Triploidy induction through cntd1 knockout in red crucian carp (Carassius auratus red var.)

Triploid fish are distinguished by their rapid growth rate, enhanced resistance, and bisexual sterility, making them highly desirable for breeding purposes. A recent innovation in triploid production involves the utilization of cntd1 (cyclin N-terminal domain containing 1) knockout in zebrafish. In this study, this method was successfully applied to red crucian carp (RCC) through the implementation of the clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9 (CRISPR/Cas9) technique. Analysis of the cntd1 knockout F0 generation in RCC revealed phenotypes similar to those observed in Cntd1-deficient zebrafish. The male F0 RCC exhibited severely inhibited spermatogenesis characterized by pink testes and watery semen. Subsequent examination indicated markedly reduced spermatozoa production, with numerous spermatocytes arrested at the metaphase of meiosis and enriched apoptotic signals in the testes. In contrast, females of the F0 generation spawn normally, but their offspring displayed developmental deformities and variable ploidy levels during embryogenesis. Upon maturation, the offspring were categorized into diploids and triploids based on DNA fluorescence values per cell and chromosome numbers. As previously reported, the erythrocytes of triploids displayed karyorrhexis and dumbbell-shaped nuclei. The current study demonstrates that the method of ploidy regulation based on cntd1 knockout is adaptive to various species of fish and holds great potential for use in polyploid breeding.