301 EFFECT OF THE MII-AGE AND ACTIVATION PROTOCOL ON THE PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES

Abstract

The completion of porcine oocyte nuclear maturation (MII) in vitro, characterized by the time of polar body extrusion, starts at about 32 h of maturation and lasts more than 12 h. This leads to the simultaneous presence in the population of matured oocytes with differing abilities to be activated. We investigated age-dependent changes in pig oocyte maturation, activation and development in SOFaa in response to electric impulse (EL) in the presence of cytochalasin B (CB) and EL in combination with cycloheximide and cytochalasin B (EL + CHX + CB). Oocytes were matured in TCM 199 with 10% FCS, cysteine, LH, FSH (Pergovet, Serono, Geneva, Switzerland) for 36 h and then decumulated. Matured oocytes were activated at 40 and 44 h by double pulse of 30 μs DC 1, 5 kV cm⁻¹ and cultured in 5 μg mL⁻¹ CB for 4 h or by EL followed by incubation in 10 μg mL⁻¹ CHX + 5 μg mL⁻¹ CB for 4 h. According to the MII-age before activation oocytes were divided into 2 age classes: 3–7 and 7–11 h after polar body extrusion. Embryos were cultured in SOFaa in 5% CO₂, 5% O₂ at 38.5°C. The rates of cleavage, blastocyst formation and cell number of BL on Day 7 (BLD7) were recorded. Our results showed that the average rate of maturation at 44 h was 72% (n = 1377). About 50% and 87% of oocytes, that eventually matured, extruded the polar body at 37 and 40 h, respectively. The average cell number of BLD7 developed in SOFaa was 80 ± 36 (n = 52) and was not affected by activation protocol. Seventy-nine and 27% of BL had more than 50 and 100 cells per BL, respectively. Porcine oocytes activated by EL acquired their developmental competence gradually, achieving the highest rates of cleavage and blastocyst formation 7 h after polar body extrusion. By contrast, oocytes activated by EL + CHX + CB showed their maximal developmental competence earlier (3–7 h group). In conclusion, we demonstrate that electric impulse in combination with CHX + CB treatment permits earlier efficient activation of porcine oocytes (3–7 h after polar body extrusion).