255 SPERM NUCLEAR DECONDENSATION ABILITY OF IN VITRO MATURED CANINE OOCYTES

Abstract

The sperm chromatin decondensation occurs when a spermatozoon enters into an oocyte during fertilization, and the effectiveness of this process is connected to the grade of oocyte cytoplasmic maturation. In this study chromatin sperm decondensation was evaluated after IVF of canine oocytes matured in vitro (IVM), comparing different durations of maturation. Cumulus–oocytes complexes (COC) for IVM were obtained from bitch ovaries after ovariohysterectomy, selecting those COC with compact cumulus cells and a homogeneous dark cytoplasm. The COC were matured in vitro for 0, 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM HEPES, 10% FCS, 0.25 mM pyruvate, 10 IU mL^–1 of hCG, 300 IU mL^–1 of penicillin, and 20 mg mL^–1 of streptomycin at 38.5°C and 5% CO₂. Fresh ejaculates from 3 adult dogs were centrifuged, and the sperm pellet was resuspended in fert-TALP medium. In each replicate, 100-μL fert-TALP drops containing 10 to 12 IVM oocytes after each culture time were co-culture with 2.5 × 10⁶ spermatozoa mL^–1 for 24 h under culture conditions. Soon after co-culture, all oocytes were denuded from cumulus cells and fixed in 3% paraformaldehyde. The nuclear stage of the oocytes and the appearance of the sperm nucleus were determined by 4′,6-diamidino-2-phenylindole staining under a fluorescence inverted microscope. For each treatment, at least 4 replicates were performed, and the data were compared statistically by chi-square test, using InfoStat Professional Program. A total of 800 oocytes were evaluated, and the percentages of oocytes with sperm penetration were 58% (138/238), 61% (108/177), 72% (118/165), and 70% (153/220) at 0, 48, 72, and 96 h of IVM, respectively. The percentage of sperm nuclear decondensation at each time point significantly increased up to 72 h of culture, showing 12, 34, 81, and 85% of sperm nuclei deconsated, respectively. The percentages of nuclear maturation also increased (P ≤ 0.05) with time, showing 0, 8, 20, and 27% of oocytes at second metaphase (MII) stage at 0, 48, 72, and 96 h of culture. The percentage of MII stage was much lower than that of chromatin decondensation in all maturing groups. These results suggest that canine oocytes matured in vitro are able to decondense the sperm chromatin during IVF, and this ability increases up to 72 h of culture. Nevertheless, cytoplasmic maturation, as evaluated by sperm chromatin decondensation, in canine oocytes matured in vitro may not be completely connected with nuclear development.