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The culling of male day-old chicks from layer strains is an ethically questionable procedure and therefore requires the exploration of alternatives. We previously described a method for endocrine in ovo-sexing of domestic chicken via analysis of the allantoic fluid. In this respect sex identification takes place before the onset of embryonic pain perception. Aim of the present study was the evaluation of said technique on a larger scale in a commercial hatchery. To examine possible influences of the procedure on chicken health and production parameters, rearing and performance data of hatched female chicken were also analysed. The trial was performed with fertile eggs from the layer strains Lohmann Brown (LB) and Lohmann Selected Leghorn (LSL). Allantoic fluid was withdrawn on day 8 + 4 h or 9 + 4 h of incubation and analysed via a competitive enzyme-linked immunosorbent assay measuring the oestrone sulphate concentration. Subsequently, the embryonic gender was assigned using a statistically established cut off value. Sexing accuracy averaged at 84% (day 8 + 4 h) and increased to 98% (LB) and 100% (LSL) for day 9 4 h old embryos. Compared to an untreated control group, the hatching rate of the experimental group was reduced by 1.4 to 3.5 points of percentage (LB) and 12.7 points of percentage (LSL) due to sampling of allantoic fluid. Hatching weight of the day-old chicks did not differ between both groups. Monitoring of the post hatching performance showed that sampling of the allantoic fluid has only negligible effects on the hens. Although weight distinctions between control and experimental group were observed during the rearing period, the adult hens' laying performance, egg weight and body live weight did not differ significantly between the groups. These results demonstrate that the described method for in ovo-sexing is a potential and ethically justifiable alternative to the culling of day-old male layer chicks. The basic requirements for a practical implementation are fulfilled: The technique allows an accurate and reliable sexing and causes no reduction in the production performance of adult hens. The reduction of the hatching rate due to the sampling of allantoic fluid is an important factor for additional research. Further investigations are necessary to develop technical devices for a cost-effective implementation of this method in commercial hatcheries. [ABSTRACT FROM AUTHOR]