Sexing domestic chicken before hatch: A new method for in ovo gender identification

Male chicks are an unwanted by-product when producing laying hens. The common practice to kill them directly after they have hatched gives rise to ethical concerns worldwide. The aim of this study was to develop an endocrine method to determine the sex of domestic chicken before hatch. On Days 7 to 10 of incubation, the allantoic fluid from brown layers' eggs (n = 750) was analyzed via enzyme immunoassay for their content of estradiol, estrone sulfate, and testosterone in order to detect gender differences. We successfully established a reliable method for in ovo sex identification on Day 9 of incubation by estrone sulfate measurement in the allantoic fluid. Female embryos displayed significantly higher hormone levels in the allantoic fluid than males (female: median = 0.312 ng/mL; male: median = 0.110 ng/mL; P ≤ 0.001). Our method allows the sexing of domestic chicken at a very early stage of embryonic development, even before the onset of pain perception. The possibility to eliminate eggs containing male embryos on Day 9 of incubation represents a vast improvement compared with culling day-old chicks.