Novel insights into eugenol anaesthesia in poeciliid larvae: Preliminary study of gill histology

The increasing use of fish in research emphasizes the need for effective and safe anaesthetic protocols, particularly for early life stages. Eugenol has been proposed as a promising anaesthetic, yet information regarding its effects on Cnesterodon decemmaculatus larvae remains scarce. This study assessed anaesthesia and recovery times, as well as gill tissue integrity, in larvae exposed to different eugenol concentrations. A randomized controlled trial was conducted using four treatment groups (6, 12.5, 25 and 50 mgL^-1) and a control group (10 mgL^-1 ethanol). Significant differences were observed in both anaesthesia and recovery times across treatments (p < 0.05). Morphometric analyses showed that branchial filament area was significantly smaller in all eugenol-treated groups compared to the control (p ≤ 0.02), and filament perimeter was also significantly reduced in all treatments (p < 0.001). Regarding necrosis, larvae exposed to 6 and 50 mgL^-1 exhibited significantly more necrotic foci than controls (p < 0.001). Necrosis was highest at 6 mgL^-1, decreased at 12.5 and 25 mgL^-1 (p < 0.01) and increased again at 50 mgL^-1 (p < 0.001). No significant differences were observed for oedema, congestion, lamellar fusion, epithelial lifting or vacuolization (p ≥ 0.08). However, oedema was higher at 6 mgL^-1 than at 12.5 mgL^-1 (p = 0.02), and congestion was lower at 12.5 mgL^-1 than 25 mgL^-1 (p = 0.04). Overall, the results indicate that 12.5 mgL^-1 eugenol provides effective anaesthesia with minimal histological alterations, representing the optimal concentration for use in C. decemmaculatus larvae.