Introduction
Nile tilapia (Oreochromis niloticus), a globally significant aquaculture species, plays a critical role in addressing food security due to its high nutritional value and adaptability to diverse farming environments (Shafiujjaman et al., 2024). However, the intensification of aquaculture practices has introduced stressors that adversely affect fish health and increase vulnerability to diseases (Li et al., 2022). Conventional solutions like antibiotics and chemical therapeutics, while effective, raise concerns about environmental degradation, drug resistance, and food safety. Functional feed additives, particularly those derived from natural sources, have emerged as promising alternatives for enhancing fish health and immune function. White button mushrooms (Agaricus bisporus), recognized for their rich bioactive compound profile—including polysaccharides, phenolics, and antioxidants—have shown potential in modulating immune responses and oxidative stress (Habib et al., 2021). Despite the demonstrated benefits of mushrooms in aquaculture, the molecular mechanisms underlying their effects, particularly on cytokine-mediated immune responses, remain underexplored. This study evaluates the impact of dietary supplementation with A. bisporus powder on antioxidant activity, immune parameters, and cytokine gene expression in Nile tilapia, providing valuable insights into its potential as a sustainable aquafeed additive (Habib et al., 2021; Harikrishnan et al., 2018).
Methodology
White button mushrooms were sourced locally, cleaned, sliced, dried at 50°C for 24 hours, and ground into powder. Experimental diets were formulated to include 0%, 0.5%, 1%, and 2% mushroom powder (Amiri et al., 2018; Hoseinifar et al., 2019). Diets were adjusted to maintain iso-nitrogenous and iso-caloric properties and pelletized into uniform 2 mm pellets for feeding. A total of 240 juvenile Nile tilapia, weighing approximately 3.1±0.3 g, were distributed randomly into 12 aquaria (250 liters) in a completely randomized design. Each treatment was replicated thrice, with 20 fish per aquarium. The fish were fed their respective diets to apparent satiation four times daily over 56 days. Water quality parameters, including temperature (28.01±0.10°C), pH (7.13±0.20), and dissolved oxygen (5.23±0.18 mg/L), were maintained within optimal ranges. At the conclusion of the feeding trial, six fish per treatment group were anesthetized using 0.3 mL/L of 2-phenoxyethanol. Blood samples were collected via caudal vein puncture for serum isolation. Immune tissues, specifically the head kidney and spleen, were aseptically excised and stored in RNA later solution for subsequent molecular analysis. Antioxidant enzyme activities, including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx), as well as total antioxidant capacity (T-AOC), were quantified using commercial kits. Lipid peroxidation was assessed by measuring serum malondialdehyde (MDA) levels. Immune parameters, including serum bactericidal activity, immunoglobulin M (IgM) concentrations, and lysozyme levels, were evaluated using standard methodologies. RNA was extracted from head kidney and spleen tissues and reverse-transcribed into cDNA. Quantitative PCR (qPCR) was used to measure the expression of immune-related cytokines, including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β). Relative expression levels were normalized to β-actin, and fold changes were calculated using the 2−ΔΔCt method. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test for pairwise comparisons. Results were presented as mean ± standard deviation, with statistical significance set at p<0.05.
Results
Dietary supplementation with mushroom powder significantly enhanced antioxidant enzyme activities, with the 1% and 2% treatments showing the greatest increases in CAT, SOD, and GPX activities compared to the control group (p<0.05). Total antioxidant capacity (T-AOC) was similarly elevated in these groups, while serum MDA levels, indicative of lipid peroxidation, were markedly reduced. These results underscore the potent antioxidant properties of A. bisporus, attributed to its phenolic and flavonoid compounds, which act as radical scavengers and reduce oxidative stress. Comparable studies have demonstrated the role of mushroom-derived antioxidants in enhancing oxidative resilience in fish, including common carp and rainbow trout. Mushroom supplementation significantly increased serum bactericidal activity, IgM concentrations, and lysozyme levels. The 1% and 2% inclusion levels elicited the strongest responses (p<0.05), reflecting improved non-specific immune defenses. Polysaccharides such as beta-glucans, abundant in mushrooms, are known to activate macrophages and stimulate the release of immune mediators, enhancing humoral immunity. The observed immune improvements align with previous findings in aquaculture species, where mushroom-based diets augmented pathogen resistance and overall health. The expression of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in head kidney and spleen tissues was significantly upregulated in fish fed mushroom-enriched diets, particularly at the 1% and 2% inclusion levels (p<0.05). Cytokines play a pivotal role in coordinating immune responses, mediating inflammation, and facilitating pathogen clearance. The enhanced expression observed in this study likely results from mushroom-derived polysaccharides interacting with toll-like receptors on immune cells, triggering downstream signaling pathways. These findings corroborate previous studies that reported similar cytokine upregulation in fish fed functional diets containing prebiotics or plant-derived compounds. The dual antioxidant and immunomodulatory effects of A. bisporus are attributed to its diverse bioactive compounds, including phenolics, flavonoids, and beta-glucans. These molecules neutralize reactive oxygen species (ROS), enhance the activity of antioxidant enzymes, and stimulate immune responses. Additionally, mushrooms may influence gut microbiota composition, indirectly bolstering immune function. While this study provides compelling evidence of the benefits of A. bisporus, further research is warranted to isolate specific bioactive compounds and elucidate their precise mechanisms of action. The findings of this study highlight the potential of white button mushroom powder as a sustainable alternative to antibiotics and chemical treatments in aquaculture. By enhancing both antioxidant defenses and immune responses, mushroom supplementation can improve fish health, reduce disease incidence, and mitigate environmental risks associated with conventional therapeutics. Functional diets incorporating natural additives like A. bisporus align with the goals of sustainable aquaculture, promoting health and productivity without compromising ecological integrity.
Discussion and conclusion
This study demonstrates the efficacy of dietary supplementation with white button mushroom powder in enhancing the antioxidant and immune systems of Nile tilapia. The 1% and 2% inclusion levels yielded the most significant improvements in antioxidant enzyme activities (CAT, SOD, GPX), total antioxidant capacity (T-AOC), and reductions in serum MDA levels. Immune parameters, including bactericidal activity, IgM concentrations, and lysozyme levels, were also significantly enhanced. Furthermore, mushroom supplementation upregulated the expression of cytokines IL-6, TNF-α, and IL-1β, indicating robust immunomodulatory effects. These findings underscore the potential of A. bisporus as a functional feed additive, offering a natural and sustainable approach to improving fish health and reducing reliance on chemical therapeutics in aquaculture. Future research should focus on identifying and characterizing the specific bioactive compounds responsible for these effects and optimizing their application in aquafeeds.
Conflict of interest
We wish to confirm that there are no known conflicts of interest associated with this study.
Acknowledgment
The authors are grateful to the Fisheries Lab at Science and Research Branch Institute and the Aquatics Production & Trade Union of Iran for their kind cooperation.