Intracytoplasmic sperm injection in bovine: Effects of oocyte activation, sperm pretreatment and injection technique

Intracytoplasmic sperm injection (ICSI) is a very important technique for t reating male subfertility and for basic research. The efficiency of ICSI in bovine is very limited because of the necessity for additional oocyte acti vation before or after the ICSI procedure. In this study, we compared the e ffects of seven different protocols on activation and fertilization rates o f bovine oocytes after ICSI and on their subsequent development under in vi tro conditions. The protocols include 1) different chemical activation of o ocytes, 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 th is treatment for oocyte activation, 59% of oocytes were activated and 31% o f oocytes were fertilized using dithiothreitol (DTT) pretreated spermatozoa and Piezo-driven injection. Using the protocols with the same oocyte activ ation or activation with calcium ionophore (Ca-I) and cycloheximide (CHX), nonpretreated sperm, and conventional injection technique, early cleavage r ate (79.6% and 77.6%, respectively) were significantly (P < 0.01) higher wh en compared with all other protocols. The latter protocol resulted in 8% bl astocyst and 90% of the obtained blastocysts were found to be diploid. Our results demonstrate that activation of oocytes, sperm treatment, and inject ion technique separately or together could improve the success of bovine IC SI. (C) 2000 by Elsevier Science Inc.