236 PARTHENOGENETIC ACTIVATION OF IN VITRO-MATURED OVINE OOCYTES WITH STRONTIUM

Abstract

The objective of the present experiments was to examine whether strontium could activate in vitro-matured ovine oocytes. Oocytes were collected and matured as previously described (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). Briefly, selected cumulus–oocyte complexes were cultured in modified TCM-199 medium supplemented with 20% sheep serum and hormones for 22–23 h, at 39°C, 5% CO₂ in air. Matured oocytes were randomly divided into four groups and treated as follows: (1) cultured in 10 mm strontium + 5 μg mL^–1 cytochalasin B in Ca²⁺-free CZB medium for 4–5 h; (2) electrically activated in Ca²⁺-containing medium, then cultured in 10 mm strontium + 5 μg mL^–1 cytochalasin B in Ca²⁺-free CZB medium for 4–5 h; (3) electrically activated in Ca²⁺-containing medium and then cultured in SOF medium containing 5 μg mL^–1 cytochalasin B for 4–5 h; and (4) electrically activated in Ca²⁺-free medium and then transferred into SOF medium + 5 μg mL^–1 cytochalasin B for 4–5 h. This experiment was repeated three times. Activation rates based on the number of pronuclear formations/the number of oocytes cultured were 96.7% (147/152), 95.9% (116/121), 75.9% (101/133), and 43.0% (56/107) in Groups 1–4, respectively. After 7 days of culture in SOF medium, 26.8%, 33.3%, 19.6%, and 0% of oocytes in Groups 1, 2, 3, and 4 developed to the blastocyst stage, respectively. Significant differences in blastocyst rate were observed across these groups except between groups 1 and 2 (P < 0.01). However, there were no significant differences in mean number of nuclei/blastocyst across Groups 1, 2, and 3 (P > 0.05). Our results demonstrated that in vitro-matured ovine oocytes can be effectively activated with strontium alone, resulting in an activation rate of 96.7% and a blastocyst rate of 26.8% (blastocysts/oocytes). Also, a combination of strontium and electrical pulses could benefit sheep oocyte activation and embryo development to the blastocyst stage (95.9% and 33.3%, respectively). We conclude that strontium is an effective activator for sheep oocyte activation and it could be used for sheep nuclear transfer.

Table 1. Parthenogenetic development of oocytes activated by SrCl²⁺ and electrical pulses