Generating YY supermales and validating amh as the sex-determining gene in salt-tolerant pink tilapia

Monosex production in aquaculture enhances growth, feed efficiency, and market value while reducing aggression and uncontrolled reproduction. However, the generation of monosex populations remains labour-intensive and costly. In this study, we developed a sex-linked DNA marker on chromosome 23 to distinguish XX, XY, and YY genotypes in salt-tolerant pink tilapia (Oreochromis spp.). Using this marker, we optimized a protocol to generate YY supermales for producing all-XY progeny. By feeding larvae with 17?-estradiol (E2, 120 mg/Kg in feed) from 5 to 30 days post-hatch (dph), 214 XY females were obtained. Crosses between 10 XY females and 10 XY males produced 231 YY males across 10 families. Subsequent matings of five YY males with five XX females yielded five all-XY populations, which exhibited an average body weight increase of 9.93% compared to mixed-sex groups and 29.30% compared to all-female groups. While in four XY populations, all fish were male, one XY family produced only 85.3% phenotypic males, suggesting variation in sex determination mechanisms and environmental effects. Furthermore, qRT-PCR and Crispr/Cas9-mediated knockout experiments confirmed amh as a major sex-determining gene in pink tilapia. Together, these findings establish a practical and efficient strategy for improving salt-tolerant pink tilapia aquaculture through the production of fast-growing, all-XY populations.