The use of proper anesthesia in zebrafish research is essential to ensure fish welfare and data reliability. However, anesthesia long-term side effects remain poorly understood. The purpose of this study was to assess anesthesia quality and recovery in adult zebrafish using different anesthetic protocols and to determine possible long-term effects on the fish activity and anxiety-like behaviors after anesthesia. Mixed-sex adult AB zebrafish were randomly assigned to five different groups (Control, 175 mg/L of tricaine methanesulfonate [MS222], 45 mg/L of clove oil, 2 mg/L of etomidate, and 5 mg/L of propofol combined with 150 mg/L of lidocaine) and placed in the respective anesthetic bath. Time to lose the equilibrium, response to touch and to caudal fin pinch stimuli, and recovery after anesthesia administration were evaluated. In addition, after stopping anesthesia, respiratory rate, activity, and anxiety-like behaviors in the novel tank test were studied. Overall, all protocols proved to be adequate for zebrafish anesthesia research as they showed full recovery at 1 h, and only etomidate had minor effects on fish behavior in the novel tank, a validated test for anxiety.
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