Abstract
Background:
When fish are immunosuppressed by handling stress, water quality, parasitism, or population density, they develop Aeromonosis. Aeromonas hydrophila is a Gram-negative bacterium that is found in water and can infect fish, frogs, and mammals, including humans. A. hydrophila is a major pathogen in freshwater aquaculture, causing significant economic losses and raising concerns about antimicrobial resistance due to excessive antibiotic use. Coconut oil, rich in lauric acid, has potential as a natural immunomodulatory feed additive.

Aim:
This study aimed to investigate the antibacterial effects of coconut oil against A. hydrophila through in vitro assays, assess its impact on zebrafish mortality and gut microbiota in vivo, and explore changes in immune-related protein profiles using SDS-PAGE analysis.

Method:
A post-test-only control group design was applied, utilizing 225 zebrafish divided into five groups. Three experimental groups received coconut oil-enriched diets at doses of 1000, 2000, and 4000 mg/kg over a 60-day period, followed by immersion challenges with c (10⁸ CFU/mL). Antibacterial activity was evaluated using the disc diffusion method. Gut microbiota, including lactic acid bacteria, were isolated and identified on selective media, and protein expression profiling was conducted via SDS-PAGE.

Results:
Coconut oil did not demonstrate direct antibacterial effects against A. hydrophila up to 100% concentration. Nevertheless, zebrafish in groups T1 (1000 mg/kg) and T3 (4000 mg/kg) exhibited lower mortality rates compared to the positive control. Gut microbiota analyses identified A. hydrophila, Staphylococcus aureus, Pseudomonas aeruginosa, and Lactobacillus spp. SDS-PAGE revealed bands corresponding to proteins at approximately 85–90 kDa (likely HSPs), 27.2–38 kDa (outer membrane proteins), and 44–48 kDa (EGF-like proteins). Notably, T1 and T3 groups showed stronger EGF-like bands and a more stable gut microbiota profile, marked by elevated counts of Lactobacillus spp.

Conclusion:
Coconut oil lacks direct bactericidal activity against A. hydrophila but enhances host resistance by stabilizing gut microbiota and promoting the expression of defensive proteins such as HSP90 and EGF-like molecules. These findings highlight the potential of coconut oil as a non-antibiotic functional feed additive for sustainable aquaculture. Further large-scale and long-term studies are warranted to validate its efficacy.

Key words: Aeromonas hydrophila, Coconut oil, Defensive proteins, Immunomodulation, Gut microbiota