Guidelines for Euthanasia of Rodent Fetuses and Neonates
The following guidelines and general references are suggested to assist NIH intramural Animal
Care and Use Committees in reviewing proposals which involve the use of rodent fetuses orneonates.1-22 In all cases, the person performing the euthanasia must be fully trained in theappropriate procedures.
The AVMA Guidelines for the Euthanasia of Animals: 2013 Edition states that “Scientific data
indicate that mammalian embryos and fetuses are in a state of unconsciousness throughout
pregnancy and birth.” It also states that “The precocious young of guinea pigs remain
insentient and unconscious until 75% to 80% of the way through pregnancy and remain
unconscious until after birth due to chemical inhibitors” and “embryos and fetuses cannot
consciously experience feelings such as breathlessness or pain. Therefore, they also cannot
suffer while dying in utero after the death of the dam, whatever the cause.” 2 There is also
literature which indicates that there is the likelihood that pain may be perceived relative to theneural development in mouse, rat and hamster fetuses over 15 days, and in guinea pig fetusesover 35 days and that behavioral responses to sensory stimulation do occur. 18-22
1.

Fetuses - Mouse, Rat, Hamster, and Guinea Pig Fetuses to Birth: When fetuses(mouse, rat & hamster > E15, or guinea pigs > E35) are required for study, euthanasia
of individual fetuses may be induced by acceptable physical methods of euthanasia,
such as decapitation with surgical scissors or cervical dislocation. Although physicalmethods are considered the most effective and humane because of the speed,
intraplacental injection of pentobarbital may be justified for procedures which requirepreservation of anatomical and histological mouse fetal structures or avoid hypoxia
associated with physical methods. 5
When fetuses are not required for study, the method chosen for euthanasia of apregnant dam should ensure rapid cerebral anoxia to the fetus with minimal disturbanceto the uterine milieu minimizing fetal arousal.7 Methods for euthanasia of the dam
should follow recommendations of the ARAC Guidelines for Euthanasia of Rodents Using
Carbon Dioxide and the AVMA Guideline for the Euthanasia of Animals.
Alternatively, if the mother is euthanized as described above, the uterus with the pups or
the pups with the amniotic sac intact can be removed from the dam, however, it may
take 1 hour or longer before the fetuses are dead.8 If, at any point, the fetuses areallowed to breathe, then they must be euthanized by methods which may include
decapitation, cervical dislocation, hypothermia, anesthesia followed by exsanguination,or chemical anesthetic overdose. If believed to be unconscious, fetuses may be immersed
in liquid nitrogen. When chemical fixation of the whole fetus is required, fetuses must be
euthanized prior to immersion.

2. Neonates (newborn animals that are breathing): Maturation of nociceptors and thedevelopment of excitatory and inhibitory receptor systems occur during the period justprior to birth and into the second week of postnatal life.10-14 Resistance to hypoxia at this3

age results in a prolonged time to unconsciousness when CO2 is used as a euthanasia
agent.2,4,15 If CO2 is used, a secondary physical method of euthanasia is required to ensure
death (e.g. cervical dislocation, decapitation, bilateral pneumothorax). Death must be
verified after euthanasia and prior to disposal. 14
3. Fetuses and Altricial Neonates (note NOT Guinea Pigs): Hypothermia is an acceptable
method of euthanasia for fetuses and altricial neonates up to 7 days of age as long as
direct contact with ice/cold surfaces is avoided. Injectable anesthetic agent overdose canalso be used.
4. Mouse, Rat, and Hamster Neonates up to 10 days of age: Acceptable methods foreuthanasia include: injection of chemical anesthetics (e.g., pentobarbital), decapitation or
cervical dislocation. Additionally, these animals are sensitive to inhalant anesthetics;
e.g., CO2, or isoflurane from a vaporizer (used with appropriate safety considerations)although prolonged exposure, up to 50 minutes may be necessary. 2,15-17 A secondary
physical method of euthanasia is recommended to ensure death (e.g. cervicaldislocation, decapitation, bilateral pneumothorax). Death must be verified prior todisposal. “Fetuses that are believed to be unconscious and altricial neonates <5 daysof age may be quickly killed by rapidly freezing in liquid nitrogen” 2 For neonates 5 days
or greater, immersion in liquid nitrogen may be used only if preceded by anesthesia.
2

5. Guinea Pig Neonates: Follow guidelines for adults.
2

6. Mouse, Rat and Hamster Neonates over 10 days of age: Follow guidelines for adults.
References
1. Valentim A.M., Guedes SR,Pereira AM, Antunes LM. 2015. Euthanasia using gaseous agentsin laboratory rodents. Lab Anim. Nov 24 2105
2. AVMA Guidelines for the Euthanasia of Animals: 2013 Edition
https://www.avma.org/KB/Policies/Documents/euthanasia.pdf
3. Artwohl J, et al. 2006. Report of the ACLAM task force on rodent euthanasia. JAALAS
45(1):98-105.
4. Klaunberg B.A., O’Malley J., Clark T., Davis .JA. 2004. Euthanasia of Mouse Fetuses andNeonates. Contemp. Top. Lab. Anim. Sc. 43:(5) 29-34
5. Muñoz-Mediavilla C1, Cámara JA2, Salazar S1, Segui B1, Sanguino D3, Mulero F2, de laCueva E4, Blanco I5. Evaluation of the foetal time to death in mice after application ofdirect and indirect euthanasia methods. Lab Animal , Aug 11 2015
6. Himwich, W.A. 1962. Biochemical and neurophysiological development of the brain in theneonatal period. Int. Rev. Neurobiol. 4:117-159.
7. Mellor DJ. Galloping colts, fetal feelings, and reassuring regulations: Putting animalwelfare science into practice. 2010. J Veterinary Medical Education 37(1):94-100.
8. Leist KH, Grauwiler J. Fetal pathology in rats following uterine-vessel clamping on day 14 ofgestation. Teratology 1974;10:55-67.
9. Committee on Guidelines for the Use of Animals in Neuroscience and Behavioral
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11.
12.
13.

14.

15.
16.
17.
18.

19.
20.
21.

22.

Research. 2003. Guidelines for the Care and Use of Mammals in Neuroscience andBehavioral Research, p.102-108. National Academies Press, Washington, D.C.
[http://oacu.od.nih.gov/GdeMammNeuro.pdf]
Fitzgerald, M., and S. Beggs. 2001. The neurobiology of pain: developmental aspects.
Neuroscientist 7:246-257.
Gupta, A., J. Cheng, S. Wang, and G.A. Barr. 2001. Analgesic efficacy of ketorolac andmorphine in neonatal rat pups. Pharmacol. Biochem. Behav. 68:635-640.
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.
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 miceearly maturation of hair follicle afferents. J. Comp. Neurol. 436:304-323.
Office of Laboratory Animal Welfare, National Institutes of Health, U.S. Department ofHealth and Human Services. 2002. Public Health Service Policy on Humane Care and Useof Laboratory Animals - Clarification Regarding Use of Carbon Dioxide for Euthanasia of
Small Laboratory Animals. [http://grants.nih.gov/grants/guide/notice-files/NOT-OD-02062.html]
Pritchett K, et al. Euthanasia of neonatal mice with carbon dioxide. Comparative Med,
55(3):275-281, 2005.
Pritchett K. Euthanasia of neonatal rats with carbon dioxide. JAALAS, 48(1):23-27,2009.
Singer, D. 1999. Neonatal tolerance to hypoxia: a comparative-physiologicalapproach.Comp. Biochem. Physiol. 123:221-234.
Takada, S.H., C.A.G. Sampaio, W. Allemandi, P.H. Ito, L.F. Takase, M.I. Nogueira. 2011. A
modified rat model of neonatal anoxia: Development and evaluation by pusleoximetry,arterial gasometry, and Fos immunoreactivity. Journal of Neuroscience Methods, 198(2011) 62-69.
Himwich WA. 1962. Biochemical and neurophysiological development of the brain in theneonatal period. Int Rev Neurobiol 4:117-119
Yi DK, Barr GA. 1997. Formalin-induced c-fos expression in the spinal cord of fetal rats. Pain
73:347-354
Copploa DM, Millar LC, Chen CJ, VAndenbergh JG. 1997. Chronic cocaine exposure affects
stimulus-induced but not spontaneous behavior of near-term fetus. Pharmacol Biochem
Behav 58: 793-799.
Smotheraman WP, Robinson SR. 1985. The rat fetus in its environment: behavioral
adjustments to novel, familiar, aversive and conditioned stimuli present in utero. Behav
Neurosci 99:521-530.

Approved - 02/12/97
Revised - 11/10/98, 03/27/02, 10/13/04, 12/14/05, 10/10/07, 05/12/10, 03/09/11,04/10/13, 06/22/16
Archived – 9/23/2020

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