Allantoic fluid-based qPCR for early-onset in ovo sexing

Abstract The culling of day-old male chickens remains an important welfare issue in the poultry industry. Several governments ( e.g. , Germany, France or Italy) have prohibited this practice, pushing the hatcheries to look for alternatives. Although different solutions exist for solving this problem, sex determination during embryo’s incubation (so called in ovo sexing) is considered the most suitable both for consumers and the industry since it aligns with the increasing demand for ethical and sustainable agriculture. However, to be applied in the market, in ovo sexing technologies have to meet a number of requirements, such as being compatible with all egg colors and early developmental stages, while maintaining 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 (present in both males and females) between incubation days 6 and 9. By utilizing quantitative polymerase chain reaction (qPCR) as analysis method and allantoic fluid (AF) as sample, our study confirmed specific detection of these genes in AF, remarkably already at day 6 of development. In a blind study performed with 80 eggs, we achieved a 95% accuracy rate in sorting embryos when using the HINTW gene alone and an outstanding 100% accuracy rate when using Δλ values (difference between the HINTW and DMRT-1 qPCR cycle threshold (Ct)). Importantly, the AF sampling procedure did not reveal any significant detrimental effects on hatchability or embryo development, revealing high potential for this understudied type of sample to be used. In conclusion, the developed assay can provide more in-depth information about AF as a sample for genomic in ovo sexing and open new industrial possibilities for developing faster and cheaper assays. Research highlights A highly reliable and accurate method for in ovo sexing in early incubation stages is established. Allantoic fluid can be easily extracted with minimal invasiveness while providing the necessary genomic material for sexual sorting. HINTW was found to be present in both female and male samples due to possible maternal contamination. HINTW alone or combined with DMRT-1 gene enables early-onset sexing with 100% accuracy.