1. Humane killing – factors to take into account when selecting the most appropriate techniques Dr Penny Hawkins RESEARCH ANIMALS DEPARTMENT

2. Terminology: Euthanasia = a good death -this usually means killing to end suffering for the benefit of the individual, which is not always the case for laboratory animals Humane killing is more accurate

3. What does ‘humane killing’ mean? Directive Article 3 defines the threshold for regulation of scientific procedures as: – “a level of pain, suffering, distress or lasting harm equivalent to, or higher than, that caused by the introduction of a needle in accordance with good veterinary practice” Truly ‘humane’ killing should cause no more than threshold suffering

4. Principles of humane killing The ideal is a ‘good’ death without suffering, treating the animal with respect and minimising anxiety, discomfort, pain or distress This usually means inducing unconsciousness as rapidly as possible – but not always As with scientific procedures, it is the animal’s total experience from capture to permanent loss of consciousness that counts

5. Article 6: methods of killing “6(1). Member States shall ensure that animals are killed with minimum pain, suffering and distress. 6(3). In relation to animals covered by Annex IV, the appropriate method of killing as set out in the Annex shall be used. 6(4). Competent authorities may grant exemptions from the requirement in paragraph 3: a)To allow the use of another method provided that, on the basis of scientific evidence, the method is considered to be at least as humane; or b)When, on the basis of scientific justification, the purpose of the procedure cannot be achieved by the use of a method of killing set out in Annex IV.”

6. Annex IV: Methods of killing animals “1. In the process of killing animals, methods listed in the table below shall be used.” Many notes and requirements

7. Annex IV: exemptions from the table “1. Methods other than those listed in the table may be used: a)on unconscious animals, providing the animal does not regain consciousness before death; b)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 in accordance with 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.” These exemptions are in place for scientific reasons, for example to allow perfusion-fixation or if there is scientific justification for keeping and killing farm animals according to commercial practice Any other killing techniques required for scientific purposes, e.g. microwave, are regulated procedures and require authorisation

8. Annex IV: killing shall be completed “2. The killing of animals shall be completed by one of the following methods: a)confirmation of permanent cessation of the circulation; b)destruction of the brain; c)dislocation of the neck; d)exsanguination; or e)confirmation of the onset of rigor mortis.” This aims to prevent suffering if the killing method was not effective, for example if animals recover from CO 2 inhalation or the neck was not completely dislocated

9. Is there scientific justification that the purpose of the procedure cannot be achieved using another method (Article 6 4b) – AND do the benefits justify the harms? Is there scientific evidence that the method is as humane as those in Annex IV (Article 6 4a)? Is the right equipment, training and expertise available? Use the method, ensuring that it has been fully refined Ensure that all of these are acquired Is there a justifiable scientific requirement for a particular method? Review the methods permitted in Annex IV, considering: the experience of the animal current knowledge and approaches any welfare concerns with particular methods Choose the method that will cause least suffering between capture of the animal (or administration) and permanent loss of consciousness Choose the most effective method for confirming death, taking into account any need to preserve tissues Do not use the method, redesign project Yes - in Annex IV Yes – not in Annex IV Yes No First ask the question: Does the animal have to be killed, or could there be an alternative option?

10. Focus on Annex IV Assuming there is no scientific requirement for a particular method The most appropriate method will depend upon many factors, including: The species, stage of development and size of the animal The training and competence of staff in each technique The availability of required equipment www.ahwla.org.uk

11. Killing animals is a process Any need for capture, handling or restraint Whether the animal must be removed from the home cage Whether the animal will be mixed with other, unfamiliar animals The practical application of the technique – whether it is painful or distressing What the animal experiences between application and final loss of consciousness How death will be confirmed, and how soon

12. Decision-making criteria Welfare-related Focus on the animal’s experience Capture Handling Restraint Transport Relocation Pain Aversion to agent Suffering between administration and permanent loss of consciousness Success rate Practical issues ‘Aesthetics’ Speed Economics Welfare-related criteria should come first, to comply with the Directive

13. Anaesthetic overdose Welfare concernRefinement Often involves catching, transporting or restraining the animal Avoid or minimise these stressors, e.g. by moving (or even killing) animals in home cage Prior sedation is permitted; sedate animals if it will reduce overall stress Some anaesthetic agents can be aversive when inhaled, or irritant when injected (e.g. pentobarbital sodium, PBS is highly alkaline) Monitor animals carefully during the process, identifying causes for concern such as rearing Research which gaseous agents are least aversive and which flow rates are believed to cause least suffering for the species and strain Use buffered pentobarbital sodium MS-222 is commonly used to kill fish, but is aversive to zebrafish Aversive responses not observed when zebrafish exposed to etomidate or 2,2,2 tribromoethanol

14. Carbon dioxide – gradual fill Was thought of as ‘humane’ and ‘aesthetic’ Telemetry (EEG), physiology and behavioural studies have proven otherwise Two welfare concerns: pain and distress – CO 2 + H 2 O H 2 CO 3 H + + HCO 3 - – Distress may be dyspnoea (‘air hunger’), which is very distressing in humans and also likely to be distressing for other animals, or CO 2 -induced fear

15. Refining CO 2 delivery: flow rate Placing animals into 100 % CO 2 would kill rapidly, but be extremely painful Current thinking is that it is causes least suffering to introduce 100% CO 2 at a flow rate of 20% of the chamber volume per minute This appears to cause loss of consciousness before animals experience pain Once animals are unconscious, the CO 2 flow rate can be increased to speed the process

16. Pain is just part of the problem CO 2 causes dyspnoea, anxiety and distress and is aversive to rats and mice Massive increases in adrenaline and noradrenaline Mice and rats leave the apparatus when [CO 2 ] reaches 12 – 17 %

17. 0100200300400 Unconsciousness at around 35 % Death seconds ‘Air hunger’ at 10-15 %Pain at around 50 % CO 2 exposure timeline dyspnoea

18. Refining Co 2 delivery: reducing distress Some facilities give isoflurane or sevoflurane before switching to CO 2 Using a carbon dioxide diffuser can allow smoother distribution within the chamber compared with direct application through an inlet tube – This may reduce anxiety and distress during the process CO 2 coming from a gas cylinder may be uncomfortably cold for the animals, so a delivery system that also warms the gas is preferred After use, the chamber must be emptied of the residual CO 2 before the next animals are put into it

19. Current thinking on best practice for CO 2 Introduce CO 2 to home cages if possible Keep animals in established groups and avoid mixing unfamiliar individuals Research the benefits of anaesthetising animals first If not, then use rising concentration at 20% volume/minute, use a diffuser and warm the CO 2 for conscious animals Keep up-to-date on developments regarding the use of alternative gaseous agents

20. Cervical dislocation Believed to be humane, provided that operator is trained, competent and presented with realistic workloads But a study found that, even using most successful technique, 10 % of mice still breathed for over 3 minutes Carbone et al. (2012) PMID 22776194 http://www.ncbi.nlm.nih.gov/pubmed

21. Refining cervical dislocation Ensure that staff are trained, competent and willing to use physical methods Train using cadavers or anaesthetised animals Monitor success rates – time to cease breathing should be almost instant Decide on acceptable success rates and retrain or remove operators who cannot maintain these

22. Decapitation Decapitation is permitted for – birds under 250 g – this includes adults of some species – rodents if ‘other methods are not possible’ Kongara et al. (2014) reports around 10 seconds of EEG consistent with consciousness and pain perceptions after decapitation in rats There is insufficient evidence that decapitation is humane in post-neonatal rodents Kongara et al. (2014) PMID 24367032 http://www.ncbi.nlm.nih.gov/pubmed

23. Other methods permitted in Annex IV that are poorly evidenced with respect to welfare Carbon dioxide for birds Inert gases (Ar, N 2 ) for birds and rodents – evidence relates to domestic fowl; Ar is highly aversive to rats No guidance at all for fetal animals or cephalopods

24. Completing killing If the killing method has been competently and effectively applied, the method for completing killing and confirming death should make no difference to the animal – But if the animal has not been killed, the use of rigor mortis to confirm death may cause suffering Otherwise, the science can take precedence when choosing a method to confirm death, e.g. if brain tissue is required then ‘destruction of the brain’ will clearly not be suitable

25. Factors not relating to animal welfare ‘Aesthetics’ – because people’s feelings matter too – Staff may be uncomfortable using some physical methods Speed – only if no additional suffering is caused Economics –the money saved could be spent on welfare

26. Concluding comments Use Annex IV critically – – research into humane killing is ongoing – some of the methods are poorly evidenced or could cause suffering Do not let economic factors or convenience take precedence over animal welfare Use the same approaches as reducing suffering in procedures – – take a ‘team approach’ to refinement – keep up with the literature and current thinking – seek advice from the animal-welfare body Remember that even ‘humane’ killing is one of the ‘harms’ in the harm-benefit assessment, and part of the animal’s lifetime experience

27. Thank you