Suttner R, Zakhartchenko V, Stojkovic P, Müller S, Alberio R, Medjugorac I, Brem G, Wolf E, Stojkovic M
Department of Molecular Animal Breeding and Genetics, Ludwig-Maximilians University, Munich, Germany.
Theriogenology. 2000 Oct 1;54(6):935-48. doi: 10.1016/S0093-691X(00)00403-9.
Intracytoplasmic sperm injection (ICSI) is a very important technique for treating male subfertility and for basic research. The efficiency of ICSI in bovine is very limited because of the necessity for additional oocyte activation before or after the ICSI procedure. In this study, we compared the effects of seven different protocols on activation and fertilization rates of bovine oocytes after ICSI and on their subsequent development under in vitro conditions. The protocols include 1) different chemical activation of oocytes, 2) pretreated or nonpretreated sperm, and 3) conventional or Piezo-driven injection techniques. In all three groups, ICSI, sham-injected, and noninjected, the highest activation rates were obtained after treatment of oocytes with ionomycin followed by 6-dimethylaminopurine (6-DMAP). Using this treatment for oocyte activation, 59% of oocytes were activated and 31% of oocytes were fertilized using dithiothreitol (DTT) pretreated spermatozoa and Piezo-driven injection. Using the protocols with the same oocyte activation or activation with calcium ionophore (Ca-I) and cycloheximide (CHX), nonpretreated sperm, and conventional injection technique, early cleavage rate (79.6% and 77.6%, respectively) were significantly (P <0.01) higher when compared with all other protocols. The latter protocol resulted in 8% blastocyst and 90% of the obtained blastocysts were found to be diploid. Our results demonstrate that activation of oocytes, sperm treatment, and injection technique separately or together could improve the success of bovine ICSI.
胞浆内单精子注射（ICSI）是治疗男性生育力低下及基础研究的一项非常重要的技术。由于在ICSI操作之前或之后需要额外的卵母细胞激活，牛的ICSI效率非常有限。在本研究中，我们比较了七种不同方案对牛卵母细胞ICSI后激活率、受精率及其随后体外发育的影响。这些方案包括：1）卵母细胞的不同化学激活；2）预处理或未预处理的精子；3）传统或压电驱动注射技术。在ICSI、假注射和未注射的所有三组中，用离子霉素处理卵母细胞后再用6-二甲基氨基嘌呤（6-DMAP）处理可获得最高激活率。使用这种卵母细胞激活处理方法，使用二硫苏糖醇（DTT）预处理精子和压电驱动注射时，59%的卵母细胞被激活，31%的卵母细胞受精。使用相同的卵母细胞激活方案或用钙离子载体（Ca-I）和环己酰亚胺（CHX）激活、未预处理精子和传统注射技术时，早期卵裂率（分别为79.6%和77.6%）与所有其他方案相比显著更高（P<0.01）。后一种方案产生了8%的囊胚，且所获得的囊胚中有90%被发现为二倍体。我们的结果表明，卵母细胞激活、精子处理和注射技术单独或共同作用可提高牛ICSI的成功率。
