不同化学激活方法和电激活参数对猪体外成熟卵母细胞早期孤雌发育的影响

Effect of different chemical activation methods and electro-activation parameters on early development of porcine oocytes matured and parthenogenetically activated in vitro

利用屠宰场猪卵巢卵母细胞,在体外成熟培养44～48h后,对核成熟卵母细胞进行激活。试验1为不同化学激活方法：10%乙醇＋10mg/L放线菌酮组、2.5mmol/L氯化锶＋10mg/L放线菌酮组、5μmol/L离子霉素＋2.5mmol/L6-二甲氨基嘌呤（6-DMAP）组、200μmol/L硫柳汞＋8mmol/L二硫苏糖醇组和对照组（不用任何激活剂）。试验2为用不同电场强度和脉冲时程进行电激活之后放入胚胎培养液中进行体外培养7d。结果显示,（1）4种不同化学激活方法处理卵子的1原核形成率、2原核形成率和原核形成率都显著比对照组高（P〈0.05）,其中离子霉素＋6-DMAP组的2原核形成率和总原核形成率最高,分别为（23.1±3.5）%和（65.2±3.5）%,显著高于其他处理组（P〈0.05）;（2）4种不同化学激活方法处理组的囊胚率都比对照组高（P〈0.01）,其中离子霉素＋6-DMAP组的卵裂率及囊胚率最高,分别为（46.6±18.5）%和（5.6±4.2）%;（3）本试验所用的4种不同化学激活方法对猪4-细胞孤雌胚SDS-PAGE电泳的蛋白质表达图谱没有显著影响;（4）电场强度为1.7kV/cm、脉冲时程为50、70μs时猪体外成熟卵母细胞激活效果最好,卵裂率、囊胚率、囊胚细胞数分别为：（77.4±9.7）%、（12.4±3.7）%、（17.6±5.9）%和（75.1±10.6）%、（12.3±2.6）%、（19.1±8.1）。以上结果说明：本试验所用的4种化学激活方法均能有效激活猪体外成熟卵母细胞,其中离子霉素＋6-DMAP的激活效果最理想;在本实验室条件下,采用1.7kV/cm的电场强度、50～70μs的脉冲时程均能有效地激活猪体外成熟卵母细胞;本试验所用的4种不同化学激活方法对猪4-细胞孤雌胚SDS-PAGE电泳的蛋白质表达图谱没有显著影响。

The objectives were to investigate the effect of different chemical activation methods and electro-activation parameters of porcine oocytes matured and parthenogenetically activated in vitro. Porcine oocytes aspirated from ovaries in local abattoir were matured in vitro for 44 - 48 h. Oocytes with the first polar body were activated by： （ 1 ） different chemical methods that is 10% ethanol ＋ 10 mg/L cycloheximide （CHX） ;2.5 mmol/L SrC12 ＋ 10 mg/L CHX;5 μmol/L ionomycin ＋ 2.5 mmol/L 6- dimethylaminopurine （6-DMAP）;200 μmol/L Thimemsal ＋ 8 mmol/L dithiothreitol （DIT）; the control-no artificial activation （Expt. 1 ）;（2） different pulse strength and duration （Expt. 2）, and then were cultured for 168 h in embryo culture medium. The results indicated： （ 1 ） the rates of pmnuclear formation of all treatment groups were higher than that of the control （ P 〈 0.05）, with the pmnuclear formation rate of ionomycin ＋ 6-DMAP group to be the highest; （2） the blastocyst rates of all treatment groups were higher（ P 〈 0.01 ） than that of the control;（3） the highest rates of cleavage and blastocyst was obtained in ionomycin ＋ 6-DMAP group; （4）the SDS-PAGE protein expression pmf＇des of porcine 4-cell parthenogenetic embryos obtained from oocytes activated by the four chemical activation methods were not significantly different; （5）the efficiency of electro-actlvation of porcine oocytes matured in vitro was the best when the pulse strength was 1.7 kV/cm and the pulse duration was 50 μs or 70 μs. It is concluded that： （1） all of the chemical activation protocols used in this study can enhanc the blastocyst development of porcine oocytes, and the combination of ionomycin and 6-DMAP is the ideal artificial activation method for porcine oocytes; （2） the SDS-PAGE protein expression profiles of porcine g-cell parthenogenetic embryos obtained from oocytes activated by the four chemical activation methods are not significantly different; （3） at our laboratory, the optima/electric stimulation parameter for porcine oocytes matured in vitro is 1.7 kV/cm pulse strength and 50μs or 70 μs pulse duration.