Animal Ethics Committee, Faculty of Health Sciences, University of Cape Town
Standard Operating Procedures
Gas Euthanasia of Rats and Mice
AEC002: Standard Operating Procedure: Gas Euthanasia of Rats and Mice
1. Purpose
The purpose of this Standard Operating Procedure (SOP) is to ensure that the humane killing of rats and mice is
performed with the minimum possible discomfort, pain or distress to the animals; that death is reliably induced
and confirmed by an appropriate method; and that the procedure does not endanger human health or safety.
This SOP describes euthanasia using gas, i.e. carbon dioxide gas (100% CO2), with or without prior general
anaesthesia of the animals using halothane or isofluorane volatile anaesthetic gas (5% in air)1-3.
2. Responsibility
Only appropriately trained personnel, who have been authorised by the UCT Faculty of Health Sciences (FHS)
Animal Ethics Committee (AEC), may perform euthanasia.
3. Principles
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Euthanasia must always be performed in a separate room, so that other animals cannot see, hear or smell the
animals being euthanased.
Do not delay euthanasia once animals have been taken to the euthanasia room.
Cages for euthanasia must not be stacked on top of each other, since this can block airflow into the cages.
Individual species must always be euthanased separately.
If animals from different cages are to be euthanased together, combine them only in the euthanasia chamber
(i.e. do not combine them in one cage, before placing them in the euthanasia chamber). Always ensure that
animals are compatible and will not fight in the chamber, e.g. do not combine males from different cages.
Moribund or sick animals must be euthanased separately from healthy animals. Sick animals may need to be
kept in the euthanasia chamber for a longer time, due to altered respiration.
All unweaned animals (younger than 21 days) must stay with the lactating female until the time of euthanasia.
Neonates (i.e. mice or rats younger than 15 days of age) can hold their breaths for a very long time and
have a high tolerance to hypoxia (i.e. oxygen deficiency), so they must be exposed to anaesthetic gas and/or
CO2 for a long time, i.e. for at least 45 minutes in total.
Carbon dioxide (100% CO2) must be supplied as a compressed gas. Dry ice may not be used as CO2 source.
The euthanasia chamber must allow good visibility of all animals. Do not overcrowd the chamber. Ensure
that all animals are able to make normal postural adjustments. The maximum number of animals to place
inside the euthanasia chamber depends on the size of the chamber and the size of the animals, as below:
Euthanasia chamber volume
2 litres
10 litres
10 litres
Euthanasia chamber volume
10 litres
10 litres
10 litres
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Mouse weight
12 to 18 grams
20 to 23 grams
> 30 grams
Rat weight
50 to 100 grams
250 to 300 grams
350 to 400 grams
Number of mice
10
20
10
Number of rats
8
5
4
After euthanasia, the death of each animal must be confirmed by one of the following methods:
1. Decapitation (e.g. guillotine; sharp scissors in neonates)
2. Dislocation of the neck (i.e. manual breaking of the upper neck)
3. Exsanguination (i.e. bleeding out, e.g. by cardiac puncture; eye enucleation)
4. Permanent cessation of the circulation (e.g. removal of the heart; transect aorta)
5. Destruction of the brain (e.g. crush cranium with strong artery forceps in neonates)
6. Confirmation of the onset of rigor mortis (i.e. generalised muscle stiffness) with blue mucous membranes.
This may require waiting for 10-60 minutes after the euthanasia procedure has been completed.
February 2013
AEC002
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Animal Ethics Committee, Faculty of Health Sciences, University of Cape Town
Standard Operating Procedures
Gas Euthanasia of Rats and Mice
Method 1:
Euthanasia using halothane or isofluorane anaesthetic, followed by CO2 gas
1. Ensure that the euthanasia chamber is clean, and that there is sufficient gas in the CO2 cylinder (100% CO2)
to complete the procedure (confirm by opening the regulator valve on the cylinder).
2. Place the animals inside the euthanasia chamber.
3. Halothane and isofluorane anaesthetics must be handled in an appropriately ventilated chemical fume hood,
biosafety cabinet, or in a system with an active gas-scavenging device, to prevent human exposure to the
fumes (chemical hazards). Always wear gloves and appropriate protective clothing (i.e. a lab coat).
4. Aspirate 3 ml (three millilitres) of halothane or isofluorane anaesthetic (100% liquid) into a disposable
pipette. Expel the anaesthetic onto a swab (or cotton wool), attach the swab to the inside of the lid of the
euthanasia chamber, and place the lid firmly onto the chamber (with the swab on the inside). Ensure that the
animals are not in direct contact with the anaesthetic liquid, since this is irritating to the animals.
5. Start the timer (i.e. clock).
6. Closely observe the animals inside the chamber until all animals are completely recumbent and obviously
deeply anaesthetised. Animals must remain exposed to the anaesthetic in the euthanasia chamber for at least
the following length of time (confirm on the timer):
 Mice (older than 21 days of age): 5 minutes
 Rats (older than 21 days of age): 6 minutes
 Neonates, i.e. mice or rats younger than 15 days of age: Leave animals for at least 10 minutes in the chamber
with halothane or isofluorane, then place an additional 1 ml of halothane or isofluorane liquid on the
swab, and close the lid again for a further 30 minutes (i.e. 40 minutes total anaesthetic exposure time).
7. Introduce CO2 gas into the euthanasia chamber. Open the CO2 flowmeter in order to deliver 100% of the
euthanasia chamber’s volume per minute (i.e. to fill the chamber with CO2 over 1 minute). The correct
CO2 flowrate to use depends on the size (volume) of the euthanasia chamber, as in the table below:
Euthanasia chamber volume
2 litres
5 litres
7.5 litres (e.g. type II long cage)
10 litres
8.
9.
10.
11.
12.
13.
14.
15.
16.
CO2 flowrate to use
2 litres per minute
5 litres per minute
7.5 litres per minute
10 litres per minute
Start the timer.
Allow 1 minute for the euthanasia chamber to fill with CO2 (at above flowrates). Close off the flowmeter.
Leave the animals inside the closed euthanasia chamber for a further 5 minutes.
Open the euthanasia chamber, and confirm the death of each animal by one of the following methods:
 Decapitation (e.g. guillotine; sharp scissors in neonates)
 Dislocation of the neck (i.e. manual breaking of the upper neck)
 Exsanguination (i.e. bleeding out, e.g. by cardiac puncture; eye enucleation)
 Permanent cessation of the circulation (e.g. removal of the heart; transect aorta)
 Destruction of the brain (e.g. crush cranium with strong artery forceps in neonates)
 Confirmation of the onset of rigor mortis (i.e. generalised muscle stiffness) with blue mucous membranes.
This may require waiting for 10-60 minutes after the above euthanasia procedure has been completed.
Dispose of the animals in the prescribed manner for the facility.
Turn the euthanasia chamber upside down for 3 minutes to allow CO2 to flow out of the chamber (CO2 is
heavier than air), and then clean and dry the chamber before any other animals are euthanased.
Once all animals have been euthanased, close off the regulator valve on the CO2 cylinder.
Once all animals have been euthanased, clean and dry the euthanasia chamber, turn it upside down and store
the chamber in this position (with the lid removed).
If you experience any difficulties or have any concerns regarding the technique, speak to the veterinarian or
registered laboratory animal technologists of the UCT Research Animal Facility.
February 2013
AEC002
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Animal Ethics Committee, Faculty of Health Sciences, University of Cape Town
Standard Operating Procedures
Gas Euthanasia of Rats and Mice
Method 2:
Euthanasia using a slow rising concentration of CO2 gas
1. Ensure that the euthanasia chamber is clean, and that there is sufficient gas in the CO 2 cylinder (100% CO2)
to complete the procedure (confirm by opening the regulator valve on the cylinder).
2. Place the animals inside the euthanasia chamber and close the lid firmly.
3. Introduce CO2 gas into the euthanasia chamber. Open the CO2 flowmeter to deliver 20% of the euthanasia
chamber’s volume per minute (i.e. to fill the chamber with CO2 over 5 minutes). Higher CO2 flowrates must
not be used since this causes distress and pain to the animals. The correct CO2 flowrate to use depends on the size
(volume) of the euthanasia chamber, as in the table below:
Euthanasia chamber volume
2 litres
5 litres
7.5 litres (e.g. type II long cage)
10 litres
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
CO2 flowrate to use
400 ml per minute
1 litre per minute
1.5 litres per minute
2 litres per minute
Start the timer (i.e. clock).
Allow 5 minutes for the chamber to fill with CO2 (confirm on the timer). Close off the flowmeter.
Leave the animals inside the closed euthanasia chamber for a further 5 minutes (confirm on the timer).
Neonates (i.e. mice or rats younger than 15 days of age) must be left in the chamber for at least 45 minutes.
Open the euthanasia chamber, and confirm the death of each animal by one of the following methods:
 Decapitation (e.g. guillotine; sharp scissors in neonates)
 Dislocation of the neck (i.e. manual breaking of the upper neck)
 Exsanguination (i.e. bleeding out, e.g. by cardiac puncture; eye enucleation)
 Permanent cessation of the circulation (e.g. removal of the heart; transect aorta)
 Destruction of the brain (e.g. crush cranium with strong artery forceps in neonates)
 Confirmation of the onset of rigor mortis (i.e. generalised muscle stiffness) with blue mucous membranes.
This may require waiting for 10-60 minutes after the above euthanasia procedure has been completed.
Dispose of the animals in the prescribed manner for the facility.
Turn the euthanasia chamber upside down for 3 minutes to allow CO2 to flow out of the chamber (CO2 is
heavier than air), and then clean and dry the chamber before any other animals are euthanased.
Once all animals have been euthanased, close off the regulator valve on the CO2 cylinder.
Once all animals have been euthanased, clean and dry the euthanasia chamber, turn it upside down and store
the chamber in this position (with the lid removed).
If you experience any difficulties or have any concerns regarding the technique, speak to the veterinarian or
registered laboratory animal technologists of the UCT Research Animal Facility.
References
1
2
3
Hawkins P, P. L., Golledge H, Leach M, Banzett R, Coenen A, Cooper J, Danneman P, Flecknell P, Kirkden R,
Niel L & Raj M. Newcastle Consensus Meeting on Carbon Dioxide Euthanasia of Laboratory Animals (2006).
James Artwohl, P. B., Brian Corning, Susan Stein. Report of the ACLAM Task Force on Rodent Euthanasia
(2005).
AVMA Panel on Euthanasia. AVMA Guidelines on Euthanasia (2007).
February 2013
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