48 The impact of leucine supplementation in milk replacer on muscle proteome and metabolism in neonatal dairy calves

Abstract Abstract: Introduction Early postnatal nutrition plays a critical role in regulating muscle growth and metabolism. Leucine, a branched-chain amino acid, activates the mTOR signaling pathway and promotes protein synthesis, yet its specific effects on the muscle proteome in neonatal dairy calves remain unclear. Objective To investigate the influence of leucine supplementation in milk replacer (MR) in muscle proteomic profiles at day 28 of treatment. Methods Thirty-five newborn Holstein heifer calves were randomly assigned to one of three treatment groups: Control (no added amino acids), Leucine-supplemented (5% in MR), and Alanine-supplemented (isonitrogenous to Leucine). Calves received equal MR volumes for 56 days. Muscle biopsies collected on day 28 underwent proteomic analysis using Data-Independent Acquisition (DIA) via Orbitrap Exploris 480, with protein quantification performed using Spectronaut. Protein–protein interaction (PPI) networks and Gene Ontology (GO) enrichment were analyzed with STRING v12.5. Results In the Leucine vs. Control group, 25 proteins were upregulated (P ≤ 0.05), and 15 were upregulated in Leucine vs. Alanine, with enrichment in amino acid and nucleotide metabolism, suggesting enhanced protein synthesis and cell proliferation (FDR ≤ 0.05). Steroid hormone response proteins (dehydroepiandrosterone, estradiol, progesterone) and caseins were enriched in both comparisons (FDR ≤ 0.05), implying a role for hormonal regulation in muscle development. In the leucine-supplemented groups, proteins associated with catabolic processes were downregulated (FDR ≤ 0.05), suggesting a shift towards anabolic metabolism. Conclusion Leucine supplementation promoted an anabolic muscle environment, enhancing proteome remodeling and metabolic efficiency. (Supported by SBARE)