Effects of plant extract formulations on anxiety-like behavior, sociability, and gut microbiota diversity in paracetamol-treated adult zebrafish

Plant extract-based formulations may influence behavior and gut microbiota, but their effects following acute paracetamol (PAR) exposure remain unclear. This preliminary study evaluated the behavioral effects of two formulations, coded MOD and TEL, and explored TEL-associated microbiota changes in adult zebrafish. Sixty fish were randomized to six groups (10/group). PAR exposure (5 mM for 24 h) was followed by formulation treatment (500 mg/L for five days), alongside untreated, PAR-only, and formulation-only controls. Behavior was assessed using the novel tank test (NTT) and social behavior test. Full-length 16S ribosomal ribonucleic acid (rRNA) gene sequencing was performed in untreated, PAR-only, and PAR-plus-TEL groups (three fish/group). Compared with PAR alone, MOD increased swimming distance and velocity during social testing (p = 0.028 and p = 0.003, respectively), whereas TEL reduced vertical crossings in the NTT (p = 0.035) and time spent near social stimuli (p = 0.0033). Reductions in freezing duration with both formulations were not statistically significant compared with PAR alone. No significant differences in alpha or beta diversity were detected, and no genus-level differences remained significant after multiple-testing correction. These exploratory findings suggest formulation- and test-dependent behavioral responses without statistically supported microbiota changes. Larger studies with replicated tanks and deeper sequencing are needed to clarify these associations.