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Abstract Mycoplasma hyopneumoniae, the primary pathogenic bacterium causing enzootic pneumonia, significantly affects worldwide swine production. The infection is usually persistent and bacterial identification and isolation of M. hyopneumoniae in clinical samples are challenging due to the fastidious requirements for its growth. Hence, new practical surveillance tools that improve or complement existing diagnostics on M. hyopneumoniae are desirable, especially in early infection. The objective of this study was to identify potential metabolite markers of early M. hyopneumoniae infection in pigs through metabolomics analysis. Samples obtained from pigs in a previous M. hyopneumoniae experimental infection were used in this study. Briefly, two pigs served as mock inoculated controls and ten pigs were intra-tracheally inoculated with M. hyopneumoniae. Sera, laryngeal swabs (LS), and tracheo-bronchial lavage fluid (TBLF) were collected from all pigs at 0, 2, 5, 9, 14, 21 and 28 days post-inoculation (dpi). Bronchial swabs (BS) were collected post-mortem at 28 dpi. Mycoplasma hyopneumoniae infection was confirmed by PCR in LS, TBLF and BS. Serum metabolites were profiled using high-resolution liquid chromatography–mass spectrometry (LC–MS) analysis. Metabolite markers were identified by structural analysis following multivariate analysis of LC–MS data. The results showed that M. hyopneumoniae infection time-dependently altered the serum levels of selective amino acids and fatty acids. α-Aminobutyric acid and long-chain fatty acids were markedly increased at 14 and 21 dpi in inoculated pigs (p |