LAUSR

Simple Summary The increased popularity of zebrafish as a research model calls for appropriate refinement of procedures, such as finding the best anaesthetic protocol to use in adult zebrafish in a laboratory setting. The scarce literature available regarding aversion described the most used anaesthetic (MS222) as aversive to adult zebrafish; therefore, the study of alternatives is urgent. Thus, we studied the impact of three anaesthetic alternatives (a combination of propofol with lidocaine, clove oil, and etomidate), and the standard anaesthetic (MS222) on zebrafish aversion and on cortisol levels. Contrary to what was expected, MS222 did not result in clear signs of aversion, while only etomidate generated a similar profile to the aversive substance (hydrochloric acid). Our results suggested that all the anaesthetic protocols except for etomidate were valid candidates for use in a laboratory setting, although none were innocuous. Abstract The use of zebrafish (Danio rerio) as an animal model is growing and occurs in a wide range of scientific areas. Therefore, researchers need better and more appropriate anaesthetics for stressful and/or painful procedures to prevent unpleasant experiences. Thus, we aimed to study if adult zebrafish displayed aversion-associated behaviours (conditioned place aversion) and alterations in cortisol levels when exposed to equipotent concentrations of MS222, propofol/lidocaine, clove oil, or etomidate. Adult AB zebrafish (mixed-sex, N = 177) were randomly assigned to MS222 (150 mg/L), Propofol/Lidocaine (5 mg/L propofol + 150 mg/L lidocaine), Clove Oil (45 mg/L), or Etomidate (2 mg/L) groups. The conditioned place aversion test was used to assess behavioural aversion. Only etomidate resulted in a similar aversion to the positive control group (HCl; pH = 3). Cortisol levels were measured 5 and 15 min after loss of equilibrium. Etomidate induced low levels of cortisol by impairing its synthesis, whereas all the other groups had similar cortisol levels. Based on our data, etomidate was ruled out as an alternative to MS222, as it showed an aversive profile. The remaining protocols were not innocuous, displaying a weak aversive profile when compared to the positive control. In conclusion, a combination of propofol with lidocaine, clove oil, and MS222 were valid candidates for use as anaesthetic protocols.