Humane methods of killing EU Module 6.1

Question 1

Can methods other than those listed in Annex IV be used for killing?

Answer 1

Other methods can be used if a method from Annex IV is unsuitable because of the requirements of a particular research project, but the alternative method used must be approved as part of the program of research work.

Question 2

What are potential scenarios where other methods can be used?

Answer 2

on unconscious animals, providing the animal does not regain consciousness before death;
on animals used in agricultural research, when the aim of the project requires that the animals are kept under similar conditions to those under which commercial farm animals are kept; these animals may be killed following the requirements laid down in Annex I to Council Regulation (EC) No 1099/2009 of 24 September 2009 on the protection of animals at the time of killing

Question 3

What can captive bolt be used on?

Answer 3

Reptiles (only to be used on large reptiles), Rabbits, Large Mammals

Question 4

What can electrical stunning be used on?

Answer 4

Fish, amphibians, birds, rabbits, dogs, cats, ferrets, foxes, large mammals.

Question 5

Which method should you choose?

Answer 5

The method chosen for killing our animals humanely must result in the minimum amount of pain and distress. This requires not only the careful choice of method and its correct application, but also the careful management of animals before and during the procedure. In many instances, the scientific requirements of a study must be considered alongside animal welfare concerns to ensure that death is as free from pain and distress as possible.

Question 6

How can death be confirmed?

Answer 6

confirmation of permanent cessation of the circulation
destruction of the brain
dislocation of the neck
exsanguination
confirmation of the onset of rigor mortis

Question 7

What can’t CO2 be used on foetuses and neonates

Answer 7

As they are very resistant to the effects of carbon dioxide

Question 8

What should we consider when killing pregnant animals?

Answer 8

When euthanasing both the mother and the foetuses, if possible, the mother should be humanely killed, and the foetuses die in the uterus without becoming conscious.

Question 9

Can CO2 be used on Rabbits?

Answer 9

carbon dioxide and inhalant anaesthetics seem more irritant to cause more distress in rabbits.

Question 10

Which methods can effect scientific results?

Answer 10

For example, some methods could directly affect blood biochemistry (e.g. many anaesthetics)
Tissues and organs can also be affected by the hypoxia and acidosis that results when using a method that causes death relatively slowly (e.g. carbon dioxide).
Finally, physical methods of euthanasia could damage tissues that are needed after the animal has been killed.

Question 11

How quickly are animals killed using Cervical Dislocation?

Answer 11

<10-15 seconds

Question 12

How quickly are animals killed using Exposure to Carbon Dioxide (gradual fill, e.g. 30-70% of chamber volume/min)?

Answer 12

5-10 minutes

Question 13

How quickly are animals killed using Overdose of Anaesthetic by intravenous injection?

Answer 13

< 10 seconds

Question 14

How quickly are animals killed using Overdose of Anaesthetic by intraperitoneal injection?

Answer 14

5-10 minutes

Question 15

How quickly are animals killed using Overdose of Anaesthetic by inhalation (at normal concentration for induction of anaesthesia)?

Answer 15

20-30 minutes

Question 16

How quickly are animals killed using Concussion by striking the cranium?

Answer 16

< 5 seconds

Question 17

How does cervical dislocation kill animals?

Answer 17

This technique aims to cause extensive and irreversible damage and disruption to the cervical spinal cord and brainstem by dislocating and compressing the cervical vertebrae.

Question 18

Reasons against cervical dislocation?

Answer 18

Haemorrhage into the lungs can occur shortly after death, and the brainstem and cerebellum can be damaged, as well as the cervical spine.

Question 19

How is Exposure to Carbon Dioxide carried out?

Answer 19

Animals are placed in a chamber and exposed to a rising concentration of carbon dioxide. The gas produces tissue acidosis and loss of consciousness through a direct effect on CNS function, followed by death from respiratory and cardiac failure

Question 20

Reasons against Exposure to Carbon Dioxide?

Answer 20

Because death occurs relatively slowly, tissue hypoxia and marked acidosis will occur because of the effects of carbon dioxide. Although carbon dioxide is inexpensive, specialist equipment for euthanasia using the agent is relatively costly.

Question 21

How is Overdose of Inhalation Anaesthetic carried out?

Answer 21

Animals are exposed to a rising concentration of inhalational anaesthetic agent (usually isoflurane). The process is similar to inducing anaesthesia, but the concentration of vapour delivered is maintained at 5%, and exposure to this concentration eventually produces cessation of respiration and circulation and death. Other inhaled agents have similar effects. Note that it can take 30-60 minutes before cardiac arrest occurs.

Question 22

Reasons against Overdose of Inhalation Anaesthetic.

Answer 22

Using inhaled methods of euthanasia generally results in death without any tissue damage, although some changes in lung histopathology can result, although this occurs more frequently if carbon dioxide is used after induction of anaesthesia with isoflurane.

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.

Inhalational anaesthetics are effective euthanasia agents in neonatal rodents.

Reptiles, amphibia and diving birds can hold their breath for very prolonged periods, so administering euthanasia agents by inhalation is not recommended.

Question 23

How is Overdose of injectable anaesthetic carried out?

Answer 23

After injection into the abdomen, the anaesthetic is gradually absorbed into the bloodstream and carried to the brain, producing slow loss of consciousness.

Pentobarbital is the most commonly used agent in laboratory rodents because of its long shelf life and rapidity of action.

If the overdose of an anaesthetic agent is administered intravenously, then loss of consciousness and death is very rapid.

Question 24

What are reasons against Overdose of injectable anaesthetic?

Answer 24

Many of the anaesthetic agents used are subject to legal controls relating to their acquisition, storage and use. 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.

The anaesthetic agent will be present in the tissues following euthanasia, and some evidence of peritoneal irritation may be present when anaesthetics have been injected by this route.

Question 25

How is concussion carried out?

Answer 25

triking the cranium, either by impact with a hard surface such as a bench top 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 and the degree of damage to the brain, so death should be ensured either by cervical dislocation or rapid exsanguination.

Question 26

Reasons against Concussion.

Answer 26

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.

Question 27

What methods can be used for other small rodents (e.g. Hamster, gerbil, guinea pigs)?

Answer 27

Annex IV permits the use of all of the techniques described above for rats and mice. Adult hamsters and gerbils may have a bodyweight of 110-140g, so although permitted under Annex IV, cervical dislocation may be technically more difficult, and sedation of the animals may be advisable. Similarly, cervical dislocation in adult guinea pigs, which generally weigh 600-1200g, is even more difficult to carry out humanely, so prior sedation or anaesthesia is recommended.

Question 28

What methods are used for Dogs, cats, ferrets and non-human primates?

Answer 28

Although Annex IV includes the killing of neonatal dogs, cats and ferrets by concussion, this method would rarely, if ever, be used. Similarly, killing these species with electrical stunning requires special apparatus which would only be used in very few establishments in exceptional circumstances. In research laboratories, these species would typically be killed by anaesthetic overdose, usually administered by intravenous injection. If required, a sedative agent may be given first - this would usually be required with Old World primates because of difficulties associated with physical restraint.

Question 29

What methods are used for Farm animals (pigs, sheep and cattle)?

Answer 29

When housed in a research facility, these species would usually be sedated and then killed with an intravenous injection of an anaesthetic agent. When used in agricultural research, and when the project goals require it, then these animals can be killed using recognised humane slaughter techniques,

Question 30

What methods are used for fish?

Answer 30

As with other species. the method of euthanasia used will vary depending on the fish species, its stage of development and the scientific purpose of the project. Annex IV lists three techniques, an overdose of anaesthetic, concussion and electrical stunning using specialised apparatus. Overdose of anaesthetic, usually by immersion, is the only method practicable in the most widely used species, zebrafish (Danio rerio), especially when killing juvenile fish. The most widely used anaesthetic is MS-222, which should be buffered with NaHCO3. A recent comparison of anaesthetics (von Krogh et al., 2021(opens in a new tab)) concluded that the preferred method was 1g/L lidocaine hydrochloride with 2 g/L NaHCO 3 and 50 mL/L 96% ethanol, which induced immobility in less than 10 s and with only 2% of adult zebrafish displaying aversive behaviours during treatment.

Question 31

What methods are used for Reptiles and Amphibia?

Answer 31

Annex IV lists anaesthetic overdose or concussion as methods of euthanasia. However, given the wide range of species used in research, and the different stages of development requiring euthanasia, it is strongly advised that advice is obtained as to the most appropriate agents and techniques to use. Although intra-coelomic pentobarbital is usually effective, the time taken for this and other anaesthetics to produce loss of consciousness, anaesthesia, and death are highly variable.

Question 32

What rate of fill is used for Exposure to carbon dioxide by gradual fill?

Answer 32

30-70% of chamber volume per minute

Question 33

Can captive bolt be used for sheep?

Answer 33

Yes

Question 34

What issues can be encountered when overdose of pentobarbital, administered intraperitoneally is used?

Answer 34

Injection of pentobarbital may cause some pain Correctly checked Intraperitoneal administration can have a high failure rate