亮甲酚蓝筛选对广西巴马小型猪孤雌激活及克隆重构胚早期发育的影响

Effect of brilliant cresyl blue selection on parthenogenetic activation and early development of handmade cloning embryos of Bama minipig in Guangxi

【目的】探讨亮甲酚蓝(BCB)染色是否可以筛选出发育潜能优秀的卵母细胞。【方法】猪卵丘卵母细胞复合体(COCs)在体外成熟培养前用13μmol/LBCB染色,根据卵母细胞胞质蓝色的有无分为阳性(BCB+,蓝色)和阴性组(BCB-,不着色),以在不含BCB的改良杜氏磷酸盐缓冲液(m-DPBS)中处理90min为控制对照组,直接培育作空白对照,体外成熟培养后统计各组卵母细胞的核成熟率及孤雌激活胚胎、克隆重构胚胎(供体细胞来自广西巴马小型猪)的早期发育率。【结果】BCB+组卵母细胞的核成熟率(80.8%)显著高于BCB-组(44.2%)和空白对照组(72.4%);BCB+组的孤雌囊胚形成率(32.9%)显著高于BCB-组(10.1%),但与对照组(25.8%)之间无显著差异;BCB+组显微操作核移植胚胎的囊胚发育率(14.1%)显著高于BCB-组(3.4%),但与对照组之间无显著差异;各组手工克隆胚胎发育率之间无显著差异。【结论】BCB染色筛选出的卵母细胞具有较好的核成熟发育潜能,但未能显著改善广西巴马小型猪显微操作及手工克隆胚胎的早期发育。

[Objective ]The aim of this research was to evaluate whether the oocyte with superior developmental competence could be selected via brilliant cresyl blue （BCB） staining. [Method]Cumulus oocyte complexs （COCs） of Bama minipig were stained with BCB （13 μmol/L） before in vitro maturation （IVM）, according to the colour of oocyte cytoplasm, COCs were divided into BCB- （colourless cytoplasm） group and BCB＋ group （coloured cytoplasm）, taking the COCs incubated in m-DPBS without BCB for 90 minutes before IVM was treated control group while the directly cultured IVM was treated as blank control. After IVM, oocyte nuclear maturation rate, the developmental rates of parthenogenetic activation （PA）, and handmade cloning （HMC） embryos in each group of Guangxi Bama minipig were calculated. [ Result ] Oocyte nuclear maturation rate in BCB＋group （80.8%） was significantly higher than that in BCB- group （44.2%） and control group （72.4%）. The blastocyst rate of PA in BCB＋group was significantly higher than that in BCB- group （32.9% VS 10.1%）, but not significantly different from the blastocyst rate of the control group （25.8%）. The blastocyst rate of NT in BCB~ group was significantly higher than that in BCB- group （14.1% VS 3.4%）, but not significantly different than the blastocyst rate of the control group. There was no significant difference in blastocyst rate of HMC among BCB＋ group, BCB- group and the control group. [Conclusion]The positive porcine COCs selected with BCB staining displayed superior developmental competence of nuclear maturation, but this method could not significantly improve the developmental rate of PA, micromanioulation, and HMC embryos at early stage.