1. Fetuses up to 14 days in gestation: Neural development at this stage is minimal and pain perception is considered unlikely. 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 fetuses at this stage of development.
  2. Fetuses 15 days in gestation to birth: The literature on the development of pain pathways suggests the possibility of pain perception at this time. Whereas fetuses at this age are resistant to inhalant anesthetics, such as isoflurane or CO2, euthanasia may be induced by the skillful injection of chemical anesthetics or by a physical method of euthanasia, such as cervical dislocation or decapitation with surgical scissors.

When chemical fixation of the whole fetus is required, fetuses should be anesthetized prior to immersion in or perfusion with fixative solutions. Anesthesia may be induced by one of the following methods: a) injection of the fetus with a chemical anesthetic, b) deep anesthesia of the mother with a chemical agent that crosses the placenta (e.g., pentobarbital), or c) hypothermia of the fetus following removal from the mother and prior to immersion in a fixative. A DLAM veterinarian should be consulted for considerations of fetal sensitivity to specific anesthetic agents. When fetuses are not required for study, the method chosen for euthanasia of a pregnant mother must ensure rapid death of the fetus (e.g., pentobarbital).


Neonatal rats and mice (P1 to P14) have been shown to be more resistant to euthanasia by CO2 overdose than adult animals, requiring prolonged exposure (Beaver et al., 2001; Klaunberg et al., 2004; Pritchett-Corning, 2009). Neonates over 14 days of age may be euthanized as per the guidelines for adults.

If CO2 narcosis is employed for P1 to P14 animals, a secondary physical method of euthanasia is required to ensure death (e.g., cervical dislocation, decapitation with a sharp pair of scissors, bilateral pneumothorax by incision of the chest). This secondary method should only be employed following CO2 exposure or administration of anesthetics such that there is no response to pain (for example, response to toe pinch) in the neonate.

Physical methods of euthanasia that are not preceded by administration of anesthetics or CO2 overdose must be documented in the animal use protocol. The AVMA Guidelines for the Euthanasia of Animals (2020 Edition) note that rat and mouse pups are born neurologically immature when compared with humans, and their afferent pain pathways are not well developed until after postnatal day 5 to 7, with cortical development occurring later. The ARC has therefore determined that personnel using physical techniques on unanesthetized neonatal mice and rats after P5 must be verified proficient in the technique prior to working independently.

The use of chemical agents (such as injection of pentobarbital IP at 100 mg/kg, or inhalation of 32% isoflurane) is preferred as an alternative to CO2 inhalation in neonates. Be aware that prolonged exposure to isoflurane may also be needed to euthanize the animal, and a secondary physical method of euthanasia is required.

Approved 9/22/03; Revised 11/23/09, 9/12/11, 2/12/24