Eye pigmentation-based in-ovo chicken sexing via precision breeding

The systematic culling of male layer chicks raises ethical concerns, leading to bans in Germany and other European countries and spurring the search for reliable in-ovo sexing methods. Most existing strategies rely on integration of exogenous DNA or are limited to specific chicken strains, and none meet commercial requirements. Here, we present a broadly applicable in-ovo sexing method that avoids exogenous DNA integration. We developed precision-bred chickens with targeted disruption of the Z-linked SLC45A2 gene, which encodes a transporter essential for pigmentation. Hemizygous knockout females (ZW; SLC45A2^KO/W) exhibited eye depigmentation at embryonic day 7 (E7), whereas heterozygous knockout males (ZZ; SLC45A2^KO/+) retained normal pigmentation. This clear visual dimorphism enables accurate sexing by routine egg candling. Fertility and reproductive performance of knockouts were comparable to wild-type chickens, and genotyping confirmed 100% prediction accuracy. Unlike many current technologies, our approach requires no complex instrumentation and allows early detection during incubation. This work provides a practical and ethical solution to chick sexing, with significant advantages for commercial hatcheries. More broadly, this study illustrates the potential of precision breeding to address pressing animal welfare concerns in the modern poultry industry.