Centro de Tecnología Animal. Instituto Valenciano de Investigaciones Agrarias (CITA-IVIA), Segorbe, Castellón, Spain.
Theriogenology. 2011 Dec;76(9):1658-66. doi: 10.1016/j.theriogenology.2011.06.030.
Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca(2+) concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca(2+) concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3-66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.
注入精子核的不完全去凝聚是猪体内胞质内精子注射（ICSI）的主要问题之一。为了提高原核形成率，本研究旨在探讨在 ICSI 或人工激活后 2 小时，在卵母细胞操作和培养介质中用 0.1% Triton X-100（TX）处理精子以去除膜和/或添加 5 mM 咖啡因（CAF）对激活和胚胎发育率的影响。还分析了注射介质中包含的 4 种不同 Ca（2+）浓度对假注射后胚胎发育的影响。在实验 1 中，无论注射介质中包含的 Ca（2+）浓度如何，均未检测到对卵裂或囊胚率有显著影响。在实验 2 中，与对照组相比，用 TX 预处理精子注射的卵母细胞显示出更高的雄性原核形成率（2PN；54.1%比 36.6%）。然而，在体外胚胎发育、卵裂或囊胚率方面没有观察到差异。在实验 3 中，在微操作过程中和之后用 CAF 处理的卵母细胞与用 TX 预处理的精子一起注射，其卵母细胞激活率显著低于其他实验组（25.7%比 56.3-66.3%）。不同实验组之间的卵裂率没有差异。然而，CAF 组的囊胚率明显高于 TX+CAF 组（分别为 12.0%和 1.9%）。在第二种方法中，研究了电场强度和 CAF 处理对卵母细胞激活的影响。在实验 4 中，无论 CAF 处理如何，接受 0.6 kV/cm 电场的卵母细胞的激活率均显著高于 1.2 kV/cm 电场的卵母细胞（分别为 83.7%比 55.9%和 75.7%比 44.3%；在对照组和 CAF 组中）。在任何实验组中都没有观察到 CAF 处理的影响。总之，在没有额外激活程序的情况下，在 ICSI 前用 TX 处理精子可提高雄性原核形成率，但直到囊胚阶段才能提高胚胎发育率。当精子未经 TX 处理时，未发现咖啡因的显著作用，尽管在缺乏膜的情况下，咖啡因可避免卵母细胞激活和胚胎发育。最后，无论应用于孤雌激活的电场强度如何，咖啡因对雌性原核形成均无影响。
