50 INVESTIGATION OF THE EFFECT OF BUTYROLACTONE I AND CYCLOHEXIMIDE TREATMENT DURING IN VITRO MATURATION OF SWINE OOCYTES

Butyrolactone I and cycloheximide specifically inhibit MPF activation and prevent the resumption of meiosis. The aim of this study was to investigate the kinetics of in vitro maturation of butyrolactone I- and cycloheximide-treated swine oocytes in an attempt to produce cytoplasts for nuclear transfer. Oocytes from slaughterhouse ovaries were randomly allotted to one of 3 treatments; group 1 (n = 102 – control – 22 hours of in vitro maturation in TCM199 supplemented with 3.05 mM glucose, 0.91 mM sodium pyruvate, 10% follicular fluid, 0.57 mM cysteine, 10 ng/mL EGF, 10 IU/mL eCG and 10 IU/mL of hCG and 22 hours of culture); Group 2 (n = 191 – blocking for 10 hours in 12.5 M butyrolactone I and in vitro maturation for 44 hours); and Group 3 (n = 175 – blocking for 10 hours in 5 M cyclohexemide and in vitro maturation for 44 hours). After in vitro maturation, oocytes were fixed and stained for evaluation of meiotic division. The percentage of oocytes at metaphase II (MII) in Groups 1 and 3 (75.49% and 70.29%, respectively) were higher (P < 0.05) than Group 2 (63.35%). Based on these results and in order to increase enucleation rates, we also investigated the proximity of the first polar body (PB) with the metaphase plate (MP) in Groups 1 and 3. After in vitro maturation (36, 40, and 44 hours), oocytes were gently decumulated and incubated in microdroplets (50-µL) of bisbenzimide solution (5 g/mL) to analyze the MP and PB positions. Group 1 (control) at 44 hours of maturation (47.05% – 48/102) and Group 3 at 40, and 44 hours (60.20% – 59/98 and 55.46% – 61/110, respectively) showed similar rates, that were higher (P < 0.05) than Group 1 at 36 hours and 40 hours (4% – 4/100 and 36% – 36/100, respectively) and Group 3 at 36 hours (34.58% – 37/107). In conclusion, Group 1 at 44 hours and Group 3 at 40 or 44 hours provide the best oocytes for enucleation because they showed a high number of matured oocytes with the first polar body and the metaphase plate located proximally. This work was supported by FAPESP 02/10747-1.