Preventing chick culling in the poultry industry with a new biomarker for rapid in ovo gender screening

Chicken eggs are one of the most consumed foods worldwide. However, the practice of chicken culling in the poultry industry involves unnecessary animal suffering and finding a way to put an end to this has become a societal priority. One approach that has been propagated as acceptable is based on the selection of female eggs early in the incubation process and the devitalization of the male eggs. It is with this objective in mind that we searched for a biomarker for early gender screening in eggs. Applying an untargeted mass spectrometry approach, we profiled allantoic fluid of different day-old eggs and identified the feature 3-[(2-aminoethyl)sulfanyl]butanoic acid (ASBA) as a strong biomarker for in-ovo gender prediction for day-9 old embryos. In the present work, we describe the identification of ASBA as a new biomarker in allantoic fluid for gender screening and the optimization of a high throughput assay using acoustic droplet ejection-mass spectrometry (ADE-MS). Special attention is given to the optimization of ADE-MS compatible liquid handling and the development of the data processing to ensure a reliable gender prediction. We have been able to accurately determine the gender of day-9 eggs in a cohort of 154 samples with a prediction accuracy of 95.5%, with a throughput of 1800 samples per hour for the prototype, which may vary in production systems.