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WORKING PARTY REPORT Recommendations for 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 10 October 1996 issue of Laboratory Animals 3.5 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, , 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 enormously varying lifestyles which makes is approximately 1 min, and it very dif®cult to generalize on methods of 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 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 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 , sodium 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 before adding to water. medium ®sh are killed by inserting a rod or thumb into the mouth, holding the ®sh with It is a potent imidazole-based the opposite hand and displacing it dorsally agent with no 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. It is an imidazole-based non- Small ®sh of less than 2 cm in 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 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. Tricaine methane sulphonate (buffered MS-222) This acts by depression of the Injectable agents may be used central nervous system. This is a benzocaine but as this requires removal from water and 4 Working Party Report handling, with resultant stress, other with all ®sh (e.g. eels) and used alone it is not methods are preferable. The intraperitoneal necessarily lethal (it may only stun larger route is generally recommended. ®sh). Alternating current stimulates con- traction of skeletal, cardiac and smooth Methods acceptable for unconscious ®sh muscle, and it induces tetany, not anaesthe- sia (Summerfelt & Smith 1990). Although it Pithing For small ®sh pithing can be is used on ®sh farms by experts, often to employed in which a metal spike is pushed catch ®sh, it is not considered acceptable in into the top of the head between the eyes and the laboratory situation for euthanasia. pushed forwards and backwards to destroy the brain and proximal end of the spinal cord. Hypothermia Putting ®sh into a freezer or This method should only be carried out on crushed ice prolongs the period of con- unconscious animals. This method is con- sciousness in ®sh and does not reduce the sidered acceptable when chemical methods ability to feel pain; therefore it should not be are not appropriate for the study. used as a method of euthanasia.

Decapitation This is possible in small ®sh Fish when put into hot water but becomes problematic in larger ®sh. will press their opercula tightly to the body Decapitation of ®sh should only be carried and thus have a depot of which out under anaesthesia or after stunning as prolongs their period of consciousness. Boil- there is some doubt about immediate loss of ing water will cause extreme pain. This consciousness. Research on eels showed that method should therefore not be employed to the brain was still functioning for up to kill ®sh of any kind. 35 min after decapitation (Verheijen & Flight 2- Its main use is as an 1995) and therefore the brain should be antibiotic but is used as an anaesthetic agent immediately destroyed. This method is as well. It requires large doses to achieve acceptable only if other methods are not death with a long induction period. Some considered suitable and under the above species exhibit hyperactivity prior to loss of constraints. Spinal transection (the neck cut) consciousness. It is not considered acceptable is also unacceptable except on insensible ®sh for use for euthanasia of ®sh. (Flight & Verheijen 1993). Carbon dioxide This is not acceptable for Exsanguination This is not considered euthanasia of ®sh as it causes intense activity acceptable as a method of euthanasia as it is prior to loss of consciousness and is slow too slow and too dif®cult to locate the veins, acting. unless the ®sh is insensible. This should not be used Methods not acceptable for euthanasia of because of irritation to the mucous mem- ®sh branes as well as danger to the operator.

Removal from water This causes distress and Both have and suffering because of the length of time to the disadvantage that induction is too long. unconsciousness. Cooling prolongs this per- iod considerably. This is not an acceptable Urethane This is carcinogenic and there- method of euthanasia of ®sh (Kestin 1993, fore extremely dangerous to the operator. Kestin et al. 1991). hydrate This has a long induction period and only acts as a . Whole body crushing This is not consid- ered a humane method of euthanasia. Tertiary amyl This causes irritation during induction. Electrical stunning This could be danger- ous for the operator, if an isolated circuit is This is irritant and has a not used. Electrical stunning does not work long induction period. Euthanasia of experimental animals 5

Chlorobutanol This has the disdavantage of blood supply these animals are able to respond requiring a broad range of dosages across the to stimuli for some time. Although decapita- different species. tion, by itself, does not produce rapid un- consciousness in the severed heads of Methyl pentynol This causes stress by amphibians, rapid destruction of the brain . does extinguish responses usually thought to indicate consciousness. There is, however, a Pyridines These are dangerous to the op- remarkably intact set of somatic responses to erator. stimuli Ð long continued body movements, foot withdrawals in response to toe pinching, 3.2. Amphibians etc., as well as continued heartbeat in many There are many species of amphibians making cases for hours following brain destruction. it dif®cult to generalize on methods of This continued somatic activity is attributed euthanasia. A summary is given in Table 3. to: The skin is thin, protected by a cuticle bearing many mucus glands. This results in it being (1) prolonged tolerance of the spinal cord, generally more sensitive to physical and peripheral nerves and muscle (smooth, chemical insults than in other vertebrates. cardiac and skeletal) to hypoxic and Because amphibians are ectothermic and thus hypotensive conditions, and accustomed to ¯uctuations in body tempera- (2) a far greater degree of integration of ture, their central nervous system (CNS) is less somatic responses at the level of the sensitive to hypoxia and anoxia. Even when spinal cord instead of the brain. the cranial nerves and brain are deprived of (UFAW/WSPA 1989)

Table 2 Characteristics of methods for euthanasia of fish

Overall Ease of Operator Aesthetic rating Agent Rapidity Efficacy use safety value (1^5) Remarks

MS-222 ++ ++ ++ ++ ++ 5 Acceptable Benzocaine ++ ++ ++ ++ ++ 5 Acceptable Etomidate ++ ++ ++ ++ ++ 5 Acceptable Metomidate ++ ++ ++ ++ ++ 5 Acceptable Concussion ++ + + ++ ^ 4 Death to be confirmed Maceration ++ ++ ++ ++ + 4 Only for fish less than 2 cm in length Quinaldine ++ ++ ++ + ++ 4 Difficult to obtain in Europe Sodium ++ ++ ^ + ++ 3 May be useful for large pentobarbitone fish, intraperitoneal injection Cervical dislocation ++ ++ + ++ ^ 3 Not in large fish.To be followed by destruction of the brain Halothane + + ++ ++ ++ 2 Other methods preferable. Death to be confirmed

The following methods may only be used on unconscious fish: pithing, decapitation and exsanguination The following methods are not to be used for killing fish: removal from water, whole body crushing, electrical stunning, hypothermia, hyperthermia, 2-phenoxyethanol, carbon dioxide, diethyl ether, secobarbital, amobarbi- tal, urethane, , tertiary , tribromoethanol, , methyl pentynol, pyridines

Rapidity: ++ very rapid, + rapid, ^ slow. Efficacy: ++ very effective, + effective, ^ not effective. Ease of use: ++ easy to use, + requires expertise, ^ requires specialist training. Operator safety: ++ no danger, + little danger, ^ dangerous. Aesthetic value: ++ good aesthetically, + acceptable for most people, ^ unacceptable for many people. Rating:1^5 with 5 as highly recommended 6 Working Party Report

Recognition and con®rmation of death destruction of the brain, it may be considered Death may be recognized by cessation of an acceptable method of euthanasia. heartbeat and respiration and in cases where this is not obvious, it may be con®rmed by Chemical methods destruction of the brain. Drugs are best administered by dissolving them in the water in which the amphibians Larvae are placed. This reduces stress from handling Tadpoles and newts can be effectively killed and injection. by placing in a dish of water with MS-222 or benzocaine (dissolved in acetone). These Tricaine methane sulphonate (buffered produce rapid anaesthesia, followed by death. MS-222) This is a quick, non-irritant and humane method of killing amphibians when Adults dissolved in the water into which the Further details of methods may be found in amphibians are placed. It is recommended to Section 2. neutralize the solution with bicarbonate to When handling these species, it is impor- reduce irritation to the sensitive amphibian tant to obtain a ®rm hold, for example by skin. wearing rough textured but non-abrasive gloves or by holding them in coarse material. Benzocaine Dissolved in the water in Cooling to 3±48C will reduce metabolic and which amphibians are placed, benzocaine is locomotory processes, thus facilitating an effective drug acting on the CNS quickly handling prior to euthanasia. However, it and humanely. As benzocaine is not soluble must be remembered that cooling does not in water, it must be ®rst dissolved in acetone. reduce the ability to feel pain (UFAW/WSPA, The solution must be neutralized to avoid 1989). irritation as benzocaine reduces the pH. Sodium pentobarbitone This drug, when Physical methods injected intravenously or intraperitoneally Concussion This method, if carried out by a acts quickly on the CNS, rendering the person who is well trained in the technique is animal unconscious with little distress. an effective and humane way of stunning all However, this should only be carried out by amphibians. The hind legs should be grasped experienced people to ensure the correct site and the dorsal surface of the head struck for injection and to reduce handling to a against a hard, solid object. Alternatively, the minimum. dorsal surface of the head can be struck with a suitable instrument. Accuracy is essential T-61 Intravenously injected, or in frogs, to ensure immediate unconsciousness and injected into the dorsal lymph sac, this drug death. Death must be ensured by destroying is effective and humane for the euthanasia of the brain after concussion. amphibians.

Microwave This is an extremely quick Methods acceptable for unconscious method of euthanasia but must only be amphibians carried out by experienced personnel who know exactly where to direct the beam. Only Pithing Ensures a rapid destruction of the specialized microwave equipment designed brain resulting in immediate unconscious- for this purpose is to be used. Under no ness. This is a rapid and humane method of circumstances should domestic appliances killing amphibians if carried out by well be used for this purpose. It is not considered a trained and experienced operators. This routine method of euthanasia. method should only be carried out on unconscious animals. In some species it is Electrical stunning Frogs rendered insensi- dif®cult to bend the head forward to expose ble by electrical means may recover after the atlanto-occipital space and other meth- 10 min but if followed immediately by ods are preferable in these cases. Euthanasia of experimental animals 7

Decapitation Is acceptable only in insensi- scious, making this an unacceptable method ble amphibians as the time to unconscious- of euthanasia. ness is unknown because the nervous system is very tolerant of anoxia. Strangulation This is considered inhu- mane, and is not an acceptable method of Methods not acceptable for the euthanasia killing amphibians. of amphibians Carbon dioxide This may cause irritation Hypothermia This will make the animal to the skin, and induction takes too long, and torpid but does not reduce pain. Freezing is is therefore not considered an acceptable not acceptable, as the formation of ice method. crystals within the body tissues is likely to be extremely painful. Freezing may only be Ether This is irritant to the mucous mem- used as a method to con®rm death after branes and because of danger to the operator, another method of euthanasia has been used. it should not be used for killing amphibians.

Hyperthermia Amphibians should not be This is hepatoxic and carcino- dropped into hot or boiling water as a method genic and because of the consequent possible of euthanasia as this is extremely painful and danger to personnel, chloroform should not inhumane. be used for euthanasia.

Exsanguination Together with subsequent Volatile inhalational anaesthetics These hypovolaemic shock and anoxia this may not are not considered acceptable as they are render amphibians immediately uncon- slow to act and may be irritant to the skin.

Table 3 Characteristics of methods for euthanasia of amphibians

Overall Ease of Operator Aesthetic rating Agent Rapidity Efficacy use safety value (1^5) Remarks

MS-222 ++ ++ ++ ++ ++ 5 Acceptable Benzocaine ++ ++ ++ ++ ++ 5 Acceptable Sodium + ++ ^ + + 4 Involves handling and pentobarbitone intravenous or intraperitoneal injection Concussion ++ ++ + ++ ^ 4 Acceptable for use by experienced personnel T-61 + ++ ^ + + 3 Involves handling and intravenous injection Microwave ++ ++ ^ + ++ 3 Only for small amphibians. Not a routine procedure Electrical stunning + + + ^ ^ 2 To be followed immediately by destruction of the brain

The following methods are only to be used on unconscious amphibians: pithing and decapitation The following methods are not to be used for killing amphibians: hypothermia, hyperthermia, exsanguination, strangulation, carbon dioxide, diethyl ether, chloroform, volatile inhalational anaesthetics, chloral hydrate, keta- mine hydrochloride, chlorbutanol, , 2-phenoxyethanol, tertiary amyl alcohol, tribromoethanol and urethane

Rapidity: ++ very rapid, + rapid, ^ slow. Efficacy: ++ very effective, + effective, ^ not effective. Ease of use: ++ easy to use, + requires expertise, ^ requires specialist training. Operator safety: ++ no danger, + little danger, ^ dangerous. Aesthetic value: ++ good aesthetically, + acceptable for most people, ^ unacceptable for many people. Rating:1^5 with 5 as highly recommended 8 Working Party Report

Other agents not considered acceptable However it must be remembered that cooling include: chloral hydrate, hydro- does not reduce the ability to feel pain. chloride, chlorbutanol, methylpentynol, 2- In tortoises, turtles and terrapins, retraction phenoxyethanol, tertiary amyl alcohol, tri- of the head and protection by the carapace can bromoethanol, urethane. cause dif®culty for euthanasia. To assist in exposing the head, land tortoises can be placed in shallow, tepid water; large marine species 3.3 Reptiles may be put on a frame at 458 head up, inducing neck extension; and soft-shelled species can be Because reptiles are ectothermic and thus placed on their backs to induce neck exten- accustomed to ¯uctuations in body tem- sion. Rough textured but non-abrasive gloves perature, their central nervous system is less may be worn when handling aquatic species to sensitive to a drop in oxygen tension. Even facilitate handling. when the cranial nerves and brain are Effective restraint of the jaws and tail is the deprived of blood supply following decapita- key factor to operator safety for restraining tion, these animals are able to respond to crocodilians and this should only be done by stimuli for some time. Although decapita- experts (UFAW/WSPA 1989). tion, by itself, does not produce rapid A summary of recommendations is given unconsciousness in the severed heads of in Table 4. reptiles (Warwick 1990), rapid destruction of the brain does extinguish responses usually Recognition and con®rmation of death thought to indicate consciousness. There is, As it is dif®cult to determine whether however, a remarkably intact set of somatic reptiles are unconscious or dead, it is responses to stimuli Ð long continued body recommended that death be con®rmed by movements, foot withdrawals in response to destruction of the brain. Usually, but by no toe pinching, etc., as well as continued means always, a lack of pupillary±blink± heartbeat in many cases for hours following nictitating membrane responses, except in brain destruction. This continued somatic snakes which do not possess movable eye- activity is attributed to: lids, implies a lack of consciousness. is a reliable indicator of death as is the (1) prolonged tolerance of the spinal cord, prolonged absence of a heartbeat and/or peripheral nerves and muscle (smooth, circulation. cardiac and skeletal) to hypoxic and hypotensive conditions, and Embryos (2) a far greater degree of integration of In the case of reptiles two distinct stages somatic responses at the level of the have to be considered: the eggs and the spinal cord instead of the brain. newly-hatched individuals. For all practical (UFAW/WSPA 1989). purposes all newly hatched reptiles can be treated in the same way as adults. As reptiles Good methods of restraint are important to are born as fully developed individuals (with ensure minimal stress prior to carrying out the exception of not being able to reproduce), euthanasia. killing of the embryos at the egg stage must Particular care must be taken when hand- be carried out in a humane way to account ling venomous species, such as many types of for potentially advanced development. In snake, especially when they are not used to general eggs may succumb to high and low being handled. Padded grasping implements temperatures but some may withstand are useful in handling lizards and snakes to freezing. Hypothermia and hyperthermia ensure a ®rm but non-damaging restraint. cannot be considered acceptable because Cooling of most reptiles to 3±48C will reduce humane death cannot be guaranteed. metabolic and locomotory processes (this Drowning is not considered humane as this temperature may kill some tropical species), results in death by anoxia and is slow. Eggs thus facilitating handling prior to euthanasia. with no embryo may be frozen. Recom- Euthanasia of experimental animals 9 mended methods would include disruption of many reptiles. This method could also be the egg and killing of the embryos by dangerous to the operator and should there- injection of sodium pentobarbitone, anaes- fore only be used in ®eld conditions. A heavy thetic overdose or an appropriate physical calibre ri¯e or shotgun of a suitable calibre is method to destroy the brain or whole egg or necessary for large animals such as adult early life form. crocodiles. It is necessary to ensure that the Adults animal does not move its head prior to shooting. The Testudines must have their As the class Reptilia is varied, it is best to head exposed and held thus for accurate consider three main groups: the snakes and positioning of the gun. lizards (Squamata); tortoises, turtles and terrapins (Testudines); and crocodiles and Chemical methods alligators (Crocodilia). Larger reptiles may need to be sedated before being killed. Sodium pentobarbitone Sodium pentobar- Further details on methods may be ob- bitone is an effective and humane method of tained in Section 2. euthanasia in reptiles. The intravenous route can be used by well trained personnel. Where Physical methods intravenous injection is dif®cult the intra- Captive bolt This method may be used peritoneal route may be used but it is slower- within the laboratory situation with relative acting. Injections should not be made in- safety. It is considered an acceptable method tracardially or into the lungs as this is for large reptiles but should only be carried out regarded as painful and irritant. by experts who know exactly where to Methods acceptable for unconscious position the pistol. Care must be taken to reptiles ensure that the pistol is well maintained and is of the correct calibre and cartridge length for Pithing This may only be carried out on the species being killed. Good restraint is unconscious animals and by experienced necessary to ensure humane killing. If the bolt personnel. goes through the brain, it should kill the reptile, otherwise it may just stun the animal. Decapitation This may only be used if the Death must be ensured by destruction of the reptile has been made unconscious by other brain. methods, such as concussion, as long periods Concussion Small reptiles and those with of post-decapitation consciousness have been ®ne bone structures such as some snakes and recorded (Warwick 1990). lizards may be rendered unconscious by stunning. This involves striking the back of Methods not acceptable for euthanasia of the head of the animal with some hard reptiles implement or object, the principle being Spinal cord severance Because of the ability either to kill the animal outright or to render of reptiles to withstand anoxia and cerebral it unconscious. Ideally, the blow should be hypoxaemia, spinal cord severance is not an given with such force as to cause cessation of acceptable method of euthanasia. It has been brain activity. This must only be performed shown that crocodiles may remain conscious by people trained and experienced in hand- for up to 1 h 50 min after spinal cord ling and killing reptiles. Concussion must severance and other reptiles may remain always be followed by destruction of the conscious for similar lengths of time. brain. Shooting This is an effective method of Hypothermia This will make the animal killing most large reptiles, causing rapid and torpid but will not raise the pain threshold. substantial destruction of the brain. A high The formation of ice crystals within the body level of skill is required in order to hit the tissues is likely to be extremely painful. brain through the two brain cases found in Hypothermia is not acceptable for euthanasia. 10 Working Party Report

Hyperthermia This is not considered ac- too long to induction), chloral hydrate and ceptable as the time to unconciousness is procaine. unknown. Boiling water must never be used to kill reptiles. 3.4 Birds

Exsanguination This is not considered hu- Birds have a complex respiratory system mane due to the animal's tolerance of hypoxia. comprising the lungs and numerous air sacs with a one-way ¯ow of air. This may in¯uence the rate of absorption of inhalational agents Chloroform This has been used to kill and thus increase their ef®ciency. tortoises by injection into the peritoneal A summary of recommendations is given cavity with apparently no undesirable effects. in Table 5. Because of the potential trauma to the injected animal and danger to the operator, Recognition and con®rmation of death other methods are considered more accepta- Death may be recognized by the absence of ble. Chloroform is hepatotoxic and carcino- signs of breathing, cardiac arrest and absence genic to the operator. of re¯exes in the head (i.e cranial nerve re¯exes rather than spinal cord re¯exes). Tricaine methane sulphonate (MS-222) This Re¯exes to be checked would include has been injected intramuscularly in snakes pinching of wattles or blink re¯exes. Death and alligators. There is little information on can be ensured by destruction of the brain or the humaneness of this method and it is ensuring cessation of the heartbeat. therefore not considered acceptable. Reptiles are capable of holding their breath Embryos for a relatively long period of time and embryos from the stage at which the therefore inhalational methods such as ether, neural tube has developed into a functional halothane, en¯urane, iso¯urane and brain (450% gestation) must be destroyed methyoxy¯urane cannot be considered as humanely as they may be capable of per- practicable or humane due to slow induction. ceiving pain from that stage. The most Other agents not to be used for killing commonly used method of destroying eggs is reptiles include CO2, neuromuscular block- cooling or freezing. The recommended tem- ing agents, ketamine hydrochloride (takes perature is 548C for 4 h. Death must be Table 4 Characteristics of methods for euthanasia of reptiles

Overall Ease of Operator Aesthetic rating Agent Rapidity Efficacy use safety value (1^5) Remarks

Sodium ++ ++ ++ + ++ 5 Acceptable, but involves pentobarbitone handling Captive bolt ++ ++ ++ + + 5 Acceptable for large reptiles Concussion + + + ++ + 4 To be followed by destruction of the brain Shooting ++ ++ ++ ^ + 4 Acceptable only in field conditions

The following methods are to be used on unconscious reptiles only: pithing and decapitation The following methods are not to be used for killing reptiles: spinal cord severance, hypothermia, hyperthermia, exsanguination, chloroform, MS-222, ether, halothane, , , , carbon dioxide, neuromuscular blocking agents, ketamine hydrochloride, chloral hydrate and procaine

Rapidity: ++ very rapid, + rapid, ^ slow. Efficacy: ++ very effective, + effective, ^ not effective. Ease of use: ++ easy to use, + requires expertise, ^ requires specialist training. Operator safety: ++ no danger, + little danger, ^ dangerous. Aesthetic value: ++ good aesthetically, + acceptable for most people, ^ unacceptable for many people. Rating:1^5 with 5 as highly recommended Euthanasia of experimental animals 11 con®rmed by decapitation or some other cient to ensure that all animals are killed suitable method. In cases where the embryo immediately with no possibility of animals has been exposed for studies, decapitation is being thrown out by the revolving blades. considered an acceptable method of eutha- Chicks must be dropped one at a time down a nasia as is an overdose of anaesthetic. special chute to reduce the chances of being Disruption of the membranes and macera- thrown out by the blades in smaller appara- tion (in a macerator designed for the purpose) tuses, but larger ones have been designed to are also considered as humane methods of take larger number of chicks at a time, killing bird embryos (Bandow 1987). without this risk. This method may be aesthetically unpleasant for some operators. Adults Under no circumstances should domestic Further details on methods may be found in appliances be used. Section 2. Concussion This is carried out by a hard Physical methods blow to the head and in small birds (5250 g) this may be done by striking the head on the Cervical dislocation Cervical dislocation, if sharp edge of a table. Although not aestheti- carried out near to the head, causes damage cally pleasant it is quick and humane if carried to the lower brain region, resulting in rapid out correctly by a person trained and experi- and painless loss of consciousness. This must enced in the technique. This method may also always be followed immediately by destruc- be acceptable for small numbers of day-old tion of the brain or section of the major blood chicks. Unless suf®cient has vessels in the neck. However, research has been caused resulting in immediate death, this shown that visual evoked potentials may must be followed by destruction of the brain. remain up to 30 s after dislocation which may indicate lack of insensibility (Gregory & Microwave Small birds may be killed ra- Wotton 1990). Therefore, other methods are pidly and humanely by microwave delivered considered preferable. This method is not by specialist apparatus (Zeller et al. 1989). aesthetically pleasant as re¯exes remain Under no circumstances should domestic present for some time. This should not be appliances be used. Operators must receive used for birds of over 3 kg or some older birds specialist training in this technique to ensure where the neck is dif®cult to pull quickly. accurate direction of the microwave beam This method may be used for day-old chicks and thus humane death. This method should as long as the numbers are kept low to avoid not be considered as a routine method of human error due to tiredness (Jaksch 1981). euthanasia. The wings of the birds should be secured to avoid involuntary ¯apping (Clifford 1984). Electrical stunning Electrical stunning is commonly used in slaughterhouses but is Maceration This method may be used for generally not considered acceptable for use chicks up to 72 h old using specialist in the laboratory unless specialized equip- apparatus which contains rapidly rotating ment, safe for personnel, under legal controls, mechanically operated killing blades (Com- is used. The bird must be stunned before mission of the European Communities 1993). cardiac arrest occurs (i.e. the electrodes are Only equipment designed for this purpose placed in such a position that the brain is and complying to the standards of Council ®rst affected). Directive 93/119/EC should be used. The blades must turn at 45000 revs/min. Chemical methods Operators must be trained in the use of such Inhalational agents equipment and also have training in the maintenance of the equipment to ensure Carbon dioxide This method is used on a correct functioning at all times. The capacity large scale for chicks up to 72-h old (Clifford and design of the apparatus must be suf®- 1984). These chicks are young and relatively 12 Working Party Report

insensitive to CO2 so higher doses may be by experienced personnel. It should be needed than for adult birds. The chicks injected intraperitoneally. Some experienced should be placed in non-porous bags or operators may inject it into the foramen containers ®lled with 100% CO2 for at least magnum at the base of the skull (intrace- 10 min using a vapour feed system. A closed phalic) which is quick. system is preferable and quicker in induction than open systems. Care should be taken to T-61 Injected intramuscularly into the pec- avoid overcrowding and the levels of CO2 toral muscles of small birds, T-61 is very must be constantly monitored and main- effective. It should not be used for larger birds tained. Carbon dioxide may also be used in or poultry because it takes some time to act conjunction with argon and oxygen for and causes convulsions. poultry (Raj & Gregory 1994). The CO2 causes loss of consciousness and the argon Methods acceptable for unconscious birds causes death by hypoxia. When killing larger Decapitation This reduces the blood pres- birds with CO2, care must be taken to ensure that the chamber is completely ®lled prior to sure very quickly which may result in putting the birds into it to ensure uniform unconsciousness as well as massive trauma across the cord at the level of the brain stem levels of CO2 throughout the chamber. Older birds may ¯ap their wings after losing with an ascending and descending effect on consciousness which may not be acceptable neural activity. However, work carried out by to some operators. Gregory and Wotton (1986, 1990) on chickens shows that there are visually evoked re- Volatile inhalational anaesthetics Air or sponses for up to 30 s after decapitation. oxygen must be provided during the induc- Other methods are considered preferable tion period. Appropriate gas scavenging until further research can show that the birds equipment needs to be used with all these are made immediately insensible. agents. Pithing This is not acceptable unless the Halothane, en¯urane, iso¯urane These bird is fully anaesthetized. agents are considered acceptable for the euthanasia of most birds. They are safe for This kills birds by anoxia. Day-old personnel if used with gas scavenging appa- chicks should not be killed with nitrogen ratus and they are effective in causing because of their ability to withstand low anaesthesia and euthanasia. concentrations of oxygen. The responses by the unconscious bird may be objectionable to Carbon monoxide Carbon monoxide personnel. In general the use of nitrogen for causes rapid death as it combines with the the euthanasia of birds is not considered red blood cells in preference to oxygen, thus acceptable unless unconscious. causing hypoxia. However, it is extremely dangerous to personnel because it is not Potassium chloride This is cardiotoxic and easily detectable and should only be used by causes muscle spasms and convulsive sei- people trained in the technique and with zures, thus making it unpleasant for the appropriate gas scavenging apparatus. Only operator. It may only be used once the birds the use of commercially compressed CO are fully anaesthetized. should be used for euthanasia. Death must be con®rmed by physical means. Methods not acceptable for euthanasia of birds Injectable agents Neck crushing The neck of the small bird is Sodium pentobarbitone This is an accep- pressed against a bar, or a specialized set of table method of euthanasia for birds of all pliers, designed for the purpose, is used. It is ages. Sodium pentobarbitone causes rapid not known whether neck crushing produces and relatively stress-free death if carried out immediate unconsciousness (Gregory & Euthanasia of experimental animals 13

Wotton 1990). This method is not considered this method is not acceptable in the labora- acceptable for euthanasia as there are other tory situation. more humane methods.

Nitrous oxide Hypoxic concentrations of up Exsanguination Is not an acceptable to 100% are required to be effective and it is method of killing birds because the blood slow-acting. The bird will convulse after clots easily, possibly resulting in incomplete losing consciousness which may deter some exsanguination. operators. It is not acceptable for euthanasia of birds. Decompression (creating a vacuum) This induces dypsnoea. After 20 s at 60 mmHg the birds collapse. Because the time to uncon- Ether and chloroform These are not to be sciousness is not known and decompression used for the killing of birds due to extreme causes rapid expansion of in the air sacs operator danger and irritation to the respira- and pneumatic bones which may cause pain, tory passages of the bird.

Table 5 Characteristics of methods for euthanasia of birds

Overall Ease of Operator Aesthetic rating Agent Rapidity Efficacy use safety value (1^5) Remarks

Sodium ++ ++ + + ++ 5 Acceptable pentobarbitone T-61 ++ ++ + + ++ 4 Requires expertise: acceptable for small birds only (5250 g) Carbon dioxide ++ ++ ++ ++ + 4 Acceptable method, especially for chicks Halothane, ++ ++ ++ + ++ 4 Acceptable enflurane, isoflurane Maceration ++ ++ ++ ++ ^ 4 Acceptable for chicks up to 72 h Cervical dislocation ++ ++ ^ ++ ^ 4 Acceptable for small and young birds (5250 g) if followed by destruction of the brain Microwave ++ ++ ^ ++ + 3 To be used by experienced personnel only. Not a routine procedure Concussion ++ ++ ^ ++ ^ 3 Acceptable for birds under 250 g Carbon monoxide + + ++ ^ + 2 Danger to operator Electrical stunning + + + ^ ^ 1 Danger to operator. Other methods preferable

The following methods may only be used on unconscious birds: decapitation, pithing, nitrogen, potassium chloride The following methods are not to be used for killing birds: neck crushing, decompression, exsanguination, , diethyl ether, chloroform, , hydrogen gas, trichlorethylene, methoxyflurane, chloral hydrate, , , sulphate, ketamine and neuromuscular blocking agents

Rapidity: ++ very rapid, + rapid, ^ slow. Efficacy: ++ very effective, + effective, ^ not effective. Ease of use: ++ easy to use, + requires expertise, ^ requires specialist training. Operator safety: ++ no danger, + little danger, ^ dangerous. Aesthetic value: ++ good aesthetically, + acceptable for most people, ^ unacceptable for many people. Rating:1^5 with 5 as highly recommended 14 Working Party Report

Cyclopropane This is humane and causes administered to the dam and maintained for rapid anaesthesia, but is ¯ammable and longer to ensure that the anaesthetic has explosive in air. Because of the danger to the crossed the placenta. In many cases inhala- operator, cyclopropane is not acceptable for tional anaesthetics will not anaesthetize a euthanasia of birds. foetus. Foetuses under 4 g not anaesthetized prior to removal from the dam may be killed by Hydrogen cyanide gas This causes rapid rapid cooling in liquid nitrogen. and irreversible death by cytotoxic hypoxia but it also causes excitability and distress Neonates before death which makes it totally unac- These are newborn rodents up to 10 days old. ceptable. They may react more like embryos than Other agents not to be used include meth- adults to painful stimuli. They may be killed oxy¯urane, trichlorethylene, chloral hydrate, by decapitation or concussion. Hypothermia strychnine, nicotine, magnesium sulphate, may be considered (Phifer & Terry 1986). ketamine alone and neuromuscular blocking Carbon dioxide is not recommended as agents. neonates are more resistant to it, increasing the time to unconsciousness. Further re- search is necessary to assess which methods 3.5 Rodents are the most humane.

Rodents are the most commonly used Adults animals for experimental purposes and in- clude mice, rats, hamsters, guineapigs, ger- Further details on the methods may be found bils, shrews, and dormice. A summary of in Section 2. recommendations is given in Table 6. Physical methods Recognition and con®rmation of death When carrying out physical methods of Cessation of respiration and heartbeat, and killing rodents, consideration must be given absence of re¯exes are good indicators of to the careful handling and restraint of the irreversible death in rodents. Death may be animal prior to killing. Minimal handling con®rmed by exsanguination or extraction of and restraint is preferable. Fear and to the heart, evisceration, deep-freezing or decap- the animal may be reduced by prior sedation itation. Personnel must be trained to recognize or handling by familiar people. and ensure death when killing rodents. Concussion This is a quick and humane method of stunning rodents as long as it is Embryos performed by experienced and con®dent The time at which the neural tube develops operators. This should only be used on into a functional brain (about 60% gestation) rodents of less than 1 kg, above which must be taken as the time at which the considerable skill and sometimes a great deal foetus may perceive pain and should there- of strength are required to perform it fore be killed humanely. There is a large ef®ciently. Death must always be con®rmed. variation in degree of development at birth of various rodents. Mice and rats are totally Cervical dislocation This is a commonly dependent on the nest and have very few used and humane method of killing most layers of neuronal development in the cere- small rodents (under 150 g (Marshall et al. bral cortex, whereas guineapigs are fully 1994)) as it causes extensive damage to the developed and independent at birth. brainstem resulting in immediate uncon- If a foetus is removed from an anaesthetized sciousness and death. It is more dif®cult on mother, and is also insensible, then it may be hamsters and guineapigs due to their short killed by decapitation or removal of the heart. necks, stronger neck muscles and loose skin However, when the foetus is to be removed an over neck and shoulders. In mice and rats the increased amount of anaesthetic must be thumb and index ®nger are placed on either Euthanasia of experimental animals 15 side of the neck at the base of the skull, or, the anaesthetic. Because the liquid state of alternatively, a rod is pressed at the base of these anaesthetics are irritant, care must be the skull and then with the other hand, the taken to ensure that the cannot come base of the tail or hind limbs are quickly into contact with the chemical. Air or oxygen pulled, causing separation of the cervical must be provided during the induction vertebrae from the skull. Larger rodents and period. older rats should be sedated or stunned prior to dislocation. Death must be con®rmed as Halothane, en¯urane, iso¯urane These indicated in the section, recognition and agents act by depression of the cardiovascular con®rmation of death. and respiratory systems. They induce anaes- thesia and subsequent death. These are all Decapitation The immediate lack of circu- acceptable agents when used with appropri- lation of blood to the brain and subsequent ate gas scavenging apparatus. anoxia is thought to render the head rapidly insensible (Derr 1991). Prior anaesthetization Carbon dioxide It is recommended that a is not generally recommended as this in- minimum of 70% CO2 in oxygen or air be volves further handling and consequent used for quick loss of consciousness without stress to the animal. Other methods are hypoxia. This results in rapid anaesthesia preferable until further research can show followed by death with reduced effects from immediate unconsciousness. Specialized ap- irritation of the respiratory passages. One paratus should always be used. Care must be hundred per cent CO2 is recommended for taken to ensure that the apparatus is kept guineapigs (Noonan 1994). Only CO2 from clean and the blades are sharp. commercially available gas cylinders should be used for euthanasia of rodents. See Section Microwave This involves the use of specia- 2 for further details. lized apparatus and should only be carried out by personnel specially trained in this techni- Carbon monoxide Although this is a rela- que to ensure correct positioning of the beam. tively quick and humane method of killing When carried out correctly, it is an extremely rodents, because of the danger to the operator quick method of killing rodents and therefore it must be used with extreme caution. If very humane. Under no circumstances should chosen, it should be used with appropriate domestic appliances be used for this purpose. gas scavenging appparatus and only com- This is not a routine method of euthanasia. mercially available gas in cylinders is re- commended. The rodents must be placed Rapid freezing This is carried out by put- into a container pre®lled with at least 6% CO ting the animal into liquid nitrogen. This by volume. may only be used for foetuses and small neonates (54 g) with no fur. Larger or furred Injectable agents animals will not die immediately as it takes In larger rodents, where venepuncture is some time for the core to become frozen. possible without due stress to the animal, intravenous injection is recommended as this Chemical methods results in rapid anaesthesia and death. If When using chemical methods for euthana- venepuncture is not easily performed, intra- sia, death must be con®rmed by one of the peritoneal injection is preferred, although it methods listed above. takes longer to act and irritation of the peritoneum may occur. Under no circum- Inhalational agents stances should intrapulmonary or intracar- dial injection be given unless the animal is Volatile inhalational anaesthetics With fully anaesthetized. these agents the rodent is put into an anaesthetic chamber or an appropriate re- Sodium pentobarbitone Injected intra- ceptacle with gauze or cotton wool soaked in venously or intraperitoneally sodium pento- 16 Working Party Report barbitone acts quickly and humanely to kill well trained in intravenous injection all rodents. This is the most acceptable agent techniques. for euthanasia. All personnel must be trained in the method of injection. Sodium pento- Methods acceptable for unconscious barbitone may cause irritation of the perito- rodents neum, which can be avoided by dilution. Three times the anaesthetic dose is usually Rapid freezing This may only be used once recommended (Marshall et al. 1994, Noonan the rodent (44 g) is fully unconscious. 1994). Exsanguination This may be used once the rodent is unconscious.

T-61 T-61 acts quickly but must be injected Air embolism This may only be used on intravenously very slowly, which is not unconscious rodents as it can be painful. always easy in rodents. It should never be injected by any other route in these species. Potassium chloride This is cardiotoxic and Prior sedation may be required to assist with causes gasping, vocalizations, muscle spasms restraint during injection. Personnel must be and convulsive which make it Table 6 Characteristics of methods for euthanasia of rodents

Overall Ease of Operator Aesthetic rating Agent Rapidity Efficacy use safety value (1^5) Remarks

Halothane, enflurane, ++ ++ ++ + ++ 5 Acceptable isoflurane Sodium ++ ++ + + ++ 5 Acceptable pentobarbitone Concussion ++ ++ + ++ ^ 4 Acceptable for rodents under1 kg. Death to be confirmed by cessation of circulation Cervical dislocation ++ ++ + ++ ^ 4 Acceptable for rodents under150 g. Death to be confirmed by cessation of circulation T-61 ++ ++ ^ + ++ 4 Only to be injected intravenously Carbon dioxide + ++ ++ ++ ++ 4 Acceptable method at 470% Microwave ++ ++ ^ ++ + 3 To be used by experienced personnel only. Not a routine procedure Decapitation + + + ++ ^ 2 Other methods preferable Carbon monoxide + + + ^ ++ 2 Danger to operator Rapid freezing ^ + ++ ++ + 1 Only in small neonates (54 kg)

The following methods may only be used on unconscious rodents: rapid freezing, exsanguination, air embolism, potassium chloride, The following methods are not to be used for killing rodents: hypothermia, decompression, , drowning, nitrogen, nitrous oxide, cyclopropane, diethyl ether, chloroform, methoxyflurane, hydrogen cyanide gas, trichlor- , strychnine, nicotine, chloral hydrate, magnesium sulphate and neuromuscular blocking agents

Rapidity: ++ very rapid, + rapid, ^ slow. Efficacy: ++ very effective, + effective, ^ not effective. Ease of use: ++ easy to use, + requires expertise, ^ requires specialist training. Operator safety: ++ no danger, + little danger, ^ dangerous. Aesthetic value: ++ good aesthetically, + acceptable for most people, ^ unacceptable for many people. Rating:1^5 with 5 as highly recommended Euthanasia of experimental animals 17 unacceptable for many operators. It may Recognition and con®rmation of death only be used once the animal is fully Cessation of respiration and heartbeat, and anaesthetized. absence of re¯exes are good indicators of Ethanol This has been used injected intra- irreversible death in rabbits. Death may be peritoneally at 70% (Lord 1989). However, con®rmed by exsanguination or removal of Wallgren & Barry III (1970) state that it is the heart, evisceration, or decapitation. irritant at above 10%, making this unaccep- Personnel must be trained to recognize and table for euthanasia unless the rodent is ensure death when killing rabbits. unconscious. Embryos Methods not acceptable for euthanasia of rodents The time at which the neural tube develops into a functional brain (about 60% gestation) Hypothermia Under no circumstances must be taken as the time at which the should any rodent be killed by placing in a foetus may perceive pain and should there- freezer. Freezing may only be used as a fore be killed humanely. If an insensible method of ensuring death. foetus is removed from an anaesthetized mother it may be killed by decapitation or Nitrogen This kills rodents by hypoxia removal of the heart. However, when the making it unacceptable as it takes longer than foetus is to be removed an increased amount other agents to achieve unconsciousness. Rats of anaesthetic must be administered to the exhibit signs of panic and distress prior to dam and maintained for longer to ensure that unconsciousness (Hornett & Haynes 1984). the anaesthetic has crossed the placenta. In Nitrous oxide This kills by anoxia, is slow, many cases inhalational anaesthetics will and the rodents show signs of increased not anaesthetize a foetus. Those foetuses that activity prior to death, indicating a degree of are not removed from the dam will die of anxiety making it unacceptable as a method anoxia when the dam is killed and no further for euthanasia. method is necessary to ensure death of the foetus. Cyclopropane This is a humane and quick agent for killing rodents but it is extremely Neonates dangerous to the operator and is therefore not considered acceptable. These are newborn rabbits up to 10 days old. They may react to painful stimuli more like Ether and chloroform These are not to be an embryo than an adult. They may be killed used under any circumstances for euthanasia by decapitation and concussion. Further of rodents. They are both extremely research is necessary to assess which meth- dangerous to the operator and ether causes ods are the most humane. irritation to the respiratory passages upon inhalation. Adults The following agents are not be used for killing rodents: decompression, asphyxia, Further details on methods may be obtained drowning, trichlorethylene, methoxy¯urane, in Section 2. hydrogen cyanide gas, strychnine, nicotine, chloral hydrate, magnesium sulphate, Physical methods curariform drugs and other neuromuscular When carrying out physical methods of blocking agents. killing rabbits, consideration must be given to the careful handling and restraint of the 3.6 Rabbits animal prior to killing. Minimal handling and restraint is preferable. Fear and anxiety to A summary of recommendations is given in the animal may be reduced by prior sedation Table 7. or handling by familiar people. 18 Working Party Report

Concussion This is a quick and humane be used for this purpose. This is not a method of stunning rabbits as long as it is routine method of euthanasia. performed by experienced and con®dent operators. This involves striking the base of Rapid freezing Foetuses of under 4 g may be the head at the top of the neck in the killed by putting into liquid nitrogen. Larger occipital region. Death must always be or furred animals will not die immediately as con®rmed by cessation of circulation. it takes some time for the core to become frozen. Cervical dislocation This is a humane method of killing rabbits of under 1 kg as it Chemical methods causes extensive damage to the brainstem When using chemical methods of euthanasia, resulting in immediate unconsciousness and death must be con®rmed by one of the death. It should only be carried out by methods listed above. experienced personnel. Death must always be con®rmed by cessation of circulation. Inhalational methods Sedation may be necessary prior to disloca- tion. Volatile inhalational anaesthetics Rabbits react adversely to all gases (Green 1979) and Captive bolt This method may be useful for other methods are preferable if possible. large rabbits (over 4 kg) in limited situations With these agents the rabbit is put into an (Holtzmann 1991). Only captive bolts anaesthetic chamber or an appropriate re- designed speci®cally for use on rabbits may be ceptacle with gauze or cotton wool soaked in used. Personnel must be well trained in order the anaesthetic. Vapours are inhaled until to ensure correct positioning of the weapon. respiration ceases and death ensues. Because The bolt must penetrate about 3 cm into the the liquid state of these anaesthetics are brain (Holtzmann 1991). Death must be irritant, care must be taken to ensure that the con®rmed by ensuring cessation of rabbit cannot come into contact with the circulation. chemical. Air or oxygen must be provided during the induction period. Appropriate gas Decapitation Decapitation may be consid- scavenging equipment needs to be used with ered a humane method of killing small or all these agents. young rabbits (under 1 kg) as loss of blood supply ensures rapid loss of consciousness. Halothane, iso¯urane, en¯urane At high However, this is not possible in larger and concentrations these agents cause rapid older rabbits where the neck is too thick and anaesthesia and consequent death. These are strong for quick decapitation. all acceptable agents when used with appro- priate gas scavenging apparatus. Electrical stunning Only electric tongs designed for this purpose may be used. Care Carbon dioxide Large rabbits may become must be taken to ensure that the correct level distressed initially whilst still conscious and of current passes directly through the brain to therefore other methods are considered pre- ensure immediate unconsciousness. Death ferable if possible. One hundred per cent CO2 must be con®rmed by cessation of has been recommended by Von Cranach et al. circulation. (1991a) but may cause distress.

Microwave This involves the use of spe- Carbon monoxide Although this is a rela- cialized apparatus and should only be carried tively quick and humane method of killing out by personnel specially trained in this rabbits, because of the danger to the operator technique to ensure correct positioning of it is less acceptable for routine use. If used, it the beam. It is a quick method of killing should be used with appropriate gas scaven- small rabbits of under 300 g. Under no ging apparatus and only commercially avail- circumstances should domestic appliances able gas in cylinders should be used as fumes Euthanasia of experimental animals 19 from combustion engines are likely to be longer to act. Under no circumstances should irritant. intrapulmonary or intracardial injection be given unless the animal is fully anaesthetized. Injectable agents In rabbits, where venepuncture is possible via the ear vein (unless damaged) intravenous Sodium pentobarbitone Injected intrave- injection is recommended as this results in nously, sodium pentobarbitone acts quickly rapid anaesthesia and death. Personnel must and humanely to kill rabbits. This is the be trained in the techniques of intravenous most acceptable agent for euthanasia. Sodium and intraperitoneal injection. If venepuncture pentobarbitone may cause irritation to the is not easily performed, intraperitoneal peritoneum which may be avoided by injection is acceptable, although it takes dilution. Table 7 Characteristics of methods for euthanasia of rabbits

Overall Ease of Operator Aesthetic rating Agent Rapidity Efficacy use safety value (1^5) Remarks

Sodium pentobarbitone ++ ++ + + ++ 5 Acceptable T-61 ++ ++ ^ + ++ 4 Acceptable. Intravenous injection only Cervical dislocation ++ ++ ^ ++ ^ 4 Acceptable for rabbits under 1 kg. Sedation prior to dislocation. Death to be confirmed by cessation of circulation Captive bolt ++ ++ ^ + + 4 Requires skill. Death to be confirmed by another method Concussion ++ + ^ ++ ^ 3 Expertise required. Death to be ensured by another method Electrical stunning ++ + ++ ^ + 3 Death to be confirmed by another method Microwave ++ ++ ^ ++ + 3 To be used by experienced personnel only on small rabbits. Not a routine procedure Decapitation + + + ++ ^ 2 Acceptable for rabbits under 1 kg if other methods not possible Halothane, enflurane, ++ ++ ++ + ^ 2 Rabbits show signs of distress isoflurane Carbon dioxide + + ++ ++ + 1 Large rabbits show distress Carbon monoxide + + ++ ^ ++ 1 Danger to operator Rapid freezing + + ++ ++ + 1 Only in foetuses under 4 kg. Other methods preferred

The following methods are only to be used on unconscious rabbits: exsanguination, nitrogen, potassium chloride and air embolism The following methods are not to be used for killing rabbits: hypothermia, decompression, asphyxia, drowning, nitrous oxide, cyclopropane, diethyl ether, chloroform, trichlorethylene, hydrogen cyanide gas, methoxyflurane, chloral hydrate, strychnine, nicotine, magnesium sulphate, hydrocyanic acid, ketamine hydrochloride and neuro- muscular blocking agents

Rapidity: ++ very rapid, + rapid, ^ slow. Efficacy: ++ very effective, + effective, ^ not effective. Ease of use: ++ easy to use, + requires expertise, ^ requires specialist training. Operator safety: ++ no danger, + little danger, ^ dangerous. Aesthetic value: ++ good aesthetically, + acceptable for most people, ^ unacceptable for many people. Rating:1^5 with 5 as highly recommended 20 Working Party Report

T-61 T-61 acts quickly and humanely but dangerous to the operator and is therefore not may only be injected very slowly intrave- considered acceptable for general use. nously. It should never be injected by any Ether and chloroform These are not to be other route. Prior sedation may be considered used under any circumstances for euthanasia necessary to assist with restraint during of rabbits. They are both dangerous to the injection. Personnel must be well trained in operator and ether causes irritation to the intravenous injection techniques. Personnel respiratory passages upon inhalation. must take extreme care when handling this agent. Ketamine hydrochloride Intravenous injec- tion causes tonic contractions, accompanied Methods acceptable for unconscious by vocalizations, thus making this unaccep- rabbits table for euthanasia of rabbits.

Exsanguination This may be used to kill Other agents not to be used for killing rabbits rabbits once they are fully unconscious. include decompression, asphyxia, drowning, trichlorethylene, hydrogen cyanide gas, Nitrogen This kills rabbits by hypoxia and hydrocyanic acid, strychnine, nicotine, is therefore not considered acceptable for chloral hydrate, magnesium sulphate and euthanasia of rabbits unless they are neuromuscular blocking agents. unconscious. 3.7 Carnivores ö dogs, cats, ferrets Potassium chloride This is cardiotoxic and A summary of recommendations is given in causes gasping, vocalizations, muscle Table 8. spasms and convulsive seizures which makes it unacceptable for many operators. It may Recognition and con®rmation of death only be used once the animal is fully Cessation of respiration and heartbeat, and anaesthetized. loss of re¯exes are good indicators of irrever- Air embolism Intravenous injection of air sible death in carnivores. Death should be at 5±50 ml/kg causes rapid death but as this con®rmed by exsanguination. Personnel is accompanied by convulsions, opisthoto- should be trained to recognize and ensure nos, pupillary dilation and vocalizations, it is death when killing dogs, cats, ferrets or other not an acceptable method unless the rabbit is carnivores. fully unconscious. Embryos Methods not acceptable for euthanasia of The time at which the neural tube develops rabbits into a functional brain must be taken as the time at which the foetus may perceive pain Hypothermia Under no circumstances (30% gestation) and therefore should be killed should any rabbits be killed by placing in a humanely. If an insensible foetus is removed freezer. from an anaesthetized mother then it may be killed by decapitation or removal of the heart. Nitrous oxide This kills by anoxia, is slow However, when the foetus is to be removed an acting, and the rabbits show signs of in- increased amount of anaesthetic must be creased activity prior to death, indicating a administered to the dam and maintained for degree of anxiety. It is therefore not con- longer to ensure that the anaesthetic has sidered an acceptable method. crossed the placenta. In many cases inhala- tional anaesthetics will not anaesthetize a Methoxy¯urane This takes too long to act foetus. with a large chance of recovery. Neonates Cyclopropane This may be a humane and Neonate carnivores should generally be quick method of euthanasia, but is very treated as adults. Sodium pentobarbitone is Euthanasia of experimental animals 21

the preferred method, but CO2, cervical section recognition and con®rmation of dislocation and concussion may be consid- death. ered (Hall 1972) (see Section 2). Operators must be well trained in the physical techni- Chemical methods ques to ensure that they are correctly and In general, chemical methods of euthanasia humanely carried out. The animals must be are preferred for all dogs, cats, ferrets and exsanguinated immediately after concussion foxes. It may be preferable to sedate the or cervical dislocation. animal prior to euthanasia to reduce stress and anxiety. Adults Further details on methods are given in Inhalational methods Section 2. Volatile inhalational anaesthetics These Physical methods include halothane, iso¯urane and en¯urane. In general it is not recommended to use These are all acceptable methods of eutha- physical methods of euthanasia on carni- nasia for carnivores. Appropriate gas scaven- vores. However, where chemical agents may ging apparatus should be used to prevent interfere with the aims of the experiment, operator exposure. the following methods may be used. Re- Injectable agents straint of cats for physical methods may be If possible injection should be given intrave- dif®cult and it is recommended that all nously in order to achieve rapid anaesthesia animals be sedated prior to euthanasia. and euthanasia with minimal stress. Captive bolt Captive bolts designed speci®cally for the purpose of killing animals Sodium pentobarbitone Injected intrave- of this size may be used. Personnel must be nously, this agent provides rapid and humane trained in these techniques to ensure correct euthanasia. Intracardiac and intrapulmonary positioning of the pistol and immediate routes of injection should not be used as they death. Death must be con®rmed by cessation are extremely painful, unless under full of circulation by exsanguination. anaesthesia. All personnel must be trained in these techniques. Shooting Shooting of carnivores using a free bullet is only acceptable under ®eld conditions Secobarbital/dibucaine Secobarbital is a when no other methods can be used. Only short-acting analogue of thiamylol sodium. specialized marksmen should be used. Dibucaine is a highly toxic local anaesthetic causing rapid loss of consciousness, loss of Electrocution Ear clips are attached to respiration and cardiac arrest (Herschler et al. ensure that the current ¯ows directly 1981, Wallach et al. 1981). through the brain and death is con®rmed by passing the current through the heart. There T-61 This agent is very effective but must are two phases: stunning with 500 V shock only be injected very slowly intravenously. between the ears, followed by a lethal shock Animals must be sedated prior to adminis- at 1 kV passing from the ear to hindleg. Cats tration. It may cause convulsions in the should not be killed by electrocution due to unconscious animal, which may be aesthe- the tically unpleasant. high conductivity of their coats. Only specially designed apparatus should be used for this purpose and personnel must be Methods acceptable for unconscious well trained in this technique. Equipment carnivores must be regularly checked and maintained to ensure correct voltage. Death must be Exsanguination This may be used to kill con®rmed by one of the methods in the carnivores once they are unconscious. 22 Working Party Report

Dislocation of neck This may be used on other body cavities. It is not considered small animals under anaesthetic. Death acceptable for euthanasia of carnivores. must always be con®rmed by one of the Although carbon dioxide causes cats to methods listed above. become unconscious within 1 min, they move about the cage, licking, sneezing and Potassium chloride This may be used to trying to climb out, indicating that it may be kill unconscious carnivores. stressful. The animals also convulse which makes this method aesthetically unpleasant Methods not acceptable for euthanasia of for the operator. This is not considered carnivores acceptable as a method of euthanasia for carnivores except for neonates. Striking of chest of cats Has been suggested as a method of euthanasia but this is not considered as humane and is not to be used Carbon monoxide At concentrations above under any circumstances. 6% it is a relatively quick method of euthanasia and is recommended for the Decompression Has been used as a method killing of mustelids (Commission of the of euthanasia in the USA and Japan. It European Communities 1993). However, it probably causes much anxiety and stress to causes convulsions and vocalizations which the animals and they may experience pain may still be in the conscious phase (Chali- due to the expansion of air in the sinuses and foux & Dallaire 1983). Because of this and the

Table 8 Characteristics of methods for euthanasia of dogs, cats, ferrets, foxes

Overall Ease of Operator Aesthetic rating Agent Rapidity Efficacy use safety value (1^5) Remarks

Sodium pentobarbitone ++ ++ ^ + ++ 5 Acceptable. Intravenous injection T-61 ++ ++ ^ + + 4 Acceptable but only by slow intravenous injection under sedation Secobarbital/dibucaine ++ ++ ^ + ++ 4 Acceptable. Intravenous injection Halothane, isoflurane, ++ ++ + + ++ 4 Acceptable enflurane Captive bolt ++ ++ ^ ++ + 3 To be followed by exsanguination Electrocution ++ ++ ^ ^ ^ 3 Use only special equipment.To be followed by exsanguination Concussion ++ ++ + ++ ^ 2 Only to be used on neonates.To be followed by exsanguination Shooting ++ ++ ^ ^ ^ 1 Acceptable only in field conditions by specialized marksmen when other methods not possible

The following methods are acceptable for unconscious carnivores: exsanguination, neck dislocation and potas- sium chloride The following methods are not to be used for killing carnivores: decompression, decapitation, drowning, stran- gulation, asphyxiation, air embolism, striking chest of cats, carbon monoxide, carbon dioxide, methoxyflurane, nitrogen, nitrous oxide, trichlorethylene, hydrocyanic acid, diethyl ether, chloroform, hydrogen cyanide gas, cy- clopropane, chloral hydrate, strychnine, nicotine, magnesium sulphate and neuromuscular blocking agents

Rapidity: ++ very rapid, + rapid, ^ slow. Efficacy: ++ very effective, + effective, ^ not effective. Ease of use: ++ easy to use, + requires expertise, ^ requires specialist training. Operator safety: ++ no danger, + little danger, ^ dangerous. Aesthetic value: ++ good aesthetically, + acceptable for most people, ^ unacceptable for many people. Rating:1^5 with 5 as highly recommended Euthanasia of experimental animals 23 danger to the operator, it is not considered Recognition and con®rmation of death acceptable for experimental animals. Cessation of respiration and heartbeat, and loss of re¯exes are good indicators of Nitrogen This causes unconsciousness in irreversible death. Death should be con- dogs and cats in 1±2 min with hyperpnoea for ®rmed by exsanguination. Personnel should about 10 s before collapse. After collapse be trained to recognize and ensure death there are vocalizations, opisthonos, convul- when killing these animals. sions and gasping. Kittens and puppies are resistant to anoxia; they fall unconscious but Embryos fail to die. This is not an acceptable method. The foetuses of these large mammals are well developed at birth and therefore Ether and chloroform These are not accep- considerable care must be taken to ensure table methods of euthanasia due to irritation that they are killed humanely if removed of the respiratory passages and danger to the from the uterus. The time at which operator. euthanasia should be considered should be The following agents are also not to be used from the time at which the neural tube for killing carnivores: drowning, concussion develops into a functional brain and they (adults), decapitation, asphyxia, strangula- may thus be able to feel pain (430% tion, nitrous oxide, hydrogen cyanide gas, gestation). Foetuses may also be large and in cyclopropane, methoxy¯urane, trichlorethy- general any method used on an adult is lene, air embolism, hydrocyanic acid, chlor- considered acceptable. al hydrate, strychnine, nicotine, magnesium sulphate, and neuromuscular blocking Neonates agents. Because large mammals are born in an advanced stage of development, they should be treated as adults. 3.8 Large mammals ö pigs, sheep, goats, cattle, horses Adults Personnel using and having to kill any large Further details on methods are given in mammal must receive specialized training in Section 2. the handling, restraint and techniques of euthanasia of these animals. It is important to Physical methods avoid actions which may increase the animals' The animals must be suitably restrained in awareness of the unusual situation. The adequate devices to ensure that the animal animal is best killed in a familiar environ- remains still and calm so that the method of ment. euthanasia is accurate and quick. Personnel All operators are recommended to obtain should be quiet and handle the animals with and read EC (Council Directive 93/119/EC) care so as to reduce stress and anxiety in the (Commission of the European Communities animals. 1993) and national regulations on slaughter methods which cover most of these Captive bolt The use of captive bolt is the animals. It may be necessary to take the most acceptable physical method for large animals to approved slaughterhouses where mammals and is preferable to free bullets specialized equipment is available for humane because of operator safety. Penetrative cap- euthanasia of these animals. Euthanasia may tive bolts are preferred. Personnel must be have to be carried out by a person who has well trained in the use of captive bolt pistols been trained and holds a certi®cate under to ensure correct positioning for the species national slaughter legislation or by a veter- being killed (Universities Federation for inarian with appropriate training. Animal Welfare 1989). Care must be taken to A summary of recommendations is given ensure that the correct size bolt and cartridge in Table 9. are used and that the weapon is kept clean 24 Working Party Report and maintained in good working order. It is are considered undesirable when dealing not recommended to use captive bolt pistols with high voltages. Sheep and goats should be on adult pigs and mature bulls because of shaved in the region of the tongs to ensure their thick skulls. Death must be con®rmed good electrical contact. It should not be used immediately by exsanguination or pithing where horns make it dif®cult to position the through the hole made by the bolt. tongs correctly. Care must be taken to ensure that the animals cannot receive electrical Free bullet humane killers A humane killer shocks from contact with other animals, ®ring a free bullet is an ef®cient method of from wet surfaces, or from accidental contact killing horses, mules, donkeys and old or with the tongs. Head only or head to back hard-headed animals (Blackmore 1985, Dodd stunning across the head are both acceptable 1985, Oliver 1979). Extra care must be taken methods as they ensure immediate uncon- as it is not as safe as the captive bolt and it is sciousness. Animals must be exsanguinated therefore considered acceptable only under immediately after stunning to ensure death. ®eld conditions. All personnel must be Personnel must ensure that the correct voltage trained in these methods to ensure correct and current is used for the species of animal positioning of the weapon and that the being killed. correct calibre of weapon is used. It must be noted that the position of the weapon differs Chemical methods with each species and whether the animal is Inhalational methods horned or not. The animals tend to slump forwards when shot and therefore the opera- Volatile inhalational anaesthetics Ha- tor must take care to avoid personal damage. lothane, iso¯urane and en¯urane may be used Operators must ensure that the weapon is with an anaesthetic mask for lambs and kids. well maintained so that the chance of mis®ring is minimized. Death must be Carbon dioxide Carbon dioxide has been con®rmed immediately by exsanguination or used to kill pigs. The pigs are placed into pithing through the hole made by the bullet. large chambers that are previously ®lled with CO2 gas to above 70%. Specialized equip- Shooting Shooting using a free bullet ment only must be used. Death must be should only be done in ®eld conditions when con®rmed by exsanguination. Because pigs no other method can be used. Only specia- tend to show signs of stress other methods lized marksmen should carry this out. are considered preferable. Carbon dioxide must not be used on any other large animal. Concussion This should be done using a mechanically operated instrument which Injectable methods administers a blow to the skull without Personnel must be trained in intravenous fracture of the skull. It must be noted that injection techniques and handling and re- the position for the application of percussion straint of the animals. Animals should be stunners differs from that for the captive bolt suitably restrained and/or sedated prior to pistols. Death must be ensured by immediate euthanasia. exsanguination (within 20 s of stunning) (Blackmore 1979). Sodium pentobarbitone Injected intrave- nously, sodium pentobarbitone provides ra- Electrical stunning This method should pid euthanasia. Large volumes may be only be carried out in slaughterhouses where required for larger mammals and this may be specialized equipment is available for the made easier by the insertion of a catheter into animals being killed. It is commonly used for the jugular vein (Andrews et al. 1993). stunning pigs, sheep, calves and goats. Tongs Alternatively lower volumes of highly con- should be applied on each side of the head centrated solutions may be used, but it must between the eye and ear to span the brain. be recognized that this poses a greater danger Moist sponge rubber pads ®tted to the tongs to the operator. Excitable or nervous animals Euthanasia of experimental animals 25

Table 9 Characteristics of methods for euthanasia of large mammals

Ease Overall of Operator Aesthetic rating Agent Rapidity Efficacy use safety value (1^5) Remarks

Sodium pentobarbitone ++ ++ ^ + ++ 5 Acceptable by intravenous injection Quinalbarbitone/ ++ ++ ^ + ++ 5 Effective for horses intravenously Nupercaine Captive bolt ++ ++ + + + 5 To be followed by exsanguination Free bullet humane ++ ++ + ^ + 5 To be followed by killer exsanguination. In field conditions only T-61 ++ ++ ^ + ++ 4 Acceptable by intravenous injection Electrical stunning ++ ++ + ^ ^ 4 Use only specialized equipment. To be followed immediately by exsanguination Shooting ++ ++ ^ ^ ^ 2 Only in field conditions by a specialized marksman Concussion ++ + ^ + + 2 To be followed immediately by exsanguination Halothane, isoflurane, + + + + + 2 Recommended for lambs and kids enflurane Carbon dioxide + + ++ ++ + 1 Only for use with pigs at 470%

The following methods are acceptable only on unconscious large mammals: exsanguination, chloral hydrate and potassium chloride The following methods are not to be used for killing large mammals: carbon monoxide, methoxyflurane, tri- chlorethylene, strychnine, nicotine, magnesium sulphate, thiopentone sodium, ketamine hydrochloride, neuro- muscular blocking agents

Rapidity: ++ very rapid, + rapid, ^ slow. Efficacy: ++ very effective, + effective, ^ not effective. Ease of use: ++ easy to use, + requires expertise, ^ requires specialist training. Operator safety: ++ no danger, + little danger, ^ dangerous. Aesthetic value: ++ good aesthetically, + acceptable for most people, ^ unacceptable for many people. Rating:1^5 with 5 as highly recommended must be sedated prior to injection with Chloral hydrate This may be used intrave- sodium pentobarbitone. nously on unconscious mammals, or in conjunction with magnesium sulphate and Quinalbarbitone/Nupercaine This proprie- sodium pentobarbitone. tary mixture causes rapid and humane death in horses. This should be injected intrave- Potassium chloride This may be used to nously over a period of 5±8 s. It is not kill unconscious mammals. available in some countries. Methods not acceptable for euthanasia of T-61 T-61 is also an effective agent for large mammals euthanasia of large mammals by slow intravenous injection only. It may be neces- Carbon monoxide Some animals, including sary to sedate excitable or nervous animals pigs, show signs of severe excitation and initially. vocalizations, sometimes before uncon- sciousness, at high levels of carbon mon- Methods acceptable for unconscious large oxide. It is not an acceptable agent for mammals euthanasia. The following agents are also not acceptable Exsanguination This may be used to kill for euthanasia of large mammals: methoxy- unconscious mammals. ¯urane, trichlorethylene, strychnine, nico- 26 Working Party Report tine, magnesium sulphate, thiopentone so- humane method of killing most animals. It is dium, ketamine hydrochloride, curariform advisable in most cases to sedate the animal drugs and other neuromuscular blocking prior to euthanasia. agents. References 3.9 Non-human primates Allaby M (1991) Concise Oxford Dictionary of Personnel handling primates should be spe- Zoology. Oxford: Oxford University Press Allred JB, Berntson GG (1986) Is euthanasia of rats by cially trained for these purposes. It is decapitation inhumane? The Journal of Nutrition preferable that if primates have to be killed, 116 (9), 1859±61 that this be carried out by someone familiar Anand KJS, Hickey PR (1987) Pain and its effects in to them in order to reduce stress and anxiety. the human neonate and fetus. New England Journal For all larger primates, sedation (e.g. of Medicine 317, 1321±9 ketamine) should be administered prior to Anil MH, McKinistry JL (1991) Re¯exes and loss of euthanasia. sensibility following head-to-back electrical stun- ning in sheep. The Veterinary Record 128, 106±7 Cessation of heartbeat and respiration, and Andrews EJ, Taylor Bennett B, Clark JD, et al. 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Journal of the American Veterinary Medical Association 189 (9), 1038±9 Adults Barber BR (1972) Use of a standard autoclave for decompression euthanasia. Journal of the Institute The only recommended method for killing of Animal Technicians, 23 (3), 106±10 primates is by overdose of anaesthetic. Barocio LD (1983) Review of literature on use of T-61 Sodium pentobarbitone injected as an euthanasic agent. International Journal for intravenously is the most acceptable agent. the Study of Animal Problems 4 (4), 336±42 Baumans V, Hellebrekers LJ, Bertens APMG, Hart- Exsanguination under inhalation anaesthesia man W (1988) Evaluation of T-61 as an euthanasia is also considered acceptable, but this must agent in rabbits and dogs. 26th Scienti®c Meeting of be followed by perfusion. the Society for Laboratory Animal Science, Basel, Infants of some small species such as 13±15 September 1988 marmosets may be dif®cult to inject and this Berman E, King JB, All J, Carter HB, Rehnberg B, requires specialist expertise. Stead AG (1985) Lethality in mice and rats exposed to 2450 MHz circulatory polarized microwaves as a function of exposure duration and environmental 3.10 Other animals not commonly used factors. Journal of Applied Toxicology 5 (1), 23±31 for experiments Blackmore DK (1979) Non-penetrative percussion stunning of sheep and calves. The Veterinary As vertebrate animals vary so much in size Record 105, 372±5 and physiology, the method chosen to kill Blackmore DK (1985) Energy requirements for the any animal not included above should be penetration of heads of domestic stock and the chosen from those methods for animals that development of a multiple projectile. The Veter- inary Record 116, 36±40 are most similar biologically. Advice should Blackmore DK (1993) Euthanasia; not always eu. be obtained from a . In general, Australian Veterinary Journal 70 (11), 409±13 an overdose of sodium pentobarbitone in- Blackmore DK, Delaney MW (1988) Slaughter of jected intravenously may be considered as a Stock. A Practical Review and Guide. Publication Euthanasia of experimental animals 27

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Aquaculture and Fish Holson RR (1992) Euthanasia by decapitation: evi- Management 24, 523±8 dence that this technique produces prompt, pain- 28 Working Party Report

less unconsciousness in laboratory rodents. Neuro- dogs. Journal of the American Veterinary Medical toxicology and Teratology 14, 253±7 Association 172 (2), 149±52 Holtzmann M (1991) Tierschutzgerechtes toÈ ten von Marshall S, Milligan A, Yates R (1994) Experimental versuchskaninchen mit bolzenschuûgeraÈten. (Kill- techniques and anaesthesia in the rat and mouse. ing of experimental rabbits with captive bolt guns ANZCCART Facts sheet. ANZCCART News 7 (1) according to animal welfare regulations). Journal of 4 pp Experimental Animal Science 34, 203±6 McFarland WN, Klontz GW (1969) Anesthesia in Hornett TD, Haynes AP (1984) Comparison of carbon ®shes. Federal Proceedings 28 (4), 1535±40 dioxide/air mixture and nitrogen/air mixture for the Mikeska JA, Klemm WR (1975) EEG evaluation of euthanasia of rodents. Design of a system for humaneness of asphyxia and decapitation euthana- inhalation euthanasia. Animal Technology 35 (2), sia of the laboratory rat. Laboratory Animal Science 93±9 25 (2), 175±9 Iwarsson K, Rehbinder C (1993) A study of different Ministry of Agriculture, Fisheries and Food, Welsh euthanasia techniques in guineapigs, rats and mice. Of®ce Agriculture Department (1993) Welfare of Animal response and postmortem ®ndings. Scan- red meat animals at slaughter. Code of Practice dinavian Journal of Laboratory Animal Science 20, Moroji T, Takahashi K, Ogura K, Toishi T, Arai S 191±205 (1977) Rapid microwave ®xation of rat brain. Jaksch W (1981) Euthanasia of day-old male chicks in Journal of Microwave Power 12 (4), 273±86 the poultry industry. International Journal for the Mullenax CH, Dougherty RW (1963) Physiologic Study of Animal Problems 2 (4), 203±13 responses of swine to high concentrations of Kestin SC (1993) Pain and Stress in Fish. A report inhaled carbon dioxide. American Journal of prepared for the RSPCA, Horsham, UK Veterinary Research 24, 329±33 Kestin SC, Wotton SB, Gregory NG (1991) Effect of Noonan D (1994) The guineapig (Cavia porcellus). slaughter by removal from water on visual evoked ANZCCART Fact Sheet. ANZCCART News 7 (3), activity in the brain and re¯ex movement of 8 pp rainbow trout (Oncorhynchus mykiss). The Veter- Olfert ED, Cross BM, McWilliam AA, eds. (1993) inary Record 128, 443±6 Guide to the Care and Use of Experimental Klemm WR (1964) Carbon dioxide anesthesia in Animals. Volume 1. Canadian Council on Animal cats. American Journal of Veterinary Research 25, Care, Ontario, Canada 1201±5 Oliver DF (1979) Euthanasia of horses. The Veterinary Record 105 (10), 224±5 Lambooy E, van Voorst N (1986) Electrocution of pigs infected with noti®able diseases. The Veterinary Phifer CB, Terry LM (1986) Use of hypothermia for Quarterly 8 (1), 80±2 general anesthesia in preweanling rodents. Phy- siology and Behaviour 38, 887±90 Laursen AM (1983) Choosing between CO and 2 Quin AH (1963) Observations on a new euthanasia electrical stunning of pigs. A preliminary exam- agent for small animals (T-61). Veterinary Medicine ination of stress and ethics. In: Stunning of animals 58, 494±5 for slaughter. (Eikelenboom G, ed.). Boston: Marti- nus Nijhoff, pp 64±72 Quine JP (1980) Euthanasia by hypoxia using nitrogen. A review after 4 years of operation involving 20 500 Longair J, Finley GG, Laniel MA, et al. (1991) animals. Canadian Veterinary Journal 21, 320 Guidelines for euthanasia of domestic animals by Quine JP, Buckingham W, Strunin L (1988) Euthana- ®rearms. Canadian Veterinary Journal 32, 724±6 sia of small animals with nitrogen; comparison Lord R (1989) Use of ethanol for euthanasia of mice. with intravenous . Canadian Veter- Australian Veterinary Journal 66 (8), 268 inary Journal 29, 724±6 Lord R (1991) Humane Killing. Nature 350 (11 April), Raj M, Gregory NG (1993) Time to loss of somato- 456 sensory evoked potentials and onset of changes in Lorden JF, Klemm WR (1987) Letters on AVMA report the spontaneous electroencephalogram of turkeys about decapitation. Laboratory Animal Science during gas stunning. The Veterinary Record 133, 37 (2), 148±51 318±20 Lucke JN (1979) Euthanasia in small animals. The Raj M, Gregory NG (1994) An evaluation of humane Veterinary Record 104 (14), 316±18 gas stunning methods for turkeys. The Veterinary Lumb WV (1974) Euthanasia by noninhalant phar- Record 135, 222±3 macologic agents. Journal of the American Veter- Raj M, Gregory NG, Wotton SB (1990) Effect of carbon inary Association 165 (9), 851±2 dioxide stunning on somatosensory evoked poten- Lumb WV, Jones EW (1984) Euthanasia. In: Veterinary tials in hens. Research in Veterinary Science 49, Anesthesia 2nd edn., Philadelphia: Lea & Febiger, 355±9 pp 631±48 Raj M, Wotton SB, Gregory NG (1992) Changes in the Lumb WV, Doshi K, Scott RJ (1978) A comparative somatosensory evoked potentials and spontaneous study of T-61 and pentobarbital for euthanasia of electroencephalogram of hens during stunning with Euthanasia of experimental animals 29

a carbon dioxide and argon mixture. British toute l'eÂtude: Of®ce feÂdeÂral veÂteÂrinaire, Schwar- Veterinary Journal 148, 147±56 zenburgstr 161, 3097 Liebefeld Reilly JS, ed. (1993) Euthanasia of Animals Used for Wallach MB, Peterson KE, Richards RK (1981) Scienti®c Purposes. ANZCCART 1993. Glen Os- Electrophysiologic studies of a combination of mond. SA Australia secobarbital and dibucaine for euthanasia of dogs. Rowan AN (1986) T-61 use in the euthanasia of American Journal of Veterinary Research 42 (5), domestic animals: a survey. In: Advances in 850±3 Animal Welfare Science. Boston: Martinus Nijhoff, Wallgren H, Barry III H (1970) Actions of Alcohol, pp 79±86 Vol I, Biochemical, Physiological and Psychologi- Rowsell HC (1981) The present status of euthanasia cal Aspects. Amsterdam: Elsevier by nonanaesthetic gases. Canadian Veterinary Warrington R (1974) Electrical stunning, a review of Journal 22 (1), 8 the literature. The Veterinary Bulletin 44 (10), 617±35 Rowsell HC (1990) Euthanasia: acceptable and unac- Warwick C (1986) Euthanasia of reptiles Ð decapita- ceptable methods of killing, Ch. 21. In: Experimental tion: an inhumane method of slaughter for the class Animals in Biomedical Research, Vol. 1. pp 381±91 `Reptilia'. Canadian Veterinary Journal 27, 34 Schatzmann U, Leuenberger T, Fuchs P (1991) Jet Warwick C (1990) Crocodilian slaughter methods, injection: the possibility of using a high pressure with special reference to spinal cord severance. water jet for the stunning of slaughter pigs. Texas Journal of Science 42 (2), 191±8 Fleischwirtschaft 71, 899±901 Weisbrod KS, Dennis MD, Dong WK, Ramos B, Schatzmann U, Howard J, Pittino J, Fuchs P (1994) Jet Russell RG, Bower A (1984) Physical methods of injection for the stunning of slaughter pigs. euthanasia for rabbits. Laboratory Animal Science Fleischwirtsch International 3, 18±19 34, 516 Smith AW, Houpt KA, Kitchell RL, et al. (1986) Zeller von W, Mettler D, Schatzmann U (1989) Report of the AVMA Panel on Euthanasia. Journal Untersuchungen zur tierschutzgerechten BetaÈu- of the American Veterinary Medical Association bung des Schlachtge¯uÈ gels mit Mikrowellen 188 (3), 252±68 (2550 MHz). Deutsche TieraÈrtzliche Wochenschrift Summerfelt RC, Smith LS (1990) Anesthesia, , 96, 285±332 and related techniques. In: (Schreck CB, Moyle PB, Zimmermann M (1986) Behavioural investigations in eds). Methods for Fish Biology. Bethesda: American animals. In: Assessing pain in farm animals. Fisheries Society, pp 213±72 (Duncan IJH, Moloney V, eds). Luxembourg: Tidswell SJ, Blackmore DK, Newhook JC (1987) Commission of the European Communities, Slaughter methods: electroencephalic (EEG) studies pp 16±27 on spinal cord section, decapitation and gross Zutphen LFM van, Baumans V, Beynen AC eds (1993) trauma of the brain in lambs. New Zealand Principles of Laboratory Animal Science. Veterinary Journal 35, 46±9 Amsterdam: Elsevier Universities Federation for Animal Welfare (1989) Zwart P, Vries HR de, Cooper JE (1989) Humane Guidelines on the care of laboratory animals and methods of killing ®sh, amphibians and birds. their use for scienti®c purposes. II. Pain, analgesia Tijdschrift voor Diergeneeskunde 114 (10), 557±65 and anaesthesia. Potters Bar: UFAW UFAW/WSPA (1989) Euthanasia of Amphibians and Reptiles. Report of a Joint Universities Federation Euthanasia training materials for Animal Welfare/World Society for the Protec- tion of Animals Working Party. Potters Bar: UFAW Inter-act animal care training programmes Vanderwolf CH, Buzsaki G, Cain DP, Cooley RK, Robertson B (1988) Neocortical and hippocampal electrical activity following decapitation in the rat. This is a specially designed series of pro- Brain Research 451, 340±4 grammes produced by the Association of the Verheijen FJ, Flight WFG (1995) Commercial slaugh- British Pharmaceutical Industry (ABPI) as a ter of eel: experimental tests show methods are resource for the training of laboratory staff unacceptable. Ecology of Freshwater Fish involved in the care and use of animals under (submitted) the Animals (Scienti®c Procedures) Act 1986. Von Cranach J, Gassmann-Langmoen AB, Schatz- Topic 7 covers euthanasia. Using interactive mann U (1991a) Euthanasie bei Labornagetieren. video technology, the full series of 11 Anaesthesieabteilung der Kliniken, VeterinaÈrmedi- zinische FakultaÈt der UniversitaÈt Bern, Switzerland programmes (held on 10 laser discs) is Von Cranach J, Gassmann-Langmoen AB, Schatz- available from Mr M. Connelly, Redway mann U (1991b) Euthanasie chez les rongeurs de Interactive Video, 34 Redway, Kerridge, laboratoires. Resume. Source de reÂfeÂrence pour Maccles®eld, Cheshire SK10 5BA, UK. 30 Working Party Report

Principles of proper laboratory animal use Euthanasia interactive video programme in research To provide the trainee with an understanding This program is a basic program for of the legal requirements of Schedule 1 (UK) research investigators, technicians and sup- and the Alderley Park site regulations to be port staff. This program establishes the found in the `Humane Policy for Euthanasia appropriate foundation for use of animals in of Protected Species'. Demonstrates the biomedical research. It is a modular, correct procedure for euthanasia when using ¯exible and easy to use software template. the following methods: overdose of anaes- The program consists of a template divided thetic, physical methods, carbon dioxide. It into a series of modules containing infor- provides a means by which the trainees' mation on the following topics: regulations, understanding of the above can be assessed. ethics, euthanasia, anaesthesia and analge- Approximately 1ˆ h. A printout of the sia, pre- and post-operative care, safety, trainees' responses to questions posed disease, nutrition, alternatives and models, during the programme is available. It may be species information, USDA Animal Welfare viewed without the interactive compo- Act. The program is designed with many nent Ð this lasts about 25 min. English applications in mind. Runs on Macintosh language only. Contact Mr Bob Kemp, and IBM PC computers and requires a hard Zeneca Pharmaceuticals, Mereside, Alderley disk and 640K RAM. MTM Associates Inc., Park, Maccles®eld, Cheshire SK10 4TG, UK. PO Box 1606 Manassas, Virginia 2110, Tel: +44-1625-512726; Fax +44-1625-583074/ USA. Item no. 4211WP. 586278.

Common procedures and techniques and Using animals in research: guidelines for survival surgery: tape II (1988) investigators (1986) Handling laboratory animals in blood collec- This ®lm records in its entirety a course tion, gavage, injection and euthanasia: em- sponsored by the USDA/Agricultural Re- phasizes minimization of animal discomfort. search Service and presented to investigators Videocassette, NTSC/U-matic/VHS, English, on 25 March 1986. Speakers for the Animal 21 min. MDA-TV, University of Texas and Plant Health Service, National Institutes Cancer Centre, 151 Holcombe Houston, of Health (NIH), Public Health, and Agricul- Texas 77030, USA. Order no. 861188. tural Research Service present information on laws, policies and practices that affect the Practical methodology: reptiles part III use of research laboratory; and provide special laboratory (1988) references for those who want to learn more about speci®c procedures. Tape 1: regulatory The second of a two part series, this issues from an aphis perspective. Tape 2: ®lm programme covers laboratory techniques `Unnecessary Fuss', assembled by PETA from such as sexing, blood sampling, catheter University of Pennsylvania research docu- placement, injection sites, oral gavaging, mentation. Tape 3: principles and policies for intubation, restraint, anaesthesia and animal use in NIH extramural programmes. euthanasia for reptiles. A manual accompa- Tape 4: principles and policies for animal use nies the videocassette. Videocassette, 1/2'' in the Beltsville area. Tape 5: technical VHS, NTSC, English, 18 min. National information and training opportunities for Agricultural Library, Beltsville, Maryland animal users. Tape 6: panel discussion of 20705, USA. Videocassette no. 414 Part II. audience questions. Videocassette, U-matic, 3/4'', NTSC, English, 240 min (6 tapes). Biomethodology of the mouse National Agricultural Library, Beltsville, Maryland 20705, USA. Videocassette This video shows a close-up, detailed footage no. 186. demonstrating proper techniques of handling Euthanasia of experimental animals 31 and restraint, identi®cation, injections and Beltsville, Maryland 20705, USA. Slide blood sampling. The techniques presented no. 338, Vol. 2. are the most common, reproducible, safe and least stressful to the animal. Video, VHS The laboratory rat, biology, husbandry, (1/2'') U-matic (3/4'') standard tape formats. and research methodology (1977) MTM Associates Inc., PO Box 1606, Manassas, Virginia 2110, USA. Item This slide set describes and illustrates basic no. 3211V. anatomy and physiology of laboratory rats, discusses standard procedures for housing rats in the laboratory, and familiarizes the The mouse: handling, restraint, and other viewer with basic methodology employed for techniques (1975) manipulating rats in research. Research This programme demonstrates basic techni- methodology includes handling, restraint, cal skills required for the proper care of mice blood collection, anaesthesia and euthanasia. and their use in biomedical research. It Biological values, listed in the accompanying includes handling and restraint, injections guide. Slides, 59 slides, audiocassette, Eng- and oral administration of medicine, blood lish. National Agricultural Library, Belts- and urine collection, sexing, identi®cation, ville, Maryland 20705, USA. Slide no. 221. anaesthesia and euthanasia. A manual ac- companies the slide set. Slides, 48 slides, Biomethodology of the rat (1987) audiocassette, 12 min, English. National Demonstration of basic techniques involving Agricultural Library, Beltsville, Maryland laboratory animals including identi®cation, 20705, USA. Slide no. 227, Part 2. restraint, injection, blood withdrawal, and euthanasia. Video 1/2'' VHS, English, Biomethodology of the laboratory mouse 16 min. National Agricultural Library, (1987) Beltsville, Maryland 20705, USA. Videocas- sette no. 200. A demonstration of basic techniques invol- ving laboratory mice including identi®ca- Biomethodology of the guineapig (1987) tion, restraint, injection, blood withdrawal and euthanasia. Videocassette 1/2'', VHS Demonstration of basic techniques involving (NTSC), colour, English. National Agricul- laboratory animals including identi®cation, tural Library, Beltsville, Maryland 20705, restraint, injection, blood withdrawal, and USA. Videocassette no. 200. euthanasia. Videocassette 1/2'' VHS, English. National Agricultural Library, Beltsville, Maryland 20705, USA. Videocassette The mouse, rat and hamster (1988) no. 200. This training ®lm provides information on the humane care and use of laboratory Biomethodology of the rabbit (1987) rodents for scientists, laboratory technicians Demonstration of basic techniques involving and students. The recommendations are laboratory animals including identi®cation, consistent with the Public Health Services restraint, injection, blood withdrawal, and `guide for the care and use of laboratory euthanasia. Video 1/2'' VHS, English, animals' and regulations set by the USDA. 15 min. National Agricultural Library, Topics include housing, nutrition, environ- Beltsville, Maryland 20705, USA. Videocas- ment, record keeping, animal health care, sette no. 200. occupational safety, handling and restraint, experimental techniques, and euthanasia. A Biomethodology of the cat (1987) manual that includes a script, test and answer key accompanies the videocassette. This ®lm demonstrates to animal techni- Videocassette 1/2'', VHS (NTSC), English, cians basic techniques for handling, re- 34 min. National Agricultural Library, straining, and manipulating cats for research. 32 Working Party Report

Covers removal from caging, injection tion, injection routes, blood collection, and routes, blood collection, and euthanasia. euthanasia. Videocassette 1/2'' VHS (NTSC), Videocassette colour, English. National Agricultural 1/2'' VHS (NTSC), colour, 15 min, English. Library, Beltsville, Maryland 20705, USA. National Agricultural Library, Beltsville, Videocassette no. 336. Maryland 20705, USA. Videocassette no. 337. The non-human primates (1988)

The dog and cat (1988) This programme provides information on the humane care and use of laboratory primates This programme provides information on the for scientists, laboratory technicians, and humane care and use of laboratory dogs and students. The recommendations are consis- cats for scientists, laboratory technicians, tent with the Public Health Services `guide and students. The recommendations are for the care and use of laboratory animals' consistent with the Public Health Service and regulations set by the USDA. Topics `guide for the care and use of laboratory include housing, nutrition, environment, animals' and regulations set by the USDA. record keeping, animal health care, occupa- Topics include housing, nutrition, environ- tional safety, handling and restraint, experi- ment, record keeping, animal health care, mental techniques, and euthanasia. A occupational safety, handling and restraint, manual that includes a script, test and experimental techniques and euthanasia. A answer key accompanies the videocassette. script, test, and answer key accompany the Videocassette 1/2'' VHS (NTSC), English, videocassette. Videocassette 1/2'' VHS 29 min. National Agricultural Library, (NTSC), colour, English, 35 min. National Beltsville, Maryland 20705, USA. Slide Agricultural Library, Beltsville, Maryland no. 338, Vol 5. 20705, USA. Videocassette no. 338, Vol 4. Further information on training materials Biomethodology of the dog (1987) Information on training materials may be This ®lm demonstrates to animal techni- obtained from: cians safe and humane techniques for ma- nipulating dogs in research including (1) Dept of Laboratory Animal Science (Dr removal from caging, injection routes, blood Jan Nab, Ing T.P. Rooymans) Utrecht collection, and euthanasia. Videocassette 1/ University, Postbus 80.166, 3508 TD 2'' VHS (NTSC), colour, 15 min, English. Utrecht, The Netherlands. National Agricultural Library, Beltsville, (2) NORINA database. English language Maryland 20705, USA. Videocassette database of audiovisuals for use in the no. 335. biological sciences. Contact Karina and Adrian Smith, Laboratory Animal Unit Biomethodology of the primate (1987) Norwegian College of Veterinary Medicine, PO Box 8146 Dep., 00033 Oslo This ®lm demonstrates techniques for ma- 1, Norway. Fax +47 22 96 45 35, nipulating primates for research. It includes Telephone +47 22 96 45 74, email: manual and chemical restraint, identi®ca- [email protected].