Interactive effect of dietary formic acid and fenugreek on growth performance, immune response, and disease resistance in Nile tilapia challenged with Aeromonas hydrophila

This study investigated the interactive effects of dietary formic acid (FA) and fenugreek (FG) supplementation on growth performance, hematological and biochemical parameters, antioxidant status, innate immune response, and disease resistance in Nile tilapia ( Oreochromis niloticus ) challenged with Aeromonas hydrophila . A total of 240 juvenile fish (average initial body weight = 21.94 ± 0.04 g) were randomly allocated to four experimental groups following a 2×2 factorial design: control (no additive), FA alone (0.5%), FG alone (2%), and a combined FA+FG treatment. Fish received experimental diets for 60 days, after which all groups underwent a standardized bacterial challenge test. Significant improvements in growth were observed across all supplemented groups. Final body weight increased progressively across groups (33.92 g in controls, 43.71 g with FA alone, 47.61 g with FG alone, and 52.84 g with combined FA+FG supplementation; P < 0.001), with the combined treatment producing the greatest improvement. Similarly, the FA+FG treatment resulted in the highest specific growth rate (1.46 vs. 0.72%/day in controls) and the most favorable feed conversion ratio (1.37 vs. 1.80 in controls), with significant differences among treatments ( P < 0.001). Hematological indices (RBC, WBC, Hb, and PCV) and serum biochemical parameters (total protein, albumin, and globulin) were significantly elevated in the treatment groups relative to the control. Antioxidant enzyme activities (SOD and GPx) were markedly increased, while malondialdehyde (MDA) concentrations were significantly reduced, collectively indicating attenuated oxidative stress. Innate immune markers, including lysozyme activity and circulating cytokine levels, were significantly elevated in the supplemented groups. Furthermore, hepatic gene expression analysis revealed significant upregulation of pro-inflammatory cytokines ( IL-1β and TNF-α ) and the anti-inflammatory mediator TGF-β , suggesting improved immunomodulatory capacity. Following A. hydrophila challenge, fish fed the combined FA+FG diet exhibited the highest survival rate. These findings suggest that combined dietary supplementation with formic acid and fenugreek exerts synergistic effects on growth, physiological condition, antioxidant defense, and immune function, ultimately enhancing resistance to bacterial pathogens in Nile tilapia. The results support the potential of this dual-additive strategy as an effective, safe, and sustainable alternative to conventional antibiotic-based therapies in intensive aquaculture.