Guidelines Fish

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Guidelines for health and welfare monitoring of

fish used in research

R Johansen1 J R Needham12 D J Colquhoun3 T T Poppe4 and A J Smith1

1Norwegian School of Veterinary Science Laboratory Animal Unit PO Box 8146 Dep 0033 OsloNorway 2The Microbiology Laboratories North Harrow Middlesex HA2 7RE UK 3Section of FishHealth National Veterinary Institute PO Box 8156 Dep 0033 Oslo Norway 4Department of BasicSciences and Aquatic Medicine Norwegian School of Veterinary Science PO Box 8146 Dep 0033 OsloNorway

SummaryThe aim of this paper is to provide background material necessary for the development ofinternational guidelines for the health and welfare monitoring of fish used in research Itprovides an overview of present guidelines and discusses why more detailed and species-specific guidelines are needed A major issue within fish research is to document thesituation today and point out areas where improvements are needed

Keywords Fish health welfare monitoring guidelines

Guidelines for monitoring and reporting thegeneral health status and welfare of fish usedin research are sparse compared with thoseavailable for mammalian laboratoryanimals Despite the fact that there are morefish species than all other vertebrate speciescombined and that fish are studied in almostall biological disciplines (Powers 1989) most

guidelines for fish encompass all species andall types of research (Casebolt et al 1998)There is a great need for more species-specific guidelines for health and welfaremonitoring In some cases these guidelinesmay also have to be specific to the scientifictopic where they are to be used

The number of fish used in research isincreasing due both to the rapid expansionin the fish farming industry and an increased

use of fish as model organisms in basicresearch and chemical testing (Kane et al1996) The debate on whether to use fish ormice models started over 25 years ago (Daweamp Couch 1984) Rodent models are nowfrequently being replaced by fish models

(May et al 1987a Powers 1989 DeTollaet al 1995)

Guidelines and legislations are oftenmore liberal towards the use of fish thanmammals This can be illustrated by the lackof focus on humane endpoints in fish models(Ryder 2005) LD50 testing is for example nolonger allowed on mammals but remains in

use for fish (Braunbeck et al 2004)Even the reporting of numbers of fish usedand the type of research for which they areused is confused by a lack of common inter-national practice Harmonization in this fieldis important to avoid the transfer of researchfrom countries with high standards to thosewith lower ones In Europe all fish speciesand sizes are reported in the same statisticalgroups and the research disciplines reported

are very general Figure 1 shows for examplean analysis of the use of live fish in Norwayin 2004 This makes it difficult to monitorwhat fish are actually used for in research

Reporting of the health and welfare of fishused in research is often sparse (Brattelid ampSmith 2000) and may include generalstatements such as lsquoHealthy fish from a

Accepted 9 May 2006 r Laboratory Animals Ltd Laboratory Animals (2006) 40 323ndash340

REVIEW ARTICLE

Correspondence A J Smith Email adriansmithvethsno



commercial fish farm were randomlysampled for the experimentrsquo How the

health was monitored is often not statedInformation relating to health monitoringduring the experimental trial may also besparse often limited to statements such aslsquono clinical signs of disease were observedrsquoIn most trials no necropsies or tests areperformed to determine the health status

It is important to note that there is nointernational consensus on animal welfare

legislation and fish are often not included innational laws Some general guidelines formonitoring of laboratory animals may alsobe useful for fish (Grossblatt 1996) Generalconsiderations relevant to fish includeselection of the appropriate speciesidentification of the minimum number ofanimals required for valid results suitableliving conditions and the use of experiencedpersonnel including fish health specialists

The search for alternatives is as relevant infish as in mammals but in vitro methods infish research are sparse

Researchers are ethically bound to produceas much knowledge as possible from eachanimal used Lack of standardization ofparameters such as genotype water qualityand handling procedures often leads to

incomparable results and therefore theunnecessary use of large numbers of fishHarmonization of health and welfaremonitoring is one important factor in theprocess of standardization that is needed to

reduce fish numbers in researchLaboratory animal units may be accreditedby the Association for Assessment andAccreditation of Laboratory Animal Care(AAALAC) who rely on widely acceptedguidelines for the care and use of laboratoryanimals (Grossblatt 1996) Recommendationsfor health monitoring of rodents rabbitscats and other mammalian animals havebeen established by the Federation of

European Laboratory Animal ScienceAssociations (FELASA) (Rehbinder et al1998 Nicklas et al 2002) Thesepublications include lists of infectiousagents to be tested for suggested methods fortesting and a standardized report formWidely accepted guidelines for the care anduse of fish in research are however notcurrently available

The most recent and thorough guidelines

for fish are provided by the CanadianCouncil on Animal Care (httpwwwccacca Guidelines on the care and useof fish in research teaching and testing)These guidelines are general ones for all fishspecies in all types of research and providerecommendations for among other thingsfacilities management and husbandry Thesection on health monitoring focuses on

establishing programmes for diseasedetection written agreements with fishhealth professionals and the strategic use ofdisease control measures Detailed health-monitoring protocols are not outlinedalthough there is a table of clinical signs ofdisease to be monitored

Current guidelines usually leave it up tothe local fish health specialists to determinehow health and welfare should be monitored

and which fish should be allowed into theresearch facility (UFRC 2004 CCAC 2005Council of Europe 2006) In textbooks on thecare and use of classical laboratory fish suchas zebrafish health monitoring is oftenlimited to statements about the prevalenceof diseases and how they can be prevented(Ostrander 2000 Westerfield 2000) Few or

Laboratory Animals (2006) 40

324 R Johansen et al

Figure 1 A simple breakdown of the number of

live fish used for research in Norway 2004 Actualnumbers are provided in parentheses Most fish

were reported as having been used in the categories

lsquoBasic researchrsquo and lsquoResearch and Developmentrsquo

(RampD) More details of the type of testing for which

the fish were actually used cannot be obtained from

the statistics



no instructions are provided on what investi-gations should be performed before fish areapproved for research and how the findingsshould be interpreted and reported Standardi-zation of health and welfare monitoring is one

important part of the harmonization processthat is needed to obtain research results thatare comparable between research facilitiesFish health specialists need therefore inter-nationally accepted guidelines to ensure thatthe monitoring of health and welfare followscertain standards

A collection of guidelines for the care anduse of fish in research is available at httposlovetvethsnofish The aim of the present

paper is to provide an overview of currentguidelines on health and welfare monitoringtogether with a checklist of points to beconsidered when species-specific guidelinesare developed This paper will point out whythe monitoring of health and welfare in fishis so important but also illustrate howlimitations in our present knowledge makemonitoring difficult Present and futurepossibilities are discussed including the

need for specific guidelines for differentresearch disciplines

General considerations

Even if no infectious agent or disease isdetected the health status of different fishgroups may vary considerably An overviewof the phylogenetic background of the fish

including factors such as environmentalmonitoring and vaccination programmesshould be documented to provide the bestpossible picture of health status

Ideally experimental fish should be freefrom infectious agents and stress but this isseldom possible especially when usingfarmed fish Whether the presence of aninfectious agent will result in clinicaldisease is influenced by the general health

status of the fish which in turn is highlyinfluenced by factors in the environmentsuch as water temperature and oxygen levels(Figure 2) Subclinical infections are quitecommon in fish and the effects of suchinfections on experimental results are oftennot known The question is often notwhether an infectious agent is present or

not but how it influences the health of thefish and thereby the result of the trial Even

if the effect of a health problem infectiousagent or suboptimal condition is unknownthey should be reported to provide a totalpicture of the research situation This isimportant for comparison and interpretationof research results

Due to the lack of non-invasive testmethods health monitoring in fish isnormally performed on populations It is

therefore important to obtain representativesamples of the population If the prevalenceof the disease is low andor the sensitivity ofthe diagnostic test is low the number of fishthat have to be killed for examination maybecome too high to perform the test beforethe onset of the trial Testing of experimentalfish must therefore often be done at the endof the trial and interpretation of the resultsmay then be difficult In the worst case the

examination may show that the fish alreadyused in the trial were not suitable and theresearch results invalid

There is no doubt that the health statusand wellbeing of the research animal mayhave a major effect on research results(Melby amp Balk 1983) A history of theanimalsrsquo health and welfare throughout their


Health and welfare monitoring of fish 325

Figure 2 The relationships between fish patho-

gens and the environment Some pathogens may

cause a disease even if the environment is suitable

but suboptimal environmental factors may trigger

an infectious disease Diseases may even be causedby poor environmental factors alone



life will reveal more than tests at the time ofthe research Health and welfare monitoringof research fish should therefore start withbroodstock and eggs and continue until theresearch is finished This could be achieved

by introducing a health card system similarto that used in livestock production in somecountries

Knowledge of the cause and source ofdisease is of major importance in healthmonitoring but is often sparse Thediscovery of new fish diseases each year addsto the problem Specific guidelines musttherefore be updated continuously to adaptto new knowledge that may include health

issues detection methods medicaltreatment and the spread of pathogens Theavailability of these guidelines on theInternet allowing continuous updatingshould therefore be considered

Selection of fish

Factors to consider when selecting fish forresearch include in addition to the health

status species strain size weight andnumber of fish Different types of researchmay need different requirements for healthmonitoring prior to selection of fishIdentification of epidemiological risk factorsmay be useful when selecting farms andhealth documentation including diseasehistory vaccination strategies andtreatments need to be evaluated in relation

to the aims of the investigationFor studies on farmed fish a representativeselection from the population is often usedand this makes it easier to interpret theresearch results However there are someadvantages in establishing models usingsmaller laboratory fish to investigateproblems in farmed fish and recently a fewsuch models have been developed (LaPatraet al 2000) Our basic knowledge of the

health genomics and embryology of themost common laboratory fish species suchas zebrafish (Danio rerio) and medaka(Oryzias latipes) is more advanced than infarmed fish species and this makes themuseful in a wide range of research models(Powers 1989 Casebolt et al 1998Ostrander 2000 Alestrom 2004)

Comparison of results between trials istherefore much easier when using laboratoryfish species but the interpretation of theresults in relation to farmed fish is stilldifficult Extrapolation of results from

rodent models to human health is possiblebased upon many years of experience butsimilar knowledge on how to extrapolatethe results from for example zebrafish tosalmonids does not yet exist (Phelan et al2005)

Good production results alone do notconstitute an adequate measurement of thehealth and wellbeing of the animals Figure 3shows two Atlantic halibut of the same age

but of totally different size emphasizing theneed for both age and size to be considered asselection criteria Halibut with high growthrates have been shown to have a muchhigher prevalence of epicarditis than fishwith a lower growth rate (Johansen amp Poppe2002)

Heritability of disease resistance in fishhas been shown for bacteria (Gjedrem ampGjoslashen 1995) viruses (Okamoto et al 1993)

fungi (Nilsson 1992) and parasites (Kolstadet al 2005) Breeding programmes in forexample Atlantic salmon have led toincreased disease resistance in eachgeneration Challenge models established inone generation of fish may therefore notprovide the same results in the nextgeneration (Johansen L-H personalcommunication) Resistance to different

agents may be independent of each other ormay be positively or negatively correlated



Figure 3 Atlantic halibut Hippoglossus hippoglos-

sus from a commercial fish farm in Norway This

photograph illustrates that fish of the same age (12

months) may be of totally different sizes (2 and 40 g)



Basic knowledge of the link betweengenotype and disease resistance in fish issparse but molecular methods are nowbeing used to provide an insight into thisimportant field (Grimholt et al 2003)

Any genetic manipulation such as theproduction of an all-female population mustbe reported and its influence on the researchresults assessed (Thorgaard 1986) The acutestress response has for example been shownto differ between diploid and triploid fish(Benfey amp Biron 2000)

Most of the current guidelines for healthmonitoring of fish in research focus onquarantine of new arrivals to the research

facility (DeTolla et al 1995 Poole 1999Schwedeler amp Johnson 2000) There is greatvariation in the guidelines on how infectiousagents or diseases should be treated oncethey are detected Some state that the fishshould be free of notifiable diseases (UFRC2004 CCAC 2005) Diseases considerednotifiable vary between countries (DEFRA2003) and international notifiable fishdiseases are few (OIE 2005) Few guidelines

provide lists of common diseases that shouldbe investigated (Casebolt et al 1998) Oneset of guidelines for the use of aquaticspecies among a collection of resources forInstitutional Animal Care and UseCommittees (httpwwwnalusdagovawicpubsFishwelfareiacuchtm)recommends salt treatment and ifineffective antibiotic therapy of diseased

fish before a trial rather than exclusion ofthese stocks from research (httpwwwresearchpsueduorpareasanimalspoliciesguide10pdf) These examplesillustrate the lack of harmonization ofcriteria for allowing fish into researchfacilities

In most cases guidelines merely state thatquarantined fish should be monitored forclinical signs of disease and if a disease

problem occurs a fish health specialistshould be contacted for advice The length ofthe quarantine period is often left up to thefish health specialist (CCAC 2005) As longas these specialists are not provided withgenerally accepted guidelines therecommendations they give may varyconsiderably

The environment

Physical water parameters are numerous andfish are influenced by their environmentto a much greater degree than mammalsOptimal ranges for water parameters may

vary between different fish species andbetween the various developmental stages Itis therefore important that researchers andtechnical staff have knowledge of theappropriate environmental needs for thespecific group of fish with which they areworking Some general recommendationsthat are applicable to most fish species areavailable (May et al 1987b Klontz 1995Schwedeler amp Johnson 2000)

An overview of our knowledge on the careand use of fish as laboratory animals waspublished by Casebolt et al (1998) Theyidentified water quality as the single mostimportant element for maintaining healthyanimals and ensuring valid experimentalresults Efforts should be made to keep allparameters within acceptable ranges inorder to reduce stress Fish have the ability

to adapt to some changes in water qualitywithout stress and these ranges arecharacterized as acceptable limits Howeverrapid changes even within an acceptablerange may cause stress and should thereforebe avoided Although fish may cope withsuboptimal conditions for a short time byincreased energy use longer exposure maylead to chronic stress Measurements ofwater parameters including their fluctu-

ations and the duration of these musttherefore be documented

The main water quality parameters aretemperature oxygen saturation nitrogencompounds carbon dioxide pH and salinityA change in one of these factors may lead tofluctuation of the other parameters Otherenvironmental parameters such as light andnoise levels also need to be considered

Temperature

With the exception of a few species such astuna fish have the same body temperatureas the surrounding water Maintenance oftemperature within an optimal range istherefore of major importance and any







therefore be avoided where possible As fishmay become adapted to certain backgroundnoise and vibrations it is important that anychanges be kept to a minimum during theexperiment

Holding facilities

Different fish species and sizes havedifferent requirements for among otherparameters stocking density water flow andfeeding regime The aim should be to providethe fish with an environment allowing asnormal a behaviour as possible with aminimum of stress and aggression

Density of fish

The space provided per fish has a majoreffect on other environmental factors Highfish densities necessitate holding facilitiescapable of a high degree of environmentalcontrol High density may cause stress in thefish even if other environmental factors arewithin acceptable limits Density control

is therefore often mentioned as one of themajor factors influencing welfare ofsalmonids (Lymbery 2002) Preferred densityvaries extensively between different fishspecies and it is also important to rememberthat low density can lead to stress especiallyin fish that normally form shoals

Infectious agents

Lists of infectious agents for different fishspecies have been published in severaltextbooks (Ferguson 1989 Roberts 1989Ostrander 2000 Winton 2001) Newpathogens are constantly being discoveredand our knowledge of known pathogens isincreasing all the time Lists of pathogensand optimal detection methods are thereforea prerequisite for health monitoring The

Zebrafish Information Network (httpwwwzfinorg) provides such serviceinformation for zebrafish and similarWebsites for other fish species are needed

The Office International des Epizooties(OIE) provides a list of notifiable fishdiseases and monitors their global incidence(OIE 2005) as well as an overview of

standard diagnostic tests (OIE 2003) Thereare presently only 16 fish pathogensnotifiable to OIE but since all of these maycause serious disease they are unacceptablein fish research

The validation of detection methods andknowledge of the possibilities andlimitations of the tests used are majorfactors to take into account wheninterpreting results Knowledge of thesensitivity and specificity of the test incombination with the prevalence of theinfectious agent is often used to calculatethe number of animals necessary for testing(Nicklas et al 2002) The relatively low

sensitivity of many diagnostic methods infish is a major problem and results in manyfalse negative results This is a particularchallenge when attempting detection ofinfectious agents in subclinically infectedfish as described below Combinations ofdifferent tests are therefore frequently usedfor diagnostic purposes in fish

There are many fish diseases where thecause and pathogenesis are not fully

understood and this makes diagnosisdifficult In some cases the diagnosis has tobe based on macroscopic or microscopicchanges alone (Ferguson et al 1990) Cross-reactions are often observed when usingmethods such as immunohistochemistrywith polyclonal antisera When usingmolecular biological methods such aspolymerase chain reaction (PCR) for

detection of pathogens a discussion mayquickly arise as to whether the agentdetected is the cause of the disease or asubclinical carrier state Enzyme-linkedimmunosorbent assay (ELISA) testing forantibody response may lead to similardiscussions as to whether or not the fish isstill infected with the pathogen Challengetrials may be necessary to distinguishbetween similar strains of an agent (Santi

et al 2004) In some fish diseases electronmicroscopy has to be used (Eliassen et al2004) this is a very time consuming andoften unreliable method for detection

Since the body temperature of most fishvaries with water temperature fishpathogens often show adaptation to a widetemperature range This poses challenges





when cultivating bacteria Subclinicalbacterial infections are also relativelycommon (Hiney 1995) so bacterial detectionshould be combined with other findings tointerpret their relative importance

The source of infectious fish agents isoften not well documented and all possiblesources (fish personnel water feed orinanimate objects) should therefore beinvestigated Hygiene routines and proto-cols for preventive medicine must beindividually designed for each facility fishspecies and type of experiment

FishMost fish for research are still obtainedfrom commercial fish farms while a fewlaboratory facilities hatch and breed theirown fish As there is a risk that the fish areor have been infected by one or morepathogens before the start of the trialmonitoring should start a considerable timebefore the start of the experiment Ifpossible the parent broodfish should be

examined for vertically transmittedpathogens

Feed

Some marine fish larvae require live feedsuch as artemia rotifers and otherzooplankton in their early stages Theseorganisms may carry many types of

pathogens and good hygiene duringzooplankton production is essential Largenumbers of bacteria that are normally non-pathogenic to zooplankton have causedmajor problems such as gill inflammationenteritis and dermatitis in the production offish larvae from several species includingcod halibut and turbot These organismsmay also make the fish larvae moresusceptible to other diseases such as viral

encephalopathy and retinopathy (Johansenet al 2004)

Some fish species are still fed occasionallywith frozen or fresh fish material This canbe a major source of infection with forexample VHS-virus Mycobacterium sppand the parasite Ichthyophonus hoferi Ifpossible experimental fish should be fed

only commercial dried feed both prior to andduring the trial

It is also important to note that thenutritional status of the fish and feedingregime may have a major effect on the

outcome of infectious diseases In somecases starving fish drastically lowers themortality rate of the disease (Damsgard et al2004) Little is known about the basicmechanisms behind this starvation effectAn increased knowledge on how sick fishshould be treated and fed is important toimprove their welfare

Land-based experimental facilitiesIn land-based experimental facilities it ispossible to construct strict hygiene barriersbetween different fish groups Each tankshould be equipped with separate equipmentfor cleaning and other routine tasks Sharedequipment should be thoroughly disinfectedafter use A physical barrier between tanksmay not be necessary if there is no risk ofsplash contamination Footbaths and other

systems for disinfection andor change ofclothing should be provided if necessary aswell as systems to avoid unnecessarypersonnel traffic

Prior to initiation of the experimentpipework should be cleaned and disinfectedIncoming water may be filtered anddisinfected by ultraviolet (UV) or ozonetreatment but totally sterile water is often

not obtainable Ozone is beneficial to waterquality as it not only inactivates microorga-nisms but also oxidizes nitrite to nitrate andbreakes relatively non-biodegradablerefractory organic compounds into smallermore biogradable compounds In seawaterozone treatment may result in the release ofoxidants including toxic bromates whichmust be removed (Crecelius 1979) Ozone isdirectly toxic to fish and should be removed

by for example UV light treatment(Summerfelt et al 2004)

Testing of water for all potential pathogensis not normally feasible Tests for the totalnumber of bacteria (total cell count) or for anindicator organism before and afterdisinfection are therefore most often usedThe choice of the indicator organism needs





to be considered in relation to the fishspecies local disease situation and availabletests Norwegian legislation for examplerequires disinfectant systems capable of a3log10 reduction of Aeromonas salmonicida

subsp salmonicida and IPN-virus inresearch stations holding salmonids orfreshwater fish As far as discharged water isconcerned research stations assigned forchallenge trials in category A need to show aminimum of 5log10 reduction of IPN-viruswhile stations only assigned to challengetrials in category B and C need to show a5log10 reduction of Yersinia ruckeri

Open-water systems

Systems comprising normal farming pensare often used for large-scale experimentsboth in freshwater and saltwater Hygienicbarriers separating the fish from wildpopulations are then often impossible toconstruct As in land-based facilitiesunnecessary personnel traffic should beavoided and changing facilities provided if

considered necessary Water treatment andregulation are not possible so the risks ofinfectious disease agents gaining access tothe trial have to be taken into account

Diseases

The classical definition of a disease is a finiteabnormality of structure or function with

clinical signs (Stedman 1990) but today thedefinition is often widened to include alsosubclinical infections Situations in whichthe animals do not perform or produce atexpected levels may also be defined as adisease state (Blood amp Studdert 1993) Theterm lsquoclinical diseasersquo is therefore used hereto refer to situations where fish showclinical signs of disease

Focus should be placed not only on the

detection of disease but also on how it mayaffect the results of experimental trials Thestage and degree of disease are therefore ofmajor importance An outbreak of clinicaldisease may ruin the outcome of the trialwhile a subclinical infection or suboptimalnutrition may have only minor effectsdepending on the aim of the experiment

Detection of both non-infectious andinfectious diseases in clinically healthy fishmay require testing of a large number of fishDetection is normally easier in dead ormoribund fish so all these animals should

be examined since the findings may providevaluable information on the health status ofthe whole group

Infectious diseases

The best way to avoid an infectious diseaseis of course to avoid the infectious agent asdiscussed earlier In some cases this is notpossible and vaccination is then often used

to avoid the disease Some vaccines mayprevent an infection but most only preventor reduce the clinical manifestations ofinfection Vaccinated fish may therefore stillbe infected but the prevalence of clinicaldisease in these fish is much lowerInfections in vaccinated fish may still have amajor effect on the immune and healthstatus of the fish which in turn may have aneffect on the outcome of the research results

Monitoring of infectious agents shouldtherefore not be terminated when fish arevaccinated

Sea lice infections with concomitantdamage to the skin have a negative effect onthe general health status of the fish and mayallow entry of other pathogens It has alsobeen shown that sea lice infections cause astress reaction in the fish with a possible

subsequent suppression of the immuneresponse (Mustafa et al 1998) Sea liceinfections are therefore a good example ofconditions that may have an influence onthe general health of the fish even after thesea lice have been removed by treatmentInfections and medical treatments should berecorded for the whole lifespan of the fishand not just for a short observation periodimmediately prior to the research period

Subclinical infections

Some authors restrict the termlsquoasymptomaticrsquo to human diseases and usethe terms lsquoclinically inapparentrsquo or lsquocovertrsquofor infected animals with no signs of disease(Hiney et al 1997) The most commonly





used term in fish is lsquosubclinicalrsquo indicatingthat the fish are infected but show noclinical signs of disease Subclinicalinfections may endure for a long time andare then called persistent infections The

term chronic is used to describe a diseasethat persists for a long time with little or nochange in progression Some authors restrictthe term chronic to persistent infectionsthat eventually are cleared in contrast tolatent infections that last throughout thelifetime of the host (Flint et al 2000)

If subclinically infected fish are to be usedin experimental trials it is important todetermine the possible outcomes of the

infection It is especially important todetermine whether it may develop into aclinical disease andor if it has emerged froman acute disease If the subclinically infectedfish shed the infectious agent it is called acarrier infection and shedding may representa direct threat to the rest of the fish group Ithas been demonstrated that an infectionmay have either a positive or negativeinfluence on the outcome of an additional

infection in fish (Johansen amp Sommer 1995LaPatra et al 1995) This illustrates thedifficulties involved when attempting todetermine how an infection may influencethe research results

More information on the pathogenesis ofpersistent infections is of great importancefor developing better detection methods Forexample a recent study showed that

Atlantic halibut may be subclinicallyinfected with IPN-virus for at least 18months without showing any signs ofdisease (Gahlewat et al 2004) Reversetranscription (RT)-PCR and standard virusisolation in cell culture detected a lownumber of IPNV-infected fish compared withnew methods where adherent cells fromkidney and blood were isolated and lysedbefore passage onto cell culture (Munro et al

2004) This illustrates the complexity ofvirus detection in persistent infections andshows that a more sensitive test is oftennecessary in the persistent phase of thedisease compared with the acute phase

To increase the chances of detection astress test that triggers the subclinicalinfection into an acute disease may be used

Such a stress test has been established fordetection of subclinical infections with forexample Aeromonas salmonicida (Hiney1995) and IPN-virus (Taksdal et al 1998)

Non-infectious diseasesNon-infectious diseases may also have amajor effect on the outcome of research andmonitoring of these conditions is thereforejust as important as the infectious diseases

Malnutrition and starvation

Optimal nutrition is critical for optimalgrowth which is the main aim of fish

farming Much research has therefore beenaimed at the optimization of commercialfeed Malnutrition is therefore not acommon problem in those species that havebeen farmed for some time but may occur inspecies such as cod where the industry isstill under development Differentdevelopmental stages (for example larvaeand broodfish) and farming conditions (coldor warm water) may dictate differentnutritional regimes Optimized feed for allfish species at all ages and under allconditions is therefore important

Malnutrition is still a major problem formarine fish larvae that require livezooplankton as their first feed It is difficultto control the composition of the feed andthe nutritional value may therefore varyconsiderably Lack of pigmentation and

incomplete metamorphosis have been linkedto malnutrition in Atlantic halibut (Pittman1991) Clinical signs of malnutrition mayvary depending upon the type of deficiency(for example vitamins or proteins) andspecies (Tacon 1992)

Fish are carnivorous herbivorous oromnivorous Marine proteins may soon be inshort supply due to their inclusion in pigchicken and fish feeds so vegetable proteins

are being sought as replacements in fish feedThis may have consequences for the futurehealth and welfare of these fish

Deformities

Minor deformities may not be detected inlive fish and necropsy may be necessary If





the prevalence of the deformity is low itmay be best to examine the fish aftercompletion of the trial

Deformities in fish have been detected inmany organs The most common

deformities in salmonids are observed in thespinal cord and heart (Poppe et al 1998Baeverfjord et al 1999 Kvellestad et al2000) Fish with major deformities are easyto avoid when selecting fish for research Inother cases it may be difficult to determinewhere to draw the line between deformitiesand what is normal (Poppe et al 2003Figure 4)

Inflammations

Gills are very sensitive to any waterborneirritants and inflammation is thereforecommon Irritation may be caused byparasites particles chemicals metalsbacteria fungi or viruses Severe inflam-mation may be detected in fish with noclinical signs of disease but a stress

situation may provoke acute clinical signs infish with chronic pathological changes in thegills

Vaccination may cause mild to severeinflammatory reactions at the injection siteand pathological changes have also beendetected in other organs (Koppang et al2004 2005) These side-effects may have amajor influence on the health of the fish and

vaccinated fish should if possible not beused in research If they have to be used theyshould be examined for any side-effectsBoth the vaccine protocol and any detectedside-effects should be reported and

interpreted along with the research resultsEpicarditis (Johansen amp Poppe 2002) andcardiomyopathy syndrome (CMS) (Brunet al 2003) are other examples ofinflammatory conditions that may bedetected in fish with no clinical signs ofdisease The cause of these inflammations isstill unknown but may be because of as-yetunknown infectious agents or non-infectious causes such as malformations

malfunctions and metabolic disturbancesThis cardiac pathology must be assumed tohave a negative effect on the general healthof the fish and may therefore influenceresearch results

Severe inflammations may be detected bymacroscopic inspection of the organs whilemoderate inflammations may requirehistopathological examinations All organsshould therefore be macroscopically

examined for any signs of inflammationHistopathological examination of all organsis often not possible for economical reasonsbut some examinations need to be carriedout to provide a total picture of the healthstatus In particular histopathology of thegills may provide a good estimate of thewater quality that the fish has been exposedto before the onset of the trial

Fish welfare

The scientific study of animal welfarenecessitates an objective means for decidingwhether an animal is suffering or notSuffering includes a wide range ofunpleasant emotional states such as fearboredom pain and hunger Welfare

monitoring in animals may be performed byclinical observations or indirectly bymonitoring their environment (Wolffrom ampSantos 2005) Both health and welfare areused as general terms that may include eachother health monitoring is sometimesdefined as part of the process of ensuringanimal welfare or vice versa



Figure 4 Hearts from rainbow trout Oncorhynchus

mykiss The heart to the right has the normal

pyramid shape that provides optimal blood flow

while the heart to the left is more spherical and less

efficient as a pump Both hearts were collected at

the slaughterhouse from presumed healthy fish



Welfare in fish has not been well definedand the ability of fish to feel pain anddistress is still being debated (Rose 2002Chandroo et al 2004) The Fisheries Societyof the British Isles has published a review on

the subject giving criteria for welfare in fishand including a summary of acute andchronic stress responses (FSBI 2002) TheSociety states that there is no simple linkbetween physiological stress responses andwelfare but tertiary responses such assuppressed immune function growth andreproduction indicate chronic stress andtherefore poor welfare A recent review ofthe literature on fish welfare (Huntingford

et al 2006) concludes that fish canexperience fear-like states and that theyprobably have the capacity for suffering

The price an animal is prepared to pay toattain or escape from a situation is oftenused as an index of how the animal thrives inits environment Studies of the cognitiveability of fish have shown that they possessgreater skills than previously believed(Braithwaite 2005) However even with

better knowledge of neurophysiology andcognitive abilities in fish we will never trulyknow how a fish feels Fish should be giventhe benefit of the doubt when knowledge islacking Animal research legislation andguidelines often refer to the avoidance ofunnecessary suffering For example amethod of killing fish such as suffocationwhich is standard practice on fishing

trawlers is not acceptable in a researchsettingEven though our general knowledge of the

stress response in fish is good there are fewmethods available to document fluctuatingstress levels in populations or individual fish(Bonga 1997) The way in which diseasesaffect the wellbeing and stress level of fish isstill poorly understood (Damsgard et al2004) An apparently healthy fish in an

adequate environment is not sufficientdocumentation of good fish welfare

Behavioural changes are an important partof the stress response since they enableanimals to avoid or overcome the stressorEven though fish have restricted oppor-tunities to express their current internalstate changes in behaviour can be observed

They may for example swim faster or sloweror in different patterns Fish exhibit threemain behaviour patterns territorial schoolingor sedentary Rainbow trout for example areterritorial and therefore require space which

they defend by nipping the fins of other fish(Klontz 1995) Knowledge of this kind isessential when writing recommendationsfor fish density and tank design There areundoubtedly large species differences inoptimal stocking densities and moreresearch is needed to determine how densityaffects the individual animals Adequateknowledge of normal behaviour in thedifferent fish species together with

knowledge of how to interpret abnormalbehaviour is therefore a prequisite for goodwelfare

Fish showing signs of weakness will soonbe attacked by predators so the evolutionaryprocess has favoured behaviour that does notindicate the presence of disease and possiblyalso a higher threshold for suffering Evenfish with open wounds in the abdominalcavity and intestinal prolapse may not show

overt behavioural signs of disease Whetheror not sick fish suffer can therefore not bedetermined by behaviour alone

Possibilities for future monitoring of welfare in fish

Many of the parameters described below arecommonly used for welfare monitoring in

mammals One of the aims of this section isto explain why these parameters are not usedin fish monitoring today and indicate howthey may possibly be used in the future

Heart rate and respiration Heart andrespiration rates increase in stressful situa-tions and these parameters are thereforeoften observed when monitoring the welfare

of mammals Such information is not how-ever easy to register in fish The respiratoryrate may be monitored by looking at oper-culum ventilation rates but this is onlypossible in small glass tanks and in fish of acertain size Smart-tags that can measureheart rate and respiration in freely swim-ming fish in larger tanks are now being





investigated (ETHIQUAL EU-projecthttpwwwseafoodplusorgProject_5_2_ETHIQUAL630html) These new methodswill improve the opportunity to investigatenormal behaviour and stress responses in

fish under farming or research conditions

Blood samples Blood samples are usuallytaken from the caudal vein of fish butprocedures for sampling from many otherarteries have also been described (Black2000) Fluctuations in blood parametershave however made it more difficult tointerpret these findings in fish compared to

mammals Better understanding of thecirculatory system and stress in fish isnecessary before blood samples can be usedto a greater extent in the future

Blood parameters may for example showlarge variations depending on how the bloodsamples are obtained If 10 fish in a tank aresampled serially the stress level in the lastfish will be significantly higher than in thefirst animal (Roche amp Boge 1996) These

workers also showed that a range of stressors(osmotic thermic physical and chemical)may cause varying effects on stressparameters (cortisol blood glucosehaemoglobin and haematocrit)

The low blood pressure of fish may be oneof the main explanations for the widevariations seen in many blood parameters Inmammals blood is pumped under high

pressure directly to the body organs In fishthe blood is pumped to the gills under highpressure and then after losing pressure inthe capillary network it flows under lowpressure to the rest of the body Some vesselsare equipped with valves to ensure that theblood flows in the right direction (Figure 5)Contractions of the skeletal muscle help topump the blood through the body Venouspumps have been described in the tail gills

and the haemal canal The blood pressure inthe veins is so low that there may even benegative pressure Anaesthesia has a majorinfluence on the heart and it is important tomake sure that blood pressure is adequateduring blood sampling

Guidelines for bleeding mammals oftenquote 10 of the circulating blood volume

as a rough guide for one bleed from healthyanimals which may be repeated every 3ndash4weeks (BVAFRAMERSPCAUFAW JointWorking Group on Refinement 1993) Thereare few published estimates of circulating bloodvolume or guidelines for bleeding fish The

volume of blood in fish is normally estimatedto be 2ndash5 of body weight and the CCACguidelines recommend that no more than 1 mLblood should be sampled per kilogram bodyweight without killing the fish This is quite ahigh figure compared to mammals

The species temperature and otherparameters such as the presence of diseasemay of course influence these estimates

The amount of circulating blood in fish maybe low and may not be representative of thetotal blood volume When the fish is restingthe spleen may contain a large amount ofblood whereas this blood will be available inthe general circulation in a stressfulsituation Likewise stress will result in adiversion of blood flow from the intestinalorgans to the skeletal muscle to support thelsquofight or flightrsquo mechanisms Research in fish

is needed to provide better estimates for themaximum amount of blood that should besampled and this is especially importantwhen serial sampling is planned

Blood samples are usually taken from themain circulatory system but fish also have asecondary system with a separate capillarynetwork which so far has been detected in



Figure 5 Histology of the coronary blood vessel in

Atlantic salmon Salmo salar showing two pairs ofvalves that ensures that the blood flows in the

correct direction



gills skin and intestinal organs (Iwama ampFarrell 1998) The composition and amount ofblood in this secondary system is highlydependent upon the stress level of the fish Itmay contain very few blood cells with large

amounts of plasma if stress levels are highThe function of this system is not totallyunderstood but it is possibly related to thebalance of ions and water Stress may thereforehave a major effect on blood parameters It isalso important that water quality is within theoptimal range to avoid any switch in thecirculation due to conditions such as hypoxiahigh salinity or low pH

One solution to the problems with blood

sampling can be to sample the tank waterinstead Detection methods for steroids inthe water have been shown to correlate withthe level of steroids in the blood of the fish(Scott et al 2001 Ellis et al 2004)

The skin The first organ to show any signsof distress in mammals is often the skin andthe condition of the skin and fur are often

used as indicators of health and welfare Theskin and especially the mucus layer of fishmay also provide valuable information aboutthe wellbeing of the animal Fish used inexperimental trials should have intact skinand mucus without any signs of lesions

Changes in the environment may exert aninfluence on several factors in the mucussuch as its biochemical profile and bacterialflora (Fast et al 2002) Acute stress alone

without physical trauma or pathogens hasbeen shown to cause skin ulceration in fish(Noga et al 1998) The mucus is also the firstline of defence against several infections sodetection of changes in the mucus mayindicate the presence of pathogensImprovements in our knowledge of themucus layer may therefore be useful formonitoring both health and welfare and anEU research project has started to look intothis field (FAIR-CT98-4217 httpwwwcordisludataPROJ_FAIRACTIONeqDndSESSIONeq16683200595ndDOCeq4ndTBLeqEN_PROJhtm)

Faeces Diarrhoea or other changes in faecalconsistency or composition may be indica-

tions of poor health andor welfare In fishfaeces are voided into the water and changesare therefore not normally registered as theyare in mammals except in extreme situa-tions Fish faeces should be monitored either

by analyses of the water or by collectingintestinal samples (Black 2000) This type ofexamination is commonplace in feedingtrials and parasitological research but isseldom performed as part of a general healthinvestigation largely due to the lack ofknowledge on how to interpret the resultsFurthermore the collection of intestinalsamples may be a stressful process in itselfTo avoid unnecessary stress it should not be

used routinely but be limited to situationswhere there is concern about the healthstatus of the fish

Asymmetry and deformities Stress inbroodstock fish may result in an increasedincidence of deformities and asymmetry intheir offspring (Eriksen et al 2006) Stressorssuch as hypothermia hypoxia pathogens

suboptimal pH salinity light and malnutri-tion have been shown to cause asymmetry(Koumoundouros et al 2001) Deformitieshave been linked to poor rearing conditions(Boglione et al 2001) Further studiesare needed to establish whether or notasymmetry can be used as an indicator offish welfare

Molecular biological methods Severalresearch groups are now focusing on thedevelopment of molecular methods notonly to detect pathogens but also to inves-tigate how infectious agents and other stressfactors influence the fish (Gornati et al2005) These methods will enable newopportunities for monitoring the health andwelfare of fish Gornati et al (2005) investi-gated gene expression in fish groups reared at

different population densities and detectedchanges in six genes that will be furtherinvestigated as possible welfare indicatorsThe EU research programme lsquoWEALTH ndashWelfare and health in aquaculturersquo (httpeuropaeuintcommresearchfp6sspwealth_enhtm) focuses on detection ofmolecular biological parameters in fish that







References

Alestrom P (2004) Zebrafish model ndash from biomedi-cine to aquaculture Keynote Lecture at European

Aquaculture Society Special Publication 34 10ndash17Baeverfjord G Lein I Asgard T Rye M Storset A

(1999) Vertebral deformations induced by hightemperatures during embryogenesis in Atlanticsalmon (Salmo salar L) EAS Special Publication

27 6ndash7Benfey TJ Biron M (2000) Acute stress response in

triploid rainbow trout (Oncorhynchus mykiss) and

brook trout (Salvelinus fontinalis) Aquaculture

184 167ndash76Black MC (2000) Collection of body fluids In The

Laboratory Fish (Ostrander GK eds) BaltimoreAcademic Press 513ndash27

Blood DC Studdert P (1993) Baillie rersquos Comprehen-

sive Veterinary Dictionary 3rd edn London

Bailliere TindallBoglione C Gagliardi F Scardi M Cataudella S (2001)

Skeletal descriptors and quality assessment inlarvae and post-larvae of wild and reared gilthead

bream (Sparus aurata L) Aquaculture 192 1ndash22Bonga SEW (1997) The stress response in fish

Physiological Reviews 77 591ndash625Braithwaite VA (2005) Cognitive ability in fish Fish

Physiology 24 1ndash37Brattelid T Smith AJ (2000) Guidelines for reporting

the results of fish experiments Laboratory Ani-mals 34 131ndash5

Braunbeck T Bottcher M Hollert H et al (2004)

Towards an alternative for the acute fish LC50 testin chemical assessment the fish embryo toxicity

test goes multi-species ndash an update LINZ 1 87ndash102Brun E Poppe TT Skrudland A Jarp J (2003)

Cardiomyopathy syndrome in farmed Atlantic

salmon Salmo salar occurrence and direct finan-

cial losses for Norwegian aquaculture Diseases of

Aquatic Organisms 56 241ndash7

BVAFRAMERSPCAUFAW Joint Working Group onRefinement (1993) Removal of blood from labora-

tory mammals and birds First Report of the BVAFRAMERSPCAUFAW Joint Working Group onRefinement Laboratory Animals 27 1ndash22

Canadian Council on Animal Care (2005) Guidelineson the care and use of fish in research teaching and

testing httpwwwccaccaCasebolt DB Speare DJ Horney BS (1998) Care and

use of fish as laboratory animals current state of

knowledge Laboratory Animal Science 48 124ndash36

Chandroo KP Yue S Moccia RD (2004) An evaluationof current perspectives on consciousness and pain

in fishes Fish and Fisheries 5 281ndash95Cleef-Toedt K Kaplan L Crivello JF (2001) Killifish

metallthionein messenger RNA expression fol-lowing temperature pertubation and cadmium

exposure Cell Stress and Chaperones 6 351ndash9Council of Europe (2006) European Convention for

the Protection of Vertebrate Animals used for

Experimental and other Scientific Purposes (ETS123) Revision of Appendix A adopted by theMultilateral Consultation on 15 June 2006Strasbourg httpwwwcoeinttelegal_affairslegal_co2Doperationbiological_safety2C_use_of_animalslaboratory_animals

draft20revision20of20Appendix20AaspTopofPage

Crecelius EA (1979) Measurements of oxidants inozonized seawater and some biological reactionsJournal of Fisheries Research Board of Canada

Bulletin 36 1006ndash8Damsgard B Soslashrum U Uglestad I (2004) Effects of

feeding regime on susceptibility of Atlantic salmon(Salmo salar ) to cold water vibriosis Aquaculture

239 37ndash46Dawe CJ Couch JA (1984) Debate mouse versus

minnow the future of fish in carcinogenicitytesting National Cancer Institute Monograph 65

223ndash35DEFRA (2003) Combating Fish Disease ndash an Expla-

nation of the Controls on Notifiable Fish Diseases

in Great Britain and Advice on Steps to Prevent

the Spread of Disease UK Department for Envir-onment Food amp Rural Affairs

DeTolla LJ Srinivas S Whitaker BR et al (1995)Guidelines for the care and use of fish in researchInstitute for Laboratory Animal Research Journal

37 1ndash19Eliassen TM Solbakk IT Evensen O Gravningen K

(2004) Isolation of heart and skeletal muscleinflammation virus (HSMIV) from salmon Inter-

national Symposium on Viruses of Lower Vert-

brates (ISVLV) 19ndash22 September 2004 HakodateHokkaido Japan

Ellis T James JD Stewart C Scott AP (2004) Anoninvasive stress assay based upon measurementof free cortisol released into the water by rainbowtrout Journal of Fish Biology 65 1233ndash52

Eriksen MS Bakken M Espmark A Braastad BO

Salte R (2006) Prespawning stress in farmedAtlantic salmon Salmo salar maternal cortisolexposure and hyperthermia during embryonicdevelopment affect offspring survival growthand incidence of malformations Journal of Fish

Biology 69 114ndash29Fast MD Sims DE Burka JF Mustafa A Ross NW

(2002) Skin morphology and humoral non-specificdefence parameters of mucus and plasma inrainbow trout coho and Atlantic salmonComparative Biochemistry and Physiology 132

645ndash57Ferguson HW (1989) Systemic Pathology of Fish

Ames Iowa State University PressFerguson HW Poppe TT Speare DJ (1990) Cardio-

myopathy in farmed Norwegian salmon Diseases

of Aquatic Organisms 8 225ndash31Flint SJ Racaniello VR Enquist LW Skalka AM Krug

RM (2000) Virology Molecular Biology Pathogen-

esis and Control Washington ASM Press





FSBI (2002) Fish welfare Briefing Paper 2 Fisheries

Society of the British Isles httpwwwleacukbiologyfsbiwelfarepdf

Gahlewat SK Munro ES Ellis AE (2004) A non-destructive test for detection of IPNV-carriers inAtlantic halibut Hippoglossus hippoglossus Jour-

nal of Fish Diseases 27 233ndash9Gjedrem T Gjoslashen HM (1995) Genetic variation in

susceptibility of Atlantic salmon Salmo salar Lto furunculosis BKD and coldwater vibriosis

Aquaculture Research 26 129ndash34Gornati R Gualdoni R Cavaliere G Terova G

Saroglia M Bernardini G (2005) Molecular biologyand fish welfare a winning combination Aqua-

culture International 13 51ndash5Grimholt U Larsen S Nordmo R et al (2003) MHC

polymorphism and disease resistance in Atlantic

salmon facing pathogens with single expressedmajor histocompatibility class I and class II lociImmunogenetics 55 210ndash19

Grossblatt N (1996) Guide for the Care and Use of

Laboratory Animals Washington DC NationalAcademy Press

Hiney M (1995) Detection of stress inducible furun-culosis in salmonids vaccinated with water and oilbased furunculosis vaccines Bulletin of European

Association of Fish Pathologists 15 98ndash9Hiney M Smith P Bernoth EM (1997) Covert

Aeromonas salmonicida infections In Furuncu-

losis (Bernoth EM Ellis AE Midtlyng P Olivier GSmith P eds) London Academic Press54ndash97

Huntingford FA Adams C Braithwaite VA et al(2006) Current issues in fish welfare Journal of

Fish Biology 68 332ndash72Iwama GK Farrell AP (1998) Disorders of the

cardiovascular and respiratory systems In Fish

Diseases and Disorders Non-Infectious Disorders

(Leatherland JF Woo PTK eds) Canada CABIPublishing 245ndash78

Iwama GK Thomas PT Forsyth RB Vijayan MM(1998) Heat shock proteins expression in fishReviews in Fish Biology and Fisheries 8 35ndash56

Johansen LH Sommer A-I (1995) Multiplication ofinfectious pancreatic necrosis virus (IPNV) in headkidney and blood leukocytes isolated from Atlanticsalmon Salmo salar L Journal of Fish Diseases 18147ndash56

Johansen R Poppe TT (2002) Pericarditis and myo-carditis in farmed Atlantic halibut Hippoglossus

hippoglossus Diseases of Aquatic Organisms 49

77ndash81Johansen R Sommerset I Toslashrud B et al (2004)

Characterization of nodavirus and viral encepha-lopathy and retinopathy (VER) in farmed turbotScophthalmus maximus Journal of Fish Diseases

27 591ndash601Kane AS Gonzalez JF Reimschuessel R (1996) Fish

and amphibian models used in laboratory researchLab Animal 25 33ndash8

Klontz GW (1995) Care of fish in biological researchJournal of Animal Science 73 3485ndash92

Kolstad K Heuch PA Gjerde B Gjedrem T Salte R(2005) Genetic variation in resistance ofAtlantic salmon (Salmo salar ) to the salmonlouse Lepeoptheirus salmonis Aquaculture 247

145ndash51Koppang EO Haugarvoll E Hordvik I Aune L Poppe

TT (2005) Vaccine-associated granulomatousinflammation and melanin accumulation inAtlantic salmon Salmo salar L white muscleJournal of Fish Diseases 28 13ndash22

Koppang EO Haugarvoll E Hordvik I Poppe TTBjerkas I (2004) Granulomatous uveitis associatedwith vaccination in the Atlantic salmon Veter-

inary Pathology 41 122ndash30Koumoundouros G Divanach P Kentouri M (2001)

The effect of rearing conditions on developmentof saddleback syndrome and caudal fin deformities

in Dentex dentex (L) Aquaculture 200 285ndash304Kvellestad A Hoslashie S Thorud K Toslashrud B Lyngoslashy A

(2000) Platyspondyly and shortness of vertebralcolumn in farmed Atlantic salmon Salmo salar inNorway ndash description and interpretation of patho-logic changes Diseases of Aquatic Organisms 3997ndash108

LaPatra SE Lauda KA Jones GR (1995) Aquareovirusinterference mediated resistance to infectioushematopoietic necrosis virus Veterinary Research

26 455ndash9LaPatra SE Barone L Jones GR Zon LI (2000) Effects

of infectious hematopoietic necrosis virus andinfectious pancreatic necrosis virus infection onhematopoietic precursors of the zebrafish Blood

Cells Molecules and Diseases 26 445ndash52Lymbery P (2002) In Too Deep ndash The Welfare of

Intensively Farmed Fish Petersfield HampshireCompassion in World Farming Trust

May EB Bennett RO Lipsky MM Reimschuessel R(1987a) Using fish as models in biomedical

research Part 1 Lab Animal 16(4) 23ndash8May EB Bennett RO Lipsky MM Reimschuessel R

(1987b) Using fish as models in biomedicalresearch Part 2 Lab Animal 16(5) 25ndash31

Melby EC Balk MW (1983) The Importance of

Laboratory Animal Genetics Health and the

Environment in Biomedical Research OrlandoAcademic Press

Munro ES Gahlawat SK Ellis AE (2004) A sensitivenon-destructive method for detecting IPNV carrierAtlantic salmon Salmo salar L by culture of virus

from plastic adherent blood leucocytes Journal of Fish Diseases 27 129ndash34

Mustafa A Bower J MacWilliams C Fernandez NConboy G Burka JF (1998) Effect of sea liceinfection on macrophage functions in Atlanticsalmon Bulletin of Aquaculture Association of

Canada 2 90ndash2Nicklas W Baneux P Boot T et al (2002) Recom-

mendations for the health monitoring of rodent





and rabbit colonies in breeding and experimentalunits Laboratory Animals 36 20ndash42

Nilsson J (1992) Genetic variation in resistance of Arctic char to fungal infection Journal of Aquatic

Animal Health 4 126ndash8Noga EJ Botts S Yang MS Avtalion R (1998) Acute

stress causes skin ulceration in striped bass andhybrid bass (Morone) Veterinary Pathology 35102ndash7

OIE (2003) Manual of Diagnostic Tests for Aquatic

Animals 4th edn Paris Office International desEpizooties httpwwwoieintengnormesen_acodehtm

OIE (2005) Aquatic Animal Health Code 8th ednParis Office International des Epizooties httpwwwoieintengnormesen_acodehtm

Okamoto N Tayama T Kawanobe M Fujiki N

Yasuda Y Sano N (1993) Resistance of a rainbowtrout strain to infectious pancreatic necrosis Aquaculture 117 71ndash6

Ostrander GK (2000) The Laboratory Fish BaltimoreAcademic Press

Phelan PE Pressley ME Witten PE Mellon MT BlakeS Kim CH (2005) Characterization of snakeheadrhabdovirus infection in zebrafish (Danio rerio)Journal of Virology 79 1842ndash52

Pittman K (1991) Aspects of the early life history ofthe Atlantic halibut Hippoglossus hippoglossus

(PhD thesis) University of BergenPoole T (1999) UFAW Handbook on the Care and

Management of Laboratory Animals Amphibious

and Aquatic Vertebrates and Advanced Inverte-

brates 7th edn Essex Blackwell SciencePoppe TT Johansen R Gunnes G Toslashrud B (2003)

Heart morphology in wild and farmed Atlanticsalmon Salmo salar and rainbow trout Oncor-

hynchus mykiss Diseases of Aquatic Organisms

57 103ndash8Poppe TT Midtlyng P Sande RD (1998) Examination

of abdominal organs and diagnosis of deficient

septum transversum in Atlantic salmon (Salmosalar L) using diagnostic ultrasound imagingJournal of Fish Diseases 21 67ndash72

Powers DA (1989) Fish as model systems Science 246352ndash8

Rehbinder C Baneux P Forbes D et al (1998) FELASArecommendations for the health monitoring ofbreeding colonies and experimental units of catsdogs and pigs Laboratory Animals 32 1ndash17

Roberts RJ (1989) Fish Pathology London BailliereTindall

Roche H Boge G (1996) Fish blood parameters aspotential tool for identification of stress caused byenvironmental factors and chemical intoxicationMarine Environmental Research 41 27ndash43

Rose JD (2002) The neurobehavioral nature of fishesand the question of awareness and pain Reviews in

Fisheries Science 10 1ndash38Ryder K (2005) Challenges of applying humane end-

points in fish 2nd International Conference on the

Use of Humane Endpoints in Animal Experiments

for Biomedical Research 20ndash21 August 2005Berlin Germany

Santi N Vakharia VN Evensen O (2004) Identifica-tion of putative motifs involved in the virulence ofinfectious pancreatic necrosis virus Virology 32231ndash40

Schwedeler TE Johnson SK (2000) Responsible careand health maintenance of fish in commercialaquaculture Animal Welfare Information Centre

Bulletin 10 httpwwwnalusdagovawicnewslettersv10n310n3schwhtm

Scott AP Pinillos M Ellis T (2001) Why measuresteroids in fish plasma when you can measurethem in the water In Perspectives in Compara-

tive Endocrinology (Goos T Rastogi RK Vaudry HPierantoni R eds) Bologna Monduzzi Editore1291ndash5

Stedman TL (1990) Stedmanrsquos Medical Dictionary

25th edn Baltimore Williams amp WilkinsSummerfelt ST Sharrer MJ Hollis J Gleason LE

Summerfelt SR (2004) Dissolved ozone destructionusing ultraviolet irradiation in recirculating sal-monid culture system Aquacultural Engineering

32 209ndash23Tacon AGJ (1992) Nutritional fish pathology Mor-

phological signs of nutrient deficiency and toxicityin farmed fish FAO Fish Technical Paper 330 1ndash75

Taksdal T Ramstad A Stangeland K Dannevig BH(1998) Induction of infectious pancreatic necrosis(IPN) in covertly infected Atlantic salmon Salmo

salar L post-smolts by stress exposure by injec-tion of IPN virus and by cohabitation Journal of

Fish Diseases 21 193ndash204Thorgaard GH (1986) Ploidy manipulation and per-

formance Aquaculture 57 57ndash64Use of Fishes in Research Committee (2004) Guide-

lines for the Use of Fishes in Research MarylandAmerican Fisheries Society httpwwwfisheriesorghtmlPublic_AffairsSound_SCienceGuidelines2004shtml

Westerfield M (2000) The Zebrafish Book A Guide for

the Laboratory Use of Zebrafish (Danio rerio) 4thedn Eugene University of Oregon Press

Winton JR (2001) Fish health management In Fish

Hatchery Management (Wedemeyer G ed) Mary-

land American Fisheries Society 559ndash640Wolffrom T Santos ML (2005) Farmed Fish and

Welfare European Commision DirectorateGeneral Fisheries and Maritime Affairs 1ndash39




commercial fish farm were randomlysampled for the experimentrsquo How the

health was monitored is often not statedInformation relating to health monitoringduring the experimental trial may also besparse often limited to statements such aslsquono clinical signs of disease were observedrsquoIn most trials no necropsies or tests areperformed to determine the health status

It is important to note that there is nointernational consensus on animal welfare

legislation and fish are often not included innational laws Some general guidelines formonitoring of laboratory animals may alsobe useful for fish (Grossblatt 1996) Generalconsiderations relevant to fish includeselection of the appropriate speciesidentification of the minimum number ofanimals required for valid results suitableliving conditions and the use of experiencedpersonnel including fish health specialists

The search for alternatives is as relevant infish as in mammals but in vitro methods infish research are sparse

Researchers are ethically bound to produceas much knowledge as possible from eachanimal used Lack of standardization ofparameters such as genotype water qualityand handling procedures often leads to

incomparable results and therefore theunnecessary use of large numbers of fishHarmonization of health and welfaremonitoring is one important factor in theprocess of standardization that is needed to

reduce fish numbers in researchLaboratory animal units may be accreditedby the Association for Assessment andAccreditation of Laboratory Animal Care(AAALAC) who rely on widely acceptedguidelines for the care and use of laboratoryanimals (Grossblatt 1996) Recommendationsfor health monitoring of rodents rabbitscats and other mammalian animals havebeen established by the Federation of

European Laboratory Animal ScienceAssociations (FELASA) (Rehbinder et al1998 Nicklas et al 2002) Thesepublications include lists of infectiousagents to be tested for suggested methods fortesting and a standardized report formWidely accepted guidelines for the care anduse of fish in research are however notcurrently available

The most recent and thorough guidelines

for fish are provided by the CanadianCouncil on Animal Care (httpwwwccacca Guidelines on the care and useof fish in research teaching and testing)These guidelines are general ones for all fishspecies in all types of research and providerecommendations for among other thingsfacilities management and husbandry Thesection on health monitoring focuses on

establishing programmes for diseasedetection written agreements with fishhealth professionals and the strategic use ofdisease control measures Detailed health-monitoring protocols are not outlinedalthough there is a table of clinical signs ofdisease to be monitored

Current guidelines usually leave it up tothe local fish health specialists to determinehow health and welfare should be monitored

and which fish should be allowed into theresearch facility (UFRC 2004 CCAC 2005Council of Europe 2006) In textbooks on thecare and use of classical laboratory fish suchas zebrafish health monitoring is oftenlimited to statements about the prevalenceof diseases and how they can be prevented(Ostrander 2000 Westerfield 2000) Few or



Figure 1 A simple breakdown of the number of

live fish used for research in Norway 2004 Actualnumbers are provided in parentheses Most fish

were reported as having been used in the categories

lsquoBasic researchrsquo and lsquoResearch and Developmentrsquo

(RampD) More details of the type of testing for which

the fish were actually used cannot be obtained from

the statistics
































Selection of fish




























The environment







Temperature









Holding facilities


Density of fish



Infectious agents




















Feed




















Open-water systems



Diseases







Infectious diseases






























Deformities









Inflammations









Fish welfare
























































correct direction

























References















































































































































































































Selection of fish




























The environment







Temperature









Holding facilities


Density of fish



Infectious agents




















Feed




















Open-water systems



Diseases







Infectious diseases






























Deformities









Inflammations









Fish welfare
























































correct direction

























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Selection of fish




























The environment







Temperature









Holding facilities


Density of fish



Infectious agents




















Feed




















Open-water systems



Diseases







Infectious diseases






























Deformities









Inflammations









Fish welfare
























































correct direction

























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The environment







Temperature









Holding facilities


Density of fish



Infectious agents




















Feed




















Open-water systems



Diseases







Infectious diseases






























Deformities









Inflammations









Fish welfare
























































correct direction

























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Holding facilities


Density of fish



Infectious agents




















Feed




















Open-water systems



Diseases







Infectious diseases






























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Inflammations









Fish welfare
























































correct direction

























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Holding facilities


Density of fish



Infectious agents




















Feed




















Open-water systems



Diseases







Infectious diseases






























Deformities









Inflammations









Fish welfare
























































correct direction

























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Feed




















Open-water systems



Diseases







Infectious diseases






























Deformities









Inflammations









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correct direction

























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Open-water systems



Diseases







Infectious diseases






























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Inflammations









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Inflammations









Fish welfare
























































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Inflammations









Fish welfare
























































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References


















































































































































































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Documents

Guidelines Fish