Confirmation of death
After carrying out any of the techniques of humane killing described below, death must be confirmed. This is to ensure that animals do not recover unexpectedly after an attempt has been made to kill them. The methods that should be used are:
- Confirmation of permanent cessation of the circulation – normally confirmed by feeling for a heartbeat.
- Destruction of the brain.
- Dislocation of the neck.
- Exsanguination (for example by cutting one or more major blood vessels).
- Confirming the onset of rigor mortis – this process usually occurs 30-60 minutes after death and results in the limbs, neck and torso becoming stiff due to muscle rigidity.
- Instantaneous destruction of the body in a macerator.
Cervical dislocation (adult rodents)
The aim of this technique is to cause extensive damage and disruption to the cervical spinal cord and brainstem, by dislocating and compressing the cervical vertebrae. Mice and small rats are restrained on a non-slip surface so that they grip the surface and tend to pull away from the operator. Either the thumb and forefinger are pressed either side of the neck, at the base of the skull, and the neck dislocated, or a rod or similar blunt instrument is positioned over the neck at the base of the skull and pressed downwards. The operator’s other hand grasps the pelvis or base of the tail and applies traction to prevent the torso moving forward when pressure is applied to the neck. Different techniques are needed for larger animals and birds.
As with all methods of euthanasia, death must be confirmed after carrying out the procedure.
Making the technique as humane as possible
As with all methods of euthanasia, the animal must be handled carefully and calmly, and the person carrying out the procedure must be properly trained and confident in their ability to perform the technique quickly and effectively.
In order to prevent the animal experiencing pain, the cervical spinal cord must be disrupted rapidly. This will prevent processing of neural information from sensory nerves in the tissues overlying the spine, which will be stimulated during the process.
If conducted rapidly and effectively, the technique is humane and is listed on Schedule 1 and Annex IV for rodents under 150g and for small birds. Carrying out the procedure effectively on larger animals is more challenging, and will require extensive training on cadavers or on anaesthetised animals.
In order to prevent the animal experiencing pain, the cervical spinal cord must be disrupted rapidly. This will prevent processing of neural information from sensory nerves in the tissues overlying the spine, which will be stimulated during the process.
If conducted rapidly and effectively, the technique is humane and is listed on Schedule 1 and Annex IV for rodents under 150g and for small birds. Carrying out the procedure effectively on larger animals is more challenging, and will require extensive training on cadavers or on anaesthetised animals.
Advantages and disadvantages of this method
The method is rapid and humane when conducted correctly, and allows tissues to be obtained rapidly, and without contamination with anaesthetic agents. If conducted poorly, then death may not be instantaneous (Carbone et al, 20121). No specialised equipment or chemical agents are needed.
Haemorrhage into the lungs can occur shortly after death, and the brainstem and cerebellum can be damaged, as well as the cervical spine. Personnel may be reluctant to conduct the procedure because they find it distressing.
Haemorrhage into the lungs can occur shortly after death, and the brainstem and cerebellum can be damaged, as well as the cervical spine. Personnel may be reluctant to conduct the procedure because they find it distressing.
Safety considerations
The procedure is safe for the operator providing that the animal is handled and restrained correctly during the procedure.
Concussion by striking the cranium (followed by cervical dislocation or exsanguination) (adult rodents)
Striking the cranium, either by impact with a hard surface such as a benchtop or by striking with a blunt object will produce a very rapid loss of consciousness because of trauma to the brain. However, consciousness may return rapidly, depending upon the force of impact the degree of damage to the brain.
Making the technique as humane as possible
As with all methods of euthanasia, the animal must be handled carefully and calmly, and the person carrying out the procedure must be properly trained and confident in their ability to perform the technique quickly and effectively.
In order to prevent the animal from experiencing pain, concussion must be produced rapidly and with sufficient force. If conducted rapidly and effectively, the technique is humane and is listed on Schedule 1 and Annex IV for rodents up to 1kg bodyweight and for birds up to 250g.
In order to prevent the animal from experiencing pain, concussion must be produced rapidly and with sufficient force. If conducted rapidly and effectively, the technique is humane and is listed on Schedule 1 and Annex IV for rodents up to 1kg bodyweight and for birds up to 250g.
Advantages and disadvantages of this method
The method is rapid and humane when conducted correctly, and allows tissues to be obtained rapidly, and without contamination with anaesthetic agents. Combining concussion with immediate cervical dislocation ensures rapid loss of consciousness and rapid death. The rapid loss of consciousness caused by concussion reduces concerns related to the persistence of consciousness after the use of cervical dislocation.
No specialised equipment or chemical agents are needed. Personnel may be reluctant to conduct the procedure because they find it distressing. Trauma to the skull and brain may make this method unsuitable if the cerebral tissue is needed.
No specialised equipment or chemical agents are needed. Personnel may be reluctant to conduct the procedure because they find it distressing. Trauma to the skull and brain may make this method unsuitable if the cerebral tissue is needed.
Safety considerations
The procedure is safe for the operator providing that the animal is handled and restrained correctly during the procedure.
Overdose of carbon dioxide (adult rodents)
Animals are placed in a chamber and exposed to a rising concentration of carbon dioxide. The gas produces tissue acidosis and loss of consciousness, followed by death from respiratory and cardiac failure.
As with all methods of euthanasia, death must be confirmed after carrying out the procedure.
Making the technique as humane as possible
As with all methods of euthanasia, the animal must be handled carefully and calmly.
The carbon dioxide should be supplied from compressed gas cylinders, in a controlled way, so that animals are exposed to a gradually rising concentration of the agent. Exposure to concentrations of carbon dioxide above 40% cause a burning/painful sensation in people, and the nasal mucosa of animals is equally sensitive (reviewed in Valetentim et al, 20162). The aim of using a rising concentration is so that the animal loses consciousness before a concentration greater than 40% is reached. Flow rates of 20-25% of the chamber volume per minute should achieve this. Once consciousness has been lost, the flow rate can be increased to speed death.
Lower concentrations of carbon dioxide have been shown to be aversive to rodents, and the use of this technique is controversial – although currently permitted on Schedule 1 of A(SP)A and Appendix IV of EU 2010/63 (Valetentim et al, 20163).
The carbon dioxide should be supplied from compressed gas cylinders, in a controlled way, so that animals are exposed to a gradually rising concentration of the agent. Exposure to concentrations of carbon dioxide above 40% cause a burning/painful sensation in people, and the nasal mucosa of animals is equally sensitive (reviewed in Valetentim et al, 20162). The aim of using a rising concentration is so that the animal loses consciousness before a concentration greater than 40% is reached. Flow rates of 20-25% of the chamber volume per minute should achieve this. Once consciousness has been lost, the flow rate can be increased to speed death.
Lower concentrations of carbon dioxide have been shown to be aversive to rodents, and the use of this technique is controversial – although currently permitted on Schedule 1 of A(SP)A and Appendix IV of EU 2010/63 (Valetentim et al, 20163).
An alternate approach
An alternative approach is to use an anaesthetic agent such as isoflurane to produce a loss of consciousness, followed by the administration of carbon dioxide. Anaesthetic agents are also aversive to rodents, but less so than carbon dioxide (Wong et al, 20124). This approach is considered preferable, provided the animals have not previously been exposed to anaesthetic agents (Hawkins et al, 20165).
Whilst evidence continues to be gathered on the relative benefits of these approaches, then if carbon dioxide alone is to be used, a number of other measures are recommended. Euthanasia in the animal’s home cage, with its familiar cage mates, may reduce the stress associated with the procedure, and this consideration also applies to use of volatile anaesthetic agents (Golledge et al, 20116).
Purchase of a purpose-designed apparatus that ensures the use of appropriate flows and encourages implementation of these measures is strongly recommended.
Whilst evidence continues to be gathered on the relative benefits of these approaches, then if carbon dioxide alone is to be used, a number of other measures are recommended. Euthanasia in the animal’s home cage, with its familiar cage mates, may reduce the stress associated with the procedure, and this consideration also applies to use of volatile anaesthetic agents (Golledge et al, 20116).
Purchase of a purpose-designed apparatus that ensures the use of appropriate flows and encourages implementation of these measures is strongly recommended.
Advantages and disadvantages of this method
Use of carbon dioxide allows euthanasia of large numbers of rodents at a time, and this can be a significant practical consideration, for example when humanely killing animals no longer required as a breeding colony. When using purpose-designed apparatus, it is technically simple to undertake and leaves no chemical residues in the cadavers.
Animals lose consciousness relatively rapidly (<2min), but may take a prolonged period to die. Before losing consciousness, carbon dioxide may cause fear and anxiety (Hawkins et al, 20167). Because death occurs relatively slowly, tissue hypoxia and marked acidosis will occur because of the effects of the carbon dioxide.
Although carbon dioxide is inexpensive, specialist equipment for euthanasia using the agent is relatively costly.
Neonatal animals are insensitive to the effects of carbon dioxide, and the method should not be used on rodents aged less than 7 days.
Animals lose consciousness relatively rapidly (<2min), but may take a prolonged period to die. Before losing consciousness, carbon dioxide may cause fear and anxiety (Hawkins et al, 20167). Because death occurs relatively slowly, tissue hypoxia and marked acidosis will occur because of the effects of the carbon dioxide.
Although carbon dioxide is inexpensive, specialist equipment for euthanasia using the agent is relatively costly.
Neonatal animals are insensitive to the effects of carbon dioxide, and the method should not be used on rodents aged less than 7 days.
Safety considerations
Carbon dioxide is most unlikely to accumulate in the room environment to produce concentrations that present a hazard to personnel. Placing animals still in their home cage directly into the apparatus avoids any risk of injury during handling. The requirement to have large, heavy cylinders of carbon dioxide presents manual handling issues.
Overdose of anaesthetic – inhalational agents (adult rodents)
Animals are exposed to a volatile anaesthetic, usually at the concentration normally used to produce anaesthesia. When anaesthetising an animal, the concentration of anaesthetic would be reduced after a loss of consciousness, but if the initial high concentration is maintained this causes death by cardiac and respiratory arrest. Note that this can take up to 20-30 minutes.
As with all methods of euthanasia, death must be confirmed after carrying out the procedure.
Making the technique as humane as possible
As with all methods of euthanasia, the animal must be handled carefully and calmly. The anaesthetic should normally be delivered from a standard anaesthetic vaporiser, using oxygen or air as the carrier gas. If other methods are used (e.g. production of very high concentrations of anaesthetic using a vapour wand (Hodgson, 20078) or anaesthetic liquid placed directly onto a gauze swab) then the animal must not come into contact with liquid anaesthetic since this is very irritant to the eyes and mucus membranes.
The concentration of anaesthetic in the chamber should rise rapidly to the usual induction concentration (e.g. 5% isoflurane) so that consciousness is lost rapidly, and a prolonged period of uncoordinated movements and semi-consciousness is avoided. This requires the use of an appropriately sized chamber (less than 10l volume).
However, using purpose-built apparatus with a specialised vaporiser that can produce high volumes of anaesthetic vapour enables a much larger chamber to be used. This enables animals to be euthanased in their home cage which will reduce stress and distress caused by handling. Clearly, this is only possible if all the animals in the cage are to be euthanased.
All of the modern inhalational agents can be used, but ether is unsuitable as it is highly irritant, and explosive when mixed with air or oxygen
The concentration of anaesthetic in the chamber should rise rapidly to the usual induction concentration (e.g. 5% isoflurane) so that consciousness is lost rapidly, and a prolonged period of uncoordinated movements and semi-consciousness is avoided. This requires the use of an appropriately sized chamber (less than 10l volume).
However, using purpose-built apparatus with a specialised vaporiser that can produce high volumes of anaesthetic vapour enables a much larger chamber to be used. This enables animals to be euthanased in their home cage which will reduce stress and distress caused by handling. Clearly, this is only possible if all the animals in the cage are to be euthanased.
All of the modern inhalational agents can be used, but ether is unsuitable as it is highly irritant, and explosive when mixed with air or oxygen
Advantages and disadvantages of this method
Modern volatile anaesthetics are relatively non-irritant and less aversive to rodents than carbon dioxide. They are also relatively safe so animals can take a considerable time to die (up to 60 min). Switching to carbon dioxide exposure after the animals have lost consciousness greatly speeds up the process. However, sufficient time must be allowed for anaesthesia to deepen so that animals do not recover consciousness during exposure to carbon dioxide. Alternatively, higher concentrations of anaesthetic can be delivered, but this may cause greater aversion before a loss of consciousness.
Although the tissues will contain anaesthetic residue after death, the anaesthetic rapidly dissipates. Few effects on tissue histopathology are produced, but some pulmonary effects can be seen. Because death occurs relatively slowly, tissue hypoxia will occur.
The agents are all easily available.
Although the tissues will contain anaesthetic residue after death, the anaesthetic rapidly dissipates. Few effects on tissue histopathology are produced, but some pulmonary effects can be seen. Because death occurs relatively slowly, tissue hypoxia will occur.
The agents are all easily available.
Safety considerations
Exposure of personnel to volatile anaesthetic vapour must be avoided because of health and safety concerns. This can be achieved either by using active scavenging equipment that removes waste gas or by carrying out the procedure in a safety cabinet, with venting of the extract outside the building.
Spilling large quantities of volatiles anaesthetic (e.g. by dropping a bottle of the agent) represents a significant safety hazard. If this occurs, it is important to leave the room immediately, prevent anyone else from entering, and then following locally agreed procedures to control the spillage. In a well-ventilated room, simply leave the room unoccupied, with the door closed, and wait a few hours for the anaesthetic to evaporate and the vapour to disperse.
Compressed gas cylinders to provide a carrier gas (usually oxygen) to deliver the anaesthetic vapour are required, and these present manual handling issues, and require safe and secure storage. Check on local procedures before commencing work that requires use or handling of gas cylinders.
Spilling large quantities of volatiles anaesthetic (e.g. by dropping a bottle of the agent) represents a significant safety hazard. If this occurs, it is important to leave the room immediately, prevent anyone else from entering, and then following locally agreed procedures to control the spillage. In a well-ventilated room, simply leave the room unoccupied, with the door closed, and wait a few hours for the anaesthetic to evaporate and the vapour to disperse.
Compressed gas cylinders to provide a carrier gas (usually oxygen) to deliver the anaesthetic vapour are required, and these present manual handling issues, and require safe and secure storage. Check on local procedures before commencing work that requires use or handling of gas cylinders.
Overdose of anaesthetic – injectable agents (adult rodents)
Injectable anaesthetics can be used for euthanasia when administered at much higher doses than those used for anaesthesia. The most suitable agents are those that have a very small difference between the dose needed for anaesthesia, and the dose that will kill the animal. The most widely used agent is pentobarbital. To reduce the volume needed to carry out euthanasia, higher concentration solutions are normally used (200mg/ml, compared to 60mg/ml for anaesthesia). Pentobarbital is administered either intravenously (e.g. into a tail vein in rodents) or intraperitoneally (into the abdomen) at 3-4 times the dose used to produce anaesthesia. A high dose rate of this agent produces loss of consciousness followed by respiratory and cardiac arrest. When administered by intravenous injection, loss of consciousness and death is achieved very rapidly (within 20-30 seconds). Intraperitoneal injection results in death within 5-10 minutes.
As with all methods of euthanasia, death must be confirmed after carrying out the procedure.
Making the technique as humane as possible
As with all methods of euthanasia, the animal must be handled carefully and calmly. Personnel carrying out the procedure must be confident and competent in carrying out the injection procedure.
Intravenous administration is preferable when this is practicable, and the minor pain caused by insertion of the needle can be prevented by use of local anaesthetic cream (Flecknell et al, 19909). Using a small diameter needle may cause less discomfort on insertion through the skin, but using a small needle can limit the speed of injection of material and this can be a significant factor in large species. In small rodents, 25-27g needles are suitable for intravenous injection into the tail vein.
Intraperitoneal administration is commonly used in rats and mice since intravenous administration is more difficult than in larger species.
Pentobarbital has a high pH, and intraperitoneal injection is associated with pain, prior to a loss of consciousness. This can be prevented by mixing the pentobarbital with local anaesthetic (Svendsen et al, 200710). Because the mixture is unstable, this must be done immediately prior to using the solution.
Intravenous administration is preferable when this is practicable, and the minor pain caused by insertion of the needle can be prevented by use of local anaesthetic cream (Flecknell et al, 19909). Using a small diameter needle may cause less discomfort on insertion through the skin, but using a small needle can limit the speed of injection of material and this can be a significant factor in large species. In small rodents, 25-27g needles are suitable for intravenous injection into the tail vein.
Intraperitoneal administration is commonly used in rats and mice since intravenous administration is more difficult than in larger species.
Pentobarbital has a high pH, and intraperitoneal injection is associated with pain, prior to a loss of consciousness. This can be prevented by mixing the pentobarbital with local anaesthetic (Svendsen et al, 200710). Because the mixture is unstable, this must be done immediately prior to using the solution.
Advantages and disadvantages of this method
Intravenous administration of the anaesthetic results in rapid effects and produces a smooth and rapid loss of consciousness. Intraperitoneal injection can be associated with pain on injection (see “making the technique as humane as possible) and results in a much slower onset of loss of consciousness and slower time to death. Because death occurs relatively slowly after intraperitoneal injection, tissue hypoxia will occur.
Intravenous injection is technically challenging but reasonably easy to master. Intraperitoneal injection is technically easier. Intravenous administration requires firm restraint of the animal, which can be stressful. Intraperitoneal administration requires restraint for a shorter period.
It is possible to gain experience in both methods by practising injections in animals that have been killed using another method, immediately after their death has been confirmed.
Many of the anaesthetic agents used are controlled drugs (under the Misuse of Drugs regulations, 2001 in the UK). For example barbiturates such as pentobarbital need to be stored securely and their use recorded. Your research animal facility will be able to provide detailed guidance on local arrangements for compliance with the relevant regulations.
Intravenous injection is technically challenging but reasonably easy to master. Intraperitoneal injection is technically easier. Intravenous administration requires firm restraint of the animal, which can be stressful. Intraperitoneal administration requires restraint for a shorter period.
It is possible to gain experience in both methods by practising injections in animals that have been killed using another method, immediately after their death has been confirmed.
Many of the anaesthetic agents used are controlled drugs (under the Misuse of Drugs regulations, 2001 in the UK). For example barbiturates such as pentobarbital need to be stored securely and their use recorded. Your research animal facility will be able to provide detailed guidance on local arrangements for compliance with the relevant regulations.
Safety considerations
Inadvertent self-injection can have serious consequences (e.g. injection of a large quantity of ketamine being used to sedate a large animal, prior to euthanasia), but this is a relatively minor risk when handling the small quantities needed for euthanasia of rodents. Nevertheless, care should be taken when preparing material for injection, and when carrying out the procedure.
A more important concern relates to the potential misuse of the anaesthetic agents as drugs of abuse, and they must be stored securely and records maintained of the quantities used.
A more important concern relates to the potential misuse of the anaesthetic agents as drugs of abuse, and they must be stored securely and records maintained of the quantities used.
Summary table of methods for adult rodents
Table: Methods of Euthanasia| Method | Time to loss of consciousness | Time to death |
|---|---|---|
| Cervical Dislocation | <10-15 seconds | <10-15 seconds |
| An overdose of Carbon Dioxide (20-30% chamber volume per min) | 1-3 minutes | 5-10 minutes |
| An overdose of Anaesthetic by intravenous injection | 1-3 minutes | 5-10 minutes |
| An overdose of Anaesthetic by intraperitoneal injection | 1-3 minutes | 4-10 minutes |
| An overdose of Anaesthetic by inhalation (at normal concentration for anaesthetic induction) | 1-3 minutes | 20-30 minutes |
| Concussion by striking the cranium (followed by cervical dislocation) | <0.1 seconds | <5 seconds |
(references, Valentine et al, 201211, Cartner et al, 200712, Schoell et al, 2009, Boivin et al, 201613, Hickman et al, 2016, Kongara et al, 201314)
Neonatal Rodents and fetal animals
Neonatal and fetal animals are highly resistant to hypoxia and to the effects of carbon dioxide. Exposure to carbon dioxide is therefore not recommended as a method for these animals. Injection of an overdose of anaesthetic (e.g. pentobarbital i/p) is rapidly effective. Cervical dislocation is effective, but practically difficult to conduct in a similar way to in an adult animal because of the smaller size of neonates. Decapitation using a scalpel blade or sharp scissors is usually simpler to carry out, but it is uncertain how long consciousness persists after decapitation (Hawkins et al, 201615). Schedule 1 in the UK permits euthanasia of fetuses by cooling (to around 4 degrees C) followed by immersion in cold tissue fixative.
It has been argued that since fetal animals are not conscious whilst they remain in-utero, (Mellor and Diesch, 200616; Cambell et al, 201417), if late-pregnant animals are to be killed, and there is no scientific need to remove the fetal animals to obtain tissues, then the mother should be humanely killed, for example with an overdose of anaesthetic. The fetuses will die in utero from hypoxia, but without becoming conscious.
References
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- Valentim, A. M., Guedes, S. R., Pereira, A. M. and Antunes, L. M. (2016) ‘Euthanasia using gaseous agents in laboratory rodents’, Laboratory animals, 50(4), pp. 241–253. doi: 10.1177/0023677215618618.
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- Golledge, H.; Lukic, J.; Flecknell. P.A. The effect of behavioural state and cage environment on 1086 responses to euthanasia with isoflurane or carbon dioxide in BALB/c mice. In: Altex: 8th World 1087 Congress. 2011, Montreal, Canada: Springer Spektrum
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- Flecknell, P.A., Liles, J.H. and Williamson, H.A. (1990). The use of lignocaine-prilocaine local anaesthetic cream for pain-free venepuncture in laboratory animals. Laboratory Animals, 24, 142-146.
- Svendsen, O., Kok, L. and Lauritzen, B. (2007) ‘Nociception after intraperitoneal injection of a sodium pentobarbitone formulation with and without lidocaine in rats quantified by expression of neuronal c-fos in the spinal cord—a preliminary study.’, Laboratory animals. SAGE Publications, 41(2), pp. 197–203. doi: 10.1258/002367707780378140.
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- Cartner, Samuel C., Shayne C. Barlow, and Timothy J. Ness. “Loss of cortical function in mice after decapitation, cervical dislocation, potassium chloride injection, and CO2 inhalation.” Comparative medicine 57.6 (2007): 570-573.
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- Kongara, K., McIlhone, A., Kells, N. and Johnson, C. (2013) ‘Electroencephalographic evaluation of decapitation of the anaesthetized rat’, Laboratory animals, 48(1), pp. 15–19. doi: 10.1177/0023677213502016.
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- Campbell, M., Mellor, D. J. and Sandøe, P. (2014) ‘How should the welfare of fetal and neurologically immature postnatal animals be protected?’, Animal Welfare. Universities Federation for Animal Welfare, 23(4), pp. 369–379. doi: 10.7120/09627286.23.4.369.