In general, in vitro-matured oocytes and commercially available frozen semen are used for bovine intracytoplasmic sperm injection (ICSI), unlike ICSI in humans and experimental animals, which uses in vivo-matured oocytes and fresh semen. Bovine ICSI is also characterized by difficulties in pronuclear formation. Therefore, increasing in vitro development of produced ICSI embryos is considered to necessitate use of an artificial activation treatment after injection of motile sperm. However, because parthenogenetic embryos are found amongst produced ICSI embryos, it is necessary to establish a bovine ICSI protocol that leads to normal karyomorphism.