In Ovo Sexing and Genotyping using PCR techniques: A Contribution to the 3R Principles in Chicken Breeding

Early sex determination and genotyping of chicken embryos is crucial for ethical and resource-efficient animal research, addressing concerns about surplus animals. We developed a reproducible workflow using whole genome amplification combined with Kompetitive Allele Specific PCR (KASP) and standard endpoint PCR to perform in ovo sexing and genotyping from embryonic day four (ED4/96h) onwards. The overall efficiency improved with embryonic age. Both standard PCR and KASP provided high success for sexing and genotyping (70–100% of samples yielding a result) and accuracy (92–100%) across multiple chicken lines, including a genetically modified line. Optimal reliability and hatchability were achieved when sampling at ED7. These PCR-based techniques enable precise early identification of sex and genotype, allowing selective removal of unwanted embryonated eggs before the assumed onset of nociception. This approach supports more humane and efficient practices in chicken breeding and research, contributing to the replacement, reduction and refinement (3R) principles in animal experimentation.