First report of tetraploid induction by cold-shock treatment in olive flounder Paralichthys olivaceus

Tetraploid induction through the application of hydrostatic pressure to fish is an essential aquaculture breeding technique for large-scale triploid production. However, this method requires precise timing and expensive equipment. Tetraploid induction using cold shock may provide an alternative to hydrostaric pressure, and identifing the optimal shock initiation time could help improve induction dfficiency. Here, the optimal initiation time and duration of cold shock were assessed using olive flounder ( Paralichthys olivaceus ) as the test organism. A large-scale tetraploid induction experiment was conducted, and the induction, survival, and deformity rates of tetraploid-induced Paralichthys olivaceus samples were recorded. Ploidy stability was also analyzed. The highest tetraploid induction rate (89.0 ± 2.1 %) was achieved by exposing fertilized eggs that had been incubated at 19 ± 0.5 °C to cold shock at 55 min post-fertilization and −2.5 ± 0.5 °C for 60 min. Although the initial tetraploid induction rate was high, ploidy stability declined sharply during development until no tetraploid individuals remained at 11 months post-hatching (mph). Moreover, tissue-specific ploidy variation indicates mosaicism. Additionally, the tetraploid-induced population exhibited a low survival rate (6.8 % at 6 mph) and a high deformity rate (39.8 %). These results demonstrate that while cold shock enables efficient tetraploid induction, maintaining stable ploidy into adulthood remains a challenge. Nevertheless, this study provides a technological foundation for advancing polyploid breeding strategies in marine aquaculture. • First mitotic division occurred at 55 mpf at 19 °C in P. olivaceus. • 60-min cold shock at −2.5 °C induced 89.0 % tetraploidy at 55 mpf. • Large-scale induction showed ploidy instability and mosaicism over time. • First cold-shock-based tetraploid induction protocol in olive flounder.