Allantoic Fluid-Based qPCR for Early Onset In Ovo Sexing

The day-old male chick culling remains a welfare issue in the poultry industry. Several governments have prohibited this practice, pushing hatcheries to seek alternatives. Although different solutions exist for solving this problem, sex determination during the embryo's incubation (in ovo sexing) is considered the most suitable one among the consumers and industry. However, to be industrialized, in ovo sexing technologies must meet several requirements: compatibility with all egg colors and early developmental stages while maintaining a high hatchability rate and accuracy at low cost and high throughput. To meet these requirements, we studied the use of the sexual genes HINTW (female-specific) and DMRT-1 (both sexes) at incubation days 6-9. By utilizing the quantitative polymerase chain reaction in allantoic fluid (AF) samples, our study confirmed female-specific HINTW detection on all days without any significant detrimental effects on embryo development. We achieved 95% sexing accuracy using the HINTW cycle threshold (Ct) alone and 100% accuracy rate when using Delta lambda values (difference between the HINTW and DMRT-1 Ct). In conclusion, the developed assay can provide information about AF as a sample for in ovo sexing and open new industrial possibilities for faster and cheaper assays.