Nano-formulated humic acid modulates growth, antioxidant status, and intestinal architecture in Nile tilapia (Oreochromis niloticus)

The growing demand for sustainable aquafeeds has intensified the search for eco-friendly alternatives to synthetic antibiotics. This study evaluated conventional and nano-formulated humic acid (HA) as promising feed supplements for Nile tilapia ( Oreochromis niloticus ). Six isonitrogenous diets were formulated: control, bulk HA (H4; 4 g kg⁻ 1 ), two nano-HA supplemented diets (NH50/NH100; 50/100 mg kg⁻ 1 ), and two chitosan-loaded nano-HA supplemented diets (C-NH50/C-NH100; 50/100 mg kg⁻ 1 ). After a 60-day feeding trial, growth performance, hepatic biomarkers, immune and growth gene expression, intestinal histomorphology, antioxidant status, and digestive enzyme activities were assessed. At the end of the experiment fish were stressed by Aeromonas hydrophila injection. The results indicated that nano-HA supplementation, particularly C-NH100 significantly enhancing feed utilization efficiency ( P < 0.05 ). ALT and AST activities increased progressively across supplemented groups, with C-NH100 exhibiting the highest values. SOD and CAT activities were maximized in C-NH100 (5.04 & 3.76, respectively). In contrast, MDA levels were significantly reduced in all supplemented groups relative to control, attaining the lowest value in C-NH100 (4.62). Moreover, intestinal digestive enzymes were significantly modulated by HA supplementation. Intestinal histomorphology examination revealed that C-NH100 dietary supplementation increased the villus height by 46%, villus width by 138%, epithelial thickness by 221%, and goblet cell density by 157% relative to the control. Immune-related genes (IL8, CXC2, CC chemokines, and IgM) and growth hormone (GH) expression were markedly upregulated in C-NH100, whereas IGF-I expression exhibited a treatment-dependent response, increasing in H4 and NH100 but decreasing in NH50, C-NH50, and C-NH100 while NH100 showed comparatively lower immune expression. In contrast, the antioxidant-related gene GST showed the highest expression in C-NH100 and the lowest expression in C-NH50. Following bacterial challenge, supplemented groups descriptively exhibited lower cumulative mortality (~ 6.7% vs. 73.3% in control), consistent with a treatment-level trend toward enhanced physiological resilience. Collectively, chitosan-loaded nano-HA at 100 mg kg⁻ 1 provided improvement in feed utilization, intestinal morphology, antioxidant status, immune responses, and physiological stability under pathogenic challenge. These findings indicate that nano-HA formulations exert treatment-specific effects on growth- and immune-related molecular pathways. Collectively, these findings suggest the potential of chitosan-loaded nano-HA at 100 mg kg⁻ 1 as a sustainable functional feed additive to support health management in tilapia aquaculture.