Effects of probiotic Bacillus subtilis and B. licheniformis on water quality, growth, physiology, gene expression, and disease resistance in Sparus aurata fingerlings

This study evaluated the effects of a probiotic blend of Bacillus subtilis and Bacillus licheniformis , administered as a water additive, on the growth performance, feed efficiency, body composition, blood biochemistry, histology, gene expression, and resistance to Vibrio parahaemolyticus infection in Sparus aurata fingerlings. A total of 240 healthy fingerlings (6.10 ± 0.06 g) were distributed into 12 tanks (3 tanks per group), with 20 fish per tank. Over a period of 10 weeks, the fish were exposed to four treatments with increasing probiotic concentrations (0, 0.01, 0.02, and 0.03 g/m³), designated as Control, BSL1, BSL2, and BSL3, respectively. The water additives of BSL significantly increased the dissolved oxygen (mg/L) in a dose-dependent manner, while the values of TAN were significantly reduced by increasing the levels of BSL in the water. The NH 3 levels were the lowest in BSL2 and BSL3 compared to other groups; however, BSL1 was lower than the control group. The BSL3 group exhibited higher growth performance (final body weight, BWG, survival rate) compared to the other groups (P < 0.05). Adding BSL significantly improved the crude protein and ash content in S. aurata, while it significantly reduced the lipid content (P<0.05). BSL also significantly improved blood hematology parameters (PCV, RBCs, and Hb) and immune responses (phagocytic activity, phagocytic index, lysozyme activity, IgM, total Ig, and WBCs) in a dose-dependent manner (P<0.05) compared to the control group. Blood biochemical parameters (Total protein, albumin, globulin, and glucose), digestive enzymes (amylase and lipase) and antioxidant status (TAC, SOD, CAT) were significantly increased in a dose-dependent manner with increasing levels of probiotic in the water (P<0.05). Liver enzymes and MDA were significantly decreased by BSL-water addition (P<0.05). BSL enhanced the intestinal structure integrity of Sparus aurata . The addition of B. subtilis and B. licheniformis probiotics significantly improved the growth factors (IGF-1, IGF-2, and GHR) and immune-related genes (TNF-α, IL-1β, and IL-10) compared to the control group (P<0.05) in a dose-dependent manner. Importantly, probiotic-treated fish exhibited increased resistance to V. parahaemolyticus infection. These findings suggest that water addition of Bacillus subtilis and Bacillus licheniformis probiotics at a concentration of 0.2-0.3g/m3 improved the growth and overall health of Sparus aurata by regulating the immune responses and antioxidant status.