The present study aimed to compare the effects of butyrolactone-I (BL-I) and cycloheximide (CHX) on inhibition of germinal vesicle (GV) breakdown (GVDB) in bovine oocytes and subsequent in vitro development after in vitro maturation and fertilization. Furthermore, in experiment 2, we compared the kind of supplemented protein with CHX during inhibition of GVBD of oocytes obtained from ovaries stored for 1 day, and examined time extension of storage of oocytes. In experiment 1, bovine cumulus-oocyte complexes (COCs) collected by the aspiration of 3- to 5-mm follicles of ovaries from at a local abattoir were preincubated for 24h in TCM-199 supplemented with 100μM BL-I and 3mgmL−1 BSA or 100μLmL−1 CHX and 5% CS. As a control, fresh COCs were used without preincubation. In experiment 2, the COCs were collected from ovaries stored in phygiological saline for 1 day at 20°C. The collected COCs were preincubated for 24h in TCM-199 supplemented with 100μLmL−1 CHX and 3mgmL−1 BSA or 5% CS (CHX+BSA, CHX+CS). As a control, fresh COCs collected from ovaries stored in the same condition were used without preincubation. In both experiments, the COCs were maturated and inseminated with frozen-thawed spermatozoa. After preincubation, maturation and fertilization, some oocytes or zygotes were fixed to assess the rates of oocytes at the GV stage, MII or sperm penetration. Following insemination, the presumptive zygotes were cultured in CR1aa (Rosenkrans, C.F. Jr. et al., 1993 Biol. Reprod. 49, 459–462) supplemented with 5% CS for 8 days. Embryo development was evaluated for cleavage rates on Day 2, and for blastocyst rates on Days 7 and 8 (IVF=Day 0), respectively. To evaluate embryo quality, the total cell numbers in the blastocysts were counted by means of the air-drying method. Three replicates were carried out for each experiment. Data were analyzed by chi-square test (cleavage and blastocyst rates) and ANOVA (cell numbers). In experiment 1, there were no differences in the rates of the oocytes at the GV stage between BL-I (71.4±10.7%, mean±SD) and CHX (86.7±10.9%), but the rates of the oocytes at the MII stage for BL-I (59.6±7.4%) tended to be lower than for those in CHX (80.0±14.1%, P<0.1). The rate of MII stage for control was 67.5±18.4%, and there were no differences between control and other treatments. No differences were found in sperm penetration, normal fertilization and polyspermy after in vitro fertilization. The cleavage rate for oocytes in CHX (81.0±1.2%) was significantly higher than for those in BL-I (65.0±13.1%, P<0.01), and there was a tendency for the cleavage rate in BL-I to be lower than that of the control (75.5±4.7%, P<0.1). A significantly lower percentage of embryos cultured in BL-I (19.2±13.8%) developed to the blastocyst stage than those of embryos in the control (32.0±11.2%, P<0.05), but there were no differences in the blastocyst rate between BL-I and CHX (25.9±8.8%). Cell numbers in the blastocysts in BL-I (177.2±15.9, n=21) and CHX (191.2±12.9, n=31) were not significantly different compared to the control (198.4±14.3, n=34). In experiment 2, no significant differences were found in the cleavage rates (CHX+CS, 64.0±18.7%; CHX+BSA, 68.1±10.8% and control, 72.2±8.3%). However, the blastocyst rates in CHX+CS (4.0±7.8%) and CHX+BSA (7.7±9.2%) were significantly lower than the control (20.4±3.7%, P<0.05). These results suggested that CHX can reversibly inhibit the GVBD of bovine oocytes for 24h without compromising subsequent developmental competence after in vitro maturation, fertilization, and culture. However, COCs collected from stored ovaries for 1 day and preincubated with CHX failed to develop into blastocysts regardless of the kind of supplemented protein.
Estimation of the Optimal Timing of Fertilization for Embryo Development of In Vitro-Matured Bovine Oocytes Based on the Times of Nuclear Maturation and Sperm Penetration
