加20 μg/mL咖啡因组(P<0.05)和添加10 μg /mL肝素与10 μg/mL咖啡因协同作用组(P>0.05);结合使用肝素(20 μg/mL),添加0.0020 g/mL丙酮酸钠时,精子顶体反应率和受精率分别为78.13%和71.57%, 高于添加0.0015 g/mL和0.0025 g/mL丙酮酸钠组(P<0.05);结合使用肝素(20 μg/mL),添加0.006 g/mL BSA时,精子顶体反应率和受精率分别为80.78%和71.44%,高于添加0.001 g/mL和0.003 g/mL BSA组(P<0.05)。因此,添加适当浓度的肝素、丙酮酸钠和BSA可显著提高延边黄牛精子体外获能效果及体外受精率。     

玻璃化冷冻牛卵母细胞单精子注射后体外发育能力的研究

实验研究了不同成熟培养时间的牛卵母细胞玻璃化冷冻及胞质内单精子注射(ICSI)后的受精效果。结果表明:成熟后的新鲜牛卵母细胞按照ICSI注射方法穿刺而不注射精子组与未经穿刺的对照组相比,孤雌激活后的卵裂率、囊胚发育率及囊胚细胞数无显著差异(P>0.05);成熟培养16h(MⅠ)和23h(MⅡ)卵母细胞冷冻解冻后形态正常率均显著低于新鲜对照组(76.66%、87.33%vs100.0%)(P<0.05),冷冻解冻后二者分别成熟培养至24h,ICSI后胚胎的囊胚发育率(5.29%、14.41%)显著低于新鲜对照组(24.40%)(P<0.05);成熟培养23h与成熟培养16h的卵母细胞冷冻解冻后形态正常率及ICSI后囊胚发育率(14.41%vs5.29%)均有显著性差异(P<0.05)。实验证明,ICSI操作不会影响卵母细胞发育潜力;玻璃化冷冻影响卵母细胞解冻后形态正常率以及ICSI后胚胎的发育能力;成熟培养23h比16h的卵母细胞冷冻保存后经ICSI的胚胎发育潜力高。     

猪MII期卵母细胞的玻璃化冷冻的初步研究

试验采用3种冷冻载体(麦管、开放式拉长麦管和半麦管)对猪卵母细胞玻璃化冷冻后发育能力的影响进行研究,以探讨采用自制半麦管作为载体对猪MII期卵母细胞玻璃化冷冻后发育能力的影响.结果发现,采用半麦管法、OPS法玻璃化冷冻的猪卵母细胞的形态完整率、存活率、卵裂率分别为90.85%、60.07%、20.43%和84.15%、55.71%、14.71%,无显著差异(P＞0.05),但采用半麦管法的结果好于OPS法;与麦管法的形态完整率、存活率和卵裂率(分别为60.06%,39.64%和0)均有显著性差异(＜0.05).结论为采用半麦管法玻璃化冷冻猪卵母细胞可以稍微提高其冻后发育能力.     

水牛卵泡颗粒细胞对卵母细胞体外成熟的影响

为了系统研究颗粒细胞对水牛卵母细胞体外成熟的影响,使用颗粒细胞条件液处理或单层颗粒细胞和卵母细胞共培养的方法,探讨颗粒细胞共培养对水牛卵母细胞体外成熟和早期胚胎发育的影响.结果显示,添加颗粒细胞传代接种第2天收集的20％颗粒细胞条件液到水牛卵母细胞成熟液中能显著提高水牛卵母细胞体外成熟率和囊胚发育率(P＜0.05);然...     

Effect of Human Menopausal Gonadotropin,17β-estradiol and Epidermal Growth Factor on the Quality of in vitro Maturation Bovine Oocytes

For optimizing in vitro maturation system of bovine oocytes,we firstly examined the influence of four different hormonal regimes(FSH+LH,HMG,FSH+LH+E₂ and HMG+E₂) on oocyte maturation rates.Then we studied the effects of epidermal growth factor (EGF) in the above defined medium on bovine oocyte maturation,in vitro development and quality of parthenogenetic embryos.The cell apoptotic index of parthenogenetic blastocysts was detected by TUNEL.No significant difference was observed in maturation rates in four groups supplemented with different hormones.However,human menopausal gonadotropin (HMG) provided steady maturation results in replicates.Maturation of oocytes was promoted by supplementation with 17β-estradiol (E₂).Combination of HMG and E₂ gave rise to steady and efficient mature results.The presence of EGF at 30 ng/mL concentration significantly increased maturation rate and blastocyst rate and reduced apoptotic cells in parthenogenetic blastocysts.Therefore,the optimal oocyte maturation solution could be supplemented with 0.075 IU/mL HMG,1 μg/mL E₂ and 30 ng/mL EGF.     

白藜芦醇和褪黑素对绵羊胎儿成纤维细胞转染及卵母细胞体外成熟的影响

试验旨在利用白藜芦醇(Re)和褪黑素(MT)提高绵羊胎儿成纤维细胞电穿孔转染(电转)效率、优化卵母细胞体外成熟体系,以提高转基因动物生产效率。将绵羊胎儿成纤维细胞分为6组:对照组、添加白藜芦醇高、中、低(R^-5、R^-6、R^-7)3种浓度组及白藜芦醇和褪黑素联合添加组(R^-5M^-5、R^-6M^-7),通过电转效率和细胞凋亡情况确定电转体系优化方案;通过对细胞周期、线粒体膜电位及活性氧(ROS)的检测研究其作用机制。将绵羊卵母细胞分为对照组及白藜芦醇和褪黑素(R^-5M^-5、R^-6M^-7)联合添加组,通过绵羊卵母细胞体外成熟后卵丘细胞扩展、第一极体排出及体外受精胚胎的卵裂率检测其对卵母细胞体外成熟的作用。研究结果显示,分离的绵羊胎儿成纤维细胞P3代增殖旺盛,细胞生长曲线呈典型的S型;与对照组相比,所有试验组胎儿成纤维细胞的电转效率均显著提高(P<0.05),R^-5M^-5组电转效率最高;R^-6M^-7组显著降低细胞凋亡率(P<0.05),R^-5、R^-6、R^-5M^-5和R^-6M^-7组均显著促进细胞增殖(P<0.05);R^-6、R^-7、R^-5M^-5和R^-6M^-7组G1期细胞增加,S期细胞相对减少,但差异均不显著(P>0.05);R^-6、R^-7、R^-5M^-5和R^-6M^-7组成纤维细胞内ROS水平均显著降低(P<0.05),R^-6、R^-5M^-5和R^-6M^-7组线粒体膜电位均显著增加(P<0.05);R^-6M^-7组显著促进绵羊卵丘细胞扩展、提高卵母细胞第一极体排出率和体外受精胚胎发育能力(P<0.05)。综上,白藜芦醇和褪黑素可以优化绵羊胎儿成纤维细胞电转体系,提高卵母细胞体外成熟效率,为白藜芦醇和褪黑素在转基因动物生产中的应用提供参考依据。     

Effect of Psammaplin A on in vitro Development of Bovine Aging Oocytes

To investigate the effect of histone deacetylation inhibitor Psammaplin A (PsA) on the development of bovine aging oocytes in vitro,oocytes were randomly divided into control group,aging group and 50 mmol/L PsA treated aging group (PsA group).Immunofluorescence staining and JC-1 were used to detect the blastocyst rate of bovine oocytes after parthenogenetic activation,the number of cells in blastocysts,apoptosis,reactive oxygen species (ROS),glutathione (GSH) and mitochondrial membrane potential intensity of embryos.The results showed that the blastocyst rate of the aging group was significantly lower than that of PsA and control groups (P<0.05).The blastocyst rate of PsA group was not significantly different from that of control group (P>0.05).The number of cells in the blastocysts of control group and PsA group were significantly higher than that of aging group (P<0.05).The number of cells in the blastocysts of PsA group was not significantly different from that of control group (P>0.05).The apoptosis rate in aging group was significantly higher than that of control and PsA groups (P<0.05),the apoptosis rate of PsA group was significantly higher than that of control group (P<0.05).The GSH level of MⅡ oocytes in aging group was significantly lower than that of control and PsA groups (P<0.05).There was no significant difference in GSH level between control and PsA groups (P>0.05).The ROS level of the embryos in aging group was significantly higher than that of control and PsA groups (P<0.05).The ROS level in PsA group was significantly higher than that of control group (P<0.05).The mitochondrial membrane potential of early embryos of aging group 4-8 cells was significantly lower than that of control and PsA groups (P<0.05).The mitochondrial membrane potential intensity of control group was significantly higher than that of PsA group (P<0.05).In summary,PsA could effectively delay the aging of bovine oocytes and improve the quality of oocytes.     

GMP玻璃化冷冻未成熟牛卵母细胞核成熟及冷冻损伤试验

本试验通过荧光染色的方法建立了未成熟牛卵母细胞在体外培养过程中第1次减数分裂的各个阶段的参考判定图谱;根据这个标准来观察毛细玻璃管(GMP)玻璃化冷冻对卵母细胞核成熟和冷冻损伤的影响。结果表明,从屠宰场废弃的卵巢表面卵泡内抽取的COCs,70%处于生发泡期,12.5%生发泡开始破裂,7.5%已开始浓缩,这说明从屠宰场获得的COCs有较高的异质性;卵母细胞在成熟培养22h时收获排出第一极体的卵母细胞,可得到丰度较高的极体-胞质染色体对称、紧密相邻的成熟卵母细胞;GMP玻璃化冷冻损伤主要有2种表现形式,首先,直接影响膜结构的完整性,包括细胞膜和核膜,这可从退化的细胞比例看出(8～24h,有21.9%～27.2%的细胞处于该阶段),其次,影响CONDENSED向MⅠ期的过渡,这可从处于CONDENSED卵母细胞的比例看出(8～24h,有24.1%～34.3%的细胞处于该阶段)。