Effects of the Timing of Cumulus Cell Removal from Bovine Oocytes on Enucleation Rate and Subsequent Development after Somatic Cell Nuclear Transfer

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  • AKAGI Satoshi
    Animal Breeding and Reproduction Research Division, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Ibaraki 305-0901, Japan Animal Breeding and Reproduction Research Division, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Ibaraki 305-0901, Japan
  • SHIRAISHI Toru
    Aichi Agricultural Research Center, Aichi 480-1193, Japan Aichi Agricultural Research Center, Aichi 480-1193, Japan
  • SOMFAI Tamas
    Animal Breeding and Reproduction Research Division, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Ibaraki 305-0901, Japan Animal Breeding and Reproduction Research Division, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Ibaraki 305-0901, Japan
  • KANEDA Masahiro
    Animal Breeding and Reproduction Research Division, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Ibaraki 305-0901, Japan Animal Breeding and Reproduction Research Division, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Ibaraki 305-0901, Japan
  • HARAGUCHI Seiki
    Animal Breeding and Reproduction Research Division, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Ibaraki 305-0901, Japan Animal Breeding and Reproduction Research Division, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Ibaraki 305-0901, Japan
  • WATANABE Shinya
    Animal Breeding and Reproduction Research Division, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Ibaraki 305-0901, Japan Animal Breeding and Reproduction Research Division, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Ibaraki 305-0901, Japan

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We examined the effects of the timing of cumulus cell removal from in vitro-matured (IVM) bovine oocytes on enucleation efficiency and subsequent in vitro development after nuclear transfer (NT). Cumulus cells were removed from IVM oocytes by pipetting, by low-speed vortexing and pipetting or by high-speed vortexing at 12, 15 or 18 h of IVM, and then denuded oocytes were further cultured for 6, 3 or 0 h, respectively (18 h of IVM in total). There was no difference in the rate of extrusion of the first polar body (PB1) among the groups. The success rate of blind enucleation of oocytes denuded at 12 h (before PB1 extrusion) by high-speed vortexing (81.7%) was significantly higher than that of oocytes denuded at 18 h by high-speed vortexing (67.8%) but similar to those of oocytes denuded by pipetting after low-speed vortexing (78.6–84.1%) or pipetting alone (84.6–86.9%). After high-speed vortexing, the percentage of oocytes in which metaphase II (MII) chromosomes were located adjacent to the PB1 tended to be lower for the oocytes denuded at 18 h than that for the oocytes denuded at 12 h. In contrast, by pipetting or low-speed vortexing and pipetting, the timing of denuding did not affect the relative location of MII chromosomes. The timing and method of denuding did not affect the fusion and blastocyst formation rates of NT embryos. These results suggest that high-speed vortexing is applicable only to cumulus cell removal from oocytes prior to PB1 extrusion, while pipetting or low-speed vortexing followed by pipetting is useful regardless of the PB1 formation status and leads to successful blind enucleation of IVM oocytes.

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