305DEVELOPING AN ACTIVATION PROTOCOL FOR SOMATIC CELL NUCLEAR TRANSFER (SCNT) IN THE DOMESTIC CAT

In order to establish an activation protocol for somatic cloning in the domestic cat, we evaluated the developmental competence of cat embryos derived from in-vitro matured ova after parthenogenetic activation treatment. The quality of parthenogenetic embryos was assessed by D3 cleavage rates, D8 rates of blastocyst formation and total nucleus numbers in expanded/hatching blastocysts. Parthenogenetic activation treatments were as follows;; Treatment I: 3.0kVcm−1 (25μs, twice) in 0.3M mannitol containing 0.1mM CaCl2· 2H2O and 0.1mM MgSO4, administered to matured cat oocytes and followed by 10μgmL−1 cycloheximide +5μgmL−1 cytochalasin B in TCM 199-Earle’s salt supplemented with 0.3% BSA for 6–7h. Treatment II: The first electric stimulation was performed as described for treatment I except that the activation medium consisted of 0.3M mannitol containing Mg, but without Ca. Two hours later, pre-pulsed MII oocytes were electropulsed by applying 1.0kVcm−1 (50μs, twice, 5s apart) in 0.3M mannitol containing Ca and Mg for additional activation, followed by culture in 10μgmL−1 cycloheximide +5μgmL−1 cytochalasin B treatment in TCM 199-Earle’s salt supplemented with 0.3% BSA for 6–7h. Immature cat oocytes were obtained from ovaries by mincing/dissection and matured in vitro for 26–30h as previously described (Gomez et al., 2001, Therigenology, 55, 472). Only MII oocytes with a 1st polar body were utilized for the activation procedure after removal of cumulus cells with 0.1% hyaluronidase by gentle pipetting. A total of 1120 oocytes were collected and the overall maturation rate was 49.8% (551/1120). After parthenogenetic activation of the MII oocytes, the embryos were cultured in vitro as described previously (Pope et al., 2000, Theriogenology, 53, 163–174). The results are shown in Table 1. Treatment II resulted in significantly higher (P