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.
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Canada.
G J
GJ
Rho
B
B
Wu
S
S
Kawarsky
S P
SP
Leibo
K J
KJ
Betteridge
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002970:Cleavage Stage, Ovum; D003593:Cytoplasm; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods*; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D008845:Microinjections; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D013084:Sperm-Ovum Interactions / Q000187:drug effects / Q000502:physiology*
D007476:Ionophores; D015759:Ionomycin; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine