The production of cloned fish in the medaka (Oryzias latipes)

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Abstract
The measurement of cellular DNA content by DNA microfluorometry revealed that medaka embryos that were fertilized with normal sperm and exposed to heat shock (41°C for 3 min) or hydrostatic pressure (700 kg/cm2 for 10 min) at 85–95 min after insemination were tetraploid. Embryos fertilized with normal sperm and exposed to heat shock (41°C for 2 min at 2–3 min after insemination) were triploid. These results suggest that heat shock or hydrostatic pressure at 85–95 min after insemination arrests the first cleavage, while heat shock at 2–3 min after insemination arrests the second meiotic division.Medaka clones have been produced by the following method: Eggs from orange‐red or variegated variety were activated by UV‐irradiated, genetically impotent sperm of wild‐type fish (UV sperm). The haploid eggs obtained were diploidized by preventing the first cleavage with heat shock or hydrostatic pressure to produce homozygous females. Each of the two homozygous females was mated with vasectomized male in isotonic balanced salt solution to collect unfertilized eggs. The collected eggs were activated with UV sperm and converted from haploid to diploid by arrest of the second meiotic division with heat shock. Hatched fry of each homozygous diploid (all females) were fed with a methyltestosterone‐containing diet (40 μg/gm diet) to produce sex‐reversed males, which were mated with brood females, and thus two cloned lines were obtained.