Evaluating Intrahepatic and Intraperitoneal Sodium Pentobarbital or Ethanol for Mouse Euthanasia

Intraperitoneal (IP) injection of sodium pentobarbital (PB) is an accepted method of euthanasia for mice. However, this method has important drawbacks, including the potential for pain or misinjection. The objective of this prospective, randomized, blinded study was to determine whether intrahepatic (IH) injection of PB is more effective than IP delivery for mouse euthanasia. Secondary objectives were to: 1) determine whether IP ethanol (ET) is a suitable alternative to PB and 2) study the effect of isoflurane anesthesia on euthanasia with either PB or ET. Eighty adult CD1 mice were randomly assigned to 6 different treatment groups, were euthanized by using IP or IH injections of either PB or ET, and were either anesthetized or conscious before injection. Variables of interest were: 1) misinjection rates (based on necropsy evaluation), 2) time from injection to apnea and 3) time to cessation of heartbeat (CHB). The misinjection rate for IH injections was 93% (28/30). Two successful IH injections resulted in death within 4 s, but this method cannot be recommended due to the possibility for intrathoracic injection ( n = 4). In nonanesthetized mice, time to apnea and CHB was significantly shorter with IP ET (apnea: 72.5 s [median], CHB: 115 s) than with IP PB (apnea: 136 s, CHB: 176 s). Anesthesia at time of injection was associated with a shorter CHB time for IP PB. These data show the difficulty in achieving successful IH injections in mice, but confirm that IP ET is a viable and potentially superior alternative to IP PB. Lastly, anesthesia can shorten time to death after IP injection of PB.