PURPOSE: This study was undertaken to evaluate the optimal exposure time for nuclear envelope breakdown (NEBD) after injection of a round spermatid and to investigate the effect of oocyte activation timing on the fertilization rate and early embryo development in porcine round spermatid injection procedure. METHODS: Injected oocytes were fixed at 0.5, 1, 2, 3, and 4 h before activation, and NEBD state was examined. The three groups of oocytes were activated before and after the injection of spermatid using a single direct current pulse (100 V/mm, 50 micros): group 1) at 2 h before the injection (pre), group 2) within 0.5 h after the injection (immediate), and group 3) at 2 h after the injection (post). Activated oocytes were cultured and pronucleus formation and blastocyst development was evaluated at 15-18 h and 7-8 days after the injection, respectively. RESULTS: The proportion of oocytes with NEBD significantly increased in the groups with over 2 h of exposure time (p < 0.05) and oocyte with premature chromosome condensation began to appear 3 h after the injection. Normal fertilization and development rate to the blastocyst stage were significantly higher in the post group than in those of the pre or immediate group (p < 0.05). CONCLUSION: The optimal exposure time for NEBD is 2 h after the injection, and activation in 2 h after round spermatid injection improved the normal fertilization and early embryo development rate.
PURPOSE: This study was undertaken to evaluate the optimal exposure time for nuclear envelope breakdown (NEBD) after injection of a round spermatid and to investigate the effect of oocyte activation timing on the fertilization rate and early embryo development in porcine round spermatid injection procedure. METHODS: Injected oocytes were fixed at 0.5, 1, 2, 3, and 4 h before activation, and NEBD state was examined. The three groups of oocytes were activated before and after the injection of spermatid using a single direct current pulse (100 V/mm, 50 micros): group 1) at 2 h before the injection (pre), group 2) within 0.5 h after the injection (immediate), and group 3) at 2 h after the injection (post). Activated oocytes were cultured and pronucleus formation and blastocyst development was evaluated at 15-18 h and 7-8 days after the injection, respectively. RESULTS: The proportion of oocytes with NEBD significantly increased in the groups with over 2 h of exposure time (p < 0.05) and oocyte with premature chromosome condensation began to appear 3 h after the injection. Normal fertilization and development rate to the blastocyst stage were significantly higher in the post group than in those of the pre or immediate group (p < 0.05). CONCLUSION: The optimal exposure time for NEBD is 2 h after the injection, and activation in 2 h after round spermatid injection improved the normal fertilization and early embryo development rate.