# In ovo sexing and genotyping using PCR techniques: a contribution to the 3R principles in chicken breeding

## TL;DR

This paper introduces a PCR-based method for determining the sex and genotype of chicken embryos in ovo, supporting ethical animal research by reducing the need to hatch surplus animals.

## Contribution

A reproducible PCR-based workflow for in ovo sexing and genotyping of chicken embryos from ED4 to ED10 is developed and validated.

## Key findings

- PCR methods achieved high success rates (70–100%) and accuracy (92–100%) across embryonic stages.
- ED7 was identified as the optimal time point for reliable sampling with high hatchability.
- The protocol supports the 3R principles by preventing hatching of surplus animals before ED13.

## Abstract

Early sex determination and genotyping of chicken embryos is crucial for ethical and resource-efficient animal research. We developed a reproducible workflow using whole genome amplification combined (WGA) with Kompetitive Allele Specific PCR (KASP) and standard endpoint PCR to perform in ovo sexing and genotyping from embryonic day 4 (ED4) to ED10. Across four study phases involving various chicken lines—including a genetically modified line, Araucana crossbreeds, and commercial layers (total n = 819)—we evaluated methodological feasibility, accuracy, and hatchability. Both PCR methods demonstrated high success rates in yielding results (70–100%) and high identification accuracy (92–100%) throughout all developmental stages tested. While the overall efficiency improved with embryonic age, ED7 was identified as the optimal time point to balance reliable sampling with high hatchability. We conclude that this laboratory-scale, PCR-based protocol provides an accessible and precise method for research laboratories to identify sex and genotype simultaneously. This approach directly supports the 3R principles by preventing the hatching of surplus animals before the assumed onset of nociception (ED13).

The online version contains supplementary material available at 10.1038/s41598-026-40562-y.