Effect of Bacillus subtilis probiotics (live vs. pressure-killed) on growth performance, immune function, and biochemical indices in the rainbow trout, Oncorhynchus mykiss

The effects of different dietary probiotics, Bacillus subtilis as live and killed (paraprobiotic) and their combination, were assessed in the current research on the growth, immunity, antioxidant system, and some serum biochemical parameters in rainbow trout, Oncorhynchus mykiss. The fish were fed for 60 days across four treatment groups, each in three replicates. The diets included: 1) control diet: basal commercial diet, 2) probiotic (PRO): diet containing 1.5 × 10⁶ CFU/g live Bacillus subtilis, 3) PAR: diet containing 1.5 × 10⁶ CFU/g pressure-killed Bacillus subtilis (paraprobiotic or PAR) and 4) a combination of PRO + PAR. The growth and biochemical parameters were analyzed on days 30 and 60 of the study. Based on the findings, use of PRO and PAR in combination significantly improved growth performance (P<0.01). The combination of PRO + PAR significantly increased lysozyme and alternative complement (ACH₅₀) activities, as well as immunoglobulin (IgM) level, compared to the control group (P<0.01). The PRO + PAR group demonstrated the highest immune responses at both day 30 and day 60, indicating a synergistic effect on immune enhancement. The expression of immune-related genes (CD4, IL-β, MHC-I) was up-regulated in kidney of the treated fish, with the highest expression in PRO + PAR group (P<0.01). PAR and PRO, either separately or in combination, enhanced the activity of antioxidant enzymes (catalase, superoxide dismutase, gluthatione peroxidase) and total antioxidant capacity (TAC) levels (P<0.01). The group receiving both PRO and PAR exhibited the highest enzyme activities and TAC levels (P<0.01). Diets supplemented with PAR, PRO, and PRO + PAR induced an increase in serum albumin (ALB) and total protein (TP) levels (P<0.01). In contrast, a notable reduction was seen in triglyceride (TG), glucose (GLU), aspartate aminotransferase (AST), alanine transaminase (ALT) and creatinine (CR), with the highest decrease in PRO + PAR treatment (P<0.01). The intestinal Bacillus spp. count (BAC) exhibited an increase following PRO and PAR supplementation in the diet (P<0.01). Almost all growth, immune, and biochemical parameters were significantly higher at day 60 compared to day 30 (P<0.01). The results of this study highlight the positive effects of both live and killed Bacillus subtilis on growth, immune function, and overall health of rainbow trout. The data also indicate that the most favorable outcomes were achieved when both supplements were administered together, demonstrating their combined benefits.