Scheme design of the ENU mutagenesis in fugu. A) The number of ENU treatments in fugu. The number and timing of ENU injections was examined. ENU (concentration: 70 mg/kg body weight of fugu) was injected 3 times at weekly intervals into the intraperitoneal of fugu males at the stage before spermiation. B) Treatment with various ENU concentrations, and timing of gonadotrophin (HCG/SPH) treatment and sperm collection. Founder fish were treated with three ENU concentrations (7, 70, 120 mg/kg body weight of fugu) at the stage before spermiation. As a control, the fish were treated with buffer (0 mg/kg body weight of fugu) instead of ENU. HCG/SPH was injected into the fugu intramuscularly at 2 or 3 weeks after the last ENU injection. Sperm was collected at 3, 5, and 6 weeks after the last ENU injection.