Preisinger, R. | Search bioRxiv

bioRxiv2025

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.

developmental biology↗

bioRxiv2025

Type I and type III interferon receptor knockout chickens: Novel models for unraveling interferon dynamics in influenza infection

The rapid cross-species transmission of highly pathogenic avian influenza presents a significant zoonotic threat. Elucidating the avian interferon (IFN) system, the primary antiviral defense in chickens, is critical for controlling the virus at its source and preventing its spillover into humans and other species. We engineered type I (IFN-/{beta}) and type III (IFN-{lambda}) IFN receptor knockout chickens to dissect the role of IFNs in viral infections. Results revealed that type I IFN predominantly modulates innate immune cell populations, T cell subsets, and their contribution to antibody production following immunization under physiological conditions. In ovo and in vivo challenge experiments utilizing diverse influenza A virus strains demonstrated strain-specific roles of both IFN-/{beta} and IFN-{lambda} in orchestrating viral pathogenesis, immunological responses, and tissue-tropism effects. Notably, type I IFN was particularly crucial in the initial defense mechanisms against H3N1 avian influenza A virus infection. These novel models offer unprecedented insights into avian IFN biology within the context of avian influenza, which is essential for developing more effective strategies to prevent and control this public health challenge.

immunology↗