WORKING PARTY REPORT Recommendations for euthanasia of experimental animals: Part 1

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, Somerset TA9 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 method of killing' as `the killing of an animal with a minimum of physical and mental suffering, depending on the species'.

This report is published in two parts. Contents to Part 1 This ®rst part comprises Sections 1 Acknowledgements 294 and 2 of the report, together with a Preface 294 reading list. Section 3 of the report, 1 Introduction 295 1.1 Objectives of euthanasia 295 together with the list of all references 1.2 De®nition of terms 295 cited in both parts and details of 1.3 Signs of pain and distress 296 training materials, will be published in 1.4 Recognition and con®rmation of death 296 the January 1997 issue of Laboratory 1.5 Personnel and training 297 Animals. Reprints combining both 1.6 Handling and restraint 297 parts of the report will be available 1.7 Equipment 297 1.8 Carcass and waste disposal 298 from Mrs S E Wolfensohn, Supervisor 2 General comments on methods of of Veterinary Services, University of euthanasia 298 Oxford, Veterinary Services, c/o 2.1 Acceptable methods of University Laboratory of Physiology, euthanasia 298 2.2 Methods acceptable for Parks Road, Oxford OX1 3PT, UK. unconscious animals 305 (Tel:+44(0)1865-272545, 2.3 Methods that are not Fax:+44(0)1865-272118, acceptable for euthanasia 306 Email: [email protected]). Further reading 309

Accepted 15 February 1996 LaboratoryAnimals (1996) 30, 293^316 294 Working Party Report

Acknowledgements used in experiments and for other scienti®c purposes in assessing which method of We would like to thank the European euthanasia is the most humane and appro- Commission DGXI for providing funding for priate for the species of animal that they are this report and also Laboaratory Animals Ltd using. A brief description of each method is for publishing it and making reprints avail- given with reasons for accepting or rejecting able in order to achieve its widespread them. Details of how to carry out different distribution. methods are not provided; these may be We would like to thank the following found in references cited and in the recom- people and organizations who provided valu- mended reading list. able assistance and comments on the test: Dr Methods classi®ed as `acceptable' are those J Anderson (Animals (Scienti®c Procedures) that are considered humane for use on Inspectorate, UK Home Of®ce), Dr N conscious or lightly sedated animals. Other Baudrihaye (European Federation of Pharma- methods may be acceptable only if used on ceutical Industries' Associations), Professor J heavily sedated or unconscious animals. In Bourne (Institute for Animal Health, UK), Dr principle, all methods can be used on D Forbes (Laboratory Animal Science Asso- unconscious animals unless they are unac- ciation, UK), Professor K GaÈrtner (Medizi- ceptably dangerous to personnel or there is a nische Hochschule Hannover, Germany), Mr risk of the animal regaining consciousness J A Gregory (Institute of Animal Technology, before death occurs. Methods included under UK), Professor O HaÈnninen (Secretary those `acceptable for unconscious animals' General, ICLAS), Mrs R Harrison (UK), Dr F are those most frequently used in practice. R Homberger (University of Zurich, The last category of methods `not acceptable' Switzerland), Mr T D Hornett (Glaxo Re- are not to be used for the reasons provided in search and Development, UK), Dr K Iwarsson each case. (Karolinska Institutet, Sweden), Dr T Jenes- There are three main sections to this kog (National Board for Laboratory Animals report. Section 1 deals with general notes on (CFN), Sweden), Dr M Jennings (Royal legislative requirements of the 1986 Council Society for the Prevention of Cruelty to Directive of the EEC, general requirements of Animals, UK), Dr G Mahouy (Institut euthanasia, de®nitions of terms, and other d'HeÂmatologie, Universite de Paris, France), factors to be considered when killing experi- Professor R Murison (University of Bergen, mental animals. Section 2 provides informa- Norway), Mr P Nowlan (University of Du- tion on methods of euthanasia used for blin, Ireland), Professor C Rehbinder (Na- vertebrates and is divided broadly into tional Board for Laboratory Animals (CFN), acceptable physical and chemical methods, Sweden), Mr A Sainsbury (Institute of methods acceptable for insensible animals, Zoology, London), Professor P Schambye and those methods not considered accepta- (Board of Animal Experiments Inspectorate, ble. Section 3 covers each group of species Denmark), Dr W Scharmann (Bundesge- from ®sh to primates with general informa- sundheitsamt, Germany), Professor U tion pertaining to the species, including Schatzmann (UniversitaÈt Bern, Switzerland), recommendations on embryonic and larval Dr D Straughan (UK), Dr P Terpstra (CRC forms. Methods of euthanasia are listed and Contract Research Center, Belgium), Profes- brie¯y discussed. At the end of each species sor J E van Dijk (University of Utrecht, the section, there is a table summarizing the Netherlands), Mr D Wilkins (Eurogroup for recommendations for that species. Animal Welfare), Dr J Wong (Canadian There are, in addition, comprehensive lists Council on Animal Care). of cited references and literature recom- mended for further reading (divided into Preface general and species groups), together with information on audiovisual training materi- This report has been produced in order to als that may be used in training programmes assist personnel concerned with animals to encourage humane euthanasia practices. Euthanasia of experimental animals 295

It is recommended that all personnel read physical and mental suffering, depending on Section 1. If information is required about a the species. particular method, this may be obtained in Whilst this document provides recom- Section 2, and if information is required mendations for the euthanasia of experi- about a particular species, this may be found mental animals, it is strongly recommended in Section 3. that controls and guidelines issued in other EC directives and regulations for the eutha- nasia of animals be taken into consideration 1 Introduction (e.g. Council Directive 93/119/EC (Commis- sion of the European Communities 1993)). Animals are killed in laboratories or breeding establishments for various reasons: 1.1 Objectives of euthanasia

. at the end of an experiment or when there The primary criteria for euthanasia in terms might be continuing adverse effects; of animal welfare are that the method be . to provide blood and other tissues for a painless, achieve rapid unconsciousness and scienti®c purpose; death, require minimum restraint, avoid . when levels of pain, distress and suffering excitement, is appropriate for the age, are likely to exceed the designated level; species, and health of the animal, must . where the health or welfare of the animals minimize fear and psychological stress in the are grounds for concern; animal, be reliable, reproducible, irreversible, . when they are no longer suitable for simple to administer (in small doses if breeding; possible) and safe for the operator, and, so far . unwanted stock or those with unsuitable as possible, be aesthetically acceptable for characteristics, for example, type or sex, the operator. are not needed. 1.2 Definition of terms The Council Directive of 24 November The word euthanasia means a gentle death 1986 (Commission of the European Com- and should be regarded as an act of humane munities 1986) on the approximation of laws, killing with the minimum of pain, fear and regulations and administrative provisions of distress. the Member States regarding the protection Consciousness is the state of awareness of of animals used for experimental and other a normal animal when it can perceive stimuli scienti®c purposes (86/609/EEC) excludes from its external environment and respond in the killing of an animal from the legal the normal behaviour of an awake individual. de®nition of an experiment (Article 2(d)) if it Unconsciousness will be used to mean is carried out using the least painful method insensibility to external stimuli as would be accepted in modern practice and in accor- expected in coma or during general anaes- dance with the scienti®c purpose of collect- thesia. Two main ways of measuring insen- ing blood and other tissues from the killed sibility are to look at the physical responses animals, therefore leaving these procedures and responses in the central nervous system outside the protection of the Directive. This at the cortical level. document is designed to assist all those Pain may be de®ned as `an aversive sensory concerned with experimental animals in experience that elicits protective motor deciding which method is the most humane actions, results in learned avoidance and may and appropriate (in the context of the modify species-speci®c traits of behaviour, experiment) for killing the animal with including social behaviour' (Zimmermann which they are working. As this Directive 1986). Use of the word pain implies a protects vertebrates, this document will only conscious awareness of the stimulus and not cover euthanasia of vertebrates. Article 2(1) an unconscious re¯ex response. de®nes `humane method of killing' as the An embryo may be de®ned as an animal killing of an animal with a minimum of that is developing from a sexually fertilized 296 Working Party Report or pathenogenetically activated ovum and the animal is, in fact, conscious. In embryos which is contained within egg membranes or of the last third of their development and within the maternal body. The embryonic very young animals the peripheral as well as stage ends at the hatching or birth of the the cortical and subcortical components of young animal (Allaby 1991). the pain system are well developed, the A foetus is a mammalian embryo from the neurochemical systems are intact and func- stage of its development where its main adult tional and pain and stressor responses are features can be recognized until its birth well documented (Anand & Hickey 1987). (Allaby 1991). Pain may also be associated with deprivation A larva is considered as the stage during and/or the psychological suffering associated which it is motile and capable of feeding with poor treatment or an inadequate envir- itself, that occurs after hatching from the egg, onment. and prior to the reorganizations involved in When assessing the most humane method becoming adult (Allaby 1991). of euthanasia for any animal, sedation prior to euthanasia may be considered as a method 1.3 Signs of pain and distress of reducing possible anxiety and distress. However, a factor to consider is that this will To ensure euthanasia i.e. a gentle death, it is involve more handling which in itself may important to recognize signs of pain, fear and add to the anxiety of the animal, thus distress in the relevant species. All personnel negating the purpose of the sedative. must be trained to recognize these signs of The need to minimize fear and apprehen- suffering in the species with which they are sion must be considered in determining the working. Assessment of these factors must method of euthanaisa. Distress vocalizations, be based primarily on observations of abnor- fearful behaviour, and release of certain mal behavioural and physiological responses odours or pheromones by a frightened animal that demonstrate anxiety and fear. may cause anxiety and apprehension in Depending on the species these may others. It must be remembered that many include: vocalizations are at high frequencies and out of the human hearing range. Therefore, . distress vocalizations (not always in the whenever possible, animals should not be human audible range), present during euthanasia of others, espe- . struggling, cially of their own species. This is particu- . attempts to escape, larly important when vocalizations or release . defensive or redirected aggression, of pheromones may occur during induction . freezing/immobility response, of unconsciousness. It is also known that the . panting, last animal in a group to be removed may . salivation, become disturbed and so the last two animals . urination, defecation and evacuation of may have to be removed together. anal sacs, . pupillary dilatation, 1.4 Recognition and confirmation of . tachycardia, death . sweating, . re¯ex skeletal muscle contractions causing It is essential that all personnel are trained to shivering, tremors, or other muscular be able to recognize and con®rm death in all spasms. the species with which they are working. The most important aspects in recognition of Some of these responses can occur in death include cessation of heartbeat and unconscious as well as conscious animals. respiration, and absence of re¯exes, and in Fear may cause immobility or freezing in small laboratory animals, the lowering of the certain species, particularly rabbits and body temperature to below 258C. The meth- chickens. This immobility response should od chosen will depend on the species being not be interpreted as unconsciousness when handled. If there is any doubt about con- Euthanasia of experimental animals 297

®rmation of death, a second method should people who regularly perform euthanasia be used to kill the animal. may cause a strong sense of work dissatis- faction or alienation, which might be ex- 1.5 Personnel and training pressed by absenteeism, belligerence, or careless or callous handling of animals, along All methods of euthanasia can be badly with a high turnover rate of personnel. performed and therefore personnel carrying Coping skills for employees should be out euthanasia on animals must be suitably developed through training programmes. The trained to carry out euthanasia in the most effects of various agents and methods may be effective and humane manner. Professional subjective and based on professional judge- advice should be sought. ment, experience and intuition. Some of the Training programmes should include reported disadvantages and controversy about courses on the biology of the species to be certain practices may be based on sentiment used, suitable methods of euthanasia for each and aesthetic considerations rather than on species and national and European animal sound scienti®c data. Some physical methods welfare regulations. Training must include may be aesthetically unpleasant but quite aspects such as recognition of pain, fear, humane. The choice of method of euthanasia distress, anxiety, insensibility and death for must be based primarily on humane concerns all species to be used. Detailed courses on for the animal rather than on the sensitivities methods of euthanasia for each species must of the technician who performs the task or be provided, including assessment of the the people who observe the euthanasia. most humane and suitable methods depend- However, the opportunity must be given to ing on the species and experimental require- personnel to refuse to carry out methods of ments. The operators should be physically euthanasia they ®nd personally abhorrent. capable of carrying out various euthanasia techniques, as well as having suf®cient 1.6 Handling and restraint experience in the handling and restraint of the relevant species to minimize distress, As with other procedures applied to animals, fear and anxiety. Courses must include euthanasia usually requires some physical methods to be used to con®rm death. control over the animal. The degree of Training courses should also cover the control and kind of restraint needed will be functioning and maintenance of the equip- determined by the animal species, breed, ment to be used. Competence assessment is size, state of domestication, presence of necessary at the end of each course. painful injury or disease, degree of excite- Experienced personnel who have developed ment, and method of euthanasia. Suitable a trusting relationship with the particular control is vital to minimize pain and distress animals should be used for euthanasia of in animals, to assure safety of the person these animals as this will minimize stress performing euthanasia, and frequently to and anxiety in the animals. protect other animals and people. Gentle but All people performing euthanasia should ®rm restraint by a familiar handler, careful demonstrate professionalism and sensitivity handling, stroking, and talking during eu- for the value of animal life. The degree of thanasia often have a calming effect on many distress experienced by those people obser- animals. Where capture or restraint may ving or performing euthanasia in any form is cause pain, injury or anxiety to the animal or dependent on their backgrounds and on their danger to the operator, the prior use of personal philosophies and ethical concerns tranquillizing and immobilizing drugs may about using animals in research. The stress of be necessary. performing euthanasia is magni®ed when there are strong emotional bonds between 1.7 Equipment personnel and individual animals or when large numbers of animals are killed on a Instruments, equipment and installations regular basis. The stress experienced by used for stunning or killing animals should 298 Working Party Report be designed, constructed and maintained so life (Andrews et al. 1993, Lumb & Jones as to achieve rapid stunning and death. They 1984). should be regularly inspected and cleaned to Further details for each species group may ensure that they are in a good state of repair be obtained in Section 3. and will function correctly at all times. Blood, urine and faeces which could cause 2.1 Acceptable methods of euthanasia anxiety to subsequent animals must be removed. Physical methods These methods must cause immediate loss of consciousness through physical trauma to 1.8 Carcass and waste disposal the brain. They are most useful when pharmacological methods would interfere The possible hazards to humans when with the purpose of the experiment. While animals are known to be carrying a zoonotic physical methods may be aesthetically less agent or were treated with radioisotopes or pleasant for observers and those killing toxic chemicals must be evaluated and animals, in skilled hands they are quick and personnel handling such carcasses should certain and possibly the least distressing for take the necessary precautions to protect the animal. Specialist training is essential for themselves and others. Care should be all of these methods. These methods require taken when disposing of carcasses and other restraint which may cause extra stress for waste (for example water in which agents some animals. If possible the animal should have been dissolved) that it does not not be killed in the sight or smell of other provide any danger to others or the envir- animals. onment. Chemical methods (except carbon dioxide) must not be used on animals 2.1.1 Shooting destined for consumption or where the Shooting in the head to ensure immediate carcass may enter the food chain. Operators destruction of the brain is an effective and must ensure that they comply with na- humane way of killing large reptiles and tional and international legislation. mammals (Australian Veterinary Association 1987, Longair et al. 1991). This may be divided into two types: free bullet or captive bolt (with penetration or percussion). The 2 General comments on methods of type of weapon used must be selected euthanasia according to the species to be killed and the environment. The majority of methods that have been used to kill experimental animals are listed (a) Free bullet Special care must be taken to in this section. For those more obscure avoid danger to the operator. All personnel methods that are not mentioned it should must be trained in these techniques to ensure generally be assumed that they are not the correct positioning of the weapon to considered acceptable until they have been ensure a direct hit into the brain (Longair et carefully assessed under the criteria given al. 1991). Shooting using a free bullet must in Section 1 and have been considered not be used inside a building because of humane by a quali®ed person such as a danger to personnel from ricocheting bullets, veterinarian or the competent authority. but it may be used effectively in the ®eld by Section 1 must be consulted in conjunction skilled marksmen. When the animal can be with this section. appropriately restrained, the captive bolt Agents may cause death by three basic method is preferable as there is less danger to mechanisms: (1) hypoxia, direct or indirect; personnel. (2) direct depression of neurons vital for life A free bullet humane killer is preferred for functions; and (3) physical disruption of brain example, on horses (Blackmore 1985, Dodd activity and destruction of neurons vital for 1985, Oliver 1979). Euthanasia of experimental animals 299

(b) Captive bolt The penetrating captive small reptiles, amphibians and ®sh (Clifford bolt is an effective tool for rendering many 1984), a blow on the head may be suf®cient larger animals unconscious (Blackmore & to render the animal insensible (Green 1987). Delaney 1988, Daly & Whittington 1989, Experience and training are essential for the Green 1987, Longair et al. 1991). Large correct choice of method to be used. rabbits and dogs may also be killed in this In larger animals specialized equipment way (Dennis et al. 1988, Holtzmann 1991). such as the non-penetrating captive bolt However, it is not always effective in large must be used. The use of the hammer or pigs and mature bulls because of the poleaxe is condemned as a method of thickness and density of the skull. The stunning. These methods must always be purpose of stunning is to render the animal followed immediately by exsanguination, instantaneously insensible to pain by caus- removal of the heart or destruction of the ing concussion (Ministry of Agriculture, brain to ensure death. Training is essential Food and Fisheries 1993). The animal should for all operators. If not performed correctly, remain insensible until exsanguination is various degrees of consciousness with con- performed (Blackmore 1993). An effective comitant pain can ensue. It is dif®cult to stun may be recognized by the animal ensure consistency in performance by opera- collapsing immediately after shooting, with tors and therefore only a few animals should its body and muscles rigid and it should not be killed using this method at any one time. have a righting re¯ex. Normal rhythmic Death must be con®rmed in each animal breathing should stop, there should be a loss before the next animal is stunned. of blink re¯ex and the eyeball should point High pressure water jet has been success- outwards and not be rotated into the skull. fully used for the stunning of pigs and is an Effective stunning depends on accurate accepted method in Switzerland (Schatz- positioning of the pistol, use of the correct mann et al. 1991, 1994). strength of cartridge in relation to the species and size of the animal, the size and 2.1.3 Electrical stunning speed of the bolt and proper maintenance of the pistol. The site of penetration differs This has been used in ®sh, amphibians, birds, with each species and therefore this method dogs and other carnivores, poultry, pigs should only be carried out by suitably (Lambooy & van Voorst 1986, Laursen 1983), trained personnel. Appropriate restraint sheep, calves, goats and rabbits (Warrington must be used to prevent incorrect position- 1974). Horned animals should not be stunned ing of the pistol. The recommended pistol is using this method if the horns make it one with the bolt recessed into the muzzle dif®cult to apply the electrodes accurately. It before ®ring, rather than one where the bolt should not be used in cats due to the high extends beyond the muzzle, as the recessed conductivity of their coats (Green 1987). It is one is likely to generate a higher bolt not acceptable for use in ®sh as alternating velocity at impact. The operator should current stimulates contraction of skeletal, ensure that the bolt retracts to its full extent cardiac and smooth muscle and induces after each shot and if not, it should not be tetany, not anaesthesia. used again until it has been repaired. The Only specialized equipment should be used bolt should always be properly cleaned after for this method of euthanasia. Alternating each use. electrical current may be used to stun the animals (Breazile & Kitchell 1969) but this must be followed by another method to 2.1.2 Concussion (stunning) complete death. Alternatively, immediate This may be carried out by several means unconsciousness with cardiac arrest can be depending on the size of the animal. In caused if electrodes are applied simulta- smaller animals such as small rabbits, new- neously to the animal's head and back, but born kittens and newborn puppies, rats, the electrodes must be placed in such a mice, young guineapigs, hamsters, birds, position as to ensure that the current is 300 Working Party Report directed through the brain in order to produce Green 1987, Reilly 1993). It may be used on unconsciousness before cardiac ®brillation older rats and rabbits up to 1 kg if they are (Andrews et al. 1993). sedated or stunned prior to dislocation. The current is usually applied to the Gregory and Wotton (1990) showed that there animal's head by means of a pair of scissor- is not always immediate unconsciousness in like tongs with an electrode at the end of poultry using this method. Care must be each arm. High voltage stunners are more taken to ensure complete separation. If effective. Animals must be suitably re- carried out correctly, it should cause exten- strained so that the tongs can be accurately sive damage to the brainstem and instanta- applied. The electrodes must span the brain neous unconsciousness (Iwarsson & and be applied ®rmly so that they will not Rehbinder 1993). Death must be con®rmed slip out of position when the animal falls to by exsanguination or destruction of the brain the ground (Ministry of Agriculture, Fisheries (Blackmore 1993). and Food 1993). It may be aesthetically unpleasant for the Head to tail and head to foot stunning is operator to perform and it is recommended not acceptable as it does not cause immediate that if the operator is not totally con®dent of unconsciousness (Breazile & Kitchell 1969). performing this technique quickly and effec- Electrodes must not be applied behind the tively, that they use another method. If ears or on each side of the neck which would possible animals should be sedated or anaes- paralyse the animal without rendering it thetized prior to dislocation. unconscious, resulting in severe pain and suffering. Care must be taken to ensure that the animal does not receive an electric shock 2.1.5 Decapitation before the electrodes are correctly applied, for instance by contact with other animals being This procedure has been used for killing ®sh, stunned or having a wet skin. amphibians, birds, rodents and small rabbits. The apparatus should have a device which Decapitation involves the severing of the prevents operation if the minimum required neck of the animal, close to the head by using current cannot be passed, as well as devices a sharp instrument. The use of scissors is to measure length of time of application, discouraged unless they are suited to the voltage indicators and current level. species of animal (i.e. have suf®ciently long The signs of an effective electrical stun are blades) and the pressure is strong enough to extension of the limbs, opisthotonos (arching sever the neck in one go with ease. Decap- of the body and limb spasm), downward itation should be carried out using guillotines rotation of the eyeballs, and a tonic spasm designed specially for that purpose to ensure changing into clonic spasm with eventual rapid and quick severance in the correct muscle ¯accidity. After 15±20 s the re¯exes position (Clifford 1984). may begin to reappear and the animal may There has been much debate over the start to breathe again and therefore another length of time to loss of consciousness of the method to ensure death, such as exsangui- decapitated head in both warm and cold- nation, must be carried out immediately blooded vertebrates (Allred & Berntsen 1986, (Anil & McKinistry 1991). If the animal is Andrews et al. 1993, Blackmore 1993, not correctly stunned it may be paralysed Holson 1992, Lorden & Klemm 1987, whilst remaining fully conscious and is able Mikeska & Klemm 1975, Reilly 1993, to feel pain. Tidswell et al. 1987, Vanderwolf et al. 1988) and it has been suggested to anaesthetize or sedate the animal ®rst (Smith et al. 1986). 2.1.4 Cervical dislocation However, handling and injection of sedatives This method is used for the euthanasia of or anaesthetics prior to decapitation could ®sh, poultry, mice, young guineapigs, young increase stress prior to euthanasia and is rats and neonatal rabbits and newborn therefore not considered good for the welfare kittens and newborn puppies (Clifford 1984, of the animal. Euthanasia of experimental animals 301

In cold-blooded vertebrates the animals to a minimum to reduce stress prior to must be stunned or rendered insensible prior euthanasia. Whole body radiation has been to decapitation as they are very tolerant of successfully used on mice at temperatures of anoxia (Warwick 1986). In birds research has 47±498C with the animals dying in less than shown that there may be visually evoked 1 s (Von Cranach et al. 1991a,b) and is responses for up to 30 s after decapitation acceptable (Schatzmann, personal communi- (Gregory & Wotton 1990) which makes this cation 1995). unacceptable. In other warm-blooded ani- This is not a routine procedure for eu- mals the immediate lack of circulation of thanasia. Care must be taken as this may be blood to the brain and subsequent anoxia is dangerous to the operator (Bermann et al. thought to render the head rapidly insensible 1985). (Derr 1991) making prior stunning or seda- tion unnecessary. Use of the puntilla is not Chemical methods acceptable (Commission of the European Many anaesthetics are used in overdose as Communities 1993). euthanasia agents. An anaesthetic is an agent Use of other methods is preferred where that produces, in a controllable manner, a possible until further research can show rapid drug-induced absence of perception of all loss of consciousness. sensation. It produces unconsciousness, an- algesia, and muscle relaxation suf®cient to 2.1.6 Maceration perform procedures painlessly. Indications of This method is acceptable for the destruction anaesthetic overdose include: occurrence of of chicks up to 72 hours old which often have cardiac dysrhythmias; capillary re®ll time to be killed in large numbers (Bandow 1987, progressively slows to 3 or more seconds; Commission of the European Communities respiration slows, becomes shallow and 1993). Only macerators designed speci®cally irregular, becomes diaphragmatic, or may for this purpose must be used and under no cease; mucous membrane and skin colours conditions should domestic appliances be may be pale to cyanotic; cardiovascular, used. central nervous system, musculoskeletal, Very small ®sh (52 cm long) may be killed gastrointestinal, and ocular re¯exes are by placing down a waste disposal unit greatly diminished or cease; blood pressure (Banister, personal communication 1995). falls rapidly to produce profound hypotension (mean BP520±30 mmHg). 2.1.7 Microwave irradiation This method is used by neurobiologists as a Inhalational agents means to ®x brain metabolites without the Inhalational agents are either vaporized or loss of anatomical integrity of the brain delivered as a gas into chambers or anaes- (Moroji et al. 1977). Only specialist apparatus thetic circuits. Chambers used for delivery of (this does not include domestic microwave inhalants should be properly designed so as to ovens) designed for this purpose is to be used. ensure the even distribution of gas and to This involves focusing the microwave beam ensure that the animals are rapidly exposed precisely at a speci®c part of the brain. It is to a high concentration of the agent. They are only to be carried out on small animals such valuable for use in many small animals e.g. as amphibians, birds, mice, rats and small birds, rodents, cats and small dogs (Smith et rabbits (less than 300 g) (Zeller et al. 1989). al. 1986). As rabbits react adversely to gases This method requires specialist expertise, and show signs of excitation, other methods but when carried out correctly is humane as are preferred (Green 1979). Reptiles and death occurs in milliseconds (Andrews et al. amphibia may hold their breath resulting in a 1993, Bermann et al. 1985, Olfert et al. 1993). long induction time. Newborn animals are Care must be taken to ensure correct more resistant to hypoxia and may take positioning of the microwave beam but time longer to die: therefore other methods should taken to restrain the animal should be kept be used. 302 Working Party Report

It is important to select agents that are not it causes intense activity before loss of unpleasant to inhale because some can be consciousness and is slow acting. It should irritant and therefore be stressful. Agents not be used for cats and larger species which produce convulsions prior to uncon- because it sometimes causes excitement sciousness are unacceptable for euthanasia. (Glen & Scott 1973, Klemm 1964) and some Safety precautions should be taken when animals are averse to its pungent odour. Pigs administering inhalational agents through vocalize before losing consciousness, indi- the use of appropriate gas scavenging equip- cating a degree of distress (Gregory et al. ment. Death must be con®rmed. 1987) and other people have also indicated that it is not humane for pigs (Clifford 1984, Hoenderken 1983, Hoenderken et al. 1980, 2.1.8 Carbon dioxide Reilly 1993) despite EC and national slaugh- At concentrations above 60%, carbon diox- ter recommendations to the contrary (Com- ide acts as an anaesthetic agent and causes mission of the European Communities 1993, rapid loss of consciousness (Green 1987). It Ministry of Agriculture, Food and Fisheries is effective and humane for euthanasia of 1993). Other research indicates that the most small animals above 70%. Carbon violent reactions may be after unconscious- dioxide stimulates the respiratory centre ness (Andrews et al. 1993, Erhardt et al. 1989, which may cause anxiety and stress in the Forslid et al. 1986, Mullenax & Dougherty animal as well as being aesthetically 1963). Until further research can show any unpleasant for the observer. Carbon dioxide adverse reactions of pigs is once they are fully may form carbonic acid when in contact anaesthetized, other methods are preferable. with the nasal mucous membranes which It is not acceptable for other cold-blooded could produce a ®zzy or tingling effect vertebrates as induction is too long. Neo- which may be mildly irritating to some nates are particularly tolerant of CO2 (30± species when applied at lower concentra- 60 min to unconsciousness (van Zutphen et tions (Lucke 1979). al. 1993) depending on maturity at birth In most animals it is recommended to (those that are born more mature are less place the animals immediately into 470% tolerant of CO2). Therefore this method CO2 where the animals lose consciousness should not be used in animals less than 2 very quickly due to the narcotic effect of the weeks old. Carbon dioxide should not be used high intake of CO2 on the brain without in diving animals such as mink because of causing hypoxia (Blackshaw et al. 1988, their ability to hold their breath. Forslid et al. 1986). One hundred per cent Research has been carried out examining CO2 may cause severe dyspnoea and distress the possible advantageous effects of adding in conscious animals (van Zutphen et al. oxygen to ensure that the animals die from 1993). CO2 narcosis, rather than hypoxia (Iwarsson One hundred per cent CO2 is recom- & Rehbinder 1993). In some species there mended for chicks up to 72 hours old because appears to be a reduction in stress and they are more tolerant of CO2. Raj and anxiety, but this is accompanied by a longer Gregory (1993, 1994) and Raj et al. (1990, induction time (Blackmore 1993). Hewett et 1992) showed that the use of 60% argon in al. (1993) felt that there was no welfare conjunction with CO2 induces a rapid loss of advantage in using CO2/O2 mixtures. It may brain function in turkeys. Older birds may be dif®cult to mix gases accurately for ¯ap their wings when killed under CO2, even routine use. when comatose, which makes it aestheti- Carbon dioxide is heavier than air so cally less acceptable. Low concentrations of incomplete ®lling of a euthanasia chamber CO2 (30%) in conjunction with an inert gas is may permit tall or climbing animals to avoid considered acceptable for chickens and tur- exposure to the gas. Therefore the chamber keys. At this level, the carbon dioxide is not must be pre®lled with up to 70% CO2 before unduly pungent and it acts as an antic- placing the animals in it. However, others onvulsant. It is not recommended for ®sh as feel that it may be better to ®ll the chamber Euthanasia of experimental animals 303 once the animals have been placed in it. The detectable, it should only be used in an chambers must be designed so as to avoid appropriate gas scavenging apparatus taking injury to the animals and, if possible, have extreme care. Carbon monoxide monitors devices whereby the CO2 concentration can should be installed in the room. be easily and accurately measured. Care must be taken to limit the number of animals in a 2.1.10 Volatile inhalational anaesthetics chamber at any one time so as to maintain a When using any liquid anaesthetic care must constant CO2 concentration. be taken to ensure that it is not allowed to Carbon dioxide is non-¯ammable and non- come in contact with the animal. Suf®cient explosive and therefore presents little hazard air or oxygen should be provided during the to the operator. Fire extinguishers and solid induction period to prevent hypoxia (An- carbon dioxide are not acceptable because of drews et al. 1993). Exposure to trace concen- the lowered temperature and the noise trations of anaesthetic gases is a recognized created by the extinguisher. human health hazard and requires gas scavenging apparatus to be used in the work environment. Volatile inhalational anaes- 2.1.9 Carbon monoxide thetics are neither ¯ammable nor explosive. This causes rapid death as it combines with the red blood cells in preference to oxygen, Halothane Halothane is a commonly used thus causing hypoxia (Chalifoux & Dallaire anaesthetic agent for small laboratory 1983). There is little or no distress as there is animals and is quick acting and stress free in no smell (Blackmore 1993, Breazile & overdose for euthanasia. It has a depressant Kitchell 1969, Green 1987, Smith et al. 1986). effect on the cardiovascular and respiratory It is not acceptable for use in reptiles because systems (Green 1987). of their low metabolic rate and hypoxic tolerance. It is acceptable for small animals, En¯urane En¯urane is a commonly used but in dogs and cats vocalizations and anaesthetic agent for small laboratory ani- convulsions may occur after unconscious- mals and is quick acting and stress free in ness making it aesthetically unpleasant. overdose for euthanasia (Green 1987). It has a Death should be con®rmed by physical depressant effect on the cardiovascular and means. respiratory systems. It may be preferred to Carbon monoxide may be produced by halothane in situations where drug metabo- three methods: chemical interaction of so- lism or toxicological work is being conducted dium formate and sulphuric acid; exhaust as very little drug is metabolized in the liver. fumes from internal combustion engines; Iso¯urane Iso¯urane is a commonly used and commercially compressed CO gas. Car- anaesthetic agent which is quick acting and bon monoxide from petrol engine exhaust is stress free in overdose for euthanasia. Iso- highly irritant to respiratory tissues. To be ¯urane causes respiratory and cardiovascular considered for use in euthanasia, it must be depression. However, it has a pungent odour cooled through a water chamber and ®ltered, and must therefore not be used on animals using a scrubber unit, in order to remove the which may be able to hold their breath. It is various oxides of nitrogen and hydrocarbons, particularly useful where tissues such as oxygenates of hydrocarbons and carbon liver are to be used for toxicological or particles. Under no circumstances should microsomal studies as it undergoes no hepatic exhaust from diesel engines be used. Only metabolism. commercial CO is recommended. The ani- Agents for aquatic animals for absorption mals should only be introduced into the through the skin and gills chamber after it has been ®lled with a concentration of CO of at least 6% by 2.1.11 Benzocaine (ethyl aminobenzo- volume, supplied by a source of 100% CO. ate) As it is extremely noxious and also This agent, dissolved in acetone before dangerous to the operator because it is not adding to tank water, is an effective and 304 Working Party Report humane method of killing ®sh and amphi- tissues such as the brain. It depresses the bians. It acts by depression of the central sensory centres of the central nervous system nervous system. It has pH-independent (Summerfelt & Smith 1990). Quinaldine ef®cacy but reduces the pH of the tank water sulphate may also be used for euthanasia of which should therefore be buffered to pH 7.5 ®sh. to reduce irritation (Brown 1988, Summerfelt & Smith 1990). The breakdown time in water is about 4 h, making this agent environmen- Injectable agents tally safe and it is safe for personnel. Death Many proprietary mixtures speci®cally pre- should be con®rmed by physical means. pared for euthanasia of animals are simply triple strength anaesthetic agents, such as 2.1.12 Tricaine methane sulphonate sodium pentobarbitone, but others may have (buffered MS-222) neuromuscular blocking agents incorporated. It is essential that the animal should MS-222 is a humane and safe method of become fully anaesthetized before the neu- euthanasia for ®sh and amphibians. It has romuscular blocking agents take effect in been used for snakes and alligators by order to prevent distress to the animal. intramuscular injection but has a long Before using any agents for euthanasia, the induction period, thus increasing distress. It operator should consult the manufacturer's acts by depression of the central nervous information lea¯et with regard to dosage and system. It is soluble in both salt and fresh route of injection. In general where anaes- water but needs to be neutralized with thetic agents are used, two times the bicarbonate, imidazole, sodium hydrogen anaesthetic dose produces respiratory arrest, phosphate or sodium hydroxide to reduce while four times the dose produces cardiac irritation and tissue damage (Brown 1988). arrest when ventilated arti®cially. Three The effectiveness of MS-222 varies with times the dose usually causes death quickly species, size, temperature and water hard- and uniformly in non-ventilated animals. ness. MS-222 is unstable in sunlight and Injection may be administered by various stock solutions should be stored in brown or routes. Intravenous administration is pre- opaque bottles. It may be used in conjunction ferred because the effect is the most rapid and with quinaldine or quinaldine sulphate reliable. Intraperitoneal injection is easier to which is more effective and used in smaller administer, especially in species where the quantities than either agent used alone. veins are small and dif®cult to penetrate but it takes longer to act and may cause irritation 2.1.13 Etomidate and metomidate and transient pain and distress. Intrapulmo- These are both non-barbiturate hypnotic nic injection should be avoided because of agents that act by depression of the central discomfort to the animals. Oral and rectal nervous system. They are relatively quick routes of administration are inadvisable acting and are considered as humane agents because of prolonged onset of action, wide for killing ®sh. They are highly soluble in range of lethal doses and potential irritation water (Brown 1988, Summerfelt & Smith of tissues. Intramuscular and subcutaneous 1990). routes must not be used as they take too long to act. The intracardiac route is very painful 2.1.14 Quinaldine (2-methylquinoline) and penetration of the heart is not always This drug is commonly used for killing ®sh successful on the ®rst attempt; therefore in a humane way in the United States of these are not recommended except in in- America (USA). However it is rarely used and sensible animals. dif®cult to obtain in Europe. It must ®rst be Excitable and vicious animals should be dissolved in acetone but this has no adverse pretreated with a neuroleptanalgesic combi- effects on the animals. It has a relatively long nation, a tranquillizer, or another depressant. induction time compared to some other Trained personnel are essential for using agents. Quinaldine accumulates in lipid these methods. Euthanasia of experimental animals 305

Care must be taken when disposing of injected into the pectoral muscle, but it is carcasses because of residues in the meat. not suitable for poultry. The animal must be Care must also be taken to ensure personnel sedated prior to administration of T-61. safety. There has been concern that the curariform drug may cause cessation of respiratory 2.1.15 Barbiturates activity before the onset of unconsciousness These are the most widely used and accepted (Barocio 1983, Baumans et al. 1988, Eikmeier agents for euthanasia for most animals 1961, Quin 1963, Lumb et al. 1978, Rowan (Hatch 1982). They include barbituric acid 1986) therefore causing distress to the derivatives, oxybarbiturates (sodium pento- animal, but Hellebrekers et al. (1990) showed barbitone, secobarbital), thiobarbiturates that loss of consciousness and loss of muscle (thiopentone) and various barbiturate mix- activity in rabbits and dogs occurred simul- tures. Sodium pentobarbitone is commonly taneously, therefore making this an accep- considered the most suitable agent. They act table agent for euthanasia. The muscle by depression of the central nervous system relaxant prevents the terminal gasp reported and cause cardiac and respiratory arrest. They for the barbiturates, thus making it more cause rapid euthanasia with minimal dis- acceptable for the observer. In some dogs comfort, depending on the dose of the agent there is vocalization and muscular activity. and route of injection (intravenous is pre- This is not a conscious response but may be ferred as it is quickest). In some countries aesthetically unpleasant. It is not a con- barbiturates may only be obtained under trolled drug in many countries and may licence. therefore be easier to obtain than barbitu- rates. In other countries, such as Sweden, it is Sodium pentobarbitone This is generally not available. used either by intravenous or intraperitoneal injection of 18% (200 mg/ml) concentration in a dosage of 200 mg/kg for euthanasia. 2.2 Methods acceptable for unconscious Intravenous injection results in quicker animals death but the intraperitoneal route may be 2.2.1 Pithing simpler to perform in many species, thus reducing the stress caused by handling. This is an effective way of killing some ®sh, However, sodium pentobarbitone may cause amphibians and reptiles. This is carried out irritation of the peritoneum which can be by inserting a sharp needle through the avoided by diluting the drug. Intracardiac foramen magnum into the base of the brain injection may only be used on a fully to ensure quick brain destruction. If not anaesthetized animal as this is very painful carried out correctly and quickly, the animal and is therefore not considered acceptable. will remain conscious with subsequent pain Intracephalic injection (foramen magnum) is and distress. The animal must ®rst be effective on large birds such as poultry, but rendered unconscious by stunning or anaes- requires specialist skills. thesia. This method should only be carried out by competent personnel. 2.1.16 T-61 This agent combines a local anaesthetic 2.2.2 Rapid freezing (tetracaine HCL), a hypnotic agent and Rapid freezing has been used to minimize curariform drug (N-2-(m-methoxyphenyl)-2- enzyme activity for subsequent biochemical ethylbutyl-l-gamma-hydroxybutyramide estimations of tissues. Techniques involve: (20%), 4,4'-methylene bis-cyclohexyltri- (a) immersion of the intact animal into liquid methyl ammonium iodide (0.5%) and tetra- nitrogen; (b) decapitation and immediate caine hydrochloride (0.5%) in aqueous immersion of the head into liquid nitrogen; solution with formamide). It must only be (c) freeze blowing; (d) in situ freezing; and (e) injected intravenously and slowly as it is funnel freezing. The animals must be fully otherwise painful. In small birds it may be anaesthetized, rendered insensible or decapi- 306 Working Party Report tated prior to all freezing methods as it has control before becoming comatose, followed been shown that it may take 10±90 s to freeze by respiratory arrest. Irritation of the perito- the deep structures due to the poor thermal neum may occur. Wallgren and Barry III conductivity of the tissues surrounding the (1970) state that it is irritant at concentra- brain. It is acceptable only under speci®c tions of above 10% w/v and that the circumstances when experimental design mortality is due to unspeci®c trauma. It is requires such treatment in very small ani- not acceptable for euthanasia of vertebrates mals such as embryos or neonatal rodents unless they are anaesthetized. and rabbits (Green 1987, Van Zutphen et al. 1993). Personnel carrying out this technique 2.2.6 Chloral hydrate must be well trained and specialist equip- This acts by very slow depression of the ment is required. central nervous system. It is not acceptable for use by itself as it lacks analgesic effects, is 2.2.3 Exsanguination very slow to take effect, causes aesthetically Exsanguination should only be carried out objectionable animal movements, large vo- after the animal has been rendered insen- lumes are required and it causes irritation of sible by another method because of the the peritoneum (Breazile & Kitchell 1969, stress associated with extreme hypovolae- Hatch 1982). It may be used for large animals, mia and the pain of incising the deeper intravenously, under anaesthetic (Lumb blood vessels. An animal must not be 1974) or in combination with magnesium exsanguinated in sight or smell of other sulphate and sodium pentobarbitone (Olfert animals, using a different room if possible. et al. 1993). This is not an acceptable method of killing birds because of the tendency for the blood 2.2.7 Potassium chloride to clot and thus result in incomplete The potassium ion is cardiotoxic. Potassium exsanguination and therefore inadequate chloride causes gasping, vocalizations, mus- euthanasia. It is also not acceptable for cle spasms and convulsive seizures (Lumb reptiles and other cold-blooded vertebrates 1974). It is also unpleasant for the observer. It because of their slow metabolic rate and is unacceptable for euthanasia unless the hypoxic tolerance. animal is fully anaesthetized.

2.2.4 Nitrogen/argon 2.2.8 Air embolism

Nitrogen or argon displaces O2 and produces This involves intravenous injection of 5 to death by hypoxia. At 39%, rats become 50 ml/kg air. It has occasionally been used in unconscious only after 3 min and show signs rabbits (Weisbrod et al. 1984). It may be of panic and distress (Andrews et al. 1993). It accompanied by convulsions, opisthotonos causes unconsciousness but not death in and vocalizations (Hatch 1982). It is a very young animals. In dogs and cats uncon- painful and unreliable method and is not sciousness takes 1±2 min to occur with acceptable unless the animal is fully anaes- hyperpnoea for about 10 s before collapsing thetized. (Herin et al. 1978, Quine 1980, Quine et al. 1988, Rowsell 1981, 1990). It is therefore not 2.3 Methods that are not acceptable for an acceptable method unless the animal is euthanasia anaesthetized. 2.3.1 Decompression/vacuum 2.2.5 Ethanol This method acts by inducing cerebral This method, described by Lord (1989, 1991) hypoxia. There may be adverse physical involves the intraperitoneal injection of effects due to trapped gases in the body 500 m1 70% ethanol into mice. Ethanol cavities (e.g. sinuses, eustachian tubes) causes depression of the central nervous expanding and these could cause severe pain system. The mice show a gross loss of muscle and discomfort before the animal becomes Euthanasia of experimental animals 307 unconscious (Von Cranach et al. 1991a). with possible subsequent remaining con- There is also a chance of failure of equip- sciousness with pain, fear and distress. This ment, resulting in rapid recompression with method is not acceptable for the euthanasia severe pain and distress to the animals. of birds or any animal. Bloating, bleeding, vomiting, convulsions, urination and defecation may occur in the 2.3.6 Strangulation unconscious animal and are aesthetically This is not an acceptable method of killing unpleasant for the observer (Booth 1978, any animal due to the time taken to Hatch 1982). It may also take some time unconsciousness, and the pain, undue anxi- before unconsciousness (Barber 1972). For ety and stress that it would cause. these reasons, decompression is not accep- table as a method of euthanasia. 2.3.7 Nitrous oxide Hypoxic concentrations of almost 100% are 2.3.2 Hypothermia necessary to achieve euthanasia and it is slow Hypothermia involves the killing of animals acting therefore causing unnecessary stress. by placing them in very cold temperatures The animal will convulse after losing such as deep freezers. Hypothermia is known consciousness which reduces the accept- to act as an anaesthetic agent to a certain ability to the observer. It is not an extent (Phifer & Terry 1986). However, it is acceptable euthanasia agent. However, it not an acceptable method of euthanasia for may be used with other agents to speed the any animal. Deep freezers may only be used onset of anaesthesia. to ensure death once the animal is fully unconscious and unlikely to recover (Sum- 2.3.8 Cyclopropane merfelt & Smith 1990). Cyclopropane is a humane method of eu- thanasia for most laboratory animals as it 2.3.3 Hyperthermia produces rapid and deep anaesthesia. How- Raising the temperature in order to kill ever, it is ¯ammable in air and explosive in animals has been suggested for some cold- oxygen which makes it dangerous to the blooded vertebrates which will die above operator which reduces its acceptability as a their critical temperatures which may be general agent for euthanasia. only a few degrees above their normal activity range but this is not acceptable. 2.3.9 Ether (diethyl ether) Animals must never be dropped into boiling Ether is irritant to the mucous membranes water as it causes intense pain and a slow and at high concentrations traditionally death. found in closed containers and jars, it may be stressful to the animals as it elevates 2.3.4 Drowning/removal from water catecholamines (Blackshaw et al. 1988, Drowning is not a humane method of Breazile & Kitchell 1969, Green 1987). If used euthanasia for any vertebrate as it is slow in a vaporizer, it appeared less irritating and causes severe stress and anxiety from (Baumans, personal communication 1995). hypoxia. Removal from water for gilled It markedly elevates some blood chemicals vertebrates is not acceptable (including tad- (e.g. glucose) under high concentrations. It poles) (Kestin et al. 1991). is dangerous to the operator because of its explosive properties. It is not an acceptable 2.3.5 Neck crushing method of euthanasia. This is a method sometimes employed to kill birds. The neck of a small bird is pressed 2.3.10 Chloroform against a bar or specialized pliers or bone Chloroform acts by depression of the central calipers may be used. However, this only nervous system and causes cardiac and results in paralysis from destruction of the respiratory failure. This is not acceptable as a spinal cord and does not damage the brain euthanasia agent as it is hepatotoxic, ne- 308 Working Party Report phrotoxic and carcinogenic to the operator 2.3.16 Neuromuscular blocking agents and other animals. It causes excitement Neuromuscular blocking agents and other before loss of consciousness (Breazile & agents that do not induce loss of conscious- Kitchell 1969). Trace concentrations carried ness prior to death are not to be used for to breeding centres have been shown to euthanasia under any circumstances. seriously interfere with breeding pro- grammes in rodents (Green 1987). 2.3.17 Ketamine Ketamine is not considered acceptable as a 2.3.11 Methoxy¯urane sole agent for euthanasia as large volumes Methoxy¯urane is a commonly used anaes- would be necessary. Extensive convulsions thetic agent but is very slow acting and there and vocalizations in rabbits make it aesthe- is a high chance of full recovery even after tically unacceptable (Baneux et al. 1986, 20 min of overdose. It is dif®cult to obtain in Reilly 1993). Used in conjunction with Europe. xylazine, it may be acceptable. 2.3.18 Sedatives 2.3.12 Trichlorethylene Because of the large volumes that would be Because trichlorethylene is mainly an an- necessary to cause death, sedatives are not algesic agent and produces only light anaes- acceptable as euthanasia agents. thesia, it is not acceptable as an agent for euthanasia. It is carcinogenic, causes hyper- 2.3.19 Magnesium sulphate capnia and is dangerous to the operator. This has been used with or without sodium pentobarbitone at 80 mg/kg. It is a neuro- 2.3.13 Hydrogen cyanide gas muscular blocking agent and myocardial Hydrogen cyanide gas blocks oxygen uptake depressant, not a central nervous system causing respiratory dif®culties and violent depressant (Hatch 1982, Olfert et al. 1993). convulsions before the onset of uncon- Large volumes are required and the animals sciousness and death (Hatch 1982). It is also may exhibit muscle spasms, convulsive very dangerous to the operator. It is not seizures, vocalization, gasping breaths and acceptable for the euthanasia of any animal. defecation before death (Breazile & Kitchell 1969). The animal remains conscious until 2.3.14 2-Phenoxyethanol the brain succumbs to anoxaemic anoxia. It This agent is designed as a ®sh antibiotic but lacks analgesic or anaesthetic effects and given in large enough doses, it can kill. therefore is not an acceptable agent by itself. Dosage levels must be high and death may be 2.3.20 Other injectable anaesthetics slow, thus increasing distress for the ®sh. It causes hyperactivity in some ®sh prior to Euthanasia may be induced with many other anaesthetization (Summerfelt & Smith agents (e.g. alphaxolone/alphadolone, propo- 1990). It has a very long time of chemical fol) but because these agents have a relatively breakdown in water which makes it dif®cult wide safety margin, high doses would be to dispose of as it would be dangerous to the required, reducing their acceptibility. environment, killing bacteria in sewage 2.3.21 Other agents systems if poured down the drain. It is not Other agents not to be used include nicotine acceptable for euthanasia of ®sh. (produces serious side effects before death) and strychnine (excites the central nervous 2.3.15 Urethane system and the animal remains conscious Animals may be placed in a 1±2% solution of and in excruciating pain until it dies of urethane. It is commonly used as an anaes- suffocation) (Hatch 1982, Lumb 1974). thetic. However, it is very carcinogenic and because of this potential danger to the 2.4.22 Agents administered orally operator and problems of safe disposal, it is Agents have been added to drinking water for not acceptable (Summerfelt & Smith 1990). mass euthanasia of some animals as may Euthanasia of experimental animals 309 occur in some large scale breeding establish- Billings Ch E (1985) Effects of physical agents. In: ments. There is always a risk of some Sodeman's Pathologic Physiology, 7th edn (W animals not receiving an adequate dose, and Sodeman, ed) WB Saunders, p 1059 the time to act is generally slow. These Booth NH (1982) Inhalant anesthetics. In: Veterinary Pharmacology and Therapeutics, 5th edn, Ch. 12 substances are dangerous to the operator and (Booth NH, McDonald LE, eds). Iowa: Iowa State are not acceptable for use as agents for University Press, pp 175±202 euthanasia. Borodkin S, Macy L, Thompson G, Schmits R (1977) Stable nonaqueous pentobarbital sodium for use in 2.3.23 Narcotic analgesics laboratory animals. Journal of Pharmaceutical Sciences 66 (5), 693±5 Opiate derivates such as morphine and Brewer NR (1982) The history of euthanasia. Lab etorphine are central nervous depressants as Animal 11 (4), 17,19 well as analgesics. Overdosage causes death British Veterinary Association (1988) Killing with by depression of the respiratory centres in the kindness. Proceedings of the BVA Animal Welfare medulla. There is a large variability of Foundation's Sixth Symposium reactions by different species: some species Broom DM (1988) The scienti®c assessment of animal welfare. Applied Animal Behaviour Science 20, are rendered maniacal by large dosages of 5±19 these drugs. Because there is not much Broom DM (1991) Animal welfare: concepts and information on the humaneness of these measurement. Journal of Animal Science 69, drugs, they are not acceptable as euthanizing 4167±75 agents. Bundesamt fuÈ r VeterinaÈrwesen (1984) Blutentnahme bei Labornagetieren, Kaninchen und Katzen. In- formationsblatt Tierschutz, CH-3097 Liebefeld- Further reading Bern 15.2.1984 Bundesamt fuÈ r VeterinaÈrwesen (1985) ToÈten von Versuchstieren (Hund, Katze, Labornagetiere, General VoÈgel, Amphibien und Reptilien, Fische). Informa- Adolph EF (1969) Regulations during survival without tionsblatt Tierschutz, CH-3097 Liebefeld-Bern oxygen in infant mammals. Respiration Physiology 25.2.1985 7, 356±68 Bustad LK (1982) An educator's approach to euthana- Allert JA, Adams HR (1987) Pharmacologic consid- sia. Laboratory Animals 11, 37±41 erations in selection of tranquillizers, sedatives and Butler MM, Griffey SM, Clubb FJ, Gerrity LW, muscle relaxant drugs used in inducing animal Campbell WB (1990) The effect of euthanasia restraint. Journal of the American Veterinary technique on vascular arachidonic acid metabolism Medical Association 191(10), 1241±4 and vascular and intestinal smooth muscle con- Anon (1988) Reducing pain can magnify stress. The tractility. Laboratory Animal Science 40 (3), 277±83 Veterinary Record November 26, 559±60 Canadian Council on Animal Care (1988) AVMA Anon (1990) Barbituraatvergiftiging bij dierentuindie- 1986 Report on Euthanasia. Euthanasia methods Ð ren. Tijdschrift voor Diergeneeskunde 115(5), 241±2 cervical dislocation and decapitation with guillo- Bancroft RW, Dunn JE, II (1965) Experimental animal tine. Press release January 1988 decompressions to a near vacuum environment. Carstensen J (1978) The need for better speci®cation Aerospace Medicine 36, 720±5 of the animal model in the test situation. In: Papers Barr FM (1987) Waste anaesthetic gas exposure in and Abstracts/Symposium on Design of Experi- veterinary surgeries: a need for scavenging systems. ments and Quality of Laboratory Animals, Kuopio New Zealand Veterinary Journal 35, 68±71 5±7 October 1978. (Nevalainen T, Pelkonen K, eds) Cavaliere U, Andreano C, Raducci G, Andreoni C, Battisti GA (1984) CO2 euthanasia: Letter to the Editor/Editor's note. Laboratory Animal Science Iacovella A (1982) Intossicazione de T-61 (TanaxR). 34(3), 228 Minerva Anestestiologica 48, 861±3 Baumans V, van Herck H, Bartels H, Bertens APMG, Chatrian GE (1980) Electrophysiologic evaluation of Hoenderken R, Schlingmann F (1991) Methods of brain death: a critical appraisal. In: Electrodiagnosis euthanasia used for laboratory animals in the in Clinical Neurology. (Aminoff MJ, ed). New York: Netherlands. Abstract voordracht GV-SOLAS Churchill Livingstone, pp 525±80 (Lubeck), 9±13 September 1991 Conway CM (1965) The anaesthetic ethers. 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