Hypothermia as a Sole Euthanasia Method for Neonatal Mice (Mus musculus)

Per the AVMA euthanasia guidelines, there are no data supporting the use of hypothermia as a solitary method of euthanasia for neonatal mice. However, the use of a secondary physical method of euthanasia, such as decapitation, could be time-consuming and mentally straining on care staff, particularly if many animals need to be euthanized simultaneously. In this study, we tested hypothermia as a sole form of euthanasia in neonatal mice (day 0 to 2 and day 8 to 10 of age) in various group sizes (1, 3, 6, and 10) using an ice-water bath (4 to 5 °C), a refrigerator (4 to 7 °C), or a freezer (−22 to −10 °C). Generalized linear models were used to predict what factors influenced successful euthanasia and at what body temperature a pup was unlikely to recover. Only the freezer consistently induced anesthesia and euthanasia within an appropriate time frame for single and grouped day 0 to 2 neonates and single day 8 to 10 neonates. Histopathology of neonates collected at the time of anesthetic induction showed no evidence of lesions for any method indicating a lack of trauma and potential discomfort during the euthanasia process. With modeling, we determined that pups of the age range 0 to 2 d and 8 to 10 d had a 1% chance of survival if they reached a body temperature of 5.5 °C and 7.6 °C respectively, regardless of the hypothermia method. Conclusively, the use of a freezer as a sole method for hypothermia-induced humane euthanasia of single neonatal CD1 mice is equivalent to current AVMA acceptable hypothermia-induced euthanasia techniques: it rapidly induces unconsciousness and death, is reliable and appropriate for the selected signalment, poses little occupational hazard and environmental concerns, and has the potential of alleviating emotional fatigue.