【作者】 刘贞伟；

【导师】 李跃民；

【作者基本信息】 西南农业大学 ， 临床兽医学， 2005， 硕士

【摘要】 卵母细胞激活是核移植的关键步骤之一,卵母细胞激活率的高低直接影响核移植的效率。当前核移植效率低下,其原因之一就是受体卵母细胞未被充分激活。研究哺乳动物卵母细胞的激活及其以后的孤雌胚发育,有利于探索卵母细胞的生理反应,分析理化激活和精子激活的特征,对阐明哺乳动物卵激活、受精与发育机理等均有十分重要的意义。到目前为止,已对多种哺乳动物卵母细胞激活进行了一系列研究,但有关猪成熟卵母细胞体外激活的研究尚不够深入。本试验以体外成熟的猪卵母细胞为材料,以电脉冲、离子霉素、6-二甲氨基嘌呤（6-DMAP）、细胞松弛素B（CB）为人工刺激条件,以孤雌胚早期不同发育阶段的出现比率验证人工激活卵母细胞的效果,研究几种不同激活方法对猪体外成熟卵母细胞人工激活效果的影响,以寻找一种猪卵母细胞较优的人工激活方法,为提高猪核移植技术的成功率提供技术参考。 电激活对猪卵母细胞激活及其孤雌胚发育的影响:应用宁波新芝公司的CRY-3型细胞融合仪和配套的电极板（间距0.5mm）对猪IVM培养44～48小时的卵母细胞以不同电场强度(1.0KV·cm^-1,1.3 Kv·cm^-1,1.5 Kv·cm^-1,2.0KV·cm^-1)、不同脉冲时程（40μs,80μs,100μs,150μs）和不同脉冲次数（1次、2次、3次,每次脉冲间隔10min）进行激活。结果表明:卵母细胞的激活率和孤雌胚发育率随电场强度、脉冲宽度和脉冲次数的变化而变化。（1）固定脉冲宽度和脉冲次数选择场强组:当固定固定脉冲宽度为80μs和脉冲次数为1次,选择场强为1.3 KV·cm^-1时,激活率和卵裂率分别达到了68.75%（110/160）和74.54%（82/110）,显著高于场强为1.0KV·cm^-1实验组（44.38%,71/160;50.70%,36/71）,差异极显著（P<0.01）。场强继续升高,卵母细胞的激活率和卵裂率反而下降。场强为1.5 KV·cm^-1,2.0KV·cm^-1时,卵母细胞的激活率和卵裂率分别为56.25%（90/160）,64.44%（58/90）;54.38%（87/160）,60.92%（53/87）,显著低于场强为1.3 KV/cm实验组（P<0.05）。（2）固定场强和脉冲次数选择脉冲宽度组:在1.3 KV·cm^-1场强,1次脉冲下,脉宽80ps时,卵母细胞的激活率和卵裂率达70.63%（113/160）和75.22%（85/113）,显著高于脉宽为401μs实验组（50.63%,81/160;53.09%,43/81;P<0.05）。脉宽进一步升高并不能使卵母细胞的激活率和孤雌胚的发育率升高。脉宽为10Ops和150μs时,卵母细胞的激活率和卵裂率分别为55.63%（89/160）,67.42%（60/89）;53.13%（85/160）,57.65%（49/85）,也显著低于脉宽为80μs实验组（P<0.05）。（3）固定场强和脉冲宽度选择脉冲次数组:在场强为1.3 KV·cm^-1场强下,脉宽80μs,1次脉冲时,卵母细胞的激活率和卵裂率为71.25%（114/160）和75.44%（86/114）,显著高于2次脉冲（56.88%,91/160;60.44%,55/91）和3次脉冲（53.75%,86/160;56.98%,49/86）,差异极显著（P<0.01）。说明一次脉冲就可以使猪卵母细胞充分激活。以上结果表明:以1.3KV·cm^-1电场强度80μs的脉冲宽度,一次脉冲的电激活条件对猪卵母细胞进行刺激,可更多还原

【Abstract】 The oocytes activation is one of the key step of nuclear transfer(NT) in mammal, and the efficiency of oocytes activation affects the efficiency of NT directly. Nowadays the efficiency of NT in mammal is very low, one reason is that the oocytes are not activated enough. Therefore it is very necessary to research the oocytes activation.By now, many mammalian oocytes activation have been researched, but the study of pig oocytes activation is not enough.This study was undertaken to explore the methods ,such as electric-activation, ionomycin , 6-dimethylamin-opurine(6-DMAP), cytochalasin B(CB) for activation of the in-vitro matured porcine oocytes, and the effect on development after activation, in order to obtain an optimal method for pig in vitro-matured oocytes.The porcine oocytes matured for 44~48 hours in vitro were treated with different intensity of direct current field strengths (1.0 KVcm~-1,1.3 KV cm~-1,1.5 KV cm~-1, 2.0KVcm~-1), pulse duration (40μs, 80μs, 100μs, 150μs) , pulse times(l time,2 times,3 times). The results were as following: (l)The activation rates and development rates of the oocytes altered with the electric field strength, pulse duration and times. When pulse duration and times were fixed, the percentage of activation and cleavage in 1.3 KV cm~-1 electric strength group was respectively significantly higher than those in 1.0KV cm~-1 electric strength group (68.75%vs44.38%; 74.54%vs50.70%, P<0.01) . Electric strength was raised again, but the activation percentage and cleavage percentage started to decrease. The percentage of activation and cleavage in either 1.5 KV cm~-1 electric strength group or 2.0KV cm~-1 electric strength group was respectively significantly lower than those in 1.3 KV icm "’electric strength group(56. 25%, 64. 44%vs68.75%; 54. 38%, 60. 92%vs74.54%, P < 0. 05). (2)When the electric strength was fixed in 1.3 KV cm~-1, one time pulse, when pulse durantion was 80μs, the percentage of activation and cleavage was high to 70.63% and 75. 22%, it was significantly higher than that when the pulse durantion was 40us(50.63%, 53.09%; P < 0.05) . Pulse duration raised continuely, the activation percentage and cleavage percentage did not raise. When pulse duration was 100μs or 150μs, the percentage of activation and leavage was respectively significantly lower than pulse durantion was 80μs group(55.63%, 67.42%vs70.63%; 53.13%, 57.65%vs75.22%, P < 0.05) . (3)On condition that 1.3 KV cm~-1 for 80ns, the percentage of activation and cleavage in one time pulse group was respectively significantly higher than those in two times and three times(71.25%vs56.88%, 53.75%; 75.44%vs60.44%, 56.98%, P < 0.01). The result showed that one pulse was enough to activate pig oocytes.The porcine oocytes matured for 44~48 hours in vitro were treated with ionomycin indifferent concentration(l5μmol·L~-1, 20μmol·L~-1, 25μmol·L~-1) for different time (20min, 30 min, 40 min, 50 min) . The results were as following: The oocytes treated with ionomycin in different concentration(15μmol·L~-1. 20μmol·L~-1 25μmol·L~-1) for 40 minutes, the activation rates and cleavage rates were 57.50%, 59.78%; 60.63%, 62.89%; 58.75%, 60.64% respectively. Although the percentage of activation and cleavage in the group that 20μmol·L~-1 was the highest, but compare with the others there was no significant difference among them (P > 0.05) . The oocytes treated with 20μmol·L~-1 ionomycin for different time (20min, 30 min, 40min,50min) , the activation rates and cleavage rates were 56. 88%, 57.14%; 58.75%, 58.51%; 61.25%, 61.22%; 59.38%, 58. 95% respectively. Although the percentage of activation and cleavage in the group that 40 minutes was the highest, but compare with the others there was no significant difference between them (P > 0.05 ). The result showed that the concentration and time treated with ionomycin did not significantly effect the activation rate of porcine oocyte.The porcine oocytes matured for 44~48 hours in vitro were respectively treated with different electric pulse(1.5 KV · cm~-1, 40us, 1 time; 1.3 KV · cm~-1, 80us , 1 time; 1.0 KV · cm~-1 120μs, 1 time) , then cultured in the NCSU23 containing 6-DMAP and CB for 4 hours. The results were as following: When oocytes were first activated by electricity and then cultured in the NCSU23 containing 6-DMAP and CB, the percentage of activation and cleavage was high to 74.38%, 70.59%; 81.88%, 84.73%; 70.63%, 68.14%, it was significantly higher than the control group(64.38%, 62.14%; 70.00%, 76.79%; 60.63%, 57.73%, P < 0.05).The porcine oocytes matured for 44-48 hours in vitro were respectively treated with ionomycin (15μmol·L~-1, 50 min; 20μmol·L~-1, 40 min; 25μmol·L~-1, 30 min), then cultured in the NCSU23 containing 6-DMAP and CB for 4 hours. The results were as following: When oocytes were first activated by ionomycin and then cultured in the NCSU23 containing 6-DMAP and CB, the percentage of activation and cleavage was high to 63.13%, 62.38%; 68.13%, 68.87%; 65.00%, 64.42%, it was significantly higher than the control group(57.50%, 57.61%; 61.88%, 61.62%; 58.13%, 58.06%, P< 0.05).The porcine oocytes matured for 44-48 hours in vitro were activated by different methods of parthenogenetic activation, A: electric-activation+6-DMAP+CB; B: ionomycin+6-DMAP+ CB; C: electric-activation+ ionomycin+6-DMAP+CB. The results were as following: The percentage of activation and cleavage of group A was respectively in 81.25% and,it was significantly higher than group B(69.38%, 70.27%; P < 0.01) . The percentage of activation of group C was in 86. 88%, there was no significant difference between group A. The percentage of cleavage was high to 89. 21%, it is significantly higher than group A (83.25%, P< 0.05).In conclusion, the optimal electric stimulation conditions of porcine oocytes matured in vitro for 44-48 hours is 1.3 KV · cm~-1 direct current field strength, 80us pulse duration,one time pulse; that in the concentration of 15~25μmol·L~-1 and in the time of 20~50 min treated with ionomycin更多还原