With the purpose to establish an optimal activation procedure for nuclear transfer experiments in pig, three protocols were tested.

Cumulus-oocyte complexes (COCs) were aspirated from ovaries of prepubertal gilts. These COCs were washed in TALP-Hepes and 10 % fetal calf serum (FCS). For maturation oocytes were cultured in NCSU 23 supplemented with 10 IU/ml ECG, 10 IU/ml hCG and 10 % porcine follicular fluid for 22 h. Subsequent culture was carried out in the same medium without hormonal supplements for 22 h. After maturation cumulus cells were removed from oocytes by incubation in modified phosphate buffered saline (mPBS) and 0.1 % hyaluronidase. For activation only oocytes with a polar body were used. Three different protocols for activation were used. 1) Incubation of the oocytes in 7 % ethanol in MPM + BSA (1 mg/ml) for 5 min followed by incubation in cycloheximide (10 g/ml) and cytochalasin B (5 g/ml) in MPM+BSA for five h; 2) Incubation for 5 min in 5 mmol/L ionomycin and 75 mol/L bohemine (MW: 340) and then incubation for 5 h in MPM-BSA and bohemine. 3) Incubation of oocytes for 5 min in 5 mmol/L Ionomycin and DMAP (1.9 mmol/L) and then incubation for 5 h in DMAP. After 24 h all oocytes were fixed with acetic alcohol (1:3) for 48 h and stained with 1% aceto-orcein. Oocytes with one or more pronuclei were considered as activated. After three replicates oocyte activation rate was 46.6% (56/120), 70.4% (50/71) and 81.3% (100/123) using activation protocol 1, 2 and 3 respectively.