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)