Activation of cumulus-free equine oocytes: effect of maturation medium, calcium ionophore concentration and duration of cycloheximide exposure

Abstract

Two different culture media (TCM-199 and follicular fluid), two activation treatments (10 and 50 micromol calcium ionophore l(-1)) and three culture periods with cycloheximide were evaluated to find effective culture conditions for activation of cumulus-free equine oocytes. Oocytes were collected by scraping the follicle walls of ovaries obtained from an abattoir. Oocytes with expanded cumuli were matured at 38.2 degrees C in a humidified atmosphere of 5% CO(2) in air, in either TCM-199 with 10% fetal bovine serum (FBS) and 5 microU FSH ml(-1), or in 100% follicular fluid derived from a preovulatory follicle 24 h after injection of hCG. After 40--42 h of in vitro maturation, oocytes were denuded by gentle pipetting in TCM-199 plus 10% FBS with hyaluronidase. Oocytes with intact cytoplasmic membranes (n = 398; 94% presumed metaphase II) were treated in protein-free PBS with 10 or 50 micromol calcium ionophore l(-1) for 5 min. After washing, the oocytes were cultured in TCM-199 containing 10% FBS and 10 microg cycloheximide ml(-1) for 6 h, in cycloheximide for 6 h and then in cycloheximide-free medium for 18 h, or in cycloheximide for 24 h. The oocytes were fixed and evaluated by fluorescence microscopy. Oocytes with pronucleus I--II (dense to decondensing chromatin), pronucleus III--IV (decondensed chromatin) or progressing towards the first cleavage division were considered activated. The activation rate for oocytes matured in TCM-199 was significantly (P < 0.05) higher than for oocytes matured in follicular fluid (49% (99/204) versus 35% (60/171), respectively; P < 0.05). Culture with cycloheximide for 24 h resulted in a significantly higher rate of activation (67%, 74/111) than did the 6 h (33%, 44/136) or 6 h plus 18 h (32%, 41/128) treatments. The highest rate of activation (82%) was observed in oocytes matured in TCM-199, treated with 50 micromol calcium ionophore l(-1) and cultured with cycloheximide for 24 h.