Integrated metabolomic and gut microbiota analyses reveal TCA cycle dysfunction in broiler skeletal disorders and the protective effect of L‑malic acid.

Background: Skeletal disorders pose a significant challenge to intensive broiler production. However, the metabolic mechanisms underlying the development of skeletal abnormalities in broilers remain unclear. This study aims to reveal the underlying mechanisms through multi-omics analyses and evaluate the ameliorative effects of exogenous L-malic acid supplementation.

Results: The abnormal group exhibited reduced serum concentrations of calcium and phosphorus, as well as decreased bone calcium and phosphorus content. These changes were accompanied by deterioration in bone geometric properties and microstructural parameters. The abnormal group exhibited downregulated expression of osteogenesis-related genes, whereas the mRNA expression levels of osteoclast-related genes were significantly higher than those observed in the normal group (P < 0.05). Serum metabolomics revealed that the differentially enriched pathways were primarily associated with energy metabolism, particularly the tricarboxylic acid cycle (TCA cycle). Concurrently, the mRNA expression levels of genes encoding key glycolytic enzymes and key complexes of the electron transport chain were elevated in the abnormal group. The analysis of the gut microbiota revealed an increased abundance of harmful bacteria and a significant decrease in microbes associated with energy metabolism in the abnormal group (P < 0.05). Targeted analysis further identified TCA cycle intermediates, fumarate and malate, as robust biomarkers for discriminating skeletal health status. Early dietary supplementation with L-malic acid improved bone health in broilers by increasing TCA activity, as evidenced by enhanced bone mineralization, improved bone microstructural parameters, and an increased OPG/RANKL ratio.

Conclusions: Skeletal abnormalities in broilers are closely associated with gut microbiota dysbiosis accompanied by TCA cycle metabolic disruption. Exogenous L-malic acid supplementation improved bone health by enhancing TCA cycle-related activity, supporting a functional role of TCA cycle-related metabolism in skeletal development.