refinement of the use of non-human primates in scientific … · 2012. 10. 17. · primates are...

239

copy 2006 Universities Federation for Animal WelfareThe Old School Brewhouse Hill WheathampsteadHertfordshire AL4 8AN UK

Animal Welfare 2006 15 239-261ISSN 0962-7286

Refinement of the use of non-human primates in scientific researchPart III refinement of procedures

AE Rennie and HM Buchanan-Smith

Department of Psychology University of Stirling Stirling FK9 4LA Scotland UK Contact for correspondence and requests for reprints hmbuchanan-smithstiracuk

Abstract

There is an ethical and scientific need to minimise the harm experienced by animals used in scientific procedures and to maximisetheir well-being Welfare can be improved by the refinement of practice particularly if these refinements are applied to every aspectof the life of an animal used in the laboratory from birth to death Primates are considered likely to have a greater capacity forsuffering than other sentient species and therefore refinement of their use is particularly important The refinement of the humanimpact on laboratory-housed primates and of housing and husbandry practices are dealt with in parts I and II of this three-part reviewIn part III methods of refinement that can be applied specifically to the use of primates in procedures are summarised and discussedtogether with a description of some current practices and the scientific evidence that suggests that they should no longer be usedMethods of refinement of identification capture and restraint sampling administration of substances humane endpoints andeuthanasia are included If these methods are used taking into account species-specific differences and needs it is concluded thatharm can be minimised and primate welfare improved

Keywords animal welfare humane end-points identification non-human primates refinement scientific procedures

IntroductionIn accordance with the harmonised definition of refinementproposed by Buchanan-Smith et al 2005 the concept shouldbe considered to encompass not only the need to minimisethe degree of inhumanity where inhumanity is defined asthe infliction of distress after Russell and Burch (1992)experienced by animals used in laboratories but also theneed to promote well-being This principle should beapplied to every aspect of the life of the animal in the labo-ratory from birth to death In this way ethical concernsabout the use of animals in science can be reduced and thevalidity of the animals as scientific models increased Theapplication of refinement techniques to minimise harm andmaximise well-being of animals during use in procedures istherefore critically important Information regarding theapplication of refinement techniques to primate use ispublished in a diverse selection of journals and therefore acomprehensive summary is important In order to widenawareness refinement techniques that can be applied to allaspects of laboratory use of primates and those that applyspecifically to the housing and husbandry of these animalshave been discussed in parts I and II of this three-partreview (Rennie amp Buchanan-Smith 2006 a b) In this finalpart of the review we concentrate on methods of refinementthat can be applied specifically to the use of primates inscientific procedures in order to both increase the ability ofprimates to cope with such procedures and to reduce theirneed to cope

Refinement of proceduresIn the European Directive an experiment is defined as ldquoanyuse of an animal for experimental or other purposes thatmay cause pain suffering distress or lasting harmhellipanexperiment starts when the animal is first prepared for useand ends when no further observations are to be made forthat experimenthelliprdquo (European Union [EU] 1986 Article2[d])It is also a requirement that any experiment carried outldquohellipshall be designed to avoid distress and unnecessary painand suffering to the experimental animalshelliprdquo(EU 1986Article 7 Paragraph 4)There are many methods by which the execution of experi-mental procedures can be refined Many of these can beused to refine routine procedures carried out on the majorityof experimental animals However the potential benefits ofthese refinements will only be fully realised if they areexecuted in the context of each specific experimentalmanipulation and most importantly adapted to suit thespecies and the individuals concerned Several importantrefinements are outlined in this report in relation to theprogression of an experimental procedure from preparationthrough to termination of the experiment and re-use

IdentificationIn Europe the identification of every individual primateheld in laboratories by the least painful available method is

Universities Federation for Animal Welfare Science in the Service of Animal Welfare

240 Rennie and Buchanan-Smith

a requirement of the European Directive (EU 1986) Manyresearchers and animal care workers are able to identifyindividuals by facial features and behavioural characteris-tics (Poole et al 1999) Identification of individuals by thismethod can be carried out with a minimum of interferenceHowever the International Primatological Society (IPS)guidelines (1993) recommend that a permanent identifica-tion mark should be used so that every individual can beidentified and matched to his or her medical and researchrecords This is necessary because members of staff atresearch premises may change the care of individuals maybe transferred between staff either on the same site or ondifferent sites and because welfare inspectors must be ableto accurately match individuals with records Marking ofanimals is not considered to be an experimental procedureprovided that it is carried out by the most humane availablemethod and therefore does not have to be regulated bydesignated authorities (EU 1986) Three permanentmethods of marking have been used in laboratory primateswith varying success

Freeze brandingFreeze branding produces scar tissue and therefore whitehair growth by the application of a small branding ironcooled to around -60ordmC using dry ice or a dry ice ethanolmixture to the skin (Sherwin et al 2002) The method hasbeen used in vervet monkeys (Cercopithecus aethiops)(Griffin 1988) In cattle the method results in lower cortisoland fewer behavioural responses than hot brandingalthough the response is still greater than that in controlanimals (Schwartzkopf-Genswein et al 1997a b 1998)However hyperalgesia was produced at the site of brandingfor 7 days after treatment In order to reduce pain and tissuesensitivity sedation and local anaesthetic should be usedduring the procedure and a programme of analgesia tocontrol pain following the procedures should be consideredIn other species freeze branding produces clearly recognis-able marking however in primates difficulty in seeing thewhite brand hair in long-haired species has meant that themethod is not commonly used (Fortman et al 2002) and isnot recommended

TattooingTattooing is widely recommended in laboratory husbandryguidelines (The Canadian Council on Animal Care [CCAC]1993 IPS 1993 Baskerville 1999 Bearder amp Pitts 1999Erkert 1999 Fritz et al 1999 Visalberghi amp Anderson 1999Mendoza 1999) It is generally recommended that the tattooshould be placed in a largely hairless area of skin butdifferent areas have been recommended for differentspecies For example the inside of the ear or lips is recom-mended for prosimian species (Bearder amp Pitts 1999)whilst the inner thigh is recommended for chimpanzees(Pan troglodytes) (Fritz et al 1999) and squirrel monkeys(Saimiri spp) (Mendoza 1999) and the inner thigh or thechest is recommended for Old World monkeys (Baskerville1999) One of the main disadvantages of the technique isthat the tattoo can be difficult to read at a distance althoughthis depends on its positioning and skin pigmentation

Tattoos can even be difficult to read when the animal hasbeen captured particularly as the animal gets older as theink can fade as the animal grows (Fortman et al 2002) Thetattoo may therefore have to be renewed periodically toensure that identities are not confused (Fritz et al 1999)However it is noted that in many toxicological studies theprimates are used and killed before the fading of the tattoobecomes as issue In order to ensure the accuracy of thetattoo mark and to minimise pain the pain and distress of theprocedure all primates should be anaesthetised fortattooing Subsequently the animal must be monitored toensure that any adverse effects of the identificationprocedure are detected early on However microchips are arefinement of this identification technique

Microchip identificationSince the early 1990s subcutaneously implantedmicrochips holding unique codes for each individual havebeen used to permanently identify primates Thesemicrochips are widely used in pet animals The unique indi-vidual code on the microchip must be read using a scannerFollowing sedation the microchip is inserted under the skinusing a specially designed hypodermic needle usuallysupplied ready loaded with a microchip Insertion cantherefore be achieved very quickly The microchip may beplaced under the interscapular skin or behind the ear (Tayloret al 1993) but placement under the skin at the elbow orwrist has proven to be the most accessible for scannerreading in both long-tailed (Macaca fascicularis) andrhesus (Macaca mulatta) macaques (Wolfensohn 1993)Microchip identification has been recommended for allspecies of primates There are disadvantages to the use ofmicrochips for identification Firstly the microchips andscanning equipment are expensive and thus may signifi-cantly add to the cost of an experiment Further asmicrochips are different in Europe and the USA andCanada different readers are required Secondly themicrochips can only be picked up by the scanner when thescanner wand is around 5-10 cm away from the chip (Pooleet al 1999) Although primates can be trained to stay stillwhilst the microchip is scanned (Savastano et al 2003)untrained animals may need to be restrained in order toobtain a successful reading Finally migration of themicrochip under the skin is possible and may reduce theefficiency of identification although chips usually moveonly a short distance (approx 5 cm) (Wolfensohn 1993)

Temporary identificationBecause of difficulties with reading tattoos and microchipsthe combination of a permanent method of identificationwith a more easily distinguishable temporary method ofidentification is often used (Fortman et al 2002) Theprovision of a means of identifying the animal easily andimmediately enables the caregiver to quickly determinewhich animals they are observing and therefore todetermine whether the behaviour of that animal is normalfor that individual thus facilitating welfare assessmentEasy identification also facilitates the development of rela-tionships between the caregiver and the animal which is

copy 2006 Universities Federation for Animal Welfare

Refining the use of primates in procedures 241

thought to increase the overall standard of care (Scott 1990)Temporary methods of identification include hair dyes furclipping and collars with identity tags Care must be takenwith the choice of dye particularly in young infants toensure that its application does not cause irritation (Anon2001) and measures should be taken to minimise potentialinjury from collars and clipping equipment Collars and tagsare only appropriate for use with animals that have stoppedgrowing They are commonly used in squirrel monkeys(Mendoza 1999) callitrichids (Poole et al 1999) and owlmonkeys (Aotus spp) (Erkert 1999) and specially adaptedcollars used to help manipulate and restrain Old Worldmonkeys can be colour coded to facilitate identification ofgroup housed animals (Baskerville 1999) They must bekept clean and primates may be trained to accept collarcleaning without restraint Fur dying is commonly used toidentify dependent infants Alternatively the hair on the tailmay be clipped to distinguish between dependent infantsDye and hair clipping generally may last less than a monthThe combined use of highly visible temporary identificationmethods with permanent but less visible methods willhelp to minimise the frequency of intrusive handling andfacilitate group housing of primates of all species Howeverwhere handling is necessary for identification for examplein order to read microchips or to renew temporary markingthe effects of handling can be minimised if the animals aretrained to co-operate with the process For examplemacaques can be trained to extend a wrist to allow scanningof microchips (Wolfensohn 1993) whilst with regular inter-action with humans group housed cotton-top tamarins(Saguinus oedipus) accepted dye marking of their infantswithout capture using long cotton-tipped swabs (Hallorenet al 1989)

RestraintIn order to successfully achieve experimental objectives (egdraw blood administer test-substance) and in many cases tominimise the risk of injury to the subject or handlerrestraint is generally considered to be a prerequisite for theuse and maintenance of primates in the laboratory (Klein ampMurray 1995 Reinhardt et al 1995) Many means ofrestraining primates have been developed some of whichare specifically adapted to suit the species concernedHowever there is evidence that these methods result inchanges in physiology and behaviour (Reinhardt et al1995) The extent of these effects varies with the durationand degree of restraint (Pare amp Glavin 1986) Oftenrestraint and procedures are carried out in a room separatefrom the colony room (Reinhardt et al 1990) in order tominimise disturbance to other animals in the colony (Grantamp Doudet 2003) It is now well recognised that removalfrom the colony room and restraint by any method canconstitute an uncontrolled experimental variable (Reinhardtet al 1995) especially as it is common practice in toxi-cology research to use each animal as its own control (Landiet al 1990)

No direct reference to the use of restraint is made in theEuropean Directive (86609EEC) (EU 1986) or in any ofthe European national legislation However restraint ismentioned in the IPS International Guidelines for theAcquisition Care and Breeding of Non-human Primates(IPS 1993) and in the Canadian Council on Animal Care(CCAC) Guidelines on the Care and Use of LaboratoryAnimals (CCAC 1993) In these guidelines it is recom-mended that restraint of any sort should only be used whenno less stressful alternative is available Many of the mostcommonly used restraint techniques their relative meritsand pathological physiological and behavioural effectshave been described in recent reviews (Klein amp Murray1995 Reinhardt et al 1995 Sauceda amp Schmidt 2000) Noattempt will therefore be made to repeat these reviews herebut an overview of the most important issues will be giventogether with more recent literature

Catching techniquesBoth the impact on the primates and the safety of thepersonnel must be considered in relation to capture andhandling Capture and handling of laboratory-housedprimates requires experience and skill Several methods areregularly used within the research community acrossEurope including hand and net catching capture of animalsby containing them in their nest box or moving them intotransport cages and by attaching a pole to a collar fitted tothe animal under anaesthesia When catching by hand theuse of protective gloves has been highly recommended(Klein amp Murray 1995 Fortman et al 2002) but the weightof glove used must be carefully gauged to ensure thatexcessive pressure is not applied to the animal and that thehandler is sufficiently well protected (Sainsbury et al 1989)Species differences should also be considered Fortman et al(2002) recommend double gloving when handling macaquespecies but the use of heavy gloves to handle marmosetsmay result in damage to their delicate teeth if they attemptto bite In contrast tamarins (similar in size to marmosets)have long upper canine teeth and should not be handledwithout protective gloves (Poole et al 1999) The handlermust be aware that injury to the primate can arise as a resultof forceful removal from the cage especially when individ-uals cling to the mesh sides of the cage Sainsbury et al (1989) recommended that nets may be usedin conjunction with hand capture particularly with groupsof animals housed in large cages However this method ofcapture can result in entanglement and injury duringremoval from the net Stress resulting from net capture cancause acute diarrhoea rectal prolapse and lacerations(Luttrell et al 1994) Furthermore the method potentiallyplaces the handler in danger from both the subject and otheranimals in the cage if they must enter the home cage andchase the target animal causing fear and arousal in thewhole group (Luttrell et al 1994) Feasible less stressfulalternatives to capture are available and therefore netcapture is not recommended here Pole and collar capture and restraint is widely used in largerprimates including macaques and baboons with little risk to

Animal Welfare 2006 15 239-261


the handler (Klein amp Murray 1995) A plastic or metal collaris fitted to the animal either under anaesthesia or sedationA spring loaded pole is attached to the collar through thedoor or bars of the cage at the time of capture and the animalis moved from the pen and walked to a grooved restrainingchair into which the collar can be slotted (Klein amp Murray1995) For larger species two handlers can attach poles tothe collar increasing the level of control Whilst in this chairthe animalrsquos limbs can be restrained just as in a normalrestraint chair (see below) and the whole body is accessiblefor physical examination blood sampling and administra-tion procedures Positive reinforcement training (PRT) anddesensitisation can be used in order to get the animal toaccept capture by this method and to walk on the end of thepole using treats to reward co-operative behaviour (Markset al 2000 Sauceda amp Schmidt 2000 McGuffey et al 2002)Acceptance of the full capture and restraint procedure canbe achieved in 4 or 5 ten minute training sessions (Klein ampMurray 1995) and thus the use of pole and collar restraintwithout training must be considered unacceptable In some research establishments marmosets are capturedby trapping them in their nest box The animals move intothe box in response to the threat of capture (eg showing agloved hand or even just opening the cage) This method ofcapture reduces the risk of injury during removal from thecage and may reduce the stress of capture overall Howeverthis method of capture is considered less than ideal as it isinappropriate to use the nest box for capture The nest boxshould provide the animal with a safe retreat in which it canrest securely without the threat of capture Also themarmosets move into the nest box because of the negativelyreinforcing threat of capture thus introducing an unneces-sary source of stressIn other research establishments transport cages are used tocapture individual primates for routine examination andprocedures Different methods have been used to induce theanimals to enter the transport cage for example Luttrellet al (1994) used negative reinforcement training byshouting and waving the arms at rhesus macaques to inducethem to move into a holding pen before releasing themthrough a chute from which they could be filtered off into atransport cage After five weeks of training the animalscould reliably be caught one at a time in the transport cagePRT is preferable and has also been used to induce entry totransport cages for capture (Reinhardt 1992a b Klein ampMurray 1995 Scott et al 2003) either attached to the homecage or placed in a chute configuration Reinhardt (1992b)found that rhesus macaques trained by positive reinforce-ment would enter the transport crates in a reliable order eachtime capture was initiated This order appeared to be influ-enced more by age than dominance hierarchy but ensuredthat specific animals could reliably be caught (Reinhardt1992b) Positive reinforcement has also been used to trainmarmosets to enter transport cages Despite the relative easewith which animals can be caught by this method Line et al(1987) found that singly-housed female rhesus macaquesconfined in a transport cages for 5 minutes showed elevated

plasma cortisol 15 minutes after confinement had takenplace Also Clarke et al (1988) demonstrated that there arespecies-specific differences between rhesus long-tailed andbonnet (Macaca radiata) macaques in their physiologicaland behavioural responses to transport cage trainingFurthermore care must be taken that the design of thetransport cage enables the handler to catch the animalsafely without risk of injury For example for marmosetsthe transport box should be easily attached to the front of thecage allowing caregiverrsquos hands to be free to deliver thereward through a mesh front and simultaneously close asideways sliding door Given their weight a macaquetransport box may be most suitable on a trolley From thesestudies it cannot be concluded that training to enter atransport cage is a means of stress-free capture of labora-tory-housed primates but if carried out correctly usinghabituation and positive reinforcement (Rennie ampBuchanan-Smith 2006a) the method can significantlyreduce capture stress and must be considered the mosthumane method of capture

Manual restraintHand-restraint of primates also has risks and a high propor-tion of accidents in the laboratory occur when primates arerestrained in this way (Klein amp Murray 1995) For examplewhen restraining an adult macaque it is recommended thatat least two handlers should be involved (Reinhardt et al1995) and that the arms should be held behind the back(Sauceda amp Schmidt 2000) However shoulder joints andmuscles can be overstrained (Sainsbury et al 1989) andfracture of the humerus can occur as a result of excessiveforce used to hold the arms of the monkey (Klein amp Murray1995) Macaques may also be restrained in a cradle usingonly one arm to immobilise the animal Marmosets andtamarins should be held around the chest between thumband forefinger The thumb can be placed under the chin toprevent biting (Sainsbury et al 1989) Landi et al (1990)found that in long-tailed macaques manual restraintinduced changes in alanine aminotransferase (ALT) andaspartate aminotransferase (AST) depending on the methodof restraint ALT and AST are used as measures of liverpathology but the effects of restraint alone on these param-eters illustrates the scientific need to find alternativemethods for obtaining samples from these animals Manyother changes in physiological parameters caused bymanual restraint have been reported (see Reinhardt et al1995 for a review)

Squeeze-back cagesAnother commonly used restraint device for all largerspecies of primates used in laboratories is the squeeze backcage (Fortman et al 2002) As the name would suggest thiscaging system incorporates a manual or automaticmechanism which moves the back panel of the cage towardsthe front Thus the animal in the cage is slowly moved tothe front of the cage and can be partially or totally immo-bilised allowing access for experimental procedures



Because the method is easy to use staff can be easily trainedto use it correctly (Sauceda amp Schmidt 2000) Animals canbe restrained quickly and safely for routine procedures likeinjections and the need for physical handling is reduced(Sauceda amp Schmidt 2000) The method is not recom-mended for more complicated techniques however whenaccuracy is essential (eg intubation) (Sauceda amp Schmidt2000) Squeeze-back cages are most commonly used as ahome cage when animals are housed singly Whilst thisreduces the need for handling squeeze cages are oftendesigned to meet cage size requirements and because oftheir size and the squeeze mechanism of the cage structuraland inanimate enrichments must be kept to a minimum(Dexter amp Bayne 1994) although primates can be housed inthis system in pairs Although the method is generallyconsidered to be safe fast and relatively stress free for mostindividuals in comparison with other methods of restraintsignificant increases in cortisol and persistent decreases intestosterone have been reported following squeeze cagecapture (Reinhardt et al 1995) Blank et al (1983) found adecrease in serum prolactin accompanied by a decrease ingrowth hormone and an increase in cortisol Grant andDoudet (2003) reported behavioural signs of fear anddistress in some of their rhesus macaque subjects includingcircular pacing bouncing baring teeth and grimacing justprior to squeeze-back restraint even though their subjectshad been trained to accept the procedure However themacaques in this study were brought to a procedure roombefore restraint took place and previous studies have shownthat removal from the home cage and colony room itselfmay induce more distress than restraint in the home cage(Reinhardt et al 1990) Together this evidence suggests thatsqueeze-back cages may induce less stress than some othermethods of restraint if the cage is used as a home cageHowever in these circumstances the animal is continuallyexposed to the threat of capture within its home environ-ment Thus the use of a squeeze cage alone as the animalsrsquohome cage is not recommended As a compromise banks ofsqueeze cages can be incorporated into group housingsystems for macaques and the animals can be trainedpreferably using PRT to move into them when requiredNon-human primates can be trained to present a specificside to the front of the cage when the squeeze mechanism isstarted thus reducing the need for repositioning andreducing potential stress (Luttrell et al 1994 Reinhardt1997)

Restraint chairsRestraint chairs maintain the animal in a sitting positionduring restraint Perspex or plastic circular restraintssurround the neck and waist to hold the animal in place butallow full access to the body for experimental manipula-tions (Reinhardt et al 1995) Modern chairs can be adjustedto accommodate anatomical differences in different speciestaking account of position and length of tail position ofischial callosities crown to rump length limb length andvariations in posture (Klein amp Murray 1995) Restraintchairs have been described as lsquocomfortablersquo (Klein amp

Murray 1995) and some evidence indicates that in compar-ison with other methods of restraint including manualrestraint restraint on a board and restraint in a box or stocksthe chair is less stressful (Landi et al 1990) However chairshave also been associated with many physical problemsincluding skin abrasions necrosis of the ischial callositiesposition-dependent oedema inguinal hernias laryngeal airsacculitis and rectal prolapse (Morton et al 1987) Fracturesand decubital ulcers have also been reported (Klein ampMurray 1995) Chair restraint also has effects on physi-ology inducing persistent elevations in adrenocorti-cotrophic hormone (ACTH) and cortisol within 15 mins ofrestraint and after rhesus macaques were returned to theirhome cages (Norman amp Smith 1992 Norman et al 1994)Restraint was also shown to inhibit pituitary luteinisinghormone (Norman et al 1994) and testosterone release(Norman amp Smith 1992) in female and male rhesusmacaques respectively thus having the potential to inhibitreproduction Morrow-Tesch et al (1993) found changes inwhite blood cell counts and the percentage of neutrophilsincreased during the restraint period in rhesus macaqueswhilst the number of lymphocytes and monocytes declinedHabituation to chair restraint giving rewards for calmbehaviour can reduce the response of animals to chairrestraint (Castro et al 1981) and the inclusion of some socialcontact may reduce the stressful nature of the procedureeven more (Hennessy 1984) Smith et al (1998) showed thatin black tufted-ear marmosets (Callithrix kuhli) relocationto a novel cage produced no measurable change in urinarycortisol over a four day period provided that the move wasmade in pairs Isolated individuals showed elevated urinarycortisol in response to separation and relocation in compar-ison with baseline However empirical data on the effectsof procedures on the untested lsquobuddyrsquo animal has yet to beprovided and the use of partners to reduce stress in studyanimals should only be carried out with caution until thisinformation becomes available Where chair restraint forextended periods is unavoidable manipulation of the indi-vidualrsquos legs can help to maintain circulation reducing theeffects of the abnormal position on the animal

TetheringThe chair method of restraint allows the subject little or nofreedom of movement An alternative to chair restraint is theuse of the vest and tether system developed by Chatham(1985) This system allows continual administration andsampling via surgically implanted cannulae which areenclosed in a tether protected by a flexible stainless steelcasing (Klein amp Murray 1995) Subjects are able to lie downand turn around thus their movement within a small cage isnot impeded (Reinhardt et al 1995) Tethers can also containlead wires attached to recording devices on the exterior ofthe cage The tether is held in place in a nylon mesh orleather vestUsing tethering samples are obtained remotely so that theanimal does not need to be physically restrained and themethod may therefore be safer and less stressful for theprimate and the handler However surgery is required prior



to initiation of the study and additional time must beallowed to acclimatise the animal and ensure that the cannu-lation procedure has been successful Morton et al (1987)found that after pre-experiment conditioning without thecannula in place subjects may take as little as a week toaccept the vest and tether and stated that 95 of subjectscan be successfully adapted to the system Wheeler et al(1990) found that the cortisol response to tethering waslower than that recorded in response to chair restraint inrhesus macaques However it is likely that this difference isassociated with the presence of handlers to obtain samplesfrom chaired individuals whilst tethered animals can besampled remotely Also cortisol was measured in animalstethered for 4 weeks whilst cortisol from chair restrainedanimals was measured after 8 hours thus lower cortisol(chronically elevated) responses would be expected intethered animalsOne of the main problems with tethering is the use of a tran-scutaneous cannula which can become infected potentiallyresulting in septicaemia The best means of infectionprevention is by use of sterile procedures and the besttreatment is by complete removal of the cannula Antibioticsshould be used after cannulation and to aid recovery ifinfection occurs However septicaemia can still arisedespite best efforts to prevent its occurrence (Morton et al1987) The risk of complications with tethering are greatlyincreased by housing animals in pairs because of thepotential for interference with the vest and tether (Kinter ampJohnson 1999) For this reason animals are usually singlyhoused during the instrumentation period which in chronicstudies can last weeks or months (Crockett et al 1993) andsingle housing is viewed as unacceptable when avoidableStudies have shown that tethering is associated with chronicactivation of the adrenal cortex (Crockett et al 1993) andsympathetic nervous system (Adams et al 1988 Crockettet al 1993) despite acclimatisation procedures (Crockettet al 1993) Adaptations to the system including improvingthe fit of vests (Morton et al 1987) use of sterile procedures(Morton et al 1987) improvement of the acclimatisationprocedure (Crockett et al 1993) and the presence of andaccess to companions in an adjacent cage (Coelho et al1991) have been shown to reduce the stress induced bytethering Furthermore Reinhardt (1997) reported thatjuvenile rhesus macaques were successfully housed incompatible pairs whilst tethered If tethering is requiredsuch practices should be used where possible to refine itsuse

Other methods of restraintTubes chutes boards and boxes are further means ofrestraint that are used in laboratories to restrain non-humanprimates (eg Greig et al 2006 for marmosets) All thesemethods have been reported to induce physiological andbehavioural changes associated with stress (Reinhardt et al1995) It is clear from the literature that although allmethods of restraint can be highly stressful much of thisstress can be eliminated if the method is used sympatheti-cally with the needs of subject in mind (Fortman et al 2002)

For example Moseley and Davis (1989) adapted two tuberestraint devices for use in marmosets and owl monkeys Byusing PRT and acclimatisation procedures the subjectsapparently adapted well to the procedures without anyobvious behavioural signs of distress Animals which wereconsidered unable to adapt were not used in the studySimilarly Greig et al (2006) consider a marmoset restraintdevice to be a refinement over manual restraint as only onehandler is required (as opposed to two to three) to carry outthe procedure However no objective measurements ofbehaviour or physiological signs of stress were made inthese studies and it should be noted that assumptions aboutthe stress experienced by individuals is no substitute forempirical measurement In a study by Yasuda et al (1988)crested black macaques (Macaca nigra) were restrained inplexiglas cylindrical restraint tubes and appeared behav-iourally relaxed However glucose clearance and insulinsecretion were found to be impaired and further restraintresulted in elevations of glucose and cortisol suggesting thatanimals may in fact have experienced stress as a result ofthe restraint procedure

Section summarybull Microchipping is the best available method of permanentidentification of laboratory-housed primates PRT can beused to facilitate chip readingbull The use of a second more obvious temporary method ofmarking (eg a collar) is beneficial for rapid identificationbull Dye marking or tail-clipping is more appropriate forinfants until weaning when permanent identification isrequiredbull Restraint is often considered a prerequisite for the use ofprimates in laboratoriesbull It is also widely recognised that capture and removal fromthe colony to the procedures room and restraint canrepresent uncontrolled experimental variablesbull The use of protective gloves is recommended forpersonnel safety but care must be taken not to injure theanimals through lack of sensationbull Net capture is highly stressful resulting in marked stress-related physiological responses and often in injury Thismethod should be avoided where possiblebull The pole and collar technique can be used to capture andmove larger primates but should only be used with PRT bull Nest-box trapping is not recommended for any species asthe box is used as a secure retreat by the animals duringresting hours and should not be associated with the threat ofcapturebull Training to enter a transport cage (using PRT) is consid-ered to be the least stressful means of capture for all speciesand in New World species should be used instead of nestbox capture The design of the transport cage must be suchthat removal of the animal can be achieved without risk ofinjurybull The full time use of squeeze-back cages to facilitaterestraint and capture is considered unacceptable as these



cages are typically small limit the scope for enrichment andare associated with the constant threat of capture The use ofsqueeze back cages as part of a group housing system andthe use of positive reinforcement to train primates to enterthe system is considered a more acceptable use of thesqueeze-back systembull Chair restraint has been associated with injury and stress-related changes in physiology The use of PRT and habitua-tion as well as adaptations of design to suit the species andminimisation of the duration of restraint are essential tominimise stress associated with this restraint methodbull Tethering has been associated with a lesser degree of stressthan chair restraint but unacceptable single housing iscommonly a consequence of its use bull Most other methods of restraint are also associated withstress-related changes in physiology and behaviour Theseeffects can be minimised by sympathetic use and handlingand PRT

Sedation and anaesthesiaSedation and anaesthesia may be used to minimise theeffects of capture and restraint and are often calledlsquochemical restraintrsquo methods The agent can be deliveredusing a syringe for restrained animals or those trained topresent a limb for procedures a pole and syringe foranimals in transport crates and small cages and capturepistols rifles and blow darts for animals in large grouprooms or outside (Fortman et al 2002) Pistols and riflesexpel the anaesthetic dart very fast in order to ensure pene-tration of the skin of animals that are far away however inmost laboratory situations non-human primates are closeenough to dart using a blow pipe and the pistol and rifledarts may cause excessive bruising without increasing theaccuracy of darting (Fortman et al 2002) An overview ofthe most commonly used anaesthetics for laboratoryprimates is provided by Rensing (1999) and only asummary of key points will be reported here together withmore recent literature Ketamine is the most commonly used anaesthetic agent inprimates administered at a dose of between 5 and 40mg kg-1

(Castro et al 1981 Erkert 1999 Poole et al 1999) Accuratedosing should result in dissociative anaesthesia analgesiaand superficial sleep (Castro et al 1981) However if tooweak a dose is administered increased tonic activity ofmuscles may occur making the subject difficult to controlOverdosing can result in excitation of the central nervoussystem and cramping (Ghaly et al 2001) The advantage ofketamine anaesthesia is that it does not depress swallowbreathing and eyelid reflexes ensuring that the state of thesubject can be more easily monitored and animals will notstop breathing unexpectedly (Fortman et al 2002) A distinctdisadvantage to its use is that tolerance can develop over aperiod of time Ketamine is often used in combination withxylazine to counteract any central excitation (Erkert 1999Poole et al 1999)The use of anaesthetic agents themselves may have effectson physiological parameters The potential for effects varies

with the species and drug applied and care must be takenwhen selecting both the species and drug for each specificstudy and during interpretation of results (Reinhardt et al1995) Particular caution must also be exercised whenanaesthetics are administered to pregnant or lactatingfemales as some agents may cross through the placenta tothe foetus or be released in milk (Klein amp Murray 1995)Yasuda et al (1988) described unwanted effects of ketaminein black-crested macaques including excessive salivationslow hand and limb movements and uncoordinated eyemovements Wall et al (1985) found that ketamine injectionhad significant effects on blood cell parameters and cardio-vascular function in vervet monkeys The changes in phys-iological parameters and the behavioural effects ofanaesthetics may well be indicative of stress in non-humanprimates and thus the use of anaesthesia to minimise stressmay be counterproductive Because of the risks involvedthe use of anaesthesia either to reduce stress or to facilitateprocedures should only be considered in cases where it isnot possible to train the primate to co-operate with theprocedure voluntarily Stress associated with restraint foradministration of ketamine can be greatly reduced or elimi-nated by using PRT to encourage the individual to co-operate (Philipp 1996)

Section summarybull Ketamine is most commonly used for anaesthesia inprimates and is used both to immobilise the animal duringinvasive procedures and to reduce stress associated withrestraintbull Anaesthesia of primates is always associated with risksand it may affect physiological parameters which must betaken into account in experimental proceduresbull Where possible PRT should be used as an alternative toanaesthesia to reduce stress associated with proceduresbull Where anaesthesia is necessary to achieve the procedurePRT should be used to minimise stress associated with itsadministration

Sampling

Blood samplingBlood sampling is carried out routinely on experimentalanimals The measurement of the blood biochemistryhormones and haematological parameters are well estab-lished protocols providing reliable measures of physiolog-ical status Venous blood samples are the most commonlyused sample but arterial and cardiac samples and the use ofcannulae may also be used although some consider thatsampling by these methods requires special justification(Morton et al 1993) With poor management inexperienceunsympathetic handling and the use of the wrong or poortechnique blood sampling may become unnecessarilystressful for the animal (Morton et al 1993) and hypo-volaemia bruising haemorrhage and thrombosis may bethe result Arterial blood sampling is most commonly usedto collect large samples of blood quickly because arterialblood is under more pressure than blood in veins However



adverse effects of blood sampling are more common whenblood is collected in this way Care must be taken to ensurethat pressure is applied to puncture sites until bleeding hasdefinitely stopped If thrombi (fibrinous blood clots) form atthe site of puncture of an artery they may dislodge and cancause severe pain if they become lodged in a major vessel(eg the femoral artery) Care must be taken especially withsmaller animals that the volume of blood taken is notexcessive and that time is given for blood volumes torecover in prolonged trials It has been recommended thatfor one-off sampling 10 of the animalrsquos circulating bloodmay be taken but for repeated sampling only 1 may betaken every 24 hours (Morton et al 1993) Phlebitis (inflam-mation of the vessel) may also occur if cleanliness is notensured However excessive cleaning of the skinrsquos surfacebefore sampling may be counterproductive as clipping andshaving have the potential to increase the stressfulness ofthe procedure and repeated sterilisation of the skin canresult in inflammation dryness and discomfort (Mortonet al 1993) Under good conditions involving PRT the actual act ofvenipuncture has been achieved in Old World species withno adverse response (Priest 1991 Reinhardt et al 1991)Rhesus macaques stump-tailed macaques (Macacaarctoides) drills (Mandrillus leucophaeus) and chim-panzees (Pan troglodytes) have all been trained to acceptblood sampling in their home cages (Reinhardt 1989Reinhardt et al 1990 Priest 1991 Fritz et al 1999)Reinhardt et al (1991) compared cortisol in trained malerhesus macaques sampled in their home cage and a secondgroup of venipuncture-trained animals sampled whilstunrestrained in a restraint apparatus Animals sampled intheir home cages showed less change in cortisol than thosesampled in a restraint chair It was concluded that venipunc-ture is not stressful per se but that stress can be inducedwhen it is carried out in association with removal from thehome cage (Reinhardt et al 1991) Capitanio et al (1996)found that cortisol in rhesus macaques was unaffected eitherby the time taken to draw blood or the duration of distur-bance prior to sampling This suggests that the presence ofexperimenters in the colony room is not necessarilystressful adding weight to the argument that non-humanprimates should be trained and sampled in their home cagewherever possible However Capitanio et al (1996) did findchanges in haematological parameters independently of ahypothalamo-pituitary adrenal (HPA) axis stress responseThe authors concluded that disturbance in the colony roommay cause experimental variation in haematological param-eters but that stress is not necessarily induced Priest (1991) describes a procedure by which a diabeticadult male drill was trained to accept regular blood samplesThe drill was trained to hold a handle until the signal wasgiven to let go and a reward was given Venipuncture wasaccepted by the drill whilst holding the handle Training toaccept venipuncture is becoming more widely used in OldWorld primates and is recommended as a less stressfuloption to restraint for all Old World species (Baskerville

1999) Although training to accept laboratory procedures ispossible in New World primates (Poole et al 1999Visalberghi amp Anderson 1999 McKinley et al 2003)acceptance of venipuncture has not been achieved partiallybecause their small size makes it difficult to access bloodvessels However with training and desensitisation thestress of capture (Reinhardt 1992a b Klein amp Murray1995 Scott et al 2003) and restraint can be minimised andthus the overall stress of routine procedures can also bereduced in New World species (Moseley amp Davis 1989)Venipuncture itself may be refined by carefully selecting thegauge of hypodermic needle The gauge should be such thatthe minimum size of puncture wound is applied but thatblood will flow through the needle reasonably swiftly toavoid extending the duration of the procedure and to reducethe possibility of haemolysis of the sample (Morton et al1993) The details of suitability of different lengths andgauges of hypodermic needle for different species of labo-ratory animals are described in the BVA (AWG) FRAMEUFAW RSPCA report on the removal of blood from labo-ratory mammals and birds (Morton et al 1993)The femoral vein is considered to be the most appropriatevessel for blood sampling marmosets (Morton et al 1993Poole et al 1999) but the coccygeal (tail) vein has also beenrecommended (Morton et al 1993) The saphenous andbrachial veins are recommended for drawing blood fromOld World monkeys (Morton et al 1993 Baskerville 1999)These vessels are large enough to allow easy venous accessand to ensure that blood may be taken relatively quickly andare superficial enough to be easily accessed in conscious orlightly sedated animals (Morton et al 1993) The femoralvein has also been recommended for venipuncture in anaes-thetised heavily sedated animals or in those that are welltrained for sampling as it is located deeper below the skin(Baskerville 1999) Larger volume samples should be takenfrom the femoral vein (Baskerville 1999) Damage to blood vessels as a result of regular bloodsampling may be minimised by alternating between bloodvessels on either side However if frequent sampling isrequired some authors recommend that cannulation be usedto reduce the stress of sampling primarily by eliminatingthe need for frequent skin puncture but also to minimise theduration of handling (Wheeler et al 1990 Morton et al1993) Early cannulation techniques required that anticoag-ulant be placed in the lsquodead spacersquo of the cannula tomaintain patency This had to be removed at the start ofsampling and replaced afterwards thus increasing theduration of restraint More modern cannulae incorporatemultiple entry ports such that anticoagulant can remain inthe tube continually so that the draw-time is reduced andcannula patency is better maintained (Morton et al 1993)The use of cannulation must be carefully considered beforeundertaking the procedure as the adverse effects of thetechnique are potentially numerous In non-human primatesonly the jugular and femoral veins and arteries are recom-mended for cannulation (Morton et al 1993) Placement ofthe cannula requires anaesthesia which in itself induces



stress (Wall et al 1985) Subsequently the cannula willrequire protection from manipulation by the animal to helpprevent accidental or deliberate removal of the cannula(Morton et al 1993) Jackets with a swivel and tether havebeen used to protect cannulae long-term in primates but thismethod of restraint has been associated with long termarousal of the sympathetic nervous system (Adams et al1988) and may result in gastric ulcers (Brodie amp Hanson1966) as a result of stress Restraint of this sort usuallyrequires that animals are kept in single cages because of theassumption that cagemates will interfere with the jackets(Kinter amp Johnson 1999) although pair housing of tetheredjuvenile rhesus macaques has been achieved (Reinhardt1997) Single housing should be avoided whenever possibleas most laboratory-housed primates are highly socialspecies However cannulae have been successfully main-tained in group-housed marmosets (Morton et al 1993)Cannulae can become blocked and efforts must be made tounblock it if patency is to be restored However unblockingthe cannula may require prolonged handling thusminimising one of the potential benefits of its use Cannulaecan be a focus of bacterial infection and although the use ofaseptic conditions can minimise the risks infection canoccur resulting in thrombus formation increased bodytemperature and loss of appetite Thus the cannula mayhave to be removed and the animalrsquos involvement in theexperiment terminated

Non-invasive samplingMethods by which physiological parameters can bemeasured without venipuncture and therefore in a lessinvasive manner are gaining in popularity as validation oftheir use for a wider range of parameters progressesHormones including gonadal steroids adrenal steroidsACTH and many others can be measured in urine and faecesof non-human primates The suitability of these samplingtechniques must be considered with respect to the speciesstudied and the requirements of the experiment as the abilityto reliably detect parameters in urine or faeces is variable indifferent species and there is a time lag of variable durationbetween the appearance of parameters in the blood and theirdetection in the excreta For example in squirrel monkeyshormones are excreted predominantly in the faeces and notin urine (Visalberghi amp Anderson 1999) Although invasivemethods of collection of urine exist (including catheterisa-tion and cytocentesis) samples can be collected by posi-tioning a cup under a urinating animal or by training theanimals to urinate when and where required Training forurine collection has been used with success in marmosets(Anzenberger amp Gossweiler 1993 McKinley et al 2003)chimpanzees (Stone et al 1994 Fritz et al 1999) capuchins(Visalberghi amp Anderson 1999) and vervet monkeys(Kelley amp Bramblett 1981) Analyses of hormones in urineand faeces provide a measure of the hormone over a periodof time Thus it is particularly useful for determination ofreproductive status and for assessment of chronic arousal orstress However physiological responses to instantaneousstressors cannot be measured in this way (Lutz et al 2000)

The measurement of salivary hormones can provide a lessinvasive (although potentially intrusive) means for meas-urement of short-term responses than blood samplingAgain the potentially intrusive nature of sampling saliva canbe minimised by training non-human primates to co-operatewith the sampling procedure by positive reinforcement withreasonable reliability For example (Lutz et al 2000)collected saliva successfully from 21 out of 23 rhesusmacaques and found two potentially useful means ofobtaining the sample They found that licking a small pieceof absorbent mesh fabric and chewing a piece of cotton ropeboth produced sufficient samples although disadvantageswere identified with both methods A method for non-invasive sampling of saliva was developed and reported byCross et al (2004) In these studies marmosets weretempted to chew on a cotton wool swab by dipping the tipin banana and allowing the animal to chew on the swab for5 minutes In field conditions genetic information aboutsubject animals is commonly obtained through collection offaeces and hair samples (eg Bradley et al 2002) Such workcan also be carried out on laboratory non-human primates

TelemetryThe intrusive nature of many physiological data collectionmethods has been reduced by the use of radio- or bio-telemetry Bio-telemetry is the term used to describe thecollection of physiological data using sensors in closecontact with the animal Data picked up by the sensors isrelayed through a transmitter to a receiver The data can thusbe collected remotely without disturbing the animal Thebasic concept of this technology has been developed widelyand many methods currently exist by which this technologycan be applied in animal studies in the laboratory Non-invasive telemetry devices

Externally applied telemetric devices have been used formany years for the study of the behavioural ecology of wildanimals Transmitters are attached to the fur or skin of thetrapped animal or are designed to attach to a collar Thesystem can remain viable for periods of up to severalmonths The transmitters must either be removed by recap-turing the animal or will become detached when the animalmoults or as old skin cells slough off Externally appliedtelemetry devices may also be used in laboratory animals Inthis case the loggertransmitter is usually enclosed in ajacket worn by the animal (Kramer et al 2001) Electrodesor sensors are attached to the animal and linked up to thelogger and the data are transmitted to receivers held nearbyThis method has the advantage that the animal can movearound freely and that data may be collected 24 hours a dayAlso the equipment may be transferred between animalsand batteries can easily be changed This equipment canonly be used in larger non-human primates as well fittedvests and jackets are not available for smaller species(Visalberghi amp Anderson 1999) Also the risk of damage tothe equipment and removal of sensors is high because ofinterference with the equipment by the wearer (Kramer et al2001) Despite these problems externally applied radio-telemetry devices have been used successfully For example



Novak (2003) recently described a study in which a remotetelemetry system was used to determine heart rates insingly-housed rhesus macaques that were engaging in self-injurious behaviour It was found that monkeys engaging inself-biting behaviour had higher than normal heart ratesprior to biting and that their heart rate became even moreelevated during biting However when these animalsfinished a bout of biting their heart rates declined tobaseline levels It was argued that the act of self-biting hasa calming influence that is self-reinforcing and willtherefore persist despite changes in the environment (Novak2003) The measurement of heart rate using this method canbe carried out without difficulty despite interference withthe exact positioning of the sensor movement and the resist-ance of skin and underlying tissues However the measure-ment of some physiological parameters may require moreaccurate placement of sensors and the minimisation ofresistance in order to achieve usable results Thus thenumber of measures that may be made using externallyapplied telemetry devices is comparatively limited andsingle housing is unacceptable Therefore alternative tech-niques should be usedCapsule telemetry devices

The data-loggertransmitter component of the telemetrysystem can be made small enough to fit into a capsulealthough the data recorded by these devices tends to besimpler Such devices have been used to measure gastric pHand transit time (Barrie 1992 Mojaverian 1996) deep bodytemperature (Hoffman et al 1969) subcutaneous bodytemperature (Cilia et al 1998) The most commonly usedcapsule telemetry device is the identity microchip usedwidely in pets and in laboratory-housed animalsImplantation of these capsules may be achieved quickly byinjection through a specially designed hypodermic needlewithout sedation or surgery Restraint of the animal ishowever required Also the needle required to implant thecapsule is of a much larger gauge than that used forvenipuncture or drug administration The use of local anaes-thetic would reduce the aversive nature of the procedureitself but would increase the need and duration of handling(particularly in untrained animals) Also capsule telemetrydevices of any kind must be close to the receiver in order tobe read and thus in untrained animals handling may berequired (Kinter amp Johnson 1999)

Invasive telemetry systems

More complex transmitters and sensors have beendeveloped that can be implanted (partially or fully) surgi-cally within the animal These systems have the advantagethat minor movements of the body do not result inmovement of the sensors The sensor is applied directly tothe target area so that there is less resistance between thesensor and the tissue Such devices have been termed lsquopre-invasiversquo because they require invasive surgery in order toprovide telemetered subjects but these animals are giventime to recover before initiation of experimental protocolsthat may be both non-invasive and non-intrusive (Schnell ampGerber 1997) Partially implanted systems require initial

surgery in which the sensors electrodes or cannulae areimplanted at the required site sutured in place and thentracked back subcutaneously to the transmitter At a suitableposition (eg through interscapular skin) the wires are ledthrough the skin to be connected to the transmitter Thetransmitter can be worn by the animal in a jacket allowingunrestricted movement or the animal is restrained using atether and swivel system through which the wires run toreach the transmitter outwith the cage As with externallyapplied telemetry devices partially implanted telemetrysystems can only be used in larger primates because of diffi-culties fitting vests and jackets in smaller species(Visalberghi amp Anderson 1999) and the resulting increase inthe risk of interference with the equipment Howeverpartially implanted radio-telemetry systems have been usedsuccessfully to collect data 24 hours a day from commonmarmosets (Crofts et al 2001) Because of the transcuta-neous components of partially implanted telemetry systemsinfection and injury from interference with equipment areserious risks Because of these risks primates carryingpartially implanted telemetry systems are often housedsingly which is also likely to cause stress (Kinter ampJohnson 1999 Visalberghi amp Anderson 1999)Fully implantable devices also require surgery but hereboth the transmitter and the wires are implanted within theanimal Equipment is available for use in both large andsmall primates In marmosets (and other small primates) thetransmitter (often adapted from transmitters designed forthe similarly sized rat) is placed in the peritoneal cavity andsutured to the mucosa (Einstein et al 2000 Crofts et al2001) In larger species including rhesus Japanese andlong-tailed macaques and baboons the transmitter mayeither be placed under the interscapular skin or inserted intothe peritoneal cavity (Kaufman amp Detweiler 1999) Theelectrode wires or cannulae can then be tunnelled subcuta-neously to the appropriate position and sutured into place(Kaufman amp Detweiler 1999) Fully implantable telemetrydevices require no restraint during the test protocol andhave been successfully used to record parameters 24 hoursa day Alternation between subjects over a 24 hour periodallows multiple subjects to be housed in pairs or groups(Crofts et al 2001) These devices leave animals less opento infection as after the implantation incisions heal there isno route by which infection can cross from the externalenvironment Battery power can be saved by turning thedevice off remotely when not in use Thus working instru-mentation has been successfully maintained in commonmarmosets for up to 2 years (Crofts et al 2001) Suchdevices have been used successfully in non-human primatesto collect cardiovascular blood pressure temperaturemotor vocalisation locomotion and pH data and to recordelectrocardiograms (ECG) electromyograms (EMG) elec-troencephalograms (EEG) and electrocorticograms (ECoG)(Kinter amp Johnson 1999) The data obtained by this methodcan be used in a diversity of experiments in whichbehaviour and physiology may be monitored with orwithout manipulations For example telemetry has beenused to detect diurnal rhythms in baboons (Stark et al 1999)



and squirrel monkeys (Robinson amp Fuller 1999a b) It hasalso been used to determine the aetiology of cardiovascularchanges occurring in anticipation of locomotion in baboons(Smith et al 2000) and in response to social interaction inrhesus macaques (Aureli et al 1999) and social challenge incommon marmosets (Gerber et al 2002a b) After implan-tation in both mother and foetus radio telemetry has beenused to assess the development of neurobehavioural growthand circadian rhythms in neonatal baboons (Mirmiran et al2001) Studies have also been carried out to determinenormative values for heart rate (Schnell amp Wood 1993Kaufman amp Detweiler 1999) and ECG (Schnell amp Wood1993 Kaufman amp Detweiler 1999) blood pressure (Schnellamp Wood 1993) sleep patterns (Crofts et al 2001) and bodytemperature (Cilia et al 1998) The information obtainedusing this technique can also be used for example to accu-rately determine the effects of new procedures or drugs(Horii et al 2002) to examine psychological (Novak 2003)and physiological conditions (Kerl 1997) or to determinethe relative stressfulness of everyday husbandry procedures(Kerl amp Rothe 1996 Schnell amp Gerber 1997 Bowers et al1998 Gerber et al 2002a b) The critical advantage of thetechnique is that all measurements are taken from animalsthat can be group housed and maintained in their homeenvironment without the need for handling or restraint Forexample a fully implanted system described by Schnell andWood (1993) was used to measure blood pressure heart rateand motor activity in conscious unrestrained group-housedmarmosets over a period of 11 months Summary of the costs and benefits of the use of telemetry

The costs and benefits of the use of telemetry in animalstudies are discussed in detail in reports on the use oftelemetry by the BVAAFFRAMERSPCAUFAW JointWorking Group on Refinement (Morton et al 2003Hawkins et al 2004) In these reports it is stressed that theuse of telemetry in laboratory-housed animals will alwayshave some negative impact and the costs and benefits of itsuse must be continually reviewed throughout studies Thereis no doubt that in comparison with early efforts modernfully implanted telemetry devices are considerablyimproved Also after the implantation procedure nohandling is required and data can be obtained from lsquorestingrsquoanimals rendering the system less intrusive than traditionalmethods of data collection (Einstein et al 2000) This in turnincreases the accuracy of data collection as physiologicalparameters are unaffected by changes associated withrestraint to obtain the sample For example Schnell andGerber (1997) found that the heart rates of macaques andmarmosets trained to accept restraint for sampling were atleast 100 beats per minute faster than those measured inunrestrained telemetered animals in their home cage Bloodpressure was also lower in telemetered animals The use oftelemetry can significantly reduce the numbers of animalsrequired to obtain reliable results because of such reduc-tions in stress-related variation (Brockway et al 1993) It isalso possible to use animals for more than one study withoutthe need for further intervention

Despite these benefits the need for and extent of surgeryrequired to implant the devices in the first place cannot beignored and in primates re-use of instrumented animals iscommon because of their relative value particularly if theyare trained for operant tests These animals may be re-operated in order to replace batteries or other componentsduring the course of an experiment or series of experiments(eg Gerber et al 2002a) Schnell and Gerber (1997) foundthat marmosets involved in mild drug studies showedelevated HPA activity in comparison to baseline measure-ments despite the use of telemetry to minimise the intrusive-ness of these studies Thus it was demonstrated that therepeated re-use of animals in even very non-intrusive exper-imental protocols may introduce a certain degree of stressand therefore unwanted variation into studies Fullyimplanted telemetry devices have been found to signifi-cantly increase the reactivity of the sympathetic nervoussystem in mice (Einstein et al 2000) It is likely that sucheffects of carrying telemetry devices are less in primatesbecause the devices used are comparatively smaller inrelation to the size of the animal however such chroniceffects cannot be discounted Although the development offully implantable devices has facilitated group housing oftelemetered animals many users may continue to favoursingle housing because the majority of telemetry devicestransmit on the same frequency (Hawkins et al 2004)Hawkins et al (2004) recommend that rather than singlehousing telemetered primates they be housed in compatiblepairs with only one pair-mate being telemetered or that theanimals should be group housed and that data should becollected in turn from each device whilst the other devicesare turned off Significant benefits of using radio-telemetryand the results that can be gained from these studies must beseriously considered with respect to the considerable coststo the animal

SonophoresisCook et al (2000) reported the development of a system ofsonophoresis an innovation with the potential to consider-ably reduce the need for minor sampling procedures onlaboratory animals This sampling method works on thebasis that low-frequency ultrasound can increase the perme-ability of the skin to drugs and high molecular weightproteins Thus movement of blood constituents could beinduced in the opposite direction from blood to skin surfaceby applying an osmotic gradient to the skin and increasingskin permeability using sonophoresis The method has beenused to successfully measure glucose in humans (Tamadaet al 1995) and cortisol testosterone insulin 17β estradioland glucose in humans and sheep (Cook 2002) In order tocollect a sample a small quantity of ultrasound gel isapplied to the skin and the transdermal collection device isheld against it for a total of two minutes (Cook 2002)Results indicate that the concentrations of parametersmeasured by transdermal collection were comparable withthose measured in blood were higher than those in salivaand did not encompass the time lag (10-20 min) associatedwith salivary hormone responses (Cook 2002) Human



volunteers reported that the methods did not cause any painand thus frequent sampling is facilitated (Cook et al 2000)Although the time taken to obtain samples using thismethod is comparatively long primates could be trained toaccept this sampling technique especially as it does notcause pain

Section Summarybull Venous blood samples are the most commonly takensample from primates used in researchbull Adverse effects of blood sampling are likely to be moresevere if taken arterially Cleanliness is essential duringboth venous and arterial samplingbull Evidence indicates that stress associated with bloodsampling can be greatly reduced or eliminated by the use ofPRTbull The most appropriate sample site varies with speciesbull Some parameters can be measured in urine faeces andsaliva as well as in blood bull The invasiveness and intrusiveness of sampling by thesemethods can be reduced by PRTbull Biotelemetry can be used to collect physiological dataremotely without disturbing the animal which reducesstress associated with the experiment and because accuracyis increased reliable results can be obtained using feweranimalsbull Implanted telemetry devices allow group housing and aretherefore a refinement over those requiring single housingbull The most extensive results have been obtained usingtelemetry devices that are implanted surgically Thus thereis a conflict between the reduction of animal numbers andthe severity of the measurement techniquebull Sonophoresis is a non-invasive technique by which anextensive range of blood parameters can be measured It haspotential for use in primates and could be further refinedusing PRT

Administration The selection of the species substance to be administeredand the route of its administration is determined to a greatextent by the objectives of the experimental procedureconcerned However certain basic practices must also beconsidered in order to ensure that the principle of minimumharm is upheld A complete review of refinement techniquesfor administration of substances has been provided by theBVA (AWG)FRAMERSPCAUFAW working party(Morton et al 2001) Only an overview of the main pointswill therefore be presented hereThe nature of the selected species must be considered if forexample multiple administration sessions are to berequired Certain species (for example marmosets (Pooleet al 1999) are known to be particularly stressed by restraintand due to their small size may be more difficult to trainfor intravenous (iv) administration and other administrationprocedures than other primates The expertise of staff mustalso be considered and training and advice sought in order

to ensure that staff are capable of handling the animaladministering the substance recognising adverse effectsand initiating emergency procedures should unexpectedeffects or severity occur (Hau 1999) If the effects of anyprogramme of administration are unknown the use of pilotstudies to inform on potential consequences must be consid-ered (CCAC 1998 Wong 1998 Morton 1999 Wallace1999) Commercially available drugs (or medicines) aresupplied in a form considered suitable for the route ofadministration such that both active substance and itsvehicle are appropriate However if a substance is experi-mental or a commercially available substance is to beadministered by an unconventional route the suitability ofboth the substance the vehicle and their interaction must beconsidered with respect to the species concerned (Mortonet al 2001) Non-compatibility has the potential both toincrease the severity of the procedure and to adverselyaffect results The solubility viscosity pH irritancystability and purity of the substance must be considered andmay be affected by the frequency of administration doseand concentration and therefore the volume to be adminis-tered Some routes of administration are more stressful thanothers (Morton et al 2001) Generally the administration ofsubstances by oral or iv routes are likely to cause less irrita-tion than administration into tissues including intraperi-toneal (ip) subcutaneous and intramuscular injections(Morton et al 2001) A summary of the severity of differentroutes of administration is provided by Morton et al (2001)Most routes of administration have the potential to berefined or can be refined by using the least severe method ofadministering by that route For example intra-articularintra-cerebral and intra-tracheal routes of administrationshould only be used under anaesthetic If the primates arenot trained to accept the dose the requirement for restraintduring administration by for example iv subcutaneousintramuscular and oral routes of administration increase theoverall severity of the procedure Thus training to volun-tarily present for injection (eg Wolfensohn amp Honess 2005)or even habituation to the restraint device (Reinhardt et al1991) would greatly reduce the severity of the procedureOral administration by gavage is considered to be ofmedium severity (Morton et al 2001) partially because ofthe need for restraint in order to achieve administration andalso because poor placement of the tube has the potential toharm or kill the subject animal However in rats Wheatley(2002) found that the technique could be refined usingflexible catheters and a fixed multidirectional dosingapparatus which prevented movement of the tube onceplaced and allowed accurate dosing of substances Thisrefinement is likely to apply to primates also Extra caremust be taken when using oral gavage in marmosets as theirteeth are delicate and are easily damaged (Poole et al 1999)Alternatively substances could be incorporated into feed orfluids and offered to the animal a method considered tohave minimal impact on the animal (Morton et al 2001)However care should be taken with the use of this methodof administration as aversion to common laboratory feedscould result if an association between the consumption of



the feed with subsequent adverse effects was learned(Matsuzawa amp Hasegawa 1983) This response could subse-quently be passed on socially to other group members(Hikami et al 1990)

Section summarybull Species-specific considerations and staff expertise mustalso be considered in the design of the experimentbull Pilot studies should be carried out to evaluate methods ifnovel substances are to be administeredbull The vehicle viscosity solubility pH irritancy stabilitypurity frequency of administration dose concentration andvolume of the substance must be considered particularlywhen novel substances are administeredbull Intra-articular intra-cerebral and intra-tracheal administra-tions are considered most stressful and require anaesthesia bull Restraint is likely to result in stress and PRT should beused to reduce the stress of administration and the associ-ated restraint

SurgerySurgical procedures should be carried out under anaes-thesia using the best available animal care techniquesDuring the design phase of each experiment the timingsurgical technique and the use of analgesia (pre- and post-operatively) and anaesthesia should be carefully consideredwith reference to the best available advice in order toprevent or alleviate adverse effects The availability oftechnical staff to oversee recovery must always be ascer-tained before surgery begins and training in post-surgicaltreatment must be given The use of anaesthetics and anal-gesics is beyond the scope of this review but information onanaesthesia and analgesia is available elsewhere (eg seeFlecknell 1996 2000 Flecknell amp Waterman-Pearson2001) for overviews for all laboratory animals

Refinement of experimental endpointsIn studies of toxicity vaccines and disease the induction ofpotentially severe pain andor distress is often prerequisite(Goldberg 2000) Lethality or moribundity are accepted orrequired endpoints by law in procedures used to test andlicence new chemicals and vaccines that are carried out onprimates (Council of Europe 2001) Pain and distress arerarely treated in these studies because of the potential forinteractions with the test compound (Stokes 2000a)Alternative procedures with less severe endpoints are beingdeveloped (Lipnick et al 1995) and it is widely recognisedin the laboratory animal welfare research community thatthe incorporation of earlier endpoints into the experimentaldesign of toxicological experiments would constitute signif-icant refinement and would greatly improve welfare Non-rodent mammals used in testing is expected to be amore accurate model of the effects of a chemical in humans(Sass 2000) Primates are considered to be particularlyuseful models for human responses because their anatomyand physiology are very similar to that of humans and theyshare 85-92 of human DNA (Garg 2000) Primates aremainly used in studies of reproductive toxicology immunity

(vaccines) and disease (primarily parasitic) The UKFrance and Germany are the three principle users of non-human primates in research (Council of Europe 2003Rennie amp Buchanan-Smith 2005) Forty percent of non-human primates used in science in Europe in 2001 wereused in toxicology and safety evaluations (Council ofEurope 2003 Rennie amp Buchanan-Smith 2005) Thus theidentification validation and use of earlier endpoints whichapply specifically to primates are of the utmost importanceand should be considered a priority in these speciesThe European Directive (86609EEC) dictates that when itis deemed necessary to use animals in scientific proceduresarrangements should be made to prevent the occurrence ofpain or avoidable suffering distress or lasting harm (EU1986) Thus where more humane endpoints are fullyvalidated and accepted their use is a legal obligation Thisis not reflected in some monographs of the Council ofEurope (2001) in which both death and alternative morehumane endpoints are acceptable

Definition of humane endpoints and the principles oftheir useIn November 2000 the Organisation for Economic Co-operation and Development (OECD) published guidelineson the use of humane endpoints (OECD 2001) In thisdocument the term lsquohumane endpointrsquo was defined as ldquotheearliest indicator in an animal experiment of severe painsevere distress suffering or impending deathrdquo (OECD2000 p 10)Ultimately the aim of endpoint studies is to identifyendpoints that accurately predict death pain or distress asan outcome (Wallace 1999 OECD 2001) so that resultsmay be obtained and the experiment terminated before theanimal experiences these conditions It must be recognisedthat at present we have limited ability to predict theoutcome of toxicological tests before the onset of pain ordisease It is therefore recommended that until criteria arevalidated that can be used to predict prognoses every effortmust be made to use currently available tools to identifypain distress or suffering at or as soon as possible afteronset (OECD 2001) and steps must be taken to alleviatepain distress or suffering at this stage The early identifica-tion of these conditions will allow measures to be taken tominimise their effects

Determination of more humane endpointsImportant criteria for the determination of humaneendpoints are discussed in great detail in the literature andare recommended in the OECD guidelines on the use ofhumane endpoints (OECD 2001) Only a summary of themain points will be described here Humane endpoints must be considered in the context of theexperiment The potential benefits of the experimentaloutcome must be considered sufficiently important towarrant the induction of an expected degree of pain anddistress in the animal subject and for the proposed experi-ment to go ahead In order to effectively weigh up bothscientific and welfare consequences of a study the specific



experimental objective must be carefully considered andclearly defined (Wallace 1999) The criteria that will beused to indicate that the experimental objective has beenreached (scientific endpoints) must also be defined (Wallace2000) The experiment should be terminated as soon as the scien-tific objective has been attained (OECD 2000) Thus theearliest possible endpoint is used without jeopardising theobjective of the study and therefore reducing its benefits Itis also necessary to consider how the endpoints themselveswill be detected and whether the assessment of endpointsalone will add to the severity of the experiment (Wallace1999) (eg if more blood samples are required or the meansof measurement requires an operation (telemetry)) Theleast invasive means of detecting an endpoint must alwaysbe chosen (Olfert amp Godson 2000) Humane and scientific endpoints must also be specific tothe study species Differences in anatomy and physiologyare likely to result in variations in response to experimentalchallenges (Richmond 1999) Also species-specific diurnaland seasonal rhythms will influence biochemical andbehavioural patterns (Scharmann 1999) It must also beremembered that the time course of the response will varybetween individuals of the same species (Richmond 1999)In order to select species-specific scientific and humaneendpoints the results of previous studies using the testparameter or related parameters must be examined wherethat information is available Consultation with staff andoutside experts may also be an essential source of informa-tion (Richmond 1999) However in toxicological researchthe outcome of a particular testing procedure may beunknown and in many cases the determination of effects isthe objective In these cases the use of pilot studies with asmall number of animals examined intensively throughoutthe trial period has been strongly recommended (CCAC1998 Wong 1998 Morton 1999 Wallace 1999) In pilottrials endpoints can be determined and experience of theiruse gained morbidity time course of effects inter-sampleintervals and monitoring intervals can all be assessed Inthis way the earliest indicators of toxicological effects doserates time course and unexpected outcomes of testing canbe determined

Detection of endpointsIn the majority of scientific manipulations a lsquonormalrsquohealthy animal with potentially good welfare is compro-mised such that its state deviates from normal (Morton2000) In order to use both scientific and humane endpointseffectively we must in some way be able to recognise whenan animalrsquos condition has deviated from normal (Morton1999) Further the ability to assess the extent of thisdeviation provides us with a measure of the severity of themanipulation (Morton 1999) The assessment of the effectsof experimental procedures on laboratory animals and thusrecognition that endpoints have been reached must becarried out without the benefits of verbal self-report of painand distress The use of unprovoked and provokedbehaviour clinical signs (observable physical indicators)and biochemical markers may be objectively recorded and

used to assess deviations from normal The measurement ofall of these parameters must be based on sound backgroundknowledge of what is normal for that species and individual The occurrence of certain behaviours clinical signs andbiochemical markers are non-specific in that they occur inresponse to many experimental manipulations Theseinclude general sickness behaviours like anorexia reducedmovement and increased sleeping weight loss (as a result ofanorexia) hyper- and hypothermia and variations insystemic cortisol (general measure of the activation of theendocrine stress response) Changes of a certain magnitudein these parameters for example persistent anorexia causing10 weight loss or a reduction of body temperature by 4-6ordmCcan be used as reliable endpoints (Wallace 1999 2000 Farahet al 2001) indicating presence of general illness Howeversigns of this sort cannot be used to detect effects on specificorgan systems nor can they be used to discriminate betweenpain distress or illness More specific signs of the onset ofeffects may include guarding behaviour in response tomanipulation of a limb changes in quality of faeces (egdiarrhoea or presence of blood) cramping of limb orstomach and biochemical markers of organ damage ordisease (Poon amp Chu 1999)

Validation of endpoints In order to be scientifically and legally accepted novelendpoints must be validated against established endpoints toensure that the same degree of scientific relevance and toensure inter- and intra-laboratory reproducibility (Morton2000 Stokes 2000b) Schlede et al (2000) reviewed theclinical signs leading up to death in acute toxicity studiesincorporating 4000 rats Analysis indicated that animalsshowing convulsions lateral position tremor gasping andvocalisation died in over 90 of cases Thus these signscould be used as reliable alternative endpoints Olfert et al(1998) questioned the use of death as an endpoint in thestudy of disease They argued that by using death as anendpoint and not monitoring clinical signs as the diseaseprogresses significant differences between treatmentgroups may be missed This view is in accord with theviews of Mench (1999) who argues that particularly inchronic studies death may not in fact be caused by theexperimental variable but is simply a secondary resultcaused by dehydration or starvation If the objective of thestudy is not to assess the effects of a variable but todetermine whether or not it is toxic or disease is inducedmuch earlier endpoints may be used In disease research therate of release of acute phase proteins has proven to be prog-nostic of the severity of infection and can therefore be usedas a reliable alternative endpoint very early after infection(Olfert et al 1998) Once established it is critical that theuse of endpoints is retrospectively assessed to ensure thatthey are relevant to the study in hand and that the results ofsuch analysis are reported (Fentener van Vlissingen et al1999)

Monitoring for endpointsMany protocols are regularly used by personnel in laborato-ries to structure the monitoring procedure One of the most



frequently used in the UK is the clinical observation sheetin which possible signs of abnormality prompt the observer(Hawkins 2002) Score sheets listing clinical signs specificto the experiment are also commonly used (Hawkins 2002)The presence or absence of a sign or a score of the severityof the sign is recorded during every scheduled checkproviding an accurate record of changes in the condition ofsubjects A method of structured assessment of pain anddistress was described by Morton and Griffiths (1985) inwhich the animal is initially observed from a distancebefore being approached and finally examined clinicallyMorton later describes how these principles of assessmentcan be incorporated into the design of score sheets includingboth general and specific clinical signs (Morton 19992000) This method provides a valuable and practical tool toensure that the assessment of laboratory animals is objec-tively and clearly recorded The assessment of pain and distress in animals and how torecord it requires training Observers must have extensiveknowledge of the boundaries of normal behaviour ofanimals species and individuals on the study They mustalso be trained to recognise the critical signs indicating painand distress (Hau 1999) All members of the animal careteam (veterinarian scientist animal care worker) must beaware of their roles and responsibilities Further they mustbe aware of both the scientific and humane endpoints of thestudy and have the power and skill to execute the designatedtreatment should the endpoints be reached All personnelmust be aware of contingency plans in the event of unex-pected pain and distress (Hau 1999)Variations in the amount of abnormal behaviour recordedbetween establishments reflect differences in training andtherefore differences in the ability to detect endpoints in theexperimental situation (Schlede et al 2000) This has impli-cations for the welfare of the animals concerned In order to ensure that humane endpoints are not over-stepped observations may need to be carried out morefrequently than when death or moribundity is the endpointDuring critical periods of time (eg the first 24 hours after aprocedure has begun) the frequency of observations must befurther stepped up (Schlede et al 2000) The frequency ofobservation required may be determined during pilot trials

Use of earlier endpoints in practiceIn practice the use of humane endpoints is limited particu-larly in the testing of chemicals for use in humansHendriksen conducted an informal and unpublished surveyof research institutions that tested vaccines (Cussler et al1999) and found that many establishments were found touse death as the preferred endpoint when a less inhumanealternative was legally acceptable (Council of Europe2001) Specific descriptions of the endpoints used inresearch on primates are unusual in the literature Thereasons for this are unclear although it is likely that the needfor endpoints that are specific to the experiment in hand andthe sensitivity of the use of primates in research arecontributing factors

Endpoints can be used to significantly minimise the effectsof experimental manipulation and the welfare of laboratoryprimates may be compromised when the use of endpoints isdisregarded The following two papers illustrate thesepoints Farah et al (2001) describes the use of rodent andprimate models of schistosomiasis and describe methods bywhich the procedures can be refined and alternatives incor-porated Farah et al (2001) recommended the use of thebaboon as the main model of schistosomiasis infection inhumans because of ethical restrictions on the use of chim-panzees Numerous limits were recommended for use inexperimental manipulations Farah et al (2001) maintainedthat although few animals display clinical signs of diseasecertain limits should be applied to studies These includedrestrictions on the level of experimental infection(1000 cercariae per 5 kg baboon) dosing (several smalldoses of cercariae reduced morbidity and was a bettermodel of natural infection than one large dose) and restric-tions on the frequency of blood samples and liver biopsiesTreatment with antibiotics was provided when clinical signsof disease occurred The recommendation that animals behumanely killed using an overdose of barbiturate if theoccurrence of severe dysentery (blood in faeces) andabdominal cramping was not alleviated by 5 days oftreatment The same was recommended if weight lossreached 10 of the starting weightA second paper by Atzpodien et al (1997) described oralsafety toxicity studies of a novel non-steroidal anti-inflam-matory drug (NSAID) lsquoLornoxicamrsquo using wild-caughtlong-tailed macaques in both a 6-week dose finding studyand a 52-week study with a 4-week recovery period In theintroduction to this study it was explained that Lornoxicamhad potentially greater anti-inflammatory anti-pyretic andanalgesic activity than other NSAIDs The well-knownpotential complications of the use of NSAIDs were alsodescribed indicating that gastro-intestinal (GI) mucosaerosions and ulcerations and kidney complications were allpossible and that in serious cases in predisposed patientsthese effects could cause death In the methodology adescription of the clinical signs of NSAID treatment wasprovided including blood in faeces and weight loss associ-ated with anorexia The objective of the experiments wassimply to evaluate the oral toxicity of the drug and nohumane endpoints were defined The animals were to behumanely killed at the end of the trials and autopsiesperformed The animals were observed twice daily for novelclinical signs and for mortality Clinical signs of NSAIDtoxicity were present in the monkeys in the dose-findingstudy including faecal blood diarrhoea hypoactivityanorexia and weight loss indicative of GI lesioning Fouranimals died as a result of toxicity two of them from gutperforation and all these animals showed blood in thefaeces before they died Despite this the authors claimedthat no clinical signs of toxicity had been present In the 52-week study no clinical signs of disease were observeddespite GI lesioning found during autopsy These lesionshealed with NSAID withdrawal in a small group of animalsduring 4 weeks of recovery The results of the 52 week trial



were compromised because the wild-caught subjects wereinfected with GI parasites despite anthelmintic treatment atthe start of the study A number of GI lesions with associ-ated parasites were identified at autopsy Howeverautopsies on the recovered animals allowed discriminationof the extent to which lesions were caused by the NSAIDand not the parasites The contrasting use of endpoints inthese two European studies is dramatic and is illustrative ofthe variation in the use of more humane techniques inscience across Europe

Barriers preventing use earlier endpointsThe principal barrier preventing the more widespread uptakeof earlier endpoints in research is the potential cost Thestandard of training required to recognise the critical signs thatindicate that endpoints have been reached is much higher thanthat required to recognise death or moribundity Also thedetection of earlier endpoints requires more frequent checksand there is an associated increase in the workload required toaccomplish these checks (Cussler et al 1999) Novel endpoints must be approved for use by the OECD(Schlede et al 1999) Validation and licensing of alternativemore humane endpoints is expensive and time-consumingThe financial and time costs of this procedure are sufficientto deter scientists from undertaking this essential work(Hendriksen amp Steen 2000) To make matters worse thereis considerable prejudice against endpoints validated inother laboratories or countries The result is that the sameclinical signs are interpreted and described in different waysby different authors and in different countries and validationis repeated unnecessarily (Hendriksen 1999 Morton 1999Schlede et al 1999) In order to reduce barriers preventingthe use of humane endpoints international harmonisation oftraining (Hau 1999) and the validation and use of clinicalsigns is needed (Fentener van Vlissingen et al 1999)Published work on the use of animals in science shoulddescribe the endpoints used and how they were validatedWelfare problems encountered should also be described(Richmond 1999)

Section summarybull Currently death and moribundity are legally acceptableendpoints in obligatory toxicity and safety tests and painand distress are not routinely treated in these studiesbull Toxicology and safety evaluations are one of the main usesof primates in researchbull Humane endpoints are defined as the earliest indicator ofpain distress or ensuing deathbull Use of more humane endpoints allows animals to betreated and reduces the need for euthanasiabull Severity of endpoints should be taken into account incostbenefit analysis and the most humane methods ofdetection should be usedbull Endpoints must be species- and experiment-specificbull In novel studies advice on endpoints should be sought andif no information is available pilot studies should be used toidentify suitable early endpoints

bull Endpoints may be detected using general clinical signs orindicators specific to the studybull The scientific validity of novel endpoints must be deter-mined bull Detection of more humane endpoints requires moredetailed and more frequent monitoring and more extensivestaff trainingbull In practice the use of more humane endpoints variesconsiderably between studiesbull Barriers preventing wider uptake of more humaneendpoints include perceived financial and time investmentthe need for validation unwillingness to accept novelendpoints and poor dissemination of information

Recommendationsbull Reduced tolerance of death and moribundity as endpointswhere alternatives existbull Validation guidelines or better dissemination of methodsto widen acceptance of externally validated endpointsbull Guidelines for ethical review committees to ensureEurope-wide consistency in the use of more humaneendpointsbull A legal requirement for research to identify validate andthen use more humane endpoints

Refinement of euthanasia Euthanasia of laboratory animals may be required if animalsare found to experience an unexpected level of pain orsuffering during procedures or at the end of an experiment(EU 1986) The European Directive does not specifymethods of euthanasia but states that an animal should belsquokilled by a humane methodrsquo (EU 1986) The definition of alsquohumane method of killingrsquo is lsquothe killing of an animal witha minimum of physical and mental suffering depending onspeciesrsquo (EU 1986) In the European Convention (EuropeanCommission [EC] 1986) training of staff in methods ofeuthanasia is recommended In guidelines recognised acrossEurope overdose of anaesthesia is the only method that isconsidered acceptable for non-human primates (IPS 1993Close et al 1997 Baskerville 1999 Bearder amp Pitts 1999Erkert 1999 Fritz et al 1999 Mendoza 1999 Poole et al1999 Visalberghi amp Anderson 1999 Beaver et al 2001) In order to ensure that euthanasia is carried out humanely itis generally recommended that unconsciousness should beinduced as quickly as possible should occur prior to the lossof motor function and that no signs of pain distress or panicshould be observed (CCAC 1993 IPS 1993 Close et al1997 Home Office 1997 Beaver et al 2001) For thesereasons the most commonly recommended method ofeuthanasia for laboratory primates is iv injection of barbi-turic acid derivatives (mainly sodium pentobarbitone butalso thiopentane and secobarbital) (Baskerville 1999Bearder amp Pitts 1999 Erkert 1999 Fritz et al 1999Mendoza 1999 Poole et al 1999 Visalberghi amp Anderson1999) The use of ketamine anaesthesia prior to euthanasiais also recommended to minimise necessary restraint whilstensuring that the injection is successfully administered (IPS



1993 Erkert 1999 Poole et al 1999 Beaver et al 2001Fortman et al 2002) Euthanasia of Old World monkeys byip injection of sodium pentobarbitone was recommended asan alternative to iv administration by Baskerville (1999)Close et al (1996) recommended that ip injection may besimpler to perform and the use of this route may reducestress caused by extended handling However elsewhere theip route of administration is only recommended for theadministration of relatively large volumes of non-irritantanaesthetics to laboratory rodents because other routes arepotentially less hazardous and are more easily accessibleespecially in larger animals (Morton et al 2001) Ip admin-istration is also likely to cause more pain than iv administra-tion as it is necessary to puncture the peritoneum Overdose of barbiturates causes death by inducingdescending depression of the central nervous system (CNS)starting from the cerebral cortex resulting in loss ofconsciousness and anaesthesia (Beaver et al 2001) Death inthe unconscious animal occurs as a result of cardiac arrestwhen respiratory and cardiovascular centres are depressed(Beaver et al 2001) The onset of effects occurs very rapidlyafter iv administration (Close et al 1996) and death isinduced smoothly (Beaver et al 2001) Sodium pentobarbi-tone is also cheap and is therefore very widely used (Beaveret al 2001) although in some countries it can only beobtained under licence (Close et al 1996) Howeverconcern has been expressed about the indiscriminate use ofpentobarbitone for euthanasia particularly if administeredextravascularly (Ambrose et al 2000) In pharmacological literature evidence of hyperalgesiceffects of sub-anaesthetic doses of barbituric acid and itsderivatives were first reported in humans over 40 years ago(Dundee 1960 Neal 1965) The conclusions of these studieshave been confirmed by more recent work in mice(Carmody et al 1986 Carmody et al 1991a b) rats(Ossipov amp Gebhart 1984) and in rhesus macaques (Horiet al 1984) It has been concluded that the occurrence ofhyperalgesia following administration of low doses ofbarbiturates is the result of a mechanism involving GABAreceptors in nerve cells in the spinothalamic tract (Carmodyet al 1986) It has further been concluded that at sub-anaes-thetic doses hyperalgesia is likely to occur after administra-tion of any drug which facilitates the movement of chlorideat the GABAA receptorchloride channel complex (Ewenet al 1995) Following pentobarbitone administration atdoses as low as 20 of the anaesthetic dose nociceptivesensitivity has been reported to double (Carmody et al1986) In rats ip administration of sodium pentobarbitonefor euthanasia caused writhing and induced inflammation(Wadham cited in Ambrose et al 2000) two classicallyrecognised responses to known irritants Indeed the ipadministration of any irritant chemical can cause painswelling and adhesions (Morton et al 2001) Close et al(1996) also recognise that sodium pentobarbitone can causeirritation of the peritoneum but note that irritation can bereduced by diluting the drug Following ip administration ofany drug there is some temporal delay before an efficacious

concentration of the drug reaches the blood (although afterip administration absorption is relatively rapid) Thus for alimited period of time (dependent on dosage and size ofanimal) after ip administration of sodium pentobarbitonethe concentration of the drug in the blood will be below thatrequired to induce anaesthesia This will be exacerbated bydilution of the drug Thus ip administration of sodiumpentobarbitone has the potential to cause pain or irritation asa direct result of the injection and also to cause hyperalgesiaas a result of sub-anaesthetic doses of the drug reaching theblood The same argument is true if iv administration of thedrug is attempted but some or all of the dose is adminis-tered outwith the blood vessel as a result of poor restrainthandling or technique This evidence supports the assertionthat sodium pentobarbitone should be only be administeredintravenously and that ketamine anaesthesia should be usedto facilitate its administration (IPS 1993 Erkert 1999 Pooleet al 1999 Beaver et al 2001 Fortman et al 2002)Chloral hydrate and T-61 are euthanatising agents whichcould be used to induce death in laboratory primatesHowever chloral hydrate induces unconsciousness moreslowly than barbiturates increasing the potential require-ment for restraint and resulting in more muscular spasms(Beaver et al 2001) T-61 is a combination of an anaesthetica hypnotic and a curariform drug There is some debateconcerning the possibility that this agent may induce respi-ratory arrest before unconsciousness and for this reason thedrug is banned in the USA (Beaver et al 2001) Howeverthere is also evidence that loss of consciousness and muscleactivity occur simultaneously (Hellebrekers et al 1990)making the use of the drug more acceptable Thus T-61 isstill available in Canada and Europe (except Sweden) and isnot restricted by the same controls as barbiturates making itmore easily available (Close et al 1996) T-61 must beadministered intravenously and care must be taken not toinject the agent too quickly as this can cause pain (Beaveret al 2001) The disadvantages of the use of chloral hydrateand T-61 are generally considered to exceed those of barbi-turates (Beaver et al 2001) and sodium pentobarbitoneremains the most acceptable method of euthanasia (Closeet al 1996)When specific tissue samples are required for the experi-mental technique inhalation anaesthesia followed by exsan-guination and perfusion has been reported to be anacceptable method of euthanasia of primates (Close et al1997) Refinement of inhalation anaesthesia for primates isdiscussed in detail by Morton et al (2001) and issummarised in the lsquoadministrationrsquo and lsquorestraintrsquo sectionsabove The procedure may require short-term restrainteither manually or in a restraint chair and the use ofspecialised masks and anaesthesia equipment is necessaryAnaesthetic induction following inhalation of an anaestheticagent is slower than that induced by direct administrationinto the blood (Beaver et al 2001) Volatile inhalant anaes-thetics including halothane isoflurane desflurane andenflurane may be used for euthanasia and induce uncon-sciousness reliably and rapidly (Close et al 1996) Death is



caused by cardiovascular and respiratory centre depressionand liver metabolism of isoflurane and enflurane is onlyslight making them particularly useful in toxicology studies(Close et al 1996) However some of these agents smellstrongly and may cause breath holding thus delaying onsetof anaesthesia (Beaver et al 2001) Further evidencesuggests that both halothane and isoflurane can cause hepa-totoxicity especially if used repeatedly in close succession(De Groot amp Noll 1983 Brunt et al 1991) but this shouldnot affect their use as euthanatising agents The stress of thismethod of euthanasia may be minimised if the animals arecompetently handled and carefully trained to accept therequired restraint mask and the sound of rapidly flowinggas (Morton et al 2001) Adequate training may take up tothree weeks and unless the animals are trained to accept theprocedure for other experimental purposes the financial andtime investment in training for the one-off procedure ofeuthanasia is likely to be considered too high to be feasibleAfter anaesthesia has been induced careful monitoring isessential to ensure that an adequate level of anaesthesia ismaintained until death from exsanguination is confirmedThe use of this procedure should be limited to circum-stances where the collection of perfused tissue is absolutelynecessary for the attainment of the experimental objectiveThe training of staff is essential to achieve euthanasia bythis method

Section summarybull Euthanasia of laboratory-housed primates is required if theanimals experience pain or suffering beyond that necessaryto reach the agreed objective of the study and in some casesat the end of experimentsbull European Directive (86609EEC) (EU 1986) requiresthat the most humane methods of euthanasia are used for allanimals used in science but does not specify acceptablemethodsbull The administration of the euthanatising agent should resultin rapid loss of consciousness before death ensues Thereshould be no evidence of pain or distressbull Overdose of anaesthetic is the only method consideredacceptable for primates Ketamine can be used to minimisethe need for restraint during administrationbull Iv but not ip administration of barbiturates is recom-mendedbull Chloral hydrate and T-61 are not recommendedbull Inhalation of anaesthetics is used in studies whereperfused tissue must be collected but is not recommendedfor routine usebull Stress prior to euthanasia may be minimised by trainingprimates to co-operate with relocation to the proceduresroom and with the process of restraintbull Staff administering euthanatising agents must beextremely competent for their own and the animalsrsquo safety

Re-homing ndash an alternative to euthanasiaRe-homing or retirement is an alternative to indiscriminateeuthanasia of laboratory-housed primates on completion of

non-lethal studies or when their existence in the laboratoryis no longer required As the use of great apes in researchhas become unacceptable in Europe retirement homes havebeen found for all the laboratory-housed chimpanzees thatwere used in Europe Indeed most of the emphasis on re-homing primates has centred upon this species (Brent et al1997 Brent 2004) Re-homing of other species of labora-tory-housed primate is also becoming an option (Seelig ampTruitt 1999) Once the decision to re-home the animals hasbeen made it is imperative that the suitability of potentialhomes is rigorously assessed to ensure that standards ofhousing husbandry staff training and veterinary care aregood and that the financial stability of the establishment issecure There is no European legislation to protect re-homedprimates although each country may protect the animals innational legislation For example in the UK any person orpersons keeping animals listed under the Dangerous WildAnimals Act 1976 must have a licence The licence is onlygranted if the licensing authority is satisfied that thepremises and potential licensee are suitable The animals arealso protected under the Protection of Animals Act 1911 andthe Protection of Animals (Scotland) Act 1913 which makesit illegal to carry out acts of cruelty towards animals If asanctuary intends to allow members of the public to visitthey must also be licensed under the Zoo Licensing Act1981 and are subject to assessment by zoo inspectors andthe animals should be kept in accordance with the Standardsof Modern Zoo Practice European Directive 199922ECsimilarly requires that if animals kept in captivity are to beviewed by the public they must be licensed and require thatthe animals are kept in accordance with conservation strate-gies and housing conditions As the number of primatesretired to sanctuaries from research establishments growsthe need to regulate sanctuaries more carefully is alsoincreasing (Seelig amp Truitt 1999)

Conclusions and animal welfare implicationsRefinement of the use of primates in scientific procedurescan be achieved by avoiding alleviating or minimising theadverse effects of experimental procedures and bymaximising well-being This document provides a sampleof the types of refinements that could be incorporated intoroutine and experimental protocols With careful use basedupon knowledge of species-specific needs these refine-ments have the potential to greatly improve the welfare oflaboratory-housed primates whilst greatly reducing inhu-manity in accordance with Russell and Burchrsquos 3Rrsquos model(Russell amp Burch 1992)

AcknowledgementsWe thank Coenraad Hendriksen Mark Prescott DavidMorton for their valuable comments on a draft of this paperAnna Roberts for all her help and two anonymous refereeswhose comments substantially improved the manuscriptAER and HMB-S were supported by a grant from theEuropean Commission number QLRT-2001-00028



ReferencesAdams MR Kaplan JR Manuck SB Uberseder B andLarkin KT 1988 Persistent sympathetic nervous system arousalassociated with tethering in long-tailed macaques LaboratoryAnimal Science 38 279-281Ambrose N Wadham J and Morton DB 2000 Refinement ofeuthanasia In Balls M van Zeller AM Halder ME (eds) Progress inthe reduction refinement and replacement of animal experimentationpp 1159-170 Elsevier Science Ltd Oxford UKAnon 2001 Marking monkeys Nyanzol D Laboratory PrimateNewsletter 40 5Anzenberger G and Gossweiler H 1993 How to obtain indi-vidual urine samples from undisturbed marmoset familiesAmerican Journal of Primatology 31 223-230Atzpodien E Mehdi N Clarke D and Radhoferwelte S1997 Subacute and chronic oral toxicity of lornoxicam incynomolgus monkeys Food and Chemical Toxicology 35 465-474Aureli F Preston SD and de Waal FBM 1999 Heart rateresponses to social interactions in free-moving rhesus macaques(Macaca mulatta) A pilot study Journal of Comparative Psychology133 59-65Barrie SA 1992 Heidelberg pH capsule gastric analysis InPizzorno JE Murray MT amp Barrie SA (eds) A Textbook of NaturalMedicine JBC Publications Seattle USABaskerville M 1999 Old world monkeys In Poole T (ed) TheUFAW handbook on the care and management of laboratory animalspp 611-635 Blackwell Science Oxford UKBearder SK and Pitts RS 1999 Prosimians In Poole T (ed) TheUFAW handbook on the care and management of laboratory animalspp 542-558 Blackwell Science Oxford UKBeaver BV Reed W Leary S McKiernan B Bain FSchultz R Bennett BT Pascoe P Shull E Cork LCFrancis-Floyd R Amass KD Johnson R Schmidt RHUnderwood W Thornton GW and Kohn B 2001 2000Report of the AVMA panel on euthanasia Journal of the AmericanVeterinary Medical Association 218 669-696Blank MS Gordon TP amp Wilson ME 1983 Effects of captureand venipuncture on serum levels of prolactin growth-hormoneand cortisol in outdoor compound- housed female rhesus-mon-keys (Macaca mulatta) Acta Endocrinologica 102 190-195Bowers CL Crockett CM and Bowden DM 1998Differences in stress reactivity of laboratory macaques measuredby heart period and respiratory sinus arrhythmia American Journalof Primatology 45 245-261Bradley BJ Doran D Robbins MM Williamson E Boesch Cand Vigilant L 2002 Comparative analyses of genetic social struc-ture in wild gorillas (Gorilla gorilla) using DNA from feces and hairAmerican Journal of Physical Anthropology Supplement 34 47-48Brent L 2004 Solutions for research primates Lab Animal 33 37-43Brent L Butler TM and Haberstroh J 1997 Surplus chim-panzee crisis planning for the long-term needs of research chim-panzees Lab Animal 26 36-39Brockway BP Hassler CR and Hicks N 1993 Minimizingstress during physiological monitoring Scientists Centre for AnimalWelfare (SCAW) Bethesda USABrodie DA and Hanson HM 1966 Restraint induced gastriclesions Journal of Industrial Medicine 12 5601-5606Brunt EM White H Marsh JW Holtmann B and PetersMG 1991 Fulminant hepatic-failure after repeated exposure toisoflurane anesthesia - a case-report Hepatology 13 1017-1021

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Crockett CM Bowers CL Sackett GP and Bowden DM1993 Urinary cortisol responses of long-tailed macaques to 5 cagesizes tethering sedation and room change American Journal ofPrimatology 30 55-74Crofts HS Wilson S Muggleton NG Nutt DJ Scott EAMand Pearce PC 2001 Investigation of the sleep electrocor-ticogram of the common marmoset (Callithrix jacchus) usingradiotelemetry Clinical Neurophysiology 112 2265-2273Cross N Pines MK and Rodgers LJ 2004 Saliva sampling to assesscortisol levels in unrestrained common marmosets and the effect ofbehavioural stress American Journal of Primatology 62 107-114Cussler K Morton DB and Hendriksen CFM 1999 Humaneendpoints in vaccine research and quality control In HendriksenCFM and Morton DB (eds) Humane endpoints in animals experi-ments for biomedical research pp 95-101 Proceedings of theInternational Conference Royal Society of Medicine PressLondon UKDe Groot H and Noll T 1983 Halothane hepatotoxicity rela-tion between metabolic activation hypoxia covalent binding lipidperoxidation and liver cell damage Hepatology 3 601-606Dexter S and Bayne K 1994 Results of providing swings toindividually housed rhesus monkeys Laboratory Primate Newsletter33 9-12Dundee JW 1960 Alterations in response to somatic pain asso-ciated with anaesthesia II the effect of thiopentone and pentobar-bitone British Journal of Anaesthesia 32 407-414Einstein R Rowan C Billing R and Lavidis N 2000 The useof telemetry to refine experimental technique In Balls M vanZeller AM amp Halder ME (eds) Progress in the reduction refinementand replacement of animal experimentation pp 1187-1197 ElsevierScience Ltd Oxford UKErkert HG 1999 Owl monkeys In Poole T (ed) The UFAWhandbook on the care and management of laboratory animals pp 574-590 Blackwell Science Oxford UKEuropean Commission (EC) 1986 European Convention forthe protection of vertebrate animals used for experimental andother scientific purposes ETS 123 Strasbourg FranceEuropean Union (EU) 1986 Council Directive 86609EECParis FranceEwen A Archer DP Samanani N and Roth SH 1995Hyperalgesia during sedation effects of barbiturates and propofolin the rat Canadian Journal of Anaesthesia 42 532-540Farah IO Kariuki TM King CL and Hau J 2001 An overviewof animal models in experimental schistosomiasis and refinementsin the use of non-human primates Laboratory Animals 35 205-212Fentener van Vlissingen JM Kuijpers MHM van OostrumECM Beems RB and van Dijk JE 1999 Retrospective evalua-tion of clinical signs pathology and related discomfort in chronicstudies In Hendriksen CFM and Morton DB (eds) Humane end-points in animals experiments for biomedical research pp 89-94Proceedings of the International Conference Royal Society ofMedicine Press London UKFlecknell P 1996 Laboratory Animal Anaesthesia Academic PressLondon UKFlecknell P 2000 Refinement of laboratory animal anaesthesiaIn Balls M van Zeller AM amp Halder ME (eds) Progress in the reduc-tion refinement and replacement of animal experimentation pp 1151-1158 Elsevier Science Ltd Oxford UKFlecknell P and Waterman-Pearson A 2001 PainManagement in Animals WB Saunders London UKFortman JD Hewett TA and Taylor-Bennet B 2002 Thelaboratory non-human primate CRC Press Ltd Florida USA

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Hoffman RA George GP and Barrows WF 1969 Light syn-chronization of deep-body temperature rhythms in Macacanemestrina American Journal of Physiology 217 1487-1489Home Office 1997 Code of practice for the humane killing of ani-mals under Schedule 1 to the Animals (Scientific Procedures) Act 1986HMSO Cambridge UKHori Y Lee KH Chung JM Endo K and Willis WD 1984The effects of small doses of barbiturate on the activity of primatenociceptive tract cells Brain Research 307 9-15Horii I Kito G Hamada T Jikuzono T Kobayashi K andHashimoto K 2002 Development of telemetry system in thecommon marmoset - cardiovascular effects of astemizole andnicardipine Journal of Toxicological Sciences 27 123-130International Primatological Society (IPS) 1993 IPSInternational guidelines for the aquisition care and breeding of non-human primates Poole TB and Schwibbe M (eds) Erich GoltzeGmbH and Co KG Gottingen GermanyKaufman L and Detweiler DK 1999 A method for recordingelectrocardiograms in conscious unrestrained cynomolgus mon-keys with emphasis on maximisation of T-wave amplitudeToxicology Methods 9 285-292Kelley TM and Bramblett CA 1981 Technical Note Urinecollection from vervet monkeys by instrumental conditioningAmerican Journal of Primatology 1 95-97Kerl J 1997 Telemetrically recorded second degree heart blockin a common marmoset Laboratory Primate Newsletter 36 9-11Kerl J and Rothe H 1996 Influence of cage size and cage equip-ment on physiology and behaviour of common marmosets(Callithrix jacchus) Laboratory Primate Newsletter 35 10-13Kinter LB and Johnson DK 1999 Remote monitoring of exper-imental endpoints in animals using radiotelemetry and bioimped-ance technologies In Hendriksen CFM and Morton DB (eds)Humane endpoints in animals experiments for biomedical researchpp 58-65 Proceedings of the International Conference RoyalSociety of Medicine Press London UKKlein HJ and Murray KA 1995 Restraint In Bennett B T (ed)Non-human Primates in Biomedical Research pp 286-297 AcademicPress San Diego USAKramer K Kinter L Brockway BP Voss HP Remie R andvan Zutphen BLM 2001 The use of radiotelemetry in small lab-oratory animals recent advances Contemporary Topics inLaboratory Animal Science 40 8-16Landi MS Kissinger JT Campbell SA Kenney CA andJenkins EL 1990 The effects of four types of restraint onserum alanine aminotransferase and aspartate aminotrans-ferase in the Macaca fascicularis Journal of the American Collegeof Toxicology 9 517-523Line SW Clarke AS and Markowitz H 1987 Plasma cortisolof female rhesus monkeys in response to acute restraintLaboratory Primate Newsletter 26 1-4Lipnick RL Cotruvo JA Hill RN Bruce RD Stitzel KAWalker AP Chu I Goddard M Segal L Springer JA andMyers RC 1995 Comparison of the up-and-down conventionalLD50 and fixed-dose acute toxicity procedures Food and ChemicalToxicology 33 223-231Luttrell L Acker L Urben M and Reinhardt V 1994 Traininga large troop of rhesus macaques to cooperate during catchinganalysis of the time investment Animal Welfare 3 135-140Lutz CK Tiefenbacher S Jorgensen MJ Meyer JS andNovak MA 2000 Techniques for collecting saliva from awakeunrestrained adult monkeys for cortisol assay American Journal ofPrimatology 52 93-99

Marks D Kelly J Rice T Ames S Marr R Westfall JLloyd J and Torres C 2000 Utilizing restraint chair training toprepare primates for social housing Laboratory Primate Newsletter39 9-10Matsuzawa T and Hasegawa Y 1983 Food aversion learningin Japanese monkeys (Macaca fuscata) A strategy to avoid nox-ious food Folia Primatologica 40 247-255McGuffey LH McCully CL Bernacky BJ and Blaney SM2002 Incorporation of an enrichment program into a study proto-col involving long-term restraint in macaques Lab Animal 31 37-39McKinley J Buchanan-Smith HM Bassett L and Morris K2003 Training common marmosets (Callithrix jacchus) to cooperateduring routine laboratory procedures Ease of training and timeinvestment Journal of Applied Animal Welfare Science 6 209-220Mench J 1999 Defining endpoints The role of animal care com-mittees In Hendriksen CFM and Morton DB (eds) Humane end-points in animals experiments for biomedical research pp 133-138Proceedings of the International Conference Royal Society ofMedicine Press London UKMendoza SP 1999 Squirrel monkeys In Poole T (ed) The UFAWhandbook on the care and management of laboratory animals pp 591-600 Blackwell Science Oxford UKMirmiran M Bernardo L Jenkins SL Ma XH Brenna JTand Nathanielsz PW 2001 Growth neurobehavioural and cir-cadian rhythm development in newborn baboons PediatricResearch 49 673-677Mojaverian P 1996 Evaluation of gastrointestinal pH and gastricresidence time via the Heidelberg radiotelemetry capsule phar-maceutical application Drug Development Research 38 73-85Morrow-Tesch JL McGlone JJ and Norman RL 1993Consequences of restraint stress on natural-killer-cell activitybehavior and hormone levels in rhesus macaques (Macaca mulat-ta) Psychoneuroendocrinology 18 383-395Morton DB 1999 Humane endpoints in animal experimentationfor biomedical research ethical legal and practical aspects InHendriksen CFM and Morton DB (eds) Humane endpoints in ani-mals experiments for biomedical research pp 5-12 Proceedings ofthe International Conference Royal Society of Medicine PressLondon UKMorton DB 2000 A systematic approach for establishing humaneendpoints Institute of Laboratory Animal Research Journal 41 80-87Morton DB Abbot D Barclay R Close BS Ewbank RGask D Heath M Mattic S Poole T Seamer J SoutheeJ Thompson A Trussell B West C and Jennings M 1993Removal of blood from laboratory animals and birds LaboratoryAnimals 27 1-22Morton DB and Griffiths PHM 1985 Guidelines on the recog-nition of pain distress and discomfort in experimental-animals andan hypothesis for assessment Veterinary Record 116 431-436Morton DB Hawkins P Bevan R Heath K Kirkwood JPearce P Scott L Whelan G and Webb A 2003 Refinementsin telemetry procedures Laboratory Animals 37 261-299Morton DB Jennings M Buckwell A Ewbank R GodfreyC Holgate B Inglis I James R Page C Sharman IVerschoyle R Westall L and Wilson AB 2001 Refining pro-cedures for the administration of substances Laboratory Animals35 1-41Morton WR Knitter GH Smith PM Susor TG andSchmitt K 1987 Alternatives to chronic restraint of nonhumanprimates Journal of the American Veterinary Medical Association191 1282-1286



Moseley JR and Davis JA 1989 Psychological enrichment tech-niques and New World monkey restraint device reduce colonymanagement time Laboratory Animals 18 31-33Neal MJ 1965 The hyperalgesic action of barbiturates in miceBritish Journal of Pharmacology 24 170-177Norman RL McGlone J and Smith CJ 1994 Restraint inhibitsluteinizing hormone secretion in the follicular phase of the men-strual cycle in rhesus macaques Biology of Reproduction 50 16-26Norman RL and Smith CJ 1992 Restraint inhibits luteinizinghormone and testosterone secretion in intact male rhesusmacaques effects of concurrent naloxone administrationNeuroendocrinology 55 405-415Novak MA 2003 Self-injurious behavior in rhesus monkeys Newinsights into its etiology physiology and treatment AmericanJournal of Primatology 59 3-19OECD (Organisation for Economic Co-operation andDevelopment) 2000 Guidance document on recognitionassessment and use of clinical signs as humane endpoints OECDHealth and Safety Publications Paris FranceOECD (Organisation for Economic Co-operation andDevelopment) 2001 Guidance document on acute oral toxicitytesting No 24 OECD Environment Health and SafetyPublications Paris FranceOlfert ED and Godson DL 2000 Humane endpoints for infec-tious disease animal models Institute of Laboratory Animal ResearchJournal 41 99-104Olfert ED Godson D and Habermehl M 1998 Endpoints ininfectious disease animal models In Pain Management andHumane Endpoints Proceedings of a workshop National Academyon Animal Sciences Washington DC USAOssipov MH and Gebhart GF 1984 Light pentobarbital anes-thesia diminishes the antinociceptive potency of morphine admin-istered intracranially but not intrathecally in the rat EuropeanJournal of Pharmacology 97 137-140Pare WP and Glavin GB 1986 Restraint stress in biomedical-research - a review Neuroscience and Biobehavioral Reviews 10 339-370Philipp C 1996 Operant conditioning with the great apesProceedings of the National Conference of the AmericanAssociation of Zoo Keepers 22 156-163Poole T Hubrecht R and Kirkwood JK 1999 Marmosets andtamarins In Poole T (ed) The UFAW handbook on the care andmanagement of laboratory animals pp 559-574 Blackwell ScienceOxford UKPoon R and Chu I 1999 Urinary biomarkers as humane end-points in toxicological research Hendriksen CFM and Morton DBHumane endpoints in animals experiments for biomedical researchProceedings of the International Conference Royal Society ofMedicine Press London UKPriest GM 1991 Training a diabetic drill (Mandrillus leucophaeus)to accept insulin injections and venipuncture Laboratory PrimateNewsletter 30 1-4Reinhardt V 1989 Evaluation of long-term effectiveness of twoenvironmental enrichment objects for singly caged rhesusmacaques Lab Animal 18 365-369Reinhardt V 1992a Improved handling of experimental rhesusmonkeys In Davis H and Balfour AD (eds) The Inevitable Bondexamining scientist-animal interactions pp 171-177 CambridgeUniversity Press Cambridge UKReinhardt V 1992b Voluntary progression order in captive rhe-sus macaques Zoo Biology 11 61-66Reinhardt V 1997 Training non-human primates to cooperateduring blood collection A review Animal Technologist 48 55-73

Reinhardt V Cowley D Eisele S and Scheffler J 1991Avoiding undue responses to venipuncture in adult male rhesusmacaques Animal Technology 42 83-86Reinhardt V Cowley D Scheffler J Vertein R andWegner F 1990 Cortisol response of female rhesus-monkeys tovenipuncture in homecage versus venipuncture in restraint appa-ratus Journal of Medical Primatology 19 601-606Reinhardt V Liss C and Stevens C 1995 Restraint methodsof laboratory non-human primates a critical review AnimalWelfare 4 221-238Rennie AE and Buchanan-Smith HM 2005 Report on theextent and character of primate use in scientific proceduresacross Europe in 2001 Laboratory Primate Newsletter 44 6-12Rennie AE and Buchanan-Smith HM 2006a Refinement ofthe use of non-human primates in scientific research part I Theinfluence of humans Animal Welfare 15 203-213Rennie AE and Buchanan-Smith HM 2006b Refinement ofthe use of non-human primates in scientific research part IIHousing husbandry and acquisition Animal Welfare 15 215-238Rensing S 1999 Immobilization and anaesthesia of nonhumanprimates Primate Report 55 33-38Richmond J 1999 Criteria for humane endpoints In HendriksenCFM and Morton DB (eds) Humane endpoints in animals experi-ments for biomedical research pp 16-32 Proceedings of theInternational Conference Royal Society of Medicine PressLondon UKRobinson EL and Fuller CA 1999a Endogenous thermoregula-tory rhythms of squirrel monkeys in thermoneutrality and coldAmerican Journal of Physiology 276 R1397-R1407Robinson EL and Fuller CA 1999b Light masking of circadianrhythms of heat production heat loss and body temperature insquirrel monkeys American Journal of Physiology 276 R298-R307Russell WMS and Burch RL 1992 The principles of humaneexperimental technique Universities Federation for AnimalWelfare Wheathampstead Herts UKSainsbury AW Eaton BD and Cooper JE 1989 Restraint andanaesthesia of primates Veterinary Record 125 640-643Sass N 2000 Humane endpoints and acute toxicity testingInstitute of Laboratory Animal Research Journal 41 114-123Sauceda R and Schmidt MG 2000 Refining macaque handlingand restraint techniques Lab Animal 29 47-49Savastano G Hanson A and McCann C 2003 The develop-ment of an operant conditioning training program for the NewWorld primates at the Bronx zoo Journal of Applied AnimalWelfare Science 6 247-261Scharmann W 1999 Physiological and ethological aspects of theassessment of pain distress and suffering In Hendriksen CFM andMorton DB (eds) Humane endpoints in animals experiments for bio-medical research pp 33-39 Proceedings of the InternationalConference Royal Society of Medicine Press London UKSchlede E Diener W and Gerner I 1999 Humane endpointsin toxicity testing In Hendriksen CFM and Morton DB (eds)Humane endpoints in animals experiments for biomedical researchpp 75-78 Proceedings of the International Conference RoyalSociety of Medicine Press London UKSchlede E Gerner I and Diener W 2000 The use of humane end-points in acute oral toxicity testing In Balls M van Zeller AM HalderME (eds) Progress in the reduction refinement and replacement of animalexperimentation pp 907-914 Elsevier Science Ltd Oxford UKSchnell CR and Gerber P 1997 Training and remote monitor-ing of cardiovascular parameters in non-human primates PrimateReport 49 61-70



Schnell CR and Wood JM 1993 Measurement of blood pres-sure and heart rate by telemetry in conscious unrestrained mar-mosets American Journal of Physiology 264 H1509-H1516Schwartzkopf-Genswein KS Stookey JM Crowe TG andGenswein BMA 1998 Comparison of image analysis exertionforce and behaviour measurements for use in the assessment ofbeef cattle responses to hot iron and freeze branding Journal ofAnimal Science 76 972-979Schwartzkopf-Genswein KS Stookey JM de Passille AMand Rushen J 1997a Comparison of hot-iron and freeze brand-ing on cortisol levels and pain sensitivity in beef cattle CanadianJournal of Animal Science 77 369-374Schwartzkopf-Genswein KS Stookey JM and Welford R1997b Behaviour of cattle during hot-iron and freeze branding andthe effects on subsequent handling ease Journal of Animal Science75 2064-2072Scott L 1990 Training non-human primates meeting their behav-ioural needs In UFAW (ed) Animal Training a review and commen-tary pp 129-133 Universities Federation for Animal WelfareWheathampstead Herts UKScott L Pearce P Fairhall S Muggleton N and Smith J2003 Training non-human primates to co-operate with scientificprocedures in applied biomedical research Journal of AppliedAnimal Welfare Science 6199-208Seelig D and Truitt A 1999 Post research retirement of monkeysand other non-human primates Laboratory Primate Newsletter 38 1-4Sherwin RE Haymond S Striklan D and Olsen R 2002Freeze-branding to permanently mark bats Wildlife Society Bulletin30 97-100Smith OA Astley CA Spelman FA Golanov EV BowdenDM Chesney MA and Chalyan V 2000 Cardiovascularresponses in anticipation of changes in posture and locomotionBrain Research Bulletin 53 69-76Smith TE McGreer-Whitworth B and French JA 1998Close proximity of the heterosexual partner reduces the physio-logical and behavioral consequences of novel-cage housing inblack tufted-ear marmosets (Callithrix kuhli) Hormones andBehavior 34 211-222Stark RI Garland M Daniel SS Tropper P and Myers MM1999 Diurnal rhythms of fetal and maternal heart rate in thebaboon Early Human Development 55 195-209Stokes WS 2000a Reducing unrelieved pain and distress in lab-oratory animals using humane endpoints Institute of LaboratoryAnimal Research Journal 41 59-61Stokes WS 2000b Humane endpoints for laboratory animalsused in toxicity testing In Balls M van Zeller AM Halder ME(eds) Progress in the reduction refinement and replacement of animalexperimentation pp 897-906 Elsevier Science Ltd Oxford UK

Stone AM Bloomsmith MA Laule GE and Alford PL1994 Documenting positive reinforcement training for chim-panzee urine collection American Journal of Primatology 33 242(Abstract)Tamada J Bohannon N and Potts R 1995 Measurement ofglucose in diabetic subjects using non-invasive transdermal extrac-tion Nature Medicine 1 1198-1201Taylor L Emerson C and Wagner JL 1993 Implantablemicrochips as a means of identifying non-human primatesAmerican Association of Zoological Parks and Aquariums RegionalProceedings pp 248-253Visalberghi E and Anderson JR 1999 Capuchin monkeysIn Poole T (ed) The UFAW handbook on the care and manage-ment of laboratory animals pp 601-610 Blackwell ScienceOxford UKWall HS Worthman C and Else JG 1985 Effects of ketaminestress and repeated bleeding on the haematology of vervet mon-keys Laboratory Animals 19 138-144Wallace J 1999 Humane endpoints in cancer research InHendriksen CFM amp Morton DB (eds) Humane endpoints in animalsexperiments for biomedical research pp 79-84 Proceedings of theInternational Conference Royal Society of Medicine PressLondon UKWallace J 2000 Humane endpoints and cancer research Instituteof Laboratory Animal Research Journal 41 87-93Wheatley JL 2002 A gavage dosing apparatus with flexiblecatheter provides a less stressful gavage technique in the rat LabAnimal 31 53-56Wheeler MD Schutzengel RE Barry S and Styne DM1990 Changes in basal and stimulated growth-hormone secretionin the aging rhesus-monkey - a comparison of chair restraint andtether and vest sampling Journal of Clinical Endocrinology andMetabolism 71 1501-1507Wolfensohn SE 1993 The use of microchip implants in identifi-cation of two species of macaque Animal Welfare 2 353-359Wolfensohn S and Honess P 2005 Handbook of primate hus-bandry and welfare Blackwell Oxford UKWong JH 1998 Canadian Council on Animal Care Guidelines onchoosing an appropriate endpoint in experiments using animals inresearch teaching and testing In Pain Management and HumaneEndpoints Proceedings of a workshop National Academy onAnimal Sciences Washington DC USAYasuda M Wolff J and Howard Jr CF 1988 Effects of physi-cal and chemical restraint on intravenous glucose tolerance test incrested black macaques (Macaca nigra) American Journal ofPrimatology 15 171-180



a requirement of the European Directive (EU 1986) Manyresearchers and animal care workers are able to identifyindividuals by facial features and behavioural characteris-tics (Poole et al 1999) Identification of individuals by thismethod can be carried out with a minimum of interferenceHowever the International Primatological Society (IPS)guidelines (1993) recommend that a permanent identifica-tion mark should be used so that every individual can beidentified and matched to his or her medical and researchrecords This is necessary because members of staff atresearch premises may change the care of individuals maybe transferred between staff either on the same site or ondifferent sites and because welfare inspectors must be ableto accurately match individuals with records Marking ofanimals is not considered to be an experimental procedureprovided that it is carried out by the most humane availablemethod and therefore does not have to be regulated bydesignated authorities (EU 1986) Three permanentmethods of marking have been used in laboratory primateswith varying success

Freeze brandingFreeze branding produces scar tissue and therefore whitehair growth by the application of a small branding ironcooled to around -60ordmC using dry ice or a dry ice ethanolmixture to the skin (Sherwin et al 2002) The method hasbeen used in vervet monkeys (Cercopithecus aethiops)(Griffin 1988) In cattle the method results in lower cortisoland fewer behavioural responses than hot brandingalthough the response is still greater than that in controlanimals (Schwartzkopf-Genswein et al 1997a b 1998)However hyperalgesia was produced at the site of brandingfor 7 days after treatment In order to reduce pain and tissuesensitivity sedation and local anaesthetic should be usedduring the procedure and a programme of analgesia tocontrol pain following the procedures should be consideredIn other species freeze branding produces clearly recognis-able marking however in primates difficulty in seeing thewhite brand hair in long-haired species has meant that themethod is not commonly used (Fortman et al 2002) and isnot recommended

TattooingTattooing is widely recommended in laboratory husbandryguidelines (The Canadian Council on Animal Care [CCAC]1993 IPS 1993 Baskerville 1999 Bearder amp Pitts 1999Erkert 1999 Fritz et al 1999 Visalberghi amp Anderson 1999Mendoza 1999) It is generally recommended that the tattooshould be placed in a largely hairless area of skin butdifferent areas have been recommended for differentspecies For example the inside of the ear or lips is recom-mended for prosimian species (Bearder amp Pitts 1999)whilst the inner thigh is recommended for chimpanzees(Pan troglodytes) (Fritz et al 1999) and squirrel monkeys(Saimiri spp) (Mendoza 1999) and the inner thigh or thechest is recommended for Old World monkeys (Baskerville1999) One of the main disadvantages of the technique isthat the tattoo can be difficult to read at a distance althoughthis depends on its positioning and skin pigmentation

Tattoos can even be difficult to read when the animal hasbeen captured particularly as the animal gets older as theink can fade as the animal grows (Fortman et al 2002) Thetattoo may therefore have to be renewed periodically toensure that identities are not confused (Fritz et al 1999)However it is noted that in many toxicological studies theprimates are used and killed before the fading of the tattoobecomes as issue In order to ensure the accuracy of thetattoo mark and to minimise pain the pain and distress of theprocedure all primates should be anaesthetised fortattooing Subsequently the animal must be monitored toensure that any adverse effects of the identificationprocedure are detected early on However microchips are arefinement of this identification technique

Microchip identificationSince the early 1990s subcutaneously implantedmicrochips holding unique codes for each individual havebeen used to permanently identify primates Thesemicrochips are widely used in pet animals The unique indi-vidual code on the microchip must be read using a scannerFollowing sedation the microchip is inserted under the skinusing a specially designed hypodermic needle usuallysupplied ready loaded with a microchip Insertion cantherefore be achieved very quickly The microchip may beplaced under the interscapular skin or behind the ear (Tayloret al 1993) but placement under the skin at the elbow orwrist has proven to be the most accessible for scannerreading in both long-tailed (Macaca fascicularis) andrhesus (Macaca mulatta) macaques (Wolfensohn 1993)Microchip identification has been recommended for allspecies of primates There are disadvantages to the use ofmicrochips for identification Firstly the microchips andscanning equipment are expensive and thus may signifi-cantly add to the cost of an experiment Further asmicrochips are different in Europe and the USA andCanada different readers are required Secondly themicrochips can only be picked up by the scanner when thescanner wand is around 5-10 cm away from the chip (Pooleet al 1999) Although primates can be trained to stay stillwhilst the microchip is scanned (Savastano et al 2003)untrained animals may need to be restrained in order toobtain a successful reading Finally migration of themicrochip under the skin is possible and may reduce theefficiency of identification although chips usually moveonly a short distance (approx 5 cm) (Wolfensohn 1993)

Temporary identificationBecause of difficulties with reading tattoos and microchipsthe combination of a permanent method of identificationwith a more easily distinguishable temporary method ofidentification is often used (Fortman et al 2002) Theprovision of a means of identifying the animal easily andimmediately enables the caregiver to quickly determinewhich animals they are observing and therefore todetermine whether the behaviour of that animal is normalfor that individual thus facilitating welfare assessmentEasy identification also facilitates the development of rela-tionships between the caregiver and the animal which is
































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refinement of the use of non-human primates in scientific … · 2012. 10. 17. · primates are...

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