Guidelines for the Euthanasia of Rodent Feti and Neonates
The Report of the AVMA Panel on Euthanasia provides limited recommendations for the euthanasia of prenatal or neonatal animals. The 2007 report states: “When ovario-hysterectomies are performed, euthanasia of feti should be accomplished as soon as possible after removal from the dam.” It also states “Neonatal animals appear to be resistant to hypoxia, and because all inhalant agents ultimately cause hypoxia, neonatal animals take longer to die than adults.”1 The following guidelines are suggested to assist Animal Care and Use Committees at the NIH in reviewing proposals which involve the use of rodent feti or neonates2. In all cases, the person performing the euthanasia must be fully trained in the appropriate procedures.
Feti (unborn animals that have not breathed):
a. Mouse, Rat and Hamster Feti up to 15 days’ Guinea Pig Feti up to 34 days’ gestation:
Neural development at this stage is minimal and pain perception is considered unlikely.3,4 Euthanasia of the mother or removal of the fetus should ensure rapid death of the fetus due to loss of blood supply and non-viability of feti at this stage of development.5
b. Mouse, Rat and Hamster Feti 15 days’ gestation to birth and Guinea Pig Feti 35 days’
gestation to birth: The neural development at this stage supports the likelihood that
pain signals may be biochemically or neurophysiologically processed by the maturing
neural circuitry.3,4,6 However, recent evidence implies that feti are neither sentient nor
conscious prior to birth and thus incapable of actually perceiving pain.7,8,9 When feti are
required for study, euthanasia of individual feti may be induced by decapitation with
surgical scissors or cervical dislocation which are acceptable physical methods of
euthanasia. Alternatively, if the mother is euthanized as described in “c” below, the
uterus with the pups or the pups with the amniotic sac intact can be removed from the
dam. This results in immediate cessation of uterine blood flow and oxygen delivery to
the feti and rapid progression to an isoelectric EEG.8 If, at any point, the feti are allowed
to breathe, then they must decapitated or cervically dislocated. Rapid freezing, without
prior anesthesia, as a sole means of euthanasia is not considered to be humane1.
Animals should be deeply anesthetized prior to freezing. When chemical fixation of the
whole fetus is required, feti should be deeply anesthetized prior to immersion in, or
perfusion with, fixative solutions. Anesthesia may be effectively induced by hypothermia
of the fetus, which can be achieved by submerging the fetus (with the amniotic sac
intact) in cold (4-

c. When feti are not required for study, the method chosen for euthanasia of a pregnant
mother should ensure rapid cerebral anoxia to the fetus with minimal disturbance to the uterine milieu minimizing fetal arousal8. Recommended methods for euthanasia of the mother are CO2 exposure followed by a secondary method of euthanasia, which may include cervical dislocation, decapitation or bilateral pneumothorax. Death of the mother must be verified after euthanasia and prior to disposal. The institute veterinarian should be consulted for considerations of other euthanasia agents. Neonates (newborn animals that are breathing): Maturation of nociceptors and the
development of excitatory and inhibitory receptor systems occur during the period just prior to
birth and into the second week of postnatal life10-14. Resistance to hypoxia at this age results in
a prolonged time to unconsciousness when CO2 is used as a euthanasia agent1,5,15. A secondary
physical method of euthanasia is recommended to ensure death (e.g. cervical dislocation,
decapitation, bilateral pneumothorax). Death must be verified after euthanasia and prior to
a. Mouse, Rat and Hamster Neonates up to 10 days of age: Acceptable methods for
euthanasia include: injection of chemical anesthetics (e.g., pentobarbital), decapitation, or cervical dislocation. Additionally, these animals are sensitive to inhalant anesthetics; e.g., CO2, halothane or isoflurane (used with appropriate safety considerations) although prolonged exposure may be necessary. A secondary physical method of euthanasia is recommended to ensure death (e.g. cervical dislocation, decapitation, bilateral pneumothorax).
Immersion in liquid nitrogen may be used only if preceded by anesthesia. Similarly, anesthesia should precede immersion or perfusion with chemical fixatives. Anesthesia may be induced by inhalant or injectable anesthetics; the institute veterinarian should be consulted for appropriate agents and dosages. Alternatively, when adequately justified, hypothermia may be used to induce anesthesia in pups six days of age or less (however 3-4 days of age is more typical).16,17,18
b. Guinea Pig Neonates:
c. Mouse, Rat and Hamster Neonates over 10 days of age:
Artwohl J, et al. 2006. Report of the ACLAM task force on rodent euthanasia. JAALAS Kaufman, W. 2000. p. 227-242. In G.J. Krinke (ed.), The Laboratory Rat. Academic Press, Inc., San Diego, Calif. -induced c-fos expression in the spinal cord of fetal Klaunberg B.ANeonates. Contemp. Top. Lab. Anim. Sc. 43:(5) 29-34. Himwich, W.A. 1962. Biochemical and neurophysiological development of the brain in the neonatal period. Int. Rev. Neurobiol. 4:117-159.
Mellor DJ. Galloping colts, fetal feelings, and reassuring regulations: Putting animal-welfare science into practice. 2010. J Veterinary Medical Education 37(1):94-100. Mellor, D.J., and N.G. Gregory. Responsiveness, behavioral arousal and awareness in fetal and newborn lambs: experimental, practical and therapeutic implications. 2003. N. Z. Vet. J. 51:2-13. Committee on Guidelines for the Use of Animals in Neuroscience and Behavioral Research. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research, p.102-108. National Academies Press, Washington, D.C. [ 11. Gupta, A., J. Cheng, S. Wang, and G.A. Barr. 2001. Analgesic efficacy of ketorolac and morphine in neonatal rat pups. Pharmacol. Biochem. Behav. 68:635-640. 12. Robinson, S.E., and M.J. Wallace. 2001. Effect of perinatal buprenorphine exposure on development in the rat. J. Pharmacol. Exp. Ther. 298:797-804. 13. Woodbury, C.J., A.M. Ritter, and H.R. Koerber. 2001. Central anatomy of individual rapidly adapting low-threshold mechanoreceptors innervating the “hairy” skin of newborn mice: early maturation of hair follicle afferents. J. Comp. Neurol. 436:304-323. 14. Office of Laboratory Animal Welfare, National Institutes of Health, U.S. Department of Health and Human Services. 2002. Public Health Service Policy on Humane Care and Use of Laboratory Animals - Clarification Regarding Use of Carbon Dioxide for Euthanasia of Small Laboratory Animals. [ 15. Pritchett K, et al. Euthanasia of neonatal mice with carbon dioxide. Comparative Med, Models. 2nd Ed, Volume III. Academic Press, 2007, pp 464-465. anesthesia of neonatal rats. Lab. Anim. Sci. 47:386-395. 18. Singer, D. 1999. Neonatal tolerance to hypoxia: a comparative-physiological approach.

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