Dose-Dependent Effects of Branched-Chain Amino Acid Supplementation on Skeletal Muscle Morphology and Ultrastructure in Exercise-Trained Mice

Background: Branched-chain amino acids (BCAAs) regulate muscle protein metabolism, yet the systematic characterization of their dose-dependent morphological effects on exercised skeletal muscle remains limited. This study investigated the dose–response relationship between BCAA supplementation and skeletal muscle adaptations in exercise-trained mice. Methods: Seventy male Kunming mice were randomly assigned to seven groups (n = 10): a background group (no exercise), a control group (exercise + saline), and five exercise groups receiving BCAAs at 1–5 g/kg/day via intragastric gavage. Mice in the exercise groups performed 45 min of swimming daily (6 days/week) for 50 days. Gastrocnemius muscles were processed using hematoxylin–eosin staining, Masson trichrome staining, Gomori aldehyde fuchsin staining, and transmission electron microscopy. Data were analyzed using one-way ANOVA with Dunnett’s post hoc test. Results: BCAA supplementation increased gastrocnemius wet weight-to-body weight ratios and promoted denser fiber packing in a dose-dependent manner up to 3–4 g/kg/day. Deep-staining fiber proportion (putatively type II-like) increased progressively with BCAA concentration, plateauing at doses ≥ 3 g/kg/day, while elastic fiber content continued to rise through 5 g/kg/day. Mitochondrial size decreased as mitochondrial number increased; membrane and cristae thickness peaked at 3 g/kg/day. Sarcomere length, myofibril diameter, sarcoplasmic reticulum size, and transverse tubule diameter exhibited increasing trends. Conclusions: These findings establish a parameter-specific dose–response framework for BCAA-induced muscle remodeling. A supplemental dose of 3 g/kg/day above background dietary intake represents an effective threshold for maximizing indices of hypertrophic gains and mitochondrial structural maturation potentially indicative of functional enhancement. Higher doses (≥4 g/kg/day) elicited additional benefits in fiber density, mitochondrial proliferation, and elastic fiber content. Supplemental BCAA dosing strategies above constant background intake should be tailored to target specific structural outcomes, with functional validation required to confirm physiological relevance.