This study was conducted in two phases: first, to assess the impact of microbial fermentation on enhancing the nutritional quality of fish by-products, and second, to evaluate the effects of replacing fishmeal with these fermented by-products in the diet of Nile tilapia (Oreochromis niloticus) on growth performance, blood parameters, antioxidant indices, immunity, digestive enzyme activity, and carcass composition. In the initial phase, proteolytic activity of five bacterial strains including Bacillus subtilis (ATCC: 6051), B. licheniformis (IBRCM: 10204), Lactiplantibacillus plantarum (PTCCs: 1058 and 1745), and Lactobacillus casei (PTCC: 1608) was evaluated using growth assays in skimmed milk culture media and analyzed using Image-J software. B. licheniformis exhibited the highest proteolytic activity and was selected for fermentation. Resulting hydrolyzed proteins were characterized by peptides with molecular weights below 11 kDa. In the second phase, fishmeal was replaced with fermented by-products at five levels (0 (control), 25, 50, 75, and 100%). Two hundred ten Nile tilapia with an average weight of 2.83 ± 0.05 g were stocked in fifteen 200 L plastic tanks at three replicates, with 14 fish per tank, and fed daily at a rate of 7% of their body weight for 63 days. With increasing levels of fishmeal replacement (25% to 75%), significant improvements (p < 0.05) were observed in final weight gain, body weight gain, specific growth rate, protein production value, and protein efficiency ratio. Additionally, blood plasma concentrations of hormones T3 and T4, immunoglobulin level, the activities of complement (ACH50), and antioxidant enzymes (catalase and superoxide dismutase) increased significantly in fish fed the diets with fermented by-products compared to those of the control diet (p < 0.05). The optimal replacement levels for specific growth rate and feed conversion ratio were identified as 86.28% and 83.91%, respectively.

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