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

Abstract

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: 3.0 kV cm⁻¹ (25 μs, twice) in 0.3 M mannitol containing 0.1 mM CaCl₂· 2H₂O and 0.1 mM MgSO₄, administered to matured cat oocytes and followed by 10 μg mL⁻¹ cycloheximide +5 μg mL⁻¹ cytochalasin B in TCM 199-Earle’s salt supplemented with 0.3% BSA for 6–7 h. Treatment II: The first electric stimulation was performed as described for treatment I except that the activation medium consisted of 0.3 M mannitol containing Mg, but without Ca. Two hours later, pre-pulsed MII oocytes were electropulsed by applying 1.0 kV cm⁻¹ (50 μs, twice, 5 s apart) in 0.3 M mannitol containing Ca and Mg for additional activation, followed by culture in 10 μg mL⁻¹ cycloheximide +5μg mL⁻¹ cytochalasin B treatment in TCM 199-Earle’s salt supplemented with 0.3% BSA for 6–7 h. Immature cat oocytes were obtained from ovaries by mincing/dissection and matured in vitro for 26–30 h 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 < 0.01) D3 cleavage rates;; however, there were no significant differences in D8 blastocyst formation and total nucleus numbers. These data suggest that an additional electric activation (Treatment II) may increase the in vitro cleavage rates compared to using a fusion and electrical stimulation simultaneously (Treatment I). In addition, we demonstrated the developmental competence of domestic cat embryos derived from in vitro maturation, activation, and culture for development to the pre-implantation stage. By using these procedures for SCNT, several pregnancies were established and a healthy cloned kitten resulted in our laboratory (Shin et al., 2002, Nature, 415, 859). Therefore, this protocol can be useful, not only for prediction of the developmental competence of domestic cat oocytes matured in vitro, but also when used with SCNT to produce cloned cats.

Comparison of cleavage rates and developmental competence to blastocyst stage following parthenogenetic activation treatments in domestic cat oocytes matured in vitro