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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.

Chicken meat and eggs are important sources of food for the world population. The significant increase in food demand has pushed the food industry toward a rapid non-expensive production which in turn raises ethical issues. How chicken are cultivated and processed in food industry is no longer acceptable. Ethical and economical concerns emerging from chicken culling need to be solved in the near future. Indeed, in egg production industry, male chicken are killed at the age of 1-day post-hatching since they are not egg producers. A number of laboratory all over the world are looking for innovative non-invasive sexing methods to determine the sex of chicken in the early stages of the development before hatching. It will allow males' chicken elimination before the pain-feeling stages. In order to evaluate the efficiency of these methods, the scientific community need a reliable, easy to use and cost-effective in-ovo invasive sexing method. In this report, we developed two new invasive assays based on PCR and Q-PCR techniques respectively, which fulfil the above mentioned requirements. In the same line with other groups, we exploited the differences betweed males (ZZ) and females (ZW) chicken sexual chromosomes. We identified two genes, SWIM and Xho-I, on chromosome W and DMRT gene on chromosome Z allowing a clear discrimination between the two sexes using PCR and qPCR respectively. These two new genomic markers and their corresponding methods not only increase the accuracy but also reduce time and cost of the test compared to previously developed sexing methods. Depending on the technology available in the lab, one can choose between the two techniques requiring different machines and expertise.