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This study was conducted to evaluate the effects of dietary probiotic supplementation on growth performance, fish welfare, sensory evaluation, freshness during ice storage and changes in intestinal microbiota of rainbow trout (Oncorhynchus mykiss). The 9‐week feeding trial was conducted in cages under commercial conditions during the winter season, when water temperature decreased from 14°C to 7°C. Fish were fed either a diet without probiotic supplementation (control) or with 0.2% (2 × 109 CFU kg−1 diet) of multi‐strain probiotic bacteria (Bacillus sp., Pediococcus sp., Enterococcus sp., Lactobacillus sp.). Fish weight was recorded at 0, 2, 4, 6 and 9 weeks. Growth performance was not affected by dietary probiotic supplementation. Alternative complement activity (ACH50) was significantly higher in fish fed the diet with probiotic (58.4 ± 3.7 units mL−1) than in the control group (46.9 ± 3.1 units mL−1) (P = 0.03). Several biochemical markers were altered by the dietary treatment. 7‐ethoxyresorufin‐O‐deethylase (EROD) (P < 0.01), glutathione S‐transferase (GST) (P < 0.001), glutathione peroxidase (GPx) (P < 0.05) increased and lipid peroxidation (LP) (P < 0.01) decreased with dietary probiotic supplementation. Flesh quality and shelf‐life of fish stored on ice was not affected by dietary probiotic supplementation. Probiotic supplementation had a limited effect on the intestinal microbiota. Signal strength of the observed operational taxonomic units (OTUs) differed between the control and treatment samples and could indicate variations in the abundance of certain OTUs. Microbiota adhering to the intestinal mucus varied between sampling points, indicating that changes were associated with water temperature.

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