Metabolomic Characterization of Breast Meat from Probiotic-Supplemented Laying Hens Using Untargeted GC-MS Analysis

Probiotic supplementation may influence muscle metabolism in poultry, but its effects on the breast muscle metabolome of laying hens remain insufficiently characterized. We investigated the effects of a probiotic mixture containing Lactobacillus acidophilus KUEN 1607 and Pediococcus acidilactici KUEN 1608 using untargeted gas chromatography–mass spectrometry (GC–MS). Breast muscle samples were initially obtained from 10 control and 10 probiotic-treated hens; after exclusion of one outlier, the final dataset comprised 19 samples and 143 putatively annotated metabolites. Exploratory partial least squares–discriminant analysis (PLS-DA) suggested partial group separation, although predictive performance was limited. In body weight-adjusted limma analysis of log2-transformed abundances, ten metabolites showed nominal differences (p < 0.05), all with lower estimated abundances in the probiotic group. Only N-acetyl-D-mannosamine remained significant after Benjamini–Hochberg correction (adjusted log2 fold change = −1.641; p = 0.000072; FDR = 0.0103). However, this FDR significance was not retained when the excluded sample was included in the sensitivity analysis. Exploratory KEGG analysis of VIP-selected metabolites highlighted three pathways with significant enrichment: arginine biosynthesis (FDR = 0.0113), the citrate cycle (FDR = 0.0218), and alanine, aspartate and glutamate metabolism (FDR = 0.0401). These pathway associations do not establish altered metabolic activity or flux. N-acetyl-D-mannosamine is a candidate finding requiring independent validation in larger cohorts using targeted quantitative metabolomics.