WORKING PARTY REPORT Recommendations for euthanasia of experimental animals: Part 2 Working party: Mrs Bryony Close (Chair), Dr Keith Banister, Dr Vera Baumans, Dr Eva-Maria Bernoth, Dr Niall Bromage, DrJohn Bunyan, Professor Dr Wolff Erhardt, Professor Paul Flecknell, Dr Neville Gregory, Professor Dr Hansjoachim Hackbarth, Professor David Morton & Mr Clifford Warwick Correspondence to: Mrs B Close, Battleborough Croft, Battleborough Lane, Brent Knoll, Highbridge, SomersetTA9 4DS, UK This document was prepared for DGXI of the European Commission to be used with Directive 86/609/EEC of 24 November 1986, on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scienti®c purposes (No L 358, ISSN 0378-6978). It refers especially to Article 2(1) published by the European Commission in October 1995 which de®nes `humane methods of killing' as `the killing of an animal with a minimum of physical and mental suffering, depending on the species'. This is the second part of the working Contents to Part 2 party's report and comprises Section 3 of 3 Methods of euthanasia for each species the report, the list of all references group 2 cited in both parts and details of training 3.1 Fish 2 materials. The ®rst part, comprising 3.2 Amphibians 5 Sections 1 and 2 of the report together 3.3 Reptiles 8 with a reading list, was published in the 3.4 Birds 10 October 1996 issue of Laboratory Animals 3.5 Rodents 14 (30: 293±316). Reprints combining both 3.6 Rabbits 17 parts of the report will be available 3.7 Carnivores: dogs, cats, ferrets 20 from Mrs S E Wolfensohn, Supervisor of 3.8 Large mammals: pigs, sheep, Veterinary Services, University of goats, cattle, horses 23 Oxford, Veterinary Services, c/o 3.9 Non-human primates 25 University Laboratory of Physiology, 3.10 Other animals not commonly Parks Road, Oxford OXI 3PT, UK used for experiments 26 (Tel: +44(0)1865-272545, References 26 Fax: +44(0)1865-272118, Email: [email protected]). Euthanasia training materials 29 Accepted 15 February 1996 LaboratoryAnimals (1997) 31,1^32 2 Working Party Report 3 Methods of euthanasia for each internal ice crystals form. However, it must be species group remembered that cooling does not reduce the ability to feel pain. Section 1 must be consulted before consid- Overexposure to an anaesthetic is ®rst ering this section. manifested by a cessation of respiratory movements, followed by spasmodic overex- 3.1 Fish tension or ¯aring of the opercula. These are spaced 15±30 s apart at ®rst and then at longer There are over 20 000 species of ®sh with intervals. When the interval between spasms enormously varying lifestyles which makes is approximately 1 min, cardiac arrest and it very dif®cult to generalize on methods of death follow within a few minutes (Table 1). euthanasia. The methods listed below are meant as a guide and the operator must Recognition and con®rmation of death assess which is the best method for the Death may be recognized by cessation of respir- species being killed or obtain information ation (opercular movement) and cessation of from experts. A summary is given in Table 2. heartbeat(palpation).Deathshouldbecon®rmed Although ®sh may not have the same by destruction of the brain where possible. spinothalamic pathways as mammals for pain perception, there is evidence that they Larvae do feel pain and should therefore be killed Fish may be classed as oviparous, ovovivi- with the same care and consideration. parous or viviparous depending on whether All ®sh are sensitive to changes in the they produce eggs which hatch outside the physical and chemical parameters of their body, produce eggs which hatch inside the water (especially temperature, dissolved gas body, or give birth to free-living young levels, salinity, pH, etc.) but some species are much more tolerant of changes in any one of Table1 Stages of loss of consciousness, leading to these factors than are others. Therefore unless death in fish (after McFarland and Klontz, 1969) the species' response is known it is advisable to practise euthanasia in the ®sh's normal Level Designation Parameter(s) water. If drugs are used the water level should 0 Normal Reactive to external stimuli; be lowered to ensure rapid sedation but not so equilibrium and muscle tone much as to distress the ®sh before the addition normal of the agent. Dosing is always preferable to 1 Light Slight loss of reactivity to external injection as the latter technique involves sedation visual and tactile stimuli; handling the ®sh and thus induces stress. It equilibrium normal may be necessary to fast ®sh 24±48 h prior to 2 Deep Total loss of reactivity to external chemical euthanasia as this will permit more sedation stimuli except strong pressure; slight decrease in opercular rapid absorption by the gut and minimize the rate; equilibrium normal risk of regurgitation which could reduce the 3 Partial Partial loss of muscle tone; effect of the chemicals on the gill lamellae loss of swimming erratic; increased (Brown 1988). The tanks used should enable equilibrium opercular rate; reactive only to the operator to observe the ®sh and react strong tactile and vibrational quickly if there are signs of suffering. It is stimuli generally true that cooling will reduce the 4 Total loss of Total loss of muscle tone and equilibrium equilibrium; rapid opercular metabolic and locomotory processes, thus rate; reactive only to deep facilitating handling, but it is essential that pressure stimuli the normal temperature of the ®sh and its 5 Loss of reflex Total loss of reactivity; opercular degree of tolerance be considered. It is also reactivity movements very shallow; heart vital to note that in marine ®sh ice crystals rate very slow will form in the cells before the sea water 6 Medullary Opercular movements cease collapse immediately after gasping, freezes, thereby causing the ®sh extreme pain. followed by cardiac arrest In freshwater ®sh the water will freeze before Euthanasia of experimental animals 3 (larvae). For simplicity, it is considered that type drug and is the most effective way to kill all ®sh should be protected as soon as they most ®sh. It is soluble in both salt and fresh have hatched and the methods of euthanasia water. However, it is expensive and so may recommended for adults are considered deter some users, especially if they need to acceptable for larvae. Viviparous ®sh should kill large numbers of ®sh. Bicarbonate, be treated by immersion or injecting the imidazole, sodium hydrogen phospate or parent. sodium hydroxide should be added to neu- tralize the water (to pH 7.5) to reduce Adults irritation and tissue damage. It may be used Further details of methods may be found in in conjunction with quinaldine or quinaldine Section 2. sulphate to increase effectiveness. Physical methods Benzocaine (ethyl aminobenzoate) This acts in a similar way to MS-222 but has Concussion This involves a blow to the pH-independent ef®cacy. However, because back of the head and if carried out by it lowers the pH of the water, it should be experienced personnel is a humane method neutralized to pH 7.5. The breakdown time of euthanasia. Death must be con®rmed by in water is about 4 h, thus making this drug destruction of the brain. acceptable in terms of environmental con- tamination. As it is insoluble in water Cervical dislocation This is the breaking of benzocaine should ®rst be dissolved in the backbone near the head. Small to acetone before adding to water. medium ®sh are killed by inserting a rod or thumb into the mouth, holding the ®sh with Etomidate It is a potent imidazole-based the opposite hand and displacing it dorsally agent with no analgesic properties. It is (Clifford 1984). It is feasible and effective in highly soluble in water. Stress hormone small ®sh, but should be con®rmed by measurements of ®sh have indicated that exsanguination or destruction of the brain. etomidate may give fewer problems than MS- The stress caused by handling reduces the 222 (Zwart et al. 1989) and it is therefore acceptability of this method. It is not considered acceptable for euthanasia of ®sh. possible or humane in larger ®sh. Metomidate It is an imidazole-based non- Maceration Small ®sh of less than 2 cm in barbiturate hypnotic agent that has no analge- length may be humanely killed by placing sic properties. Used in overdose, this is down a waste disposal unit. considered acceptable for killing most species of ®sh. Chemical methods Quinaldine (2-methylquinoline) This is dif- Agents can be administered by dissolving the ®cult to obtain in Europe but is commonly chemical in the tank water. Water tempera- used in the USA for humane euthanasia. The tures often alter the ef®cacy of the drug and recommended dosages for euthanasia vary induction is often more rapid at higher depending upon species, temperature and temperatures. However, the temperature must water hardness. It accumulates in lipid tissues not be raised so that it causes any stress to the such as the brain and depresses the sensory ®sh. Drugs may also be administered by centres of the central nervous system. Qui- intramuscular or intraperitoneal injection. For naldine sulphate is also considered acceptable euthanasia anaesthetic drugs are generally as an effective euthanasia agent for ®sh. used at double or triple the recommended anaesthetic dose. In all cases, death must be Halothane This may be bubbled through con®rmed by destruction of the brain. the tank and causes anaesthesia. Death must be ensured by destruction of the brain.