Appendix 3: Guidelines for the euthanasia of fetal and neonatal mice, rats, guinea pigs and Rabbits

This is Appendix 3 of the Code of Practice for the Housing and Care of Laboratory Mice, Rats, Guinea Pigs and Rabbits.

The following guidelines are suggested to assist in reviewing proposals which involve the use of rodent and rabbit fetuses or neonates. This information can be used as guidelines for investigators, animal house staff and Animal Ethics Committees. In all cases, the person performing the euthanasia must be fully trained in the appropriate procedures.

Neural development up to 60% gestation is considered 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.¹

While hypothermia is known to act as an anaesthetic² to a certain extent, it is still a controversial method of euthanasia, as is rapid freezing (immersion in liquid nitrogen). Monitored hypothermia, followed by decapitation or rapid freezing (immersion into liquid nitrogen) to ensure euthanasia of fetuses or neonates without fur and less than 4 grams is considered acceptable by some references³. Euthanasia by CO₂ asphyxia is slow and unacceptable for neonates.

Mice and rat fetuses

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 anaesthetics including CO₂, euthanasia may be induced by the skilful injection of chemical anaesthetics. Although aesthetically unpleasant, decapitation with surgical scissors or scapel, orcervical dislocation are humane and acceptable physical methods of euthanasia.

When chemical fixation of the whole fetus is required, fetuses (with fur or greater than 4g) should be anaesthetised prior to immersion in or perfusion with fixative solutions. Anaesthesia may be induced by monitored hypothermiac² of the fetus, by injection of the fetus with a anaesthetic agent, or by deep anaesthesia of the mother with a chemical agent that crosses the placenta, such as pentobarbital. The institute veterinarian should be consulted for considerations of fetal sensitivity to specific anaesthetic agents. When fetuses are not required for study, the method chosen for euthanasia of a pregnant mother must ensure rapid death of the fetus.

Mice and rat neonates

1. Up to 10 days of age — Acceptable methods for euthanasia of neonatal mice and rats include: injection of chemical anaesthetics (such as pentobarbital), decapitation, or cervical dislocation. Anaesthesia should precede immersion or perfusion with chemical fixatives (including liquid nitrogen) if the fetus is greater than 4g. Anaesthesia may be induced by injectable anaesthetics or hypothermia and the institute veterinarian should be consulted for appropriate agents and dosages.
2. Older than 10 days — Follow guidelines as per Appendix 2.

Guinea pig and rabbit fetuses

Embryos or fetuses: Literature on the development of pain pathways suggests that there may be pain perception consistent with development of the functional brain occurs from 60% gestation. Although aesthetically unpleasant, decapitation is an acceptable method for killing these forms. It is preferable to use a guillotine for decapitation.

Guinea pig and rabbit neonates

1. Guinea pig neonates are well developed, sentient animals from birth. Rabbit neonates, in this context, are newborn rabbits up to 10 days old. Intraperitoneal injection with an overdose of an appropriate anaesthetic agent is the preferred method for euthanasia of these neonates. Cervical dislocation is an acceptable method for guinea pigs and rabbits less than 100g bodyweight.
2. Older than 10 days — Follow guidelines as per Appendix 2.

¹ 'When ovarian hysterectomies are performed, euthanasia of fetuses should be accomplished as soon as possible after removal from the dam. Neonatal animals are relatively resistant to hypoxia.' 2000 Report of the AVMA Panel on Euthanasia, JAVMA 218:688.
² Phifer CB, Terry LM. 1986. Use of hypothermia for general anaesthesia in preweanling rodent. Physiol & Behav 38:887-890.
³ 'Recommendations for euthanasia of experimental animals.' Working Party Report 1996. Laboratory Animals, 30:293-316. (refer to www.lal.org.uk)