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.

This study was conducted to evaluate the effects of roscovitine on suppression of meiosis, subsequent meiotic maturation, and cleavage rates after intracytoplasmic sperm injection of horse oocytes. Oocytes were classified as having compact or expanded cumuli (Com or Exp oocytes) and were divided into three culture groups: 30 h culture in maturation medium (30 h Mat); 54 h culture in maturation medium (54 h Mat), or 24 h culture in medium containing 66 micro mol roscovitine l(-1) and then 30 h culture in maturation medium (Ros+M). After maturation, oocytes were subjected to intracytoplasmic sperm injection and cultured in G1.2 medium for 96 h. Among oocytes fixed immediately after roscovitine culture, 26 of 31 (84%) Com oocytes and 16 of 28 (57%) Exp oocytes were at the germinal vesicle stage (P<0.05). After maturation culture, there were no differences in maturation rates or morphological cleavage rates among treatments. Among Com oocytes, significantly more embryos in the Ros+M treatment than in the 54 h Mat treatment had cleaved with > or = two normal nuclei (63 versus 36%; P<0.05); whereas among Exp oocytes, significantly more embryos in the 30 h Mat treatment than in the Ros+M treatment (63 versus 42%; P<0.05) had cleaved with > or = two normal nuclei. The average number of nuclei in embryos at 96 h was significantly higher (P<0.05) in Ros+M Com oocytes (13.5) than in any other Com or Exp group. These results demonstrate that roscovitine can reversibly maintain equine oocytes in the germinal vesicle stage for up to 24 h, and that such suppression may increase the developmental potential of Com, but not Exp, oocytes.

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.