Extruded Limnospira platensis with enzyme supplementation shifts the broiler muscle proteome toward enhanced energy metabolism pathways.

Limnospira platensis (Spirulina) is known for its nutritional and bioactive profile, but its high-level dietary inclusion effects in broilers, particularly post-extrusion and enzyme pre-treatment, remain underexplored. We investigated how 15 %L. platensis raw (MA), extruded (MAE) or extruded with pancreatin (0.20 %) and lysozyme (0.01 %) (MAEM) affects the pectoralis major proteome of Ross 308 broilers. Birds were fed until day 35, then muscle proteins were prepared by filter-aided sample preparation, TMT 6-plex labelling and analysed by LC-MS/MS. Statistical analysis identified 26 differentially abundant proteins (p < 0.05, ≥ 2 peptides). MAEM samples clustered separately, showing increased abundance of NDUFA10 (Complex I; + 1.30-fold), COX5B (Complex IV; + 1.25-fold) and glycogen phosphorylase (+ 1.40-fold). Enrichment analysis revealed up-regulation of ATP synthesis, oxidative phosphorylation and glycogen catabolism in L. platensis groups, whereas control diets featured proteins linked to fatty-acid β-oxidation and myofibrillar assembly. These results indicate that extrusion combined with enzyme treatment enhances nutrient bioavailability and shifts the broiler muscle proteome toward energy-metabolism pathways without compromising performance. Data are available via ProteomeXchange (PXD064230). SIGNIFICANCE: The incorporation of Spirulina (Limnospira platensis) as a sustainable alternative to conventional feed ingredients is gaining attention in poultry nutrition. However, its specific effects on broiler's muscle proteome, as well as the impact of higher Spirulina inclusion levels on animal performance, remain largely unexplored. Herein, we investigated how extruded L. platensis, with and without enzyme supplementation, influences muscle protein expression. Results revealed a metabolic shift toward enhanced mitochondrial ATP production and glycogen metabolism when L. platensis was incorporated into the diets, suggesting improved energy efficiency and nutrient utilisation. These findings provide valuable insight into the biological mechanisms underlying muscle development in broilers fed microalgae-based diets. More broadly, they highlight the potential of combining pre-treated microalga ingredients as a strategy to optimise animal performance and health in the framework of poultry production.