Evaluation of a mixture of medetomidine, alfaxalone and butorphanol as an alternative drug for euthanasia in mice.

Euthanasia agents should induce a rapid and painless loss of consciousness, followed by cardiopulmonary arrest and subsequent brain death. Injectable drugs such as pentobarbital sodium are commonly used for laboratory rodents due to their quick and smooth action. However, the discontinuation of pharmaceutical-grade pentobarbital sodium and secobarbital sodium in Japan, along with a global shortage of pentobarbital in late 2020, has increased the demand for new injectable euthanasia drugs. In Japan, the combination of medetomidine, midazolam, and butorphanol (MMB), as well as a newer formulation in which midazolam is replaced with alfaxalone (MAB), have been widely used as balanced anesthesia for rodents. To evaluate their potential as alternative euthanasia agents in mice, we compared mortality rates and the time intervals to the loss of the righting reflex, respiratory arrest, and cardiac arrest following anesthetic administration. An intraperitoneal injection of MAB at five times the anesthetic dose induced death within 10 min with the loss of the righting reflex, respiratory arrest, and cardiac arrest occurring at 1.5 min, 4 min, and 9 min respectively, in all mice, which was comparable to those observed with 300 mg/kg of secobarbital. In contrast, none of the mice administered MMB at five times the anesthetic dose experienced cardiopulmonary arrest within 30 min. Intraperitoneal overdose of MAB induces rapid and irreversible death, supporting its potential use as an effective euthanasia agent in mice.