Muscle hyperplasia and hypertrophy dynamics in rainbow trout ( <i>Oncorhynchus mykiss</i> ): impact of high plant-based protein and additive mixtures on muscle physiology

The effects of high plant-based proteins (PP) used as alternative protein sources in aquafeeds on muscle cellularity and myogenic factors of rainbow trout, <i>Oncorhynchus mykiss</i>, remain unclear. This study explored muscle fibre growth phases and the impact of two additive mixtures (A) in high-PP diets on muscle physiology. Over a seven-month trial, 2000 fish (2·22 g) were divided into four groups (five replicates each) and fed isonitrogenous (fry, 46 %; fingerling, 44 %; and grow-out, 42 % crude protein) and isolipidic (20 % lipid) diets: control (30 % fishmeal), PP, PP + A1 (krill meal, taurine, selenium) and PP + A2 (proline, hydroxyproline, vitamin C). Sampling for muscle histology and myogenic gene expression was conducted at ten sampling points from Day 0 to Day 214. Muscle histology (fibre distribution: small, 0-20 μm; small-medium, 20-60 μm; large-medium, 60-100 μm and large, ≥ 100 μm diameter) revealed four growth phases: hyperplasia (2·2-15 g), hypertrophy (15-50 g), hyperplasia (50-150 g) and hypertrophy (150-350 g). MyoD2 and myogenic regulatory factor 4 (MRF4) were upregulated during hyperplasia, while myostatin 1 (MSTN1)/myostatin 2 (2) and reduced Paired box 7 indicating growth inhibition and fewer satellite cells. The PP diet without additives altered fibre recruitment, while PP + A2 enhanced hypertrophy, increasing large (> 100 μm) fibres. Additive mixtures modulated myogenic gene expression, with PP + A2 promoting MyoD2, myogenin and MRF4 and reducing MEF2A/C, contrary to known hypertrophy markers. PP + A1 and PP + A2 diets reduced MSTN1 expression, potentially mitigating growth inhibition. Additive supplementation in PP diets alleviates negative impacts on muscle cellularity and myogenic regulation. The identified growth phases provide insights for precision nutrition, supporting improved feeding strategies for sustainable aquaculture.