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(347) COMBINED ACTIVATION OF PORCINE OOCYTES INJECTED WITH NUCLEI OF FETAL MINIATURE PIG CELLS AND THEIR SUBSEQUENT DEVELOPMENT IN VITRO. Fuchinoue, Kohei1,2, Kawahara, Manabu1, Wakai, Takuya1, Sasada, Hiroshi1, Kyono, Koichi2, Sato, Eimei1, 1 Laboratory of Animal Reproduction, Sendai, Japan2 Ladies Clinic Kyono, Furukawa, Japan ABSTRACT- Since a technique for in vitro maturation of oocytes has been developed in several animals, in vitro matured oocytes may be used as a potential recipient for production of animal cloning by xenoreconstruction. In addition, when cloned embryos are produced by nuclear transfer, activation of reconstituted embryos is crucial for their subsequent development, which requires an optimal protocol to be established for specific species. In this study, to produce reconstituted embryos derived from miniature-pigs, porcine oocytes that were collected from ovaries were cultured in vitro in NCSU23 and subjected as recipient oocytes for the experiments. Donor cells were established from fetal miniature-pig fibroblast cells. Nuclear transfer was performed by injection of nuclei of fetal miniature-pig cells into porcine oocytes using a piezo-drived manipulator. Reconstituted embryos were activated by combined stimuli and cultured for subsequent development. In experiment 1, activation of oocytes by combination with either of electricity + cycloheximide, ionomycin + electricity, or ionomycin + electricity + cycloheximide resulted in 60.5, 60.0 and 77.4% in cleavage, respectively, whereas the corresponding developmental rates to blastocyst stage were 9.3, 20.0 and 19.4%, respectively. In experiment 2, reconstituted embryos that were activated by combination of ionomycin + electricity + cycloheximide showed 43.8% in cleavage, of which 15.6% developed to blastocysts with blastomeres ranged between 25 and 57. These results suggest that combined activation may be a superior protocol for production of reconstituted embryos derived from miniature-pigs by nuclear transfer using pig oocytes. KEY WORDS: miniature-pig, nuclear transfer, activation |
