Integrated Proteomics and Metabolomics Can Shed Light on Potential Biomarkers of Inflammation and Infection in Chicken Embryos as an Experimental Model

Salmonella Pullorum (SP) infection has been proposed as an experimental model of inflammation in late-stage chicken embryos (CEs). We refined this model by inoculating 9.5 day embryos via allantois with a low SP dose and evaluating outcomes at 24 and 48 h postinfection. This approach aims to provide a framework for systematically exploring early molecular events triggered by infection and inflammation while using the CE as an ethically acceptable and biologically relevant model of systemic inflammation. Clinical parameters (mortality and weight), serum proteomics, and untargeted metabolomics were assessed. Proteomics was performed by LC–MS/MS, followed by PANTHER and KEGG functional enrichment; metabolomics was performed to assess serum alterations. Mortality increased at 48 h postinfection (30%), and surviving embryos showed a trend toward reduced weight. Proteomic profiling revealed an increase in overall protein concentrations in the infected group compared to the control, despite high zero inflation, with α-2-macroglobulin significantly upregulated at 48 h, indicating an endogenous immune response. For pathway-level enrichment, Bayesian analysis suggested subtle effects on proteome dynamics. Metabolomics identified time-dependent changes, including increased levels of α-tocopherol-related and phenylalanine-derived metabolites and decreased levels of oxidized fatty acid derivatives at 48 h. These findings support the CE model before 12 days as an ethically refined and sensitive system for studying acute inflammatory responses.