Comparative study of tricaine methanesulfonate (MS-222) and eugenol as euthanasia agents in zebrafish (Danio rerio) as an experimental model

In experimental procedures inevitably leading to the sacrifice of animals, suitable measures should be taken to minimise their pain and suffering as much as possible, as well as to prevent any modification or masking in the experimental results obtained. An overdose of anaesthetic is the method of euthanasia most employed in fish, since it is effective and easy to apply. Our objectives were to compare the efficacy of eugenol and of tricaine methanesulfonate (MS-222) as euthanasia agents in zebrafish, and to make a histological evaluation of the possible effects derived from their application. The concentrations established for eugenol were 0.25 and 0.35 mg/mL, and those for MS-222 were 0.25 and 0.50 mg/mL, for both the buffered solution and the non-buffered one. Eugenol turned out to be a stronger euthanasia agent than MS-222 in zebrafish, presenting with significantly shorter euthanasia times. However, the exposure of the fish to euthanasia doses of eugenol triggered branchial alterations, in addition to serious lesions and changes in their nerve tissue. The results obtained with MS-222 also revealed a marked branchial alteration derived from its use. In this respect, the addition of a buffer to the MS-222 solution enhanced the effectiveness of the drug, with significantly shorter euthanasia times being achieved than with the non-buffered solution, and diminished the severity of the lesions described. We therefore determined that the buffered MS-222 solution is the most effective, reliable and safest method of euthanasia for use in research on zebrafish.