The culling of day-old male chicks is an animal welfare issue within the laying hen industry that raises substantial ethical concern. Alternative methods are sought to pre-select males during embryonic development. This method is called in ovo sexing and allows more humane male culling. A robust and non-invasive in ovo color sexing technique was developed and validated in this research. To this end, visible-near-infrared (vis-NIR) point spectroscopy was used, which has advantages over state-of-the-art hyperspectral imaging in terms of accuracy and cost. Two independent experiments were each conducted on a batch of 600 Isa Brown eggs. These eggs were individually illuminated on d 8 to 14, and d 18 of incubation by a halogen lamp and the signal was measured in the vis-NIR range from 300 to 1,145 nm. Next, optimal preprocessing strategies were applied and partial least squares discriminant analysis (PLS-DA) models were built and further optimized after performing a forward interval partial least squares (FiPLS) for variable selection. Results demonstrated that d 12 is too early for vis-NIR in ovo sexing, resulting in a prediction accuracy of 86.49%. However, after 13 d of incubation, an accuracy of 97.78% was obtained, increasing to 99.52% on d 14. Furthermore, these accuracies were higher than earlier reported percentages from hyperspectral imaging and successful sexing was expedited from d 14 to d 13. Moreover, prediction improvement up to 99.05% was obtained on d 13 by correcting for the variability in eggshell properties using d 0 eggshell corrections. Applying the method on d 18 resulted in a lower accuracy of 94.62% due to stronger light attenuation by the growing embryos. Finally, a reduced spectral range of 749 to 861 nm was found to be sufficient for correct classification of 98.46% of the eggs. This paves the way for high-throughput and cost-efficient usage of smaller and cheaper spectrophotometers in commercial hatcheries.
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