SVH AEC SOP.26
EUTHANASIA OF MICE AND RATS
BACKGROUND
The humane killing of mice and rats for culling or part of a scientific investigation must be
conducted either by experienced staff, or under the supervision of experienced staff.
Mice and rats should be killed either away from other mice and rats or as a familiar group in
their home cage. The methods below are rapid, free of signs of pain or distress, reliable and
minimally distressful to be person performing the procedure.
In many protocols experimental animals are anaesthetized in order to collect tissues, perfuse
etc before euthanasia is performed. This method, is aneaesthesia without recovery, is the
preferred method of euthanasia whenever practical.
FOETAL EUTHANASIA
General Advice

Foetuses: <15 days of gestation
Death of the mother results in death of foetus as foetal development of the heart and
vascular system is not completed until E 15.

Foetuses: E15 days to birth
The literature suggests there is a possibility of pain perception from this stage and
newborn pups are resistant to oxygen deprivation.
PROCEDURES
Methods of Foetal Euthanasia
1. Carbon Dioxide Asphyxiation
Place foetuses removed from the uterus in a carbon dioxide chamber and keep in the
chamber for several minutes after they are cyanotic and all signs of life absent. This could
be up to 10 minutes. This should be immediately followed with a physical method
(decapitation or hypothermia).
2. Decapitation with sharp scissors or surgical blade
A dedicated pair of heavy-duty sharp scissors is the easiest and safest method.
SOP. 26 Euthanasia In Mice and Rats
Author: Dr Sue Peirce
App: 7 June 2006
When chemical fixation of the foetus is required for histology, the foetus should be
anaesthetized with monitored hypothermia or deep anaesthesia of the mother with barbiturate
(Pentobarbital, Lethabarb 200mg/kg), prior to removal of the foetus. Hypothermia is achieved
by placing pups on a non-stick surface in a freezer for several minutes or immersion in slushy,
crushed ice.
EUTHANASIA BY AGE
1.
NEONATES UP TO 5 DAYS OF AGE
1.1 Anaesthesia by carbon dioxide inhalation or immersion in crushed ice, followed by
decapitation with sharp scissors is the most effective method of euthanasia
1.2 Overdose of anesthetics agent by intraperitoneal injection is also acceptable
a. Pentobarbital (Lethabarb, 200mg/kg)
b. Ketamine (300mg/kg) + Xylazine (30mg;kg)
1.3 Cervical dislocation using a thumb and forefinger squeeze to break neck can be used
if options 1 and 2 above are not available.
2.
MORE THAN 5 DAYS OF AGE TO ADULT
2.1 Carbon dioxide inhalation
2.2 Overdose of anaesthetic agent by intraperitoneal injection
2.3 Cervical dislocation if less than 150g
2.4 Decapitation after anaesthesia, using a guillotine, is acceptable for particular tissue
preservation techniques
2.1 Carbon Dioxide Inhalations Using the Euthanasia Cabinet in Biological Resources
Centre
Carbon dioxide is a frequently used euthanasia agent for laboratory rats and mice and
is the most efficient method of euthanasia, Inspiration of high concentrations of carbon
dioxide can be distressing. A recommended procedure is to place animals into a
chamber that contains room air and then introduce the carbon dioxide. There is no
conclusive evidence that adding oxygen to the carbon dioxide makes the procedure less
stressful to the animals
A fill rate of 20% of the chamber volume per minute carbon dioxide added to existing
room air in the chamber should be appropriate to achieve a balanced gas mixture to
fulfill the objective of rapid unconsciousness with minimal distress to the animals.
SOP. 26 Euthanasia In Mice and Rats
Author: Dr Sue Peirce
App: 7 June 2006
Place the box of mice in the euthanasia cabinet and fill with carbon dioxide for 1 minute.
The anaesthetic machine induction box can also be used for a smaller number of mice.
The euthanasia chamber should be kept clean.
2.1.1 For mice
Place the mouse box, with gas delivery hose directly into mouse box, into the
euthanasia cabinet or other sealed container, and turn CO2 gas on full for 1 minute. If
more than one mouse box, remove lids, turn the CO2 gas full for 1 minute. Leave boxes
in cabinet for 3-5 minutes. Check mice are dead before disposal.
2.1.2 For rats
Place rats in container with gas full on for 2 minutes, leave in CO2 atmosphere for a
further 6-8 minutes. Check rats are dead before disposal.
Warning: Euthanized animals should not be exposed to room air until death has occurred as
the effects of CO2 can be quickly reversed in the presence of oxygen.
Signs of death include:
 cessation of breathing
 cyanosis
 absence of heart beat
 cessation of any movement when exposed to room air
Confirmation of death should not be based on a single sign.
An additional method of euthanasia (decapitation, cervical dislocation pneumothorax, terminal
cardiac bleeding) may be used to confirm death of animal following CO2 inhalation
2.2 Overdose of Anaesthetic Agent
The injection can be administered intracardiac (mouse and rat) or intravenously (rat), if the
mouse or rat is anaesthetised prior to overdose of anaesthetic agent
a. Pentobarbital (Lethabarb, 200mg/kg)
b. Ketamine (300mg/kg) + Xylazine 30mg/kg
2.2.1 Pentobarbital
As some barbiturate euthanasia solutions can be an irritant to tissues the solutions are
diluted prior to injection. This is not necessary if the animal anaesthetized prior to
euthanasia
Dose rate for euthanasia solution of sodium pentobarbital (Lethabarb) diluted 1 in 2
with water or saline.
SOP. 26 Euthanasia In Mice and Rats
Author: Dr Sue Peirce
App: 7 June 2006
The following volumes administer approximately 400mg/kg:
Mouse <20g
Rat
0,05ml i/p
20-30g
0,1ml i/p
250g
0,25ml i/p
500g
0,5ml i/p
2.2.2. Kethamine: Xylazine mixture
Administered at three the anaesthetic dose – approx 300mg/kg Ketamina and 30 mg/kg
Xylasine
Mouse/ 20g
0,6ml K and 0,03ml X i/p or s/c
Rat/100g
0,3ml K and 0,15ml X i/p or s/c
REFERENCES
Bureau of Animal Welfare: Code of practice for the housing and care of laboratory mice, rats,
guinea pigs and rabbits. Victorian Government, Department of Primary Industries, 2004
University of Melbourne Animal Welfare Committee: Recommended methods of euthanasia
for common laboratory animals, 2004.
Euthanasia of Mouse Foetuses and Neonates, Klaunberg, BA, O’Malley J. Clark T, Davis J.
Contemporary Topics Vol 43, 5 Sept 2004.
Report of the AVMA Panel on Euthanasia 2000, JAVMA Vol 218, Nº 5, March 2001
Report of the ACLAM Task Force on Rodent Euthanasia 2005.
Comparison of Carbon Dioxide, Argon and Nitrogen for Inducing Unconsciousness or
Euthanasia of Rats, Sharp J, Azar T & Lawson D, Journal of American Association for
Laboratory Animal Science Vol 45 Nº 2 March 2006.
SOP. 26 Euthanasia In Mice and Rats
Author: Dr Sue Peirce
App: 7 June 2006