Abstract
Despite global efforts, minimizing the culling of one-day hatched male chicks remains a critical priority in the poultry industry due to significant socio-economic concerns. To address this issue, various molecular assays have been developed for in ovo sex determination, enabling the identification and elimination of male embryos at early developmental stages. However, due to their complexity associated with ensuring high precision, the requirement for advanced infrastructures and the time-intensive nature of current methods, these assays have yet to achieve widespread commercialization. In this study, we developed two novel digital readout assays employing PCR, LAMP and RPA techniques, which were evaluated for sensitivity, specificity and robustness using 82 nine-day-old chick embryos. Our data demonstrate that both newly developed PCR-based assays accurately and reliably determined the sex of all 82 chick embryos. Moreover, the LAMP- and RPA-based assays produced comparable results while offering the advantage of isothermal amplification, enabling detection through naked-eye colorimetric and/or fluorescence-based readouts. Notably, these assays operate within a shorter time frame, with LAMP completing amplification in 20 min at 65 °C and RPA in 30 min at 37 °C. These newly developed assays substantially simplify experimental settings while offering faster and more affordable sexing methods. By addressing critical challenges associated with in ovo sexing, they contribute to the advancement of non-invasive in ovo sexing techniques, facilitating their potential commercialization in the future.