Aim of the Study
When performing euthanasia, the method has to induce loss of consciousness and death with a minimum of pain and distress. Also, the time required to induce loss of consciousness is an important parameter (1). A former study in rats showed that intraperitoneal injection of pentobarbital for euthanasia is associated with pain (2). The aim of this study is to investigate if low dose pentobarbital without or high dose pentobarbital with a local analgesic, induces sign of pain and distress when used for euthanasia of laboratory mice.

Materials and Methods
14 NMRI female mice were randomized after weight into 2 groups, receiving 0.5 ml pentobarbital 50 mg/ml or 0.5 ml pentobarbital 200 mg/ml with lidocaine 20 mg/ml intraperitoneally. Signs of pain and discomfort were measured by semi-quantitative scoring of activity, respiration rate, signs of pain (back arcing, urination/defecation and vocalization) during injection or in the absorption phase. Further time from the injection until the animal died (respiratory and cardiac arrest) was measures. The statistical analysis was performed by Graph Pad Prism 6.0. Semi-quantitative data were analyzed by Mann-Whitney U-test. Absorption time was analyzed by Student´s t-test. The study was approved by the Danish National Animal Experiments Inspectorate.

Results
The results showed no significant difference in activity between groups. Most animals showed no changes in activity after injection. In both groups most animals hyperventilated after injection. Only few animals showed signs of pain after injection in both treatment groups. The time until death was significant longer in animals receiving pentobarbital 50 mg/ml, compared to the group of animals receiving pentobarbital 200 mg/ml. It took in average 1 min longer for animals in the low concentration group than the animals receiving the high concentration group before death occurred.

Conclusion
From the results we can conclude that a high concentration of pentobarbital in combination with lidocaine causes the mice to be euthanized more quickly without a higher degree of discomfort and pain. We recommend that euthanasia of laboratory mice is performed with pentobarbital 200 mg/ml with lidocaine 20mg/ml instead of pentobarbital 50 mg/ml.

1. Leary S, Underwood W, Anthony R, Cartner S. AVMA Guidelines for the Euthanasia of Animals: 2013 Edition.
2. Zatroch KK, Knight CG, Reimer JN, Pang DSJ. Refinement of intraperitoneal injection of sodium pentobarbital for euthanasia in laboratory rats (Rattus norvegicus). BMC Vet Res. 2017;13(1): 4-10.