Evaluation of the Effectiveness of Eugenol and MS-222 as Anesthetics in Zebrafish in Repeated Exposures and Post-Anesthesia Behaviour

The increasing use of the zebrafish (<i>Danio rerio</i>) in scientific experiments has made it necessary to implement anesthesia protocols guaranteeing minimum pain and suffering for these animals and ensuring the reliability of the results obtained from their research. Therefore, we aimed to compare the effectiveness of two anesthetics, eugenol and MS-222, in consecutive administrations and evaluate the zebrafish behaviour after repeated anesthesia. Thus, several zebrafish were anaesthetized with eugenol, MS-222, and buffered MS-222 three times repeatedly with a 24-h interval between each exposure. The induction and recovery periods were also timed. Their swimming frequency was determined after each exposure to assess their behaviour after the anesthesia. Anesthesia induction was quicker with eugenol compared to MS-222. However, eugenol presented longer recovery times, which were prolonged after each exposure. Also, the swimming frequency was reduced after each anesthesia with eugenol. The buffered version of MS-222 was more efficacious than the non-buffered one. Both versions of MS-222 did not affect the swimming frequency. Based on these findings, we recommend the utilization of MS-222 buffered rather than eugenol when repeated, brief-duration anesthesia is necessitated for a study.