Assessment of Pain Associated with the Injection of Sodium Pentobarbital in Laboratory Mice (Mus musculus)

The AVMA Guidelines for the Euthanasia of Animals considers injection of barbiturates to be an acceptable method of euthanasia in rodents but states there is a potential for pain when administered intraperitoneally. This study examined the potential for pain in mice by assessing visceral pain after intraperitoneal administration and acute pain by using a paw-lick test. Male and female mice (n=160) intraperitoneally received a euthanizing dose of sodium pentobarbital at a concentration of 5, 50, or 390 mg/mL and were observed for writhing, peritoneum-directed behaviors (PDB), loss of righting reflex, and time to death. Writhing was not observed in any animal. There was no significant difference in the number of mice exhibiting PDB or in the rate of PDB for responders receiving either saline or the 390-mg/mL solution. There was a significant treatment effect on time, with greater concentration and dose resulting in more rapid loss of righting reflex and death. In the second set of experiments, the same solutions were injected subcutaneously into the plantar hindpaw of male and female mice (n= 84). The number of responders, latency until the first lick, and the number of licks per responder were recorded. The number of responders was increased in the 50-mg/mL group; however, there was no difference in latency or the number of licks per responder. These results show that intraperitoneal injection of sodium pentobarbital for euthanasia in mice did not result in increased behavioral signs of pain, and animals lose consciousness more rapidly than the onset of pain seen in the pawlick test. Therefore, although sodium pentobarbital is capable of inducing inflammation, euthanasia through intraperitoneal administration is rapid and does not result in overt signs of pain when compared with injection of saline.