Overall experimental scheme of artificial insemination using frozen medaka sperm. Artificial insemination using frozen medaka sperm is carried out in the following three steps: (1) Collect mature unfertilized eggs from adult females in balanced salt solution (BSS). (2) Thaw frozen sperm. The cryotube containing sperm-filled capillaries is transferred in a Dewar flask from a storage tank. The capillary is picked and placed on dry ice in a styrofoam box. The sperm is thawed and suspended in BSS in the lid of a 0.5-mL tube. (3) Inseminate using thawed sperm. Unfertilized eggs are inseminated with the sperm suspension in the well of a 24-well plate. Fertilized eggs are sorted from unfertilized eggs and collected in BSS.