ミニブタ体細胞クローン胚の作出に用いる融合および活性化用培地中におけるカルシウム濃度の最適化

概要

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The effects of Ca2+ concentration in activation medium and cytochalasin B treatment after activation on the parthenogenetic development of pig oocytes were examined. In addition, cloned embryos derived from miniature pig somatic cells were activated under optimal conditions and the effects of Ca2+ in fusion medium on the development of embryos after activation was examined. When oocytes were activated in 0.1 mM Ca2+ and then treated with cytochalasin B, the blastocyst formation rate (28.6%) was significantly higher than those activated in 0-0.05 or 1.0 mM (11.0-18.3%). Treatment with cytochalasin B decreased the second polar body extrusion rate of activated oocytes. The presence or absence of Ca2+ in fusion medium did not affect the fusion rate of miniature pig somatic cells with recipient oocytes. A few cloned embryos developed to the blastocyst stage (2.7-9.0%) without an additional activation treatment. On the other hand, significantly more embryos developed to the blastocyst stage after activation treatment when they were fused in the absence (28.9%) of Ca2+ rather than the presence (16.5%) of it. These results show that the highest blastocyst formation rate for miniature pig cloned embryos is obtained when donor cells and recipient oocytes are fused in the absence of Ca2+ and then activated in 0.1 mM Ca2+ and treated with cytochalasin B.