A combined treatment of ionomycin with ethanol improves blastocyst development of bovine oocytes harvested from stored ovaries and microinjected with spermatozoa.

Regardless of the presence of sperm-borne oocyte-activating factors, activation of bovine oocytes with exogenous activation stimuli is required for further development after intracytoplasmic sperm injection (ICSI). The current study was designed to develop a new activation regimen for improving the blastocyst yield after ICSI of bovine oocytes harvested from ovaries stored at 10 to 12 degrees C for 24h. After ICSI, oocytes were treated with 5microM ionomycin for 5 min, 7% ethanol for 5 or 10min, ionomycin followed by ethanol (5 or 10 min), ionomycin followed by 10 microg/mL cycloheximide for 5h, or ionomycin followed by 1.9 mM 6-dimethylaminopurine for 3h. Across the activation regimens, the cleavage rates of ICSI oocytes (45% to 77%) were higher than those of parthenogenetically activated oocytes (11% to 21%; P<0.05). Activating the ICSI oocytes with ionomycin plus ethanol improved the blastocyst yield (29% to 30%) compared with that of nontreated oocytes (12%; P<0.05), but the other regimens did not improve the blastocyst yield (9% to 18%; P>0.05). Higher blastocyst yields were due to increasing the proportion of ICSI oocytes that passed through the early postfertilization events until cleavage. None of the regimens have any adverse effect on the quality of the blastocysts regarding the total cell number or the proportion of the inner cell mass cells. Thus, a new activation regimen using two triggers for single calcium increase effectively improved blastocyst yield after bovine ICSI using oocytes harvested from stored ovaries.