Activation regimens to prepare bovine oocytes for intracytoplasmic sperm injection

Activation of bovine oocytes to produce a single haploid pronucleus in preparation for intracytoplasmic sperm injection (ICSI) has been investigated with various combinations of ionomycin and 6-dimethylaminopurine (DMAP). Effects were evaluated by immunocytochemical staining, chromosomal analysis and assessment of development in vitro. Oocytes matured in vitro were exposed to: ionomycin alone (single or repeated treatments, Groups 1 and 2 respectively), ionomycin followed by DMAP (immediately or after a 3-h delay, Groups 3 and 4), or no treatment (control, Group 5). They were then co-cultured in M199 with bovine oviductal epithelial cells. Activation rates were not significantly different among groups but significantly fewer oocytes in Group 3 extruded a second polar body than in Groups 1, 2, and 4. Most parthenotes (60% to 80%) in Groups 1, 2, and 4 were haploid, whereas 82% in Group 3 were mixoploid or polyploid. Most of the parthenotes (88%) in Group 4 formed a single pronucleus besides extruding the second polar body and were therefore more suitable for ICSI than those of Groups 1 and 2 in which condensed chromosomes predominated. The respective rates of oocyte cleavage in Groups 1 to 4 were 24%, 36%, 70%, and 75%; corresponding blastocyst rates were 1%, 5%, 17%, and 8%. There were significantly fewer cells in the parthenotes of Groups 1, 2, and 4 than of Group 3, or of embryos produced by in vitro fertilization. Thus, delaying the addition of DMAP after ionomycin decreases chromosomal abnormalities and produces a high proportion of activated oocytes suitable for ICSI.