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Animals 6 (9): 50 (2016)
University of British Columbia. Animal Welfare Program
Hawkins, Penny
Prescott, Mark J.
Carbone, Larry
Dennison, Ngaire
Johnson, Craig
Makowska, I. Joanna
Marquardt, Nicole
Readman, Gareth
Weary, Daniel M.
Golledge, Huw D. R.
Millions of laboratory animals are killed each year worldwide. There is an ethical, and in many countries also a legal, imperative to ensure those deaths cause minimal suffering. However, there is a lack of consensus regarding what methods of killing are humane for many species and stages of development. In 2013, an international group of researchers and stakeholders met at Newcastle University, United Kingdom to discuss the latest research and which methods could currently be considered most humane for the most commonly used laboratory species (mice, rats and zebrafish). They also discussed factors to consider when making decisions about appropriate techniques for particular species and projects, and priorities for further research. This report summarises the research findings and discussions, with recommendations to help inform good practice for humane killing.
animalsConference ReportA Good Death? Report of the Second NewcastleMeeting on Laboratory Animal EuthanasiaPenny Hawkins 1, Mark J. Prescott 2,*, Larry Carbone 3, Ngaire Dennison 4,†, Craig Johnson 5,I. Joanna Makowska 6, Nicole Marquardt 7, Gareth Readman 8, Daniel M. Weary 6 andHuw D. R. Golledge 91 Research Animals Department, Royal Society for the Prevention of Cruelty to Animals (RSPCA),Wilberforce Way, Southwater, Horsham, West Sussex RH13 9RS, UK; penny.hawkins@rspca.org.uk2 National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs),Gibbs Building, 215 Euston Road, London NW1 2BE, UK3 Laboratory Animal Resource Center, University of California, San Francisco, CA 94143, USA;larry.carbone@ucsf.edu4 Animals in Science Regulation Unit, Home Office Science, 1st Floor Peel NE, 2 Marsham Street,London SW1P 4DF, UK; n.dennison@dundee.ac.uk5 Institute of Veterinary, Animal and Biomedical Sciences, Private Bag 11 222, Massey University,Palmerston North 4442, New Zealand; c.b.johnson@massey.ac.nz6 Animal Welfare Program, University of British Colombia, 2357 Main Mall, Vancouver, BC V6T 1Z4, Canada;makowska@interchange.ubc.ca (I.J.M.); dan.weary@ubc.ca (D.M.W.)7 Institute of Pharmacology and Toxicology, Freie Universität Berlin, Koserstraße 20, Berlin 14195, Germany;marquardt@berliner-fortbildungen.de8 School of Biology, University of Plymouth, Smeaton 008D, Drakes Circus, Plymouth PL4 8AA, UK;gareth.readman@plymouth.ac.uk9 Universities Federation for Animal Welfare (UFAW), The Old School, Brewhouse Hill, Wheathampstead,Hertfordshire AL4 8AN, UK; golledge@ufaw.org.uk* Correspondence: mark.prescott@nc3rs.org.uk; Tel.: +44-207-611-2226; Fax: +44-207-611-2260† Current address: Biological Services, University of Dundee, MSI/WTB/JBC complex,Dundee DD1 5EH, UK.Academic Editor: Clive J. C. PhillipsReceived: 8 December 2015; Accepted: 11 August 2016; Published: 23 August 2016Simple Summary: Millions of laboratory animals are killed each year worldwide. However, thereis a lack of consensus regarding what methods of killing are humane for many species and stagesof development. This report summarises research findings and discussions from an internationalmeeting of experts and stakeholders, with recommendations to inform good practice for humanekilling of mice, rats and zebrafish. It provides additional guidance and perspectives for researchersdesigning projects that involve euthanasing animals, researchers studying aspects of humanekilling, euthanasia device manufacturers, regulators, and institutional ethics or animal care anduse committees that wish to review local practice.Abstract: Millions of laboratory animals are killed each year worldwide. There is an ethical, and inmany countries also a legal, imperative to ensure those deaths cause minimal suffering. However,there is a lack of consensus regarding what methods of killing are humane for many species and stagesof development. In 2013, an international group of researchers and stakeholders met at NewcastleUniversity, United Kingdom to discuss the latest research and which methods could currently beconsidered most humane for the most commonly used laboratory species (mice, rats and zebrafish).They also discussed factors to consider when making decisions about appropriate techniques forparticular species and projects, and priorities for further research. This report summarises the researchfindings and discussions, with recommendations to help inform good practice for humane killing.Animals 2016, 6, 50; doi:10.3390/ani6090050 www.mdpi.com/journal/animalsAnimals 2016, 6, 50 2 of 28Keywords: animal welfare; carbon dioxide; euthanasia; humane killing; mouse; rat; refinement;3Rs; zebrafish1. IntroductionThe vast majority of animals used in research and testing worldwide are killed, either becausetheir tissues or organs are required at the end of a study, because a humane endpoint has beenreached, or because they are surplus to requirements (e.g., due to overbreeding or if they do not have adesired genotype). There are ethical and legal reasons for ensuring that laboratory animals are killedas humanely as possible. However, there is debate regarding the relative humaneness of, and bestpractice for, a number of commonly used techniques, such as carbon dioxide (CO2).In recognition of this, a meeting on carbon dioxide euthanasia of laboratory animals was held atthe Newcastle University in 2006, with support from the UK’s National Centre for the Replacement,Refinement and Reduction of Animals in Research (NC3Rs) and Laboratory Animals Limited. Thisinitial meeting aimed to bring together scientists who had researched the use of CO2 for killinglaboratory rats and mice, to help inform best practice for CO2 euthanasia. Additional aims were toidentify what research was needed to further identify and evaluate welfare issues associated with theuse of CO2, and to consider whether any preferable alternatives were currently available.The initial Newcastle meeting report [1] set out the majority view of the meeting participants.It identified animal welfare problems with CO2 killing and made recommendations for good practice,but could not recommend any other inhaled agents apart from volatile anaesthetics, with the caveatthat the aversiveness of these can vary. A list of recommendations was made for future research,to identify good practice and evaluate potential alternatives to CO2.The 2006 report raised the profile of the debate regarding CO2, prompting a number ofestablishments to review their CO2 administration protocols, or to induce general anaesthesia withan inhaled anaesthetic agent before switching to CO2. A further outcome of the meeting was theannouncement by the NC3Rs of a call for applications for a Strategic Award aimed at answeringsome of the identified research needs, which was ultimately made to Dr Huw Golledge of NewcastleUniversity. A component of the NC3Rs award included funding to run a second meeting on laboratoryanimal euthanasia, to provide an update on progress, which is the subject of this report.Since the 2006 meeting, many regulations and/or guidelines governing the use of humane killingtechniques for laboratory animals have been revised, including those issued by the European Union [2]and its Member States, the American Veterinary Medical Association [3] and the Canadian Council onAnimal Care [4]. Some key changes include:• the use of CO2 should only be in a rising concentration [2,3];• the recommendation that CO2 should not be used as a sole killing agent on conscious animals [4];• addition of the use of inert gases as an “acceptable” technique for rodents [2];• a presumption that decapitation is only acceptable when no other method is possible [2];• the opposite presumption that the only welfare concern with decapitation and cervical dislocationis the skill of the person [3].The impact of these policy changes on animal welfare is variable. For example, as reviewed below,administering CO2 in a rising concentration (as opposed to pre-fill) will avoid pain but may increasethe duration of fear and distress relative to the pre-fill method.The overall aim of the second Newcastle Meeting, held on 9 August 2013, was to provide a forumto discuss recent research and current practice on euthanasia, so as to inform debate and policy onhumane killing (for the meeting programme, see Supplementary Materials Table S1). The specific aimswere to:• review key areas of recent research into humane killing;Animals 2016, 6, 50 3 of 28• consider how this could be interpreted and incorporated into practice;• expand the remit beyond CO2, to include recent studies that have evaluated the humanenessof some physical methods for killing rodents and the use of anaesthetics and inert gases forkilling rodents;• include zebrafish as well as mice and rats, to encompass the three most commonly usedlaboratory species;• discuss special considerations when killing neonatal animals;• incorporate extended discussion sessions to explore ethical issues and criteria for judging thehumaneness of different techniques and devices.Demographic information, participants’ views and information on current practices wereobtained using an interactive, electronic polling system (Turning Technologies, LLC). The audience of90 participants included researchers, veterinarians, facility managers, animal technologists, researchregulators, and staff of animal welfare and 3Rs organisations. Most participants worked withinacademic institutions. Two thirds were based in the UK and one sixth in other EU member states,with the remainder from the USA, Canada, Japan and New Zealand. Four fifths of the audience weredirectly involved in the euthanasia of animals used in research; mostly rats and mice but also fish andbirds. Participants directly involved in euthanasia reported the use of a variety of techniques for miceand rats (Figure 1) and fish (Figure 2).Animals 2016, 6, 50 3 of 26  expand the remit beyond CO2, to include recent studies that have evaluated the humaneness of some physical methods for killing rodents and the use of anaesthetics and inert gases for killing rodents;  include zebrafish as well as mice and rats, to encompass the three most commonly used laboratory species;  discuss special considerations when killing neonatal animals;  incorporate extended discussion sessions to explore ethical issues and criteria for judging the humaneness of different techniques and devices. Demographic information, participants’ views and information on current practices were obtained using an interactive, electronic polling system (Turning Technologies, LLC). The audience of 90 participants included researchers, veterinarians, facility managers, animal technologists, research regulators, and staff of animal welfare and 3Rs organisations. Most participants worked within academic institutions. Two thirds were based in the UK and one sixth in other EU member states, with the remainder from the USA, Canada, Japan and New Zealand. Four fifths of the audience were directly involved in the euthanasia of animals used in research; mostly rats and mice but also fish and birds. Participants directly involved in euthanasia reported the use of a variety of techniques for mice and rats (Figure 1) and fish (Figure 2). Figure 1. Meeting participants’ responses to the questions: “Which methods do you use to kill mice/rats?”. Figure 2. Meeting participants’ responses to the question: “Which methods do you use to kill fish?”. Figure 1. Meeting participants’ responses to the q i s: “Which metho s d you use to killmice/rats?”.Animals 2016, 6, 50 3 of 26  expand the remit beyond CO2, to include recent studies that have evaluated the humaneness of some physical methods for killing rodents and the use of anaesthetics and inert gases for killing rodents;  include zebrafish as well as mice and rats, to encompass the three most commonly used laboratory species;  discuss special considerations when killing neonatal animals;  incorporate extended discussion sessions to explore ethical issues and criteria for judging the humaneness of different techniques and devices. Demographic information, participants’ views and i formation on current practices were obtained usi g an inte active, electronic polling system (Turning Technolog es, LLC). The audience of 90 participants included researchers, veterinarians, facility ma agers, anim l technologists, research regulators, and staff of animal welfare and 3Rs organisations. Most participants worked within academic institutions. Two thirds were based in the UK and one sixth in other EU member states, with the remainder from the USA, Canada, Japan and New Zealand. Four fifths of the audience were directly involved in the euthanasia of animals used in research; mostly rats and mice but also fish and birds. Participants directly involved in euthanasia reported the use of a variety of techniques for mice and rats (Figure 1) and fish (Figure 2). Figure 1. Meeting participants’ responses to the questions: “Which methods do you use to kill mice/rats?”. Figure 2. Meeting participants’ responses to the question: “Which methods do you use to kill fish?”. Figure 2. Meeting participants’ responses to the q esti : “ hich methods d you use to kill fish?”.Animals 2016, 6, 50 4 of 28Section 2 of this report summarises the speakers’ presentations and sets out the majority views ofthe meeting participants on some selected topics, which were obtained using the interactive electronicpolling system during the discussion sessions. A summary of the main points and areas for furtherresearch is given in Section 3 and conclusions and recommendations are set out in Section 4. The overallaim is to provide information and points to consider for a range of audiences worldwide, includingresearchers designing projects that may involve euthanising animals, researchers studying aspects ofhumane killing (e.g., evaluating the physiology of the killing process or the humaneness of differenttechniques), veterinarians, animal technologists, euthanasia device manufacturers, regulators, andinstitutes that wish to review local practice and training for humane killing (e.g., via review by theethics or animal care and use committee).2. Summary of the MeetingThe welfare implications of any humane killing technique will depend upon the individualanimal’s species, strain and stage of development. The meeting focused on inhaled agents such asanaesthetics and CO2, decapitation and cervical dislocation in mice and rats, and addition of agentsto tank water in zebrafish, as these techniques and species account for the majority of killing inthe laboratory.Euthanasia techniques ultimately cause death by three basic mechanisms: (i) direct depression ofneuronal activity necessary for life function; (ii) hypoxia; or (iii) physical disruption of brain activity.By definition, welfare concerns can occur only during the period of consciousness; what happensbetween the complete loss of consciousness and death should not be an issue because an animalcannot suffer during this period. However, for some methods there may be a period of full or partialconsciousness for some time after the initial insult has occurred.The animal’s experience between the commencement of the killing technique, and the final loss ofconsciousness, is only one consideration when determining the relative humaneness of a euthanasiaprotocol. Humane killing is a process during which the animal may experience a number of stressful,distressing or painful events, depending on the technique, including capture, handling, restraint,separation from cage-mates, exposure to unfamiliar conspecifics, transport, relocation to a differentenclosure [5–7] and pain (e.g., due to injection, or the chemical properties of the euthanasia agent).The potential for recovery from the technique is also an important criterion for determining thehumaneness of some methods. Although all currently available techniques will inevitably involvesome degree of pain and/or distress, due consideration of all of the above elements should help todetermine the most humane method. The sub-sections below should be read in this context, althoughthe focus is on reducing suffering around application of the technique.2.1. Inhaled Agents in RodentsMost laboratory rodents are killed using inhalation techniques. European Directive 2010/63/EUpermits two inhalation techniques for rodents: a gradually rising concentration of CO2 or inert gases(argon or nitrogen) (The use of inert gases for killing laboratory animals is not permitted in the UK,because they are considered to be aversive (argon) or there is insufficient evidence that they arehumane (nitrogen)). In addition, inhaled anaesthetics may be used to anaesthetise animals beforeswitching to CO2 to complete the euthanasia process. The AVMA euthanasia guidelines also state thatinhaled anaesthetics or a gradual fill of CO2 (the latter with or without premedication with inhaledanaesthetics) are acceptable for rodents, but lists nitrogen and argon amongst unacceptable methods,unless animals are anaesthetised [3].2.1.1. Aversion to CO2 and Inhaled AnaestheticsOf the many studies examining responses of rodents to CO2, all show that the agent is aversive.Given that rats and mice consistently avoid CO2 when they are able to, they are likely to experiencedistress if they cannot escape, as is the case during euthanasia. At the meeting, Joanna MakowskaAnimals 2016, 6, 50 5 of 28reviewed the results from a wide cross-section of studies that consistently demonstrate that miceand rats show aversion to CO2 even if this involves foregoing a desirable food reward or exposingthemselves to another aversive stimulus.The approach-avoidance paradigm has been used to evaluate mouse and rat aversion to CO2,argon and inhaled anaesthetics. Mice and rats exposed to a rising concentration of CO2 leave a testchamber at the cost of abandoning food rewards when levels rise to approximately 12%–18% [8–10].With O2 supplementation, rats will stay until the concentration reaches around 21% CO2 [11]; however,as noted by the study authors, this reduction is not enough to warrant recommending the use of CO2and O2 mixtures for killing rats. Even animals that have been deprived of food for 24 h (and hence arehighly motivated to access the food reward) will escape the chamber when CO2 reaches approximately12%–18% [12]. This research supports earlier work demonstrating that CO2 is aversive [1].One potential criticism of the approach-avoidance paradigm is that CO2 may affect taste receptors,making the sweet food reward less attractive. Aversion-avoidance testing cannot be subjectedto this criticism as it compares motivation to avoid the gas with motivation to avoid an open,brightly lit chamber (known from a large body of work to be fear inducing in rodents). As withthe approach-avoidance tests, mice and rats tested using the aversion-avoidance paradigm never stayin a chamber filling with CO2 and instead escape to the brightly lit open chamber they otherwiseavoid [13,14]. Thus across a large number of peer-reviewed studies, using a range of species, strainsand testing methods, we see a highly consistent aversion response to concentrations of CO2 muchlower than are required for anaesthesia.Evidence also indicates that exposure to volatile anaesthetics is a refinement over exposure toCO2. In approach-avoidance testing, most naïve rodents choose to remain in a chamber (with a foodreward) filling with isoflurane, sevoflurane or halothane until they are ataxic, and some rodents neverleave [15–17]. Similarly, in aversion-avoidance testing, most naïve rodents tested with these agentsremain in the dark chamber until they lose consciousness, rather than escaping to the brightly litcompartment [13–15].However, all available evidence indicates that repeated exposure to the volatile anaesthetics ismore aversive than the initial exposure. A series of studies have shown that mice and rats are morelikely to escape the test chamber, and to do so more quickly, on second and subsequent exposures tovolatile anaesthetics [13–15]. In contrast, aversion to CO2 does not significantly increase on subsequentexposure [14]. This could be a “floor effect”, in which aversion to CO2 is already maximal and cannotincrease further. Alternatively, it could reflect the innate nature of aversion to CO2, a naturally occurringsubstance to which rodents may have evolved to be averse, as opposed to isoflurane, a synthetic agentwith a pungent odour (to humans, at least) which could serve as a potent unconditioned stimulus.The similarity in odour of isoflurane and sevoflurane may explain the generalization of the conditionedresponse to isoflurane to sevoflurane.So, although the volatile agents can be an important refinement over CO2 (and anoxia), theseagents cannot be considered ideal; some animals choose to escape the test chamber when exposedto these agents within both approach-avoidance and aversion-avoidance paradigms. The availableevidence indicates that isoflurane and sevoflurane appear to be equally aversive to mice and rats,and that halothane is more aversive [15,16]. Most of the literature relates to volatile anaestheticsdelivered using a precision vaporiser, but these agents can also be administered using the so-called“drop method” whereby the agent is delivered as a liquid (typically absorbed onto a swab) andvaporised rapidly within the chamber, allowing a much higher concentration to be achieved than themaximum output from a vaporiser (typically 5% for isoflurane). In this case, it may be possible to killanimals in a relatively short period of time; however, mice exposed to a rapidly rising concentration ofisoflurane using the “drop technique” showed a higher level of aversion than those exposed using avaporiser [13].On the basis of published aversion studies available at the time (2006), the previous Newcastlemeeting recommended that inhalant anaesthetics be used to render animals unconscious beforeAnimals 2016, 6, 50 6 of 28switching to CO2. (Because of their wide safety margins, it is time consuming to kill animals withisoflurane [18] or halothane, so a secondary method is sometimes preferred for the sake of efficiencyand safety). However, subsequent aversion studies have indicated that this may not necessarily be arefinement if animals have previously been exposed to inhalant anaesthetics (see below).It is important to note that the time from unconsciousness to death has no bearing on animalwelfare; as emphasised above, welfare concerns cease once the animal has been rendered unconscious.However, there can be serious welfare concerns, if poor technique results in some animals recoveringconsciousness before the secondary method takes effect. A study by Moody et al. [19], using an adaptedchamber that accommodates an investigator’s hand yet is still airtight, has enabled righting and pedalwithdrawal reflexes to be monitored in mice. This study showed that, with a flow rate of 17% chambervolume per minute of 5% isoflurane in O2, mice become recumbent after approximately 35 s fromthe beginning of exposure. However, the righting reflex is present until a mean of 45 s has elapsed,and the pedal withdrawal reflex is not lost until a mean of 75 s. On this basis and with a cautioussafety margin (mean + 3 S.D.), when using the above administration protocol and CO2 exposure asthe secondary method, CO2 should not be applied until at least 80 s after the onset of recumbency toprevent recovery of consciousness, which has been seen when switching from isoflurane to high flowrates of CO2 immediately after loss of consciousness [20].To summarise findings in this field to date, in aversion-avoidance tests both mice and rats willalways choose exposure to light over exposure to CO2, but more than 50% of mice and rats willtolerate first exposure to an inhalant anaesthetic until they become recumbent. However, both speciesalso show evidence of learned aversion to inhaled anaesthetic agents. The reasons for this are as yetunknown, and it was suggested during discussion at the meeting that it may be due to memories ofunpleasant side effects when waking from the first exposure; but it was also suggested that nausea orthe excitation response during induction may contribute to aversion.Whatever the cause, learned aversion to inhaled anaesthetic agents is likely to present animalwelfare issues when mice and rats are repeatedly anaesthetised, for example during longitudinalstudies that involve serially imaging animals. It is also likely that the experience of euthanasia usingan inhalant anaesthetic will depend upon whether an animal has been anaesthetised before (andpotentially the interval between the anaesthetics, i.e., whether the aversion is reduced or extinguished,which has not been investigated). The way in which animals appear to generalise between isoflurane,halothane and sevoflurane suggests that using a different but chemically-related anaesthetic agentfor euthanasia will not solve the problem. All commonly used inhaled anaesthetics belong to thesame chemical family of halogenated compounds and may therefore also elicit generalised responses.There was some discussion at the meeting as to whether it might be more humane to use CO2 to killanimals if they have been exposed to inhaled anaesthetics previously, but in the aversion-avoidancetests published to date, the percentage of mice and rats staying in the dark compartment with theanaesthetic never fell to zero, as it did for CO2. Furthermore, animals previously exposed to isofluranedid not show greater aversion (on average) to isoflurane than they did to CO2. As such, currentevidence suggests that there is no welfare advantage to killing animals with CO2 if they have beenpreviously exposed to isoflurane. Even in the worst case scenario so far tested isoflurane was only asaversive as CO2.Results to date do suggest that episodes of general anaesthesia are not independent events formice and rats, and therefore the number of techniques animals undergo that require general anaesthesiawith inhaled anaesthetic agents (e.g., surgery or imaging) should be minimised and factored intoharm-benefit analyses. More studies are required to elucidate the magnitude of aversion in animalsexposed to isoflurane more than twice, and the effect on the magnitude of aversion of longer or shortintervals between anaesthesia.Aversion to an agent is generally interpreted as indicating that forced exposure (beyond thepoint of aversion) is distressing, but some caveats were discussed by Huw Golledge. Behaviouralsigns of apparent distress during forced exposure can be difficult to interpret; for example, someAnimals 2016, 6, 50 7 of 28behavioural responses during exposure to volatile anaesthetics (such as increased locomotor activity,e.g., running) may not be consciously perceived by the animal as distressing. Similarly, a freezingresponse to a fear-inducing agent (such as CO2) can be mistakenly interpreted as a lack of distress.Correlating these specific behaviours with physiological parameters can help to determine their valueas indicators of adverse physiological stress or distress [21]. Even some avoidance responses maybe difficult to interpret; for instance, a wide range of taxa avoid exposure to high concentrations ofCO2, even the nematode worm C. elegans [22], so an avoidance response per se may not be strongevidence for a consciously felt negative effect. Of course, the more sophisticated testing methods(aversion-avoidance and approach-avoidance), where animals must trade off one motivation foranother, cannot be so easily dismissed. However, even in these cases results could be confoundedif, for instance, the test agents affected the animal’s motivation to retrieve a reward as opposed tocausing distress. Furthermore, physiological indicators of stress can be elevated by eustress, or can beelevated in unconscious animals, and therefore cannot be unequivocally linked to negative emotionalstates. Ideally, therefore, physiological and behavioural measures should be combined. Evidence thatan inhaled agent is capable of causing psychological distress (and would cause distress if exposurewas inescapable) is significantly strengthened where behaviours which could indicate distress isobserved at concentrations that have been shown to induce physiological signs of stress, and where theagent can be shown to cause an animal to either forego a reward, or expose themselves to a stressfulenvironment, in order to avoid the agent. Where aversion-avoidance or approach-avoidance studiesresult in significantly lower signs of aversion, there is good reason to argue that the behavioural signsof excitation may not be representative of distress. In such cases, any observed signs of physiologicalstress could represent stress that is not correlated with a negative affective state, e.g., that arising fromincreased locomotor activity.A behavioural testing approach that has been successfully applied in zebrafish (see Section 2.4)is the conditioned place aversion paradigm. This test relies on the animal’s memory of the effectsof exposure to the agent. The animal makes an association between the affective state induced bythe agent (how they feel when they are exposed to the chemical, gas or anaesthetic vapour) andenvironmental cues. That is, there is a Pavlovian association between the agent (the unconditionedstimulus) and the environment (the conditioned stimulus). The choices that an animal makes inthe absence of the test agent can thus provide insight into each animal’s memory of their subjectivestate during exposure to the agent. Development of conditioned place aversion therefore relies onthe animal’s emotional experiences and is not subject to the same confounds as other behaviouralapproaches discussed in the preceding paragraph.The conditioned place preference/aversion paradigm also employs protocols for exposing theanimals to the test agent, when they are being conditioned, that are tailored to match conditions duringthe euthanasia process. Rats are handled immediately before the procedure, the fill rates are identicalto those used in practice, and animals are conditioned for 90 s, until they are on the brink of losingconsciousness. Furthermore, during conditioning exposures the animal is confined to a chamber andexposed to the agent, unlike approach-avoidance or aversion-avoidance studies where the animal isfree to terminate exposure by leaving the chamber at any time. The experience of animals undergoingconditioning therefore mimics much more closely the experience of animals undergoing euthanasia.In an unpublished study, Golledge et al. repeatedly exposed male Lister hooded rats to CO2(20% chamber volume per minute flow rate, 2.4 L/min), isoflurane (5%, delivered in 100% oxygen,2.4 L/min) and argon (chamber prefilled to 98%) to the brink of losing consciousness. These animalshad no opportunity to escape from the chamber at any time. The same animals were exposed toair at the same flow rate but in a different chamber. Control and exposure chambers were madedistinctive using both visual and tactile cues (Figure 3). Following six conditioning exposures to eachof the chambers, the animals were allowed to move freely between the two and their preferenceswere measured. All three agents caused significant conditioned place aversion. This suggests thatrats repeatedly exposed to all three agents have some memory of negative affect during exposure,Animals 2016, 6, 50 8 of 28consistent with the results reviewed earlier showing that repeat exposure is aversive. Golledge calledfor more research to document the extent of suffering when animals are exposed to different euthanasiaagents. This may require the development of and use of more sophisticated testing methods. Thesefindings also underline the need to search for alternative agents for both anaesthesia and euthanasia.Animals 2016, 6, 50 8 of 26 were measured. All three agents caused significant conditioned place aversion. This suggests that rats repeatedly exposed to all three agents have some memory of negative affect during exposure, consistent with the results reviewed earlier showing that repeat exposure is aversive. Golledge called for more research to document the extent of suffering when animals are exposed to different euthanasia agents. This ay requir the development of and use of more sophisticat d testing methods. These findings also underline the need to search f r alternative agents for both anaesthesia a euth nasia. Figure 3. Example of apparatus for conditioned place preference/aversion paradigm. The right and left cage compartments have differently patterned and textured floors and walls to ensure the animal can discriminate between them. 2.1.2. Pain and CO2 Huw Golledge summarised the evidence from the neuroscience, psychology and physiology literature that exposure to CO2, above a certain concentration, is likely to be painful for animals (see [1] for a review). The mechanism for this is well known; carbonic acid is formed when gaseous CO2 encounters mucous membranes or other moist tissues. Humans find CO2 painful at concentrations over around 50% [23], and rodents have similar nociceptors to humans, at a similar density, in their nasal and ocular epithelia. These nociceptors respond to CO2 at similar concentrations to human nociceptors [24]. There are long-standing concerns that CO2 killing would be painful for animals [25]; for those placed in a pre-filled chamber this would certainly be the case, but using gradual fill probably avoids causing pain. For example, with a fill rate of 20% chamber volume per minute, rats lose consciousness at 156 ± 5 s (Golledge, unpubl. obs.), at which point the concentration of CO2 is 39%, which is likely to be before the gas reaches levels liable to cause pain (Figure 4). Figure 3. Example of apparatus for conditioned place preference/aversion paradigm. The right andleft cage compartments have differently patterned and textured floors and walls to ensure the animalcan discriminate between them.2.1.2. Pain and CO2Huw Golledge summarised the evidence from the neuroscience, psychology and physiologyliterature that exposure to CO2, above a certain concentration, is likely to be painful for animals (see [1]for a review). The mechanism for this is well known; carbonic acid is formed when gaseous CO2encounters mucous membranes or other moist tissues. Humans find CO2 painful at concentrationsover around 50% [23], and rodents have similar nociceptors to humans, at a similar density, in theirnasal and ocular epithelia. These nociceptors respond to CO2 at similar concentrations to humannociceptors [24].There are long-standing concerns that CO2 killing would be painful for animals [25]; for thoseplaced in a pre-filled chamber this would certainly be the case, but using gradual fill probably avoidscausing pain. For example, with a fill rate of 20% chamber volume per minute, rats lose consciousnessat 156 ± 5 s (Golledge, unpubl. obs.), at which point the concentration of CO2 is 39%, which is likely tobe before the gas reaches levels liable to cause pain (Figure 4).Animals 2016, 6, 50 9 of 28Animals 2016, 6, 50 9 of 26 Figure 4. The induction of loss of consciousness during 20% per minute filling with CO2. Time line for loss of consciousness in rats exposed to CO2 at 20% chamber volume per minute. Animals lose consciousness after approximately 156 s. Bars below the x-axis indicate periods where the animals would be conscious or unconscious. Shaded area shows the likely time/concentration where CO2 could cause pain (which occurs after loss consciousness in this case). Levels above 5% may cause anxiety and include a significant duration where the animal would still be conscious. 2.1.3. Fear and Anxiety with CO2 Carbon dioxide is known to be aversive, at concentrations far below those that cause pain; this aversion is speculated to be due to increased anxiety associated with the inability to escape. There are thus welfare concerns associated with gradual fill because of the long duration when conscious animals are exposed to aversive concentrations of CO2 (see Section 2.1.1 above and Figure 4). Mice can detect CO2 at near atmospheric concentrations (0.04%) and actively avoid levels of just 0.2% [26], whereas CO2 concentrations of up to 30% are odourless to humans [27]. In the mouse, specialised olfactory neurons have been identified that depolarise in response to CO2, with a detection threshold of 0.1%. These neurons have monosynaptic connection to the olfactory bulb in the brain, suggesting a mechanism for CO2 detection and avoidance in mice which is not shared with humans [26]. Another potential source of suffering is CO2-induced fear, which occurs in both humans and rats. A single breath of 35% CO2 can cause panic in susceptible humans, and brief exposure to normoxic 20% CO2 (The composition of the gas was: 20% CO2, 21% O2, 59% N2) activates “panic/defence” related brain circuits in rats, such as the hypothalamus, rostroventrolateral medulla (RVM) and periaqueductal gray (PAG) [28]. Inhalation of normoxic gas mixtures containing just 7.5% CO2 causes anxiety in normal human volunteers if inhaled for longer periods of time [29] and hypervigilance to threat stimuli (e.g., increased orienting towards, and slower orienting away from, the perceived threat) [30]. When inhaling hypercarbic gas, people report feeling irritable, nervous, anxious, worried and “feel like leaving”. The anxiety is ameliorated by anxiolytic drugs such as benzodiazepines [31]. Carbon dioxide induced acidosis also evokes fear responses in mice, such as freezing behaviour which occurs at 5%–10% CO2 [32]. Fear responses occur when acidosis is sensed by the ASIC1a channel; genetically altered ASIC1a knockout mice display a significantly reduced fear response to acidosis. Neutralising the pH of the CSF in the amygdala also attenuates the fear response. The potential also exists for CO2 to cause dyspnoea, anxiety or distress via a third mechanism unrelated to pain. Also referred to as “air hunger”, this is a subjectively uncomfortable and increasingly distressing urge to breathe. Humans may suffer from dyspnoea as a result of asthma or . i l f c s i i i t filli it 2c i i r t e s t 2 c l er i t .l conscio s or unconscious. area sho s the likely time/ i 2l i ( i ft l c i i t i cas ). li t i l i ifi t r ti re t e a i al o l still be conscious.2t i cr s i t ss i it t i ilit t esc .fi l2 .22 t [ ].tifi t t l ris i r s se t 22 .potential source of suffering is CO2-induced fear, whic occurs in both humans d rats.A single breath of 35% CO2 can cause panic in susceptible humans, and brief exposure to norm xic 20%CO2 (The composition of the gas was: 20% CO2, 21% O2, 59% N2) activates “panic/defence” relatedbrai ircuits in rats, such as the hypothalamus, rostroventrolateral medulla (RVM) and peri queductalgray (PAG) [28]. Inhalation of normoxic gas mixtures containing just 7.5% CO2 causes a xiety innormal human volunt ers if i haled for longer periods of t me [29] and hyp vigilance to hreat stimuli(e.g., incre sed orienting towards, and slower orienting away f om, the perceived threat) [30]. Wheninhaling hypercarbic gas, people report feeling irritable, nerv us, anxious, worr ed nd “f el likeleaving”. The anxiety is ameliorat d by anxiolytic drugs uch as benzodiazepines [31].Carbon dioxide induced acidosis also evokes fear responses in mice, such as freezing behaviourwhich occurs at 5%–10% CO2 [32]. Fear r sponses occur wh n acidosis is sensed by the ASIC1achannel; genetically altered ASIC1a knockout mice display a significantly reduced fear response toacidosis. Neutralising he pH of the CSF in the amyg ala lso attenuates the fear response.The potential also exists for CO2 to caus dyspnoe , anxiety or distress via a third mechanismunrelated t pain. Also r ferred to as “air hung r”, this is subjectively uncomfort ble an increasinglyAnimals 2016, 6, 50 10 of 28distressing urge to breathe. Humans may suffer from dyspnoea as a result of asthma or chronic lungdiseases. Inhaling CO2 can cause dyspnoea in humans [33], and dyspnoea may also occur in rodents(see also [1]).Golledge argued that, taken together, all of the above strongly suggests that rats and mice exposedto a gradually rising concentration of CO2 will experience fear and/or anxiety even if they do notsuffer pain. Since the concentrations which cause fear are sub-anaesthetic, all animals killed with CO2are likely to experience these negative states.Nicole Marquardt presented the results of an unpublished study aiming to understandphysiological responses during exposure to inhaled agents. Responses were monitored in mice(NMRI and C57/BL6), rats (Wistar and Sprague Dawley) and Syrian hamsters exposed to varyingfill rates and concentrations of CO2, isoflurane and sevoflurane, with air as a control. The test gaseswere administered in the animals’ home cages to reduce additional sources of stress (as listed inthe introduction to this section). Catecholamine levels were assayed from trunk blood collected bydecapitating the animal immediately following the onset of anaesthesia. This study reported somespecies and strain differences; for example, 8% sevoflurane induced anaesthesia to a surgical planein 87.5% of NMRI mice but 100% of C57/Bl6 mice, and plasma noradrenaline levels were higher inhamsters compared to rats when they were exposed to 60% CO2. In general, the stress hormonesadrenaline and noradrenaline were elevated in all mice and hamsters, and most rats exposed to CO2,but much less so when rats were exposed to isoflurane or sevoflurane. Thus, these results providefurther evidence that CO2 causes stress before loss of consciousness, and that volatile anaesthetics are arefinement as they cause reduced induction of stress hormones during the conscious phase of exposure.However, it must be acknowledged that elevation of stress hormones in a conscious animal cannotbe conclusively linked to distress, since catecholamine levels have been shown to become elevated inunconscious (anaesthetised) or brain-dead animals [34,35]. Despite this, the agreement of this studywith behavioural studies showing stronger aversion to CO2 than the other tested agents does suggestthat the observed elevations may be related to consciously-experienced negative emotional states.The above studies, on humans and rodents, indicate that exposure to CO2 is likely to causestress, anxiety and hypervigilance in mice and rats. Other elements of the CO2 killing, as commonlyconducted, are also likely to be anxiogenic (e.g., capture, handling, transport to an unfamiliarenvironment and mixing with unfamiliar individuals), so CO2-induced anxiety will be in additionto that caused by the procedure in many cases. Administering agents in the home cage, or placingestablished groups in the chamber within their home cage, may minimise some of these stressors, andthus should also be considered a refinement [20]. However, even under “ideal” conditions, CO2 islikely to cause acute anxiety.Helen Valentine presented the results of her study comparing physiological and behaviouralmeasures of stress in mice killed with CO2—either with or without anxiolytic premedications(acepromazine or midazolam)—to mice anaesthetised with isoflurane. She reported that time tounconsciousness was reduced in midazolam-premedicated animals, but the stress of handling andinjecting the mice appeared to negate the benefit of premedication [36]. Corticosterone levels andinduction agitation scores were also increased in animals premedicated with midazolam. In addition,midazolam prolonged the time between unconsciousness and death, likely due to its function ofincreasing the efficiency of the inhibitory transmitter GABA, which has a neuroprotective effect [36].This will obviously not have welfare implications, but may affect some scientific protocols.Valentine concluded that signs of stress (both physiological and behavioural) were higher inisoflurane anaesthetised mice than those killed with 20% CO2, in contrast to most other studies,including those presented at the meeting by Makowska, Golledge and Marquardt. However, her studyand interpretation of the results have been criticised by others—see Makowska et al. [37]. In particular,it has been suggested that inducing anesthesia with isoflurane, then allowing recovery before exposingthe animals to CO2, has made the results unrepresentative of normal euthanasia procedures whereanimals would be allowed to become deeply anaesthetised before switching to CO2, preventingAnimals 2016, 6, 50 11 of 28recovery. The suggestion that mice made distress vocalisations following isoflurane exposure has alsobeen challenged on the basis that there is little evidence of a reliable correlation between ultrasonicvocalisations and painful or aversive stimuli in mice [38], unlike rats. See also Valentine et al. [39] fora response.2.1.4. Aversion to AnoxiaLow oxygen environments can be created by using an inert gas to displace air from the chamber.Rats and mice are very sensitive to changes in oxygen concentration in inspired air [40]. Makowskareported how, in an approach-avoidance paradigm using argon (one example of an inert gas), ratsleft the chamber at a mean oxygen concentration of 6.8% and mice at a mean of 8.6% [8,17]. In theaforementioned unpublished study by Golledge et al., a single exposure to 98% argon was foundto induce a significant conditioned place aversion, albeit weaker than that observed with repeatedconditioning. On the basis of this series of experiments, rats and mice should not be exposed to argonfor any reason, including killing.2.2. Physical Methods for Rodents—Decapitation and Cervical DislocationThere is on-going debate about the relative humaneness of inhalation versus physical methods.Physical methods can be humane within appropriate weight limits, and when effectively performedby trained and competent persons. However, potential welfare concerns and sources of sufferingwhen rodents are killed using decapitation or cervical dislocation include distress from capture andrestraint; exposure to other animals’ alarm calls or pheromones; pain or distress experienced betweenthe application of the method and unconsciousness; persistent cortical activity (which potentiallyindicates on-going consciousness) after application of the method; the possibility of operator error;consequences of operator error; and potential for recovery after incomplete cervical dislocation. Manyof these also apply to non-physical methods, but the full list of potential welfare concerns illustratesthe greater risk of suffering when physical methods are used.These concerns are reflected in legislation on the humane killing of laboratory animals. Forexample, Directive 2010/63/EU [2] permits cervical dislocation for rodents up to 1 kg (requiringsedation for animals weighing over 150 g) or concussion of the brain by striking the cranium forrodents up to 1 kg. The Directive also permits decapitation for rodents “if other methods are notpossible”, but this is not a standard method in the UK, where project authorisation is required.Concussion was not discussed within the meeting due to a lack of ongoing research, but the welfareconcerns are similar to those for the other physical methods.The AVMA Guidelines state that decapitation and cervical dislocation are both “acceptable withconditions” for mice and rats (and cervical dislocation only for rats under 200 g) [3]. Conditions includepractical factors such as training, operator skill and appropriate equipment, but special justification(e.g., from an ethics or animal care and use committee) for physical methods is not required.Larry Carbone discussed many of the issues surrounding physical techniques, including thefailure rate of cervical dislocation, and reviewed some of the science behind these codes of practiceand guidelines. With decapitation, the obvious concern is that the decapitated brain may be consciousand in pain. Some studies have made electroencephalogram (EEG) recordings in the severed heads ofmice and rats, in which animals have been anaesthetised for electrode implantation and then paralysedto allow better quality recordings to be made. A study by Mikeska and Klemm that is 40 yearsold [41] reported that six paralysed, decapitated rats had persistent “EEG activation” for 5.6–29.5 s(mean 13.6 ± 4.6 SE seconds) and a “total EEG duration” for 19.0–46.5 s. Two other rats had a shiftin polarity for over 80 s. Other studies have reported 15–20 s of EEG activity post-decapitation inanaesthetised rats, which was blocked with pre-treatment with atropine [42], while a study on awakerats reported a latency of 17 s to an isoelectric EEG [43]. However, whether these results indicateconsciousness following decapitation has been debated almost ever since the Mikeska and Klemmstudy [41]. It should be remembered that EEG can be recorded from animals under surgical anaesthesiaAnimals 2016, 6, 50 12 of 28and therefore the presence of an active EEG waveform alone is insufficient evidence to conclude thatconsciousness persists after decapitation [44]. However, the absence of EEG activity or the presenceof only a highly suppressed EEG (high amplitude, low frequency activity) does appear to stronglysuggest that consciousness has been lost and is commonly accepted to confirm adequate stunningduring slaughter of livestock species [45].On the basis of these studies on decapitated rats, the AVMA [3] urged caution when proposingto kill mice or rats by decapitation or cervical dislocation. The AVMA’s recommendations were forspecial ethics committee or IACUC (Institutional Animal Care and Use Committee) approval and forsedation or “light anaesthesia” before applying the technique.Cognizant of the uncertainties surrounding older studies of brain neurophysiology in rodentsundergoing physical methods of euthanasia, scientists have applied more modern methods. Cartneret al. examined EEG and visual evoked potentials (VEP) as evidence of brain electrophysiologyduring decapitation and cervical dislocation of mice anaesthetised with halothane and paralysed withsuccinylcholine [46]. With cervical dislocation, there was a significant decrease in VEP after 5–10 s andin EEG after 10–15 s. Durations were longer with decapitation, with a significant decrease in VEP after10–15 s and EEG after 15–20 s. The times elapsing before responses “flat line” are not given. While thiswork suggests a rapid progression to unconsciousness, Kongara et al. [44] found that EEG responsesobserved during the first 10 s following decapitation of anaesthetised rats are consistent with EEGresponses to nociception. Taken together, these newer studies suggest that decapitation may lead quiterapidly to unconsciousness, but with conscious pain perception prior to loss of sensation.With respect to cervical dislocation in rodents, the most recent AVMA guidelines [47] make thepoint that data suggest electrical activity in the brain persists for 13 s following cervical dislocationin rats, and unlike decapitation, there is no rapid exsanguination to speed the loss of consciousness.The same guidelines also acknowledge that electrical activity in the brain has been demonstrated topersist for 13 to 14 s following decapitation [41], but cite other studies which suggest that such activitydoes not imply that pain is perceived and which conclude that loss of consciousness is “rapid” postdecapitation (e.g., 2.7 s: [48]). VEPs in mice were reduced faster following cervical dislocation thandecapitation [46]. Since publication of the AVMA guidelines, a recent report by Kongara et al. [44]found that EEG responses observed during the first 10 s following decapitation of anaesthetised ratsare consistent with EEG responses to nociception.As Carbone argued, the AVMA recommendations assume that cervical dislocation is performedcompletely efficiently every time, which may be unlikely. There is the potential for non-fatal thoracicand lumbar lesions, so that animals continue to breathe, which indicates that the technique has notbeen effectively applied and there is a risk that animals may still be conscious. For example, ina study by Carbone et al. [49], mice were anaesthetised with isoflurane and randomised either toone of three cervical dislocation methods: (i) manual, anterograde (in which the operator stabilisesthe tail with one hand and pushes down and forward on the high cervical region with a twistingmotion); (ii) haemostat-assisted; or (iii) intentional thoracic dislocation (a potential accidental outcomeof cervical dislocation). Latency to respiratory arrest was measured and post-mortem radiographs andCT scans were used to ascertain where dislocations had occurred. Of the three cervical dislocationtechniques, anterograde was the most successful. Even then, however, almost 10% of the mice (2/22)continued to breathe for over 180 s. In pilot studies, some (anaesthetised) mice continued to breathefor over 15 min before being humanely killed. This is a concern because physical damage to the upperspinal cord and brainstem, as would be caused by successful cervical dislocation, should cause rapidrespiratory arrest. Whilst ongoing respiration does not indicate consciousness, cessation of breathinginduced by damage to the spinal cord and brainstem should occur during cervical dislocation, withconcomitant respiratory arrest. The failure to cause respiratory arrest is a sign that the animal hasnot been rendered brain dead and that there is a possibility of ongoing conscious perception in aninjured animal.Animals 2016, 6, 50 13 of 28This study also indicates that different methods of cervical dislocation should be explored to seewhether any are more effective than others. However, the criteria for evaluation should be chosenwith care; imaging is not the best predictor of a successful procedure as it is labour-intensive, lesionscan be hard to see and results can be difficult to interpret. Anaesthetised mice could be useful fortraining those required to carry out cervical dislocation (To reduce animal use, animals due to behumanely killed for other reasons should be used wherever possible), using time to respiratory arrestas a measure of success.It is difficult to apply the data on cervical dislocation to policy on humane killing at present.The few studies conducted to date indicate that brain waves of uncertain significance persist for up toaround 15 s and that operator error is a significant risk. In addition, it is as yet unknown how cervicaldislocation actually kills animals. The trauma high on the spinal cord leads to respiratory arrest, butthere is no significant brain injury. Due to these uncertainties, it is not currently possible to say, from awelfare aspect, how cervical dislocation compares to CO2, other inhalants or injectable agents.The following recommendations can be made, however: more research is needed on the types ofoperator error that can occur and their consequences; protocols for audits of operator success ratesshould be developed; equipment should be developed to assist with physical methods of killing(or currently available equipment should be improved as appropriate); and staff should be adequatelytrained and competent to minimise operator failure.2.3. Euthanasia of Neonatal RodentsCraig Johnson discussed the killing of neonatal animals with respect to their ability to suffer atvarious developmental stages. Most laws and guidelines that regulate animal experiments do notapply to all “animals”, but rather to those that are assumed to be capable of suffering. In this context,an animal is an organism that has the capacity to suffer because it is of a sufficiently “complex” speciesand at a relevant developmental stage. Therefore, a noxious stimulus may have a negative impact onthis animal’s welfare.There are different kinds of suffering, but this section of the report focuses on physical pain.In animals older than neonates, this has been commonly measured or inferred using EEG; forexample, dehorning in cattle produces marked effects [50]. For a review of neurophysiologicaltechniques that have been used to assess pain in animals, see Murrell and Johnson [51]. The importantquestion is whether and how age may affect the perception of pain, which depends upon the species,its reproductive strategy and developmental characteristics. There is a range of cerebral responsesto pain from neonates of very altricial species (e.g., Tammar wallaby [52]), through altricial species(e.g., rats), to precocial species (e.g., sheep [53,54]).Rats are born moderately neurologically immature, with ear canals and eyes opening between12 and 14 days postpartum. The EEG is isoelectric for the first 5–7 days, sleeping EEG differentiatesbetween REM and non-REM at around 12–18 days, their interest in objects increases by 14 days, andthey are play-fighting by 17–20 days. EEG responses to tail clamping in anaesthetised rat pups aremoderate at 12–14 days and strong from 21 to 22 days after birth [55]. These results, and the literature,suggest that conscious perception of pain may not be present before 10–12 days old, pups between 12and 18 days might be capable of some kind of conscious perception, and rats older than 18 days arecapable of conscious perception.The view of Johnson and his colleagues is therefore that the ability of neonatal rodents to sufferwhilst undergoing euthanasia is significantly diminished in comparison to later developmental stages.However, the legislation regulating animal use in the European Union [2] assumes that fetal mammalsare capable of experiencing suffering during the last third of their development, regulating their useat this stage in the same way as the use of mammals following birth. The UK Animals (ScientificProcedures) Act 1986 also gives developmental stages the “benefit of the doubt”, setting out permittedmethods of humane killing for fetal, larval and embryonic forms, including mammals, reptiles andAnimals 2016, 6, 50 14 of 28birds [56]. More research is needed into sentience in developmental stages, to better inform decisionsabout good practice for anaesthesia, analgesia and humane killing.2.4. Euthanasia of ZebrafishLarge numbers of zebrafish (Danio rerio) in laboratories worldwide are routinely anaesthetised forscientific and husbandry procedures, and as part of the euthanasia process. Two speakers, Dan Wearyand Gareth Readman, presented studies comparing responses of zebrafish to anaesthetic agents. Thereare very few specific anaesthesia protocols for species of fish (which number over 32,000) and thosethat do exist are often “historic” rather than properly evaluated for welfare impact and efficacy. Thecurrent “standard” anaesthetic used for fish is tricaine methanesulfonate or MS-222 (also known astricaine mesylate or TMS) ([57]; Figure 2).Readman described a chemotaxic test chamber developed to evaluate aversion of zebrafish tocommonly used fish anaesthetics. The chamber was virtually divided into two lanes separated by thecreation of a laminar flow within the system, and anaesthetics were introduced via one of two inlets.Agents were then introduced into one half of the tank only, and the fish could choose which half tooccupy [58]. This apparatus was used to test the aversive nature of a number of different anaestheticagents, with the assumption that fish avoid agents they found to be unpleasant.This study found that zebrafish showed aversion to propoxate, lidocaine hydrochloride,hydrochloric acid (HCl) (positive control), 2-phenoxyethanol (2-PE), MS-222 (buffered to pH 5.0using sodium bicarbonate), benzocaine, isoeugenol and quinaldine sulphate. The zebrafish showedlittle or no aversion to etomidate or tribromoethanol (TBE), but TBE can be difficult to store correctlyand readily decomposes, forming bromide which is unsafe for staff. The authors concluded thatetomidate is an appropriate and practical refinement for euthanasia of adult zebrafish. Incidentally, acost comparison of the various agents showed that etomidate was relatively inexpensive and cheaperthan MS-222.Weary presented results from a study on conditioned place avoidance responses in zebrafish tocommon anaesthetics. After only a single exposure to buffered MS-222, all fish showed conditionedplace avoidance to MS-222 but not to metomidate or clove oil [59]. These authors concluded thatetomidate/metomidate are refinements for euthanasia of adult zebrafish. However, differencesbetween the studies were seen between findings for clove oil and isoeugenol (the active ingredientin clove oil). Wong et al. [59] found no avoidance of clove oil, but Readman [58] found aversion toisoeugenol. These results could suggest a potential system difference caused by the physical/chemicalproperties of the holding water, or a strain difference within the populations used. Thus further researchis required on clove oil and components thereof (NB. Clove oil and its components are classified asrodent carcinogens by the US National Toxicology Program and, because of safety concerns, cannot beused as an anaesthetic in fish in the United States according to the US Food and Drug Administration),and it should also be noted that aversion and safety margins may well vary for other fish species.Following the presentations and discussion relating to zebrafish, 74% of those participants whovoted agreed that they would consider changing the methods they used to kill fish (shown in Figure 2)(17% voted “maybe” and 9% voted “no”). The consensus was that research on fish euthanasia shouldfocus on two main areas; more evaluations of the humaneness of some agents currently in use, andidentification of new agents and/or methods, for commonly used species (e.g., methods which areacceptable for tropical species of fish may be problematic for cold water species). A very small numberof voters felt that there was no need for further research, and that it had been easier to draw conclusionsfrom the outcomes of research into humane killing of fish than research into euthanising rodents.2.5. Standards of Evidence for Making Decisions about Euthanasia TechniquesMost people require evidence that a new euthanasia technique is “at least as humane” as currentlyused methods before they will adopt it. This is also a requirement of Directive 2010/63/EU [2].In addition, many manufacturers of devices for killing animals supply “evidence” that their productsAnimals 2016, 6, 50 15 of 28are humane. The meeting discussed what kinds of evidence are admissible and how decisions shouldbe made.Penny Hawkins discussed the kinds of factors that could be taken into account when consideringhumane killing practice at a local level, for example by a researcher wishing to ensure the mostappropriate technique is used within her project, or by an ethics or animal care and use committeeundertaking a routine review of standard practice at the establishment. Whatever the scenario,a spectrum of “standards” of evidence can be envisaged, as follows, from weakest to strongest:anecdotal evidence/opinion; unpublished data; a single publication; multiple published papers (withcompatible conclusions); expert review articles. Usually, multiple confirmatory publications or expertreviews are the minimum that should be used to justify a change in practice. However, sometimesinformed judgements can be made if studies appear to provide definitive evidence that could beevaluated within facilities; for example, Readman et al. [58] tested etomidate while Wong et al. [59]tested the analogue metomidate for zebrafish. Given the consistency in the results, despite thedifferences in agents, testing methods, etc., it seems reasonable to conclude that both agents could berefinements over the existing MS-222 for this species.Evidence for the humaneness of a euthanasia technique should include factual information todemonstrate that known stressors can be avoided (e.g., capture, handling, restraint, transport andrelocation). As success rate is an important factor, data on rates of recovery from the application ofthe technique should be obtained (e.g., for overdoses), or “death on first attempt”, which is especiallyimportant for physical methods from which animals may recover with injuries. Table 1 lists othertypes of evidence that may be required depending on the protocol.Table 1. Potential adverse effects associated with euthanasia and evidence that can be used toassess these.Adverse Effectfor the AnimalPotential Sources of Suffering orFactors to Consider EvidencePainInjectionPhysicochemical properties of agent(e.g., unbuffered PBS, H2CO3 onmucous membranes)Muscle spasms/seizuresPain from decapitationBehavioural; physical reactions, vocalisation, attention tosite(s) of painActive EEGData on duration, incidence, severity of spasms/seizures,observations or electromyogram (EMG)Aversion toinhaled agentsMolecular structure of agentConcentrationFlow rateWhether other agents used to induceanaesthesia or as additivesHighly species and strain specificBehavioural; physical reactions (e.g., escape),conditioned place preference/avoidanceInformation on properties of agent, e.g., pHNOT time to recumbency or to cease movementSufferingbetweenadministrationand deathDyspnoeaPain from injury with physical methodsAnxiety, fearInability to escape from aversive agentOther unpleasant effects of inhaled agentsBehavioural (physical reactions, vocalisation, defecation)Active EEGRespiration rate and depthCorticosteroneNOT time to recumbency or to cease movementWhen deciding between different killing techniques, all of which are compatible with scientificobjectives, humaneness (i.e., the animal’s experience) should be the primary consideration. However,if all other factors are equal when choosing between techniques, then it can be justifiable to selecta more rapid method (provided that the increased speed does not increase the intensity of pain ordistress). “Aesthetics” can be another secondary consideration; some personnel find euthanizinganimals psychologically difficult [60] and there is evidence that physical techniques are not preferredby operators [61]. It is important to allow staff to have input into the decision making process withrespect to euthanasia techniques for specific projects, but the aim should always be to choose the mosthumane method.Ngaire Dennison led a discussion concerning the standards of evidence required for regulatorychanges to allow new methods for killing certain species. Regulators, such as the UK Home Office,Animals 2016, 6, 50 16 of 28can be faced with some difficult decisions when interpreting and acting upon scientific evidence,especially if results are conflicting or they are expected to extrapolate between different species, strainsor developmental stages. The minimum information required to allow a proper consideration of amethod is a description of the method; time to final unconsciousness; time to death (both preferablybacked up with physiological data); details of ages, sexes and weights for which the method is suitable;how the technique kills; behaviours observed when the method is applied; post mortem evidenceof speed/humaneness of death; and how the method compares with other currently permissibleprotocols. Ideally, this would be published in a refereed journal and reported in accordance with theARRIVE guidelines ([62], www.nc3rs.org.uk/ARRIVE). Decisions about whether to permit alternativeeuthanasia techniques are made on the basis of the data provided, whether there is any controversyassociated with the method (in which case further information may be needed), and whether there arelikely to be any benefits to the animal (e.g., good evidence of lack of distress).Information on the suitability of the technique with respect to the science is essential forresearchers but irrelevant to the consideration of animal welfare. If there is a genuinely justifiablescientific reason for using a killing technique that is not listed as acceptable for general use underthe relevant legislation or guidelines, most regulatory systems will allow this provided that a robustscientific case is made, suffering is minimised and the procedure as a whole has successfully undergonea harm-benefit assessment.Participants were presented with a list of ten types of “scientific evidence” that might beconsidered acceptable, with the option of voting for as many as they wished. The results are shown inTable 2.Table 2. Types of “scientific evidence” that meeting participants considered acceptable for makingdecisions about the humaneness of a method for killing.Type of Evidence Number of VotersBehavioural indicators of stress, e.g., aversion 47Behavioural indicators of pain, e.g., attention to eyes or injection sites 47Positive indicators that suffering is minimal or absent 41EEG data 40Success rate 40Known properties of agents, e.g., pH, mechanism of action 35Experience of animal before euthanasia process 34Time to cease moving 28Time to death 26EMG and other activity data 19The audience discussed the value of using a combination of both physiological and behaviouralevidence when evaluating killing techniques. Behavioural indicators that were regarded as especiallyimportant were aversion to an agent “in the moment”; signs of stress or agitation (e.g., escapebehaviours); learned or conditioned aversion; and other conditioned responses such as “freezing”.Ultrasonic vocalisations were viewed as less reliable, due to the current lack of ability reliably tointerpret these, and the fact that only a limited number of species appear to vocalise as a response topain or distress.However, behaviours in the form of movement-related data, such as time to cease moving orelectromyogram (EMG), rank low in Table 2 because of general awareness that a recumbent animal maystill be aware and able to experience suffering. This demonstrates the necessity of using physiologicaldata to either help interpret behaviour, or to use as indicators of awareness and/or suffering if theanimal is no longer able to express behaviours. Participants voted for EEG data, levels of stresshormones, heart rate, blood pressure and activation of brain areas associated with negative affectivestates or stress (e.g., the HPA axis) as acceptable evidence to be taken into account when makingdecisions about killing techniques. It was noted that, although the absence of visual evoked potentialsAnimals 2016, 6, 50 17 of 28and cortical function indicate that an animal is unconscious, their presence is not necessarily a sign ofconsciousness, so care is needed when interpreting these data.Some discussion was also given to the quality of the science, and its interpretation, in studiesaiming to evaluate the humaneness of different techniques and protocols. In particular, small samplesizes and some research or reporting methods may miss or mask genuine concerns. For example,in the study of six rats killed by decapitation cited earlier [41], the mean duration of “active” EEGfollowing decapitation was 13.6 s, but the range was 5.6–29.5 s; a five-fold difference. Persistent EEGwas reported as a mean of 27.2 s, with a range of 19–465.5 s. There is no such thing as a “mean animal”,and there are clear and serious welfare implications if some animals were experiencing pain for thelonger periods. Not only are these outliers important, but a properly powered study could haverevealed further insights into the physiology of decapitation and its associated welfare (and ethical)problems. As it was, this study apparently formed the basis for policy, as discussed earlier. It is alsoworth noting that the mean active EEG has been described as “13–14 s” or “up to half a minute”depending on the viewpoint of the particular author in question.To conclude, when applying policy or using guidance notes, it is good practice to review theassociated references with respect to the experimental design, how long ago the studies were conductedand whether they are still relevant and not superseded by new knowledge, and whether they havebeen interpreted appropriately and correctly. There is a case for significant care in the analysis andpresentation of studies where the outcome is being interpreted with respect to animal welfare; theremay be a need for a slight difference from other disciplines as the outlying data represent animals’experiences that are important, so that a mean or median value is not sufficient to allow considerationof a method’s humaneness.Participants were presented with a range of suggestions for appropriate mouse and rat euthanasiaresearch directions, which they chose on the basis of the presentations and discussions at the meeting.The majority of votes were for: refinement of existing methods (e.g., home cage euthanasia with inhaledagents); identifying new techniques; gathering evidence as to whether alternatives to CO2 (such asisoflurane) might be more humane; and more evidence to inform the debate on the humaneness—orotherwise—of CO2.Commercial DevicesGolledge led a discussion on the validation of commercial devices used for killing laboratoryanimals. “Euthanasia” devices are mainly apparatus for administering inhaled agents or devices forphysically killing animals, with a recent upsurge in systems that can administer the agent in the homecage, eliminating stress associated with handling or transport. Many manufacturers have attemptedto incorporate recent research findings and recommendations, for example, with respect to fill ratesand agents.Some advantages of various systems, as cited by manufacturers, may relate to animal welfare;for example, home cage administration, automation (reducing the potential for operator error) andreliability. They may also primarily address human safety or economic interests; for example, savinglabour (multiple cages or entire racks) and technical support. However, while automation may saveresources and obviate some human error, this often comes with reduced human attention towards theanimals, which could reduce opportunities to detect and act upon avoidable suffering. From the aspectof a consumer wishing to prioritise animal welfare, these kinds of questions and issues will need to beaddressed and an informed judgement made in each case. Buying a particular system could meana significant outlay and therefore risk being locked into a particular technique, which may becomesuperseded from a welfare aspect.It will obviously not be possible to tell which device is “most humane” from a list of equivalentson the basis of manufacturers’ literature, nor is it always clear whether claims can be substantiatedwith robust data. Manufacturers’ literature often uses terms associated with good animal welfare suchas “humane” and “kind” with little direct evidence to substantiate these claims. Devices can be tested,Animals 2016, 6, 50 18 of 28but most evaluations of administration techniques are easiest to do using custom built experimentalequipment that permits relevant data to be gathered. Manufacturers generally lack the access toanimals to directly test their own apparatus, instead relying on end-users to test the apparatus in use.2.6. Input from, and Dialogue with, Ethics or Animal Care and Use CommitteesSome legislation and codes of practice suggest, or require, that ethics or animal care and usecommittees review humane killing techniques that are in use locally. The benefits of input frompersonnel such as animal technologists and veterinarians may also be emphasised; for example,the U.S. National Research Council Guide states that standardised methods of euthanasia, which arepredictable and controllable, should be developed and approved by both the attending veterinarian andIACUC [60]. The institutional Animal Welfare Body required under European Directive 2010/63/EUis also expected to include members with expertise in humane killing, and reviewing euthanasia fallswithin the Body’s main tasks [63].The audience was asked whether they had had discussions with ethics or animal care and usecommittees about killing techniques. Many reported that committees had required justification for aparticular technique, had drawn attention to a euthanasia refinement, or had wanted to discuss andreview general euthanasia practice at the facility (not in relation to any specific project). Five peoplereported that a committee had refused to permit a proposed killing technique.Thirty-four voters responded to a question on how interactions with ethics or animal care anduse committees could be improved regarding euthanasia. Of these, 80% agreed with a statement thatcommittee members needed training in understanding the animal welfare and scientific issues; 15%had never had any problems and saw no need for committees to improve; and the remainder felt thatsuch committees should not be commenting on euthanasia techniques.3. Main Points and Areas for Further ResearchThe list below summarises the topics discussed at the meeting, with the aim of helping toidentify areas that require special consideration when making informed decisions on good practice forhumanely killing mice, rats and zebrafish.3.1. Inhaled Agents in Mice and Rats• The majority of participants considered that all inhaled agents tested to date, including CO2 andinhaled anaesthetics, are aversive to some degree; animals will avoid exposure when providedwith the opportunity.• Approach-avoidance and aversion-avoidance tests have shown that, in general, isoflurane andsevoflurane are equally aversive to mice and rats, halothane is more aversive than either, and thatCO2 is inherently more aversive than any of the three other anaesthetics (see Section 2.1.1).• These tests have also shown that during first exposure to inhaled anaesthetics, more than 50%of rats and mice will choose exposure until they become recumbent rather than abandoning apreferred food reward or crossing over to a brightly lit chamber.• Results from these tests have shown evidence of learned aversion to inhaled anaesthetics; theseagents become more aversive after an initial exposure.• Conditioned place aversion tests, which rely on an animal’s memory of exposure, confirmthat repeat exposure to CO2, isoflurane and argon cause a negative affective response inrats—demonstrated as a learned aversion to the place where the substances were administered.• Anoxia (as induced by argon or other gases used to displace air) is highly aversive to rats.3.2. Carbon Dioxide in Mice and Rats• Mice have specialised olfactory neurons that respond to CO2 and, unlike humans, can smell CO2at near-atmospheric concentrations, actively avoiding levels of just 0.2%.Animals 2016, 6, 50 19 of 28• Exposure to a rising concentration of CO2 (e.g., 20% chamber volume per minute) is unlikely tobe painful for mice and rats because they lose consciousness before the chamber concentrationreaches levels associated with nociceptor activation.• However, animals exposed to gradual fill as above are still conscious when CO2 reaches levelsassociated with dyspnoea, and anxiety and fear-related behaviours.• Mice and rats always leave test chambers when the concentration of CO2 rises to 12%–18%, evenif the cost is to abandon a preferred food reward when they are food-deprived or if the cost is toenter a brightly lit chamber, which is known to be highly aversive.• Humans breathing normoxic but hypercarbic gas report feelings of fear, anxiety and irritability.In rats, breathing a gas mix of the same composition leads to increased activity in areas of thebrain involved in the initiation of fear and anxiety-associated behavioural responses.• In an evaluation of exposure to CO2, plasma levels of the stress hormones adrenaline andnoradrenaline (obtained immediately after loss of consciousness) were elevated in mice and rats.Levels were not elevated by the inhaled anaesthetic agents isoflurane or sevoflurane. CO2 canelevate catecholamine levels independent of conscious state; however, the agreement of this studywith behavioural studies showing stronger aversion to CO2 than other tested agents suggests thatthe observed elevations may be related to consciously-experienced negative emotional states.• Exposure to CO2 is thus likely to cause dyspnoea, anxiety and fear in mice, rats and possibly otherrodents. These effects will be exacerbated if the killing process also involves capture, handling,transport to an unfamiliar environment, or mixing with unfamiliar individuals.• At present, there do not appear to be welfare benefits associated with sedating mice with injectableagents before killing with CO2. A study aimed at identifying suitable injectable anxiolytics, suchas midazolam, to pre-medicate mice before CO2 killing, found that the stress of capture, handlingand administering the sedative negated the benefits.• If mice and rats are anaesthetised using an inhaled agent before switching to CO2 to completethe killing process, the change in agent should not be made until they have been recumbent for asufficiently long period to prevent rapid recovery of consciousness (for mice, evidence indicateswaiting a minimum of 80 s after animals become recumbent—see Section 2.1.1).• This evidence indicates that CO2 and agents used to induce anoxia cannot be considered humanemethods of euthanasia for rodents, so developing replacements for these agents is an essential goal.3.3. Inhaled Anaesthetic Agents in Mice and Rats• The majority of evidence shows that the use of halogenated anaesthetics to induceunconsciousness in naïve animals is a refinement over the use of CO2 or any agent used toinduce anoxia.• When administered using a precision vaporiser, volatile anaesthetics like sevoflurane andisoflurane rapidly induce unconsciousness but may take a long time to cause death in rodents.This makes these agents suitable for inducing unconsciousness (therefore minimising distressduring the period within which animals are able to experience poor welfare), but other methods,e.g., cervical dislocation or CO2 exposure, can be used to more rapidly kill animals once they havebeen anaesthetised.• Rats and mice acquire learned aversion to volatile anaesthetic agents such as isoflurane. Duringrepeat exposure to an inhaled anaesthetic, about half as many animals are willing to tolerateexposure until unconsciousness. This result indicates that any study requiring multiple exposuresto anaesthesia (e.g., for surgery or imaging, and later for euthanasia) should be consideredhigh risk.• Rats “generalise” between different inhaled anaesthetics, so using a different agent for killing willnot solve the problem of learned aversion.Animals 2016, 6, 50 20 of 28• There is a clear need to better understand aversion to inhaled anaesthetics, and in particular theeffects of repeated anaesthesia.3.4. Physical Methods for Rodents—Decapitation and Cervical Dislocation• When performed competently, physical methods may offer a rapid death, which many argue ishumane. However, there is on-going debate about the humaneness of some physical methods,including decapitation and cervical dislocation.• There is concern that a decapitated brain might be conscious and in pain. Bothelectroencephalogram (EEG) activity and visually evoked potentials (VEP) persist for someseconds in mice and rats, but the significance of this with respect to consciousness and sufferingis not yet known. Brain activity also persists following cervical dislocation.• “Cervical dislocation” can result in lower spinal dislocation and fracture without immediaterespiratory arrest, such that animals continue to breathe and may still be conscious.• In a study of operator success rate for cervical dislocation, the “anterograde” technique(see Section 2.2) proved most effective—but almost 10% of the (anaesthetised) mice continued tobreathe for over 3 min, suggesting the technique was not effective.• Good training, assessment of competence and benchmarking success rates in the use of physicaltechniques are all paramount with respect to minimising suffering.3.5. Neonates• The capacity for a neonate to suffer pain depends upon the species and the stage of neurologicaldevelopment at which individuals are born.• There is evidence that rodents lack the electrophysiological signs believed to relate to consciousbrain activity during the first few postnatal days, with consciousness appearing to emerge severaldays after birth. This suggests that neonatal rodents may lack the ability to suffer pain or distressduring this period. However, research is on-going and there is still a case for giving developmentalstages the “benefit of the doubt” and treating them as though they can suffer.3.6. Zebrafish• Avoidance responses and conditioned place-preference tests have been used to evaluate differentagents used to kill zebrafish.• Zebrafish show avoidance of propoxate, lidocaine, hydrochloric acid (HCl), 2-phenoxyethanol(2-PE), buffered MS-222 (also known as tricaine mesylate, tricane methanesulfonate, or TMS),benzocaine, isoeugenol and quinaldine sulphate. They show less evidence of avoidance ofetomidate and tribromoethanol (TBE), but TBE is difficult to store and can decompose, formingbromide which is hazardous to humans.• After just a single exposure to buffered MS-222, zebrafish will consistently avoid the chamberwhere they had encountered this agent, indicating that this experience was unpleasant for the fishand they remember this experience. Zebrafish show much less evidence of conditioned avoidanceof either metomidate or clove oil.• Together these results indicate that etomidate and metomidate could be humane alternativesfor euthanizing zebrafish. Clove oil shows promise in conditioned place aversion testing butavoidance testing shows evidence of aversion to one component (isoeugenol), suggesting thatthis agent requires more study. The most commonly used agent, MS-222, cannot be considered ahumane method for killing zebrafish.Animals 2016, 6, 50 21 of 283.7. Standards of Evidence for Making Decisions about Techniques for Humane Killing• Sound scientific evidence is essential when deciding on the most appropriate technique for aparticular species and stage of development. Usually this should be in the form of confirmatorypublications from multiple laboratories; subjective judgements are not sufficient.• It is preferable to use a combination of behavioural indicators and physiological data whenassessing techniques; for example, a recumbent animal may have an active (high frequency, lowamplitude) EEG indicating the possibility of consciousness, or an animal in a state of spasmmay have an isoelectric EEG. Supplementary information such as success rates, physicochemicalproperties of agents or the potential to minimise stressful elements of the process may alsobe relevant.• It is good practice to review the references associated with guidelines on humane killing to explore(and question if necessary) the basis for these; as with other areas of science, some guidelines maybe based on a small number of studies using few animals or fail to acknowledge the potentialimplications for animal welfare of data outliers.• Generalisation between species and strains should be avoided.• Manufacturers of “euthanasia” devices often attempt to incorporate recent research findings andrecommendations, but it is difficult to “test the humaneness” of one against another.3.8. Areas for Further Research• On-going research into mouse and rat euthanasia should focus on: identifying new techniques;refining current methods (e.g., administration of inhaled agents in the home cage); understandingthe cause of learned aversion to inhaled anaesthetics; and assessing the level of suffering causedby different euthanasia agents.• More studies are required to elucidate the magnitude of aversion in animals exposed to isofluranemore than twice, and the effect on the magnitude of aversion of longer or short intervalsbetween anaesthesia.• Research should be undertaken to evaluate when consciousness is lost after the various methods,including physical methods. If some level of consciousness persists even after recumbency (forexample), there is a potential for severe suffering.• More research is needed on types and rates of operator error when using various methods ofkilling such as cervical dislocation and decapitation, with priority given to the consequences forthe animal. Protocols for audits of operator success rate should also be developed.• The possibility that neonatal rodents have some form of consciousness needs to befurther investigated.• Research into fish euthanasia should focus on: (i) evaluations of the humaneness of some agentscurrently in use and (ii) identifying new agents and/or methods for commonly used species.4. ConclusionsMuch progress has been made since the first Newcastle meeting on euthanasia, held in 2006,towards understanding the animal welfare implications of the various killing methods. Some methodsare demonstrably less humane than others and should not be used (e.g., all available evidence showsthat inducing anoxia using inert gases is more aversive than other inhaled agents for rodents). Somemethods are likely only humane when performed correctly (e.g., cervical dislocation) and may causesignificant pain and distress if poorly performed. The overwhelming majority of evidence showsthat CO2 has a negative impact on rodent welfare. Evidence also shows that the volatile anaestheticsprovide a potential refinement over both CO2 and anoxia for killing rodents. However, some animalsshow some aversion even to the volatile anaesthetics, suggesting that the search for the ideal inhaledagent(s) should continue. Work on zebrafish has only just begun, but the two studies to date showclear and consistent evidence that etomidate/metomidate is a refinement over the use of MS-222.Animals 2016, 6, 50 22 of 28On the basis of the meeting and this report, some recommendations for actions at the local,establishment level are:• Regularly review practice for humane killing, both at the establishment (e.g., via the ethics oranimal care and use committee) and, as a researcher, within your own projects.• Be aware that killing is a process, not an event. It is important to consider the whole experience ofthe animal, from capture to permanent loss of consciousness.• When considering humane killing practice, critically review the basis for current guidelines,including the source literature.• Ensure good communications between researchers and ethics or animal care and use committees,to help ensure that scientific justifications for particular methods are understood by all, and thatcauses of suffering are recognised and minimised.• Critically consider device manufacturers’ claims regarding the humaneness of their products,reviewing the literature and seeking expert advice.• Consider using isoflurane or sevoflurane as a refinement when killing naïve rodents by inducinganaesthesia prior to introducing CO2.• If anaesthetising rodents with isoflurane or sevoflurane before changing to CO2, allow sufficienttime post-recumbency before changing the agent.• Do not use injectable anxiolytics before CO2 exposure, as this cannot be considered a refinement.• If it is not possible to change from CO2 only in the short term, make sure that the administrationprotocol reflects current thinking regarding good practice for flow rates, use of diffusers, etc.(see also [1]). Consider administering CO2 in the home cage, which will at least eliminate thestress of being moved.• Never use anoxia (e.g., as induced by argon) to kill rodents.• Be aware of concerns about physical methods, including both animal and staff welfare. If physicalmethods are used, ensure that processes are in place to ensure that staff members are trained,competent and willing to use these physical techniques, and that benchmarks for success are setand monitored.• Do not use decapitation to kill rodents routinely, as this may cause avoidable suffering.• Follow the literature on sentience in neonatal rodents, giving them the benefit of the doubt withrespect to the ability to suffer.• Use etomidate or metomidate instead of MS-222 to kill zebrafish. Clove oil may also be a suitablerefinement to the use of MS-222 but requires more research.• Keep abreast of the published literature on humane killing techniques.Supplementary Materials: The following are available online at www.mdpi.com/2076-2615/6/9/50/s1,Table S1: Meeting Agenda—Second Newcastle Meeting on Laboratory Animal Euthanasia (Newcastle University,United Kingdom, 9 August 2013).Acknowledgments: The meeting was funded by the NC3Rs as part of a Strategic Award to Huw Golledge,Newcastle University (G1000878/1).Author Contributions: Wrote or contributed to the writing of the manuscript: Penny Hawkins, Mark J. Prescott,Huw D.R. Golledge, Daniel M. Weary, I. Joanna Makowska, Gareth Readman, Nicole Marquardt, Ngaire Dennison,Larry Carbone and Craig Johnson.Conflicts of Interest: The authors declare no conflict of interest.GlossaryAcidosis an excessively acid condition of the body fluids or tissues.Affective statesemotions and other feelings in animals that are experienced as pleasantor unpleasant.Animals 2016, 6, 50 23 of 28Amygdalaa structure in the limbic system of the brain that is linked to emotionsand aggression.Anoxia absence of oxygen.Anxiogenic anxiety causing.Anxiolytic anxiety inhibiting.Approach-avoidancetesta test that uses a desirable reward (e.g., a food treat) to evaluate whetheran inhaled agent is aversive and, if so, to what extent. Animals choosewhether or not to access the reward in the presence of a range ofconcentrations of the agent, which provides a way of testing theirmotivation for the treat against their motivation to avoid the agent. Forexample, rats and mice always choose to avoid exposure to argon or CO2even when they are food deprived and a preferred treat is present,indicating that these gases are highly aversive.Aversion avoidance of a stimulus, situation or behaviour.Aversive causing avoidance of a stimulus, situation or behaviour.Aversion-avoidancetesta test that uses an animal’s motivation to avoid an unpleasant stimulus toevaluate its level of aversion to an inhaled agent. One commonly usedparadigm is for rodents to choose between a preferred, dark chamber thatis filling with a gas and an aversively brightly-lit chamber. Rats and micealways prefer the bright chamber over one filling with CO2, but somechoose the dark chamber filling with anaesthetic until theylose consciousness.Aversiveness see aversion, aversive.Chemotaxic the movement of an organism in response to a chemical stimulus.Conditionedplace aversiona commonly used technique to evaluate aversion to a specificenvironment that has been associated with a negative reward.Conditionedplace preferencea commonly used technique to evaluate preferences for stimuli that havebeen associated with a positive reward. In general, this procedureinvolves several trials where the animal is presented with the positivestimulus (e.g., food) paired with placement in a distinct environmentcontaining various cues (e.g., tactile, visual, and/or olfactory). Whenlater tested in the absence of the stimulus, approaches to and/or theamount of time spent in the compartments previously associated withthe positive stimulus serve as indicators of preference.Distressa strongly negative emotional experience, associated with stress of suchmagnitude or duration that significant changes in biological functionwould be necessary for the animal to survive if it could not easily escapethe stressor.Dyspnoea a subjective experience of breathing discomfort; “air hunger”.Electroencephalogram(EEG)a recording of the electrical activity of the brain, typically representingthe activity of the cerebral cortex in mammals.Animals 2016, 6, 50 24 of 28Electromyogram(EMG)a recording of the electrical activity produced by skeletal muscles.Eustress“good” or “beneficial” stress, occurring in response to positive events,but which may give rise to the same physiological indicators of stress asnegative events.Euthanasiafrequently used to describe the process of killing laboratory animals,euthanasia means a “good death”, which the AVMA [47] defines as adeath that occurs with “minimal pain and distress”. The term is alsooften applied to both humans and animals in the context of endingsuffering [64]. Given this, the generic use of the term “euthanasia” forlaboratory animals is questionable for two reasons. First, the humanenessof some methods used to kill laboratory animals remains in doubt, suchthat “killing” is often considered a better description of the processesused to end the lives of laboratory animals. Second, animals aresometimes killed for reasons other than ending their suffering (e.g., if anexperiment requires their tissues or if they are surplus to requirements).The term “humane killing” is therefore generally more appropriate thaneuthanasia in relation to laboratory animals.HPA axishypothalamic–pituitary–adrenal axis; the system of feedback interactionsamong the hypothalamus, pituitary gland, and adrenal glands thatcontrols reactions to stress and regulates many body processes.Humane endpointone or more predetermined physiological or behavioural signs thatdefine the point at which an experimental animal’s pain and/or distresswill be terminated, minimized or reduced by taking actions such askilling the animal, terminating a painful procedure or giving treatment torelieve pain.Humanenesscharacterised by concern or compassion for others; in the context ofmethods of humane killing, that which causes little or no pain or distress.Hypercarbic containing excess CO2.Hypoxia deficiency in the amount of oxygen reaching the tissues.Inhaled agenta substance (gas or vapour) which is administered via theinhalation route.Isoelectrichaving or involving no net electric charge or difference in electricalpotential; in EEG a signal representing no on-going brain activity.Monosynapticconnectionspassing through a single synapse (junction between two nerve cells).Neonatalrelating to the immediate period after birth. The age of neonatal rodentshas been defined as up to and including day 10, where the day of birth isday 1 [56].Nociceptor a sensory receptor (nerve) for painful stimuli.Normoxic containing the normal atmospheric concentration of oxygen (~21%).Animals 2016, 6, 50 25 of 28Olfactory bulba neural structure of the vertebrate forebrain involved in olfaction or thesense of smell.Pavlovian associationan association between a previously unrelated neutral stimulus (e.g., bell)and another stimulus (e.g., food) that reliably elicits a reaction(e.g., salivation).REM REM (rapid eye movement) sleep.Visually evokedpotential (VEP)electrophysiological responses to stimulation by either patterned orunpatterned visual stimuli.References1. Hawkins, P.; Playle, L.; Golledge, H.; Leach, M.; Banzett, R.; Coenen, A.; Cooper, J.; Danneman, P.; Flecknell, P.;Kirkden, R.; et al. Newcastle Consensus Meeting on Carbon Dioxide Euthanasia of Laboratory Animals,Newcastle upon Tyne, UK, 27–28 February 2006; pp. 1–17. 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Animal welfare
Carbon dioxide
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Mouse
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A Good Death? Report of the Second Newcastle Meeting on Laboratory Animal Euthanasia
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