Allantoic fluid-based qPCR for early-onsetin ovosexing

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 calledin ovosexing) 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 ovosexing 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 genesHINTW(female-specific) andDMRT-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 theHINTWgene alone and an outstanding 100% accuracy rate when using Δλ values (difference between theHINTWandDMRT-1qPCR 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 genomicin ovosexing and open new industrial possibilities for developing faster and cheaper assays.Research highlightsA highly reliable and accurate method forin ovosexing in early incubation stages is established.Allantoic fluid can be easily extracted with minimal invasiveness while providing the necessary genomic material for sexual sorting.HINTWwas found to be present in both female and male samples due to possible maternal contamination.HINTWalone or combined withDMRT-1gene enables early-onset sexing with 100% accuracy.

Allantoic Fluid-Based qPCR for Early Onset In Ovo Sexing

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