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4.0BloodSamplingofFinfish
4.1Introduction:
ThistemplateisintendedforusebyinstructorstotraintheDepartmentof
Fisheries&Oceans(DFO)staffandstudentsinthebloodsamplingoffinfish.
Templatesareusedtoprovidetheminimumrequirementsnecessaryinatraining
exercise,buttheinstructormayaddadditionalmaterialasdeemednecessary.
Anexperiencedinstructormustdemonstratethemethodsoutlinedinthistemplate
andtraineesmustbedeemedqualifiedincarryingoutoneormore ofthe
proceduresbeforetheyarepermittedtobloodsamplewithoutaninstructor
present.Handsontrainingofstaffisarequirementforfacilityapprovalbythe
CanadianCouncilonAnimalCare(CCAC),ofwhichDFOisamember. This
templateispartof acomprehensiveDFOScienceBranchseriesontrainingfor
usersofaquaticresearchanimals.
4.2Rationale:
Scientificstudyoftenrequiresthatfishbebloodsampledforreasonssuchas
haematology,clinicalchemistryparameters,bacteriology,parasitological
investigation,packedcellvolume,etc.Investigatorshaveanethicalobligationto
minimizethepainand/ordistressofalllaboratoryanimalsundergoinglaboratory
samplingtechniques.OnlythosemethodsdeemedacceptablebytheCCAC
shouldbeemployed.
4.3Authority:
Thestaff/consultantveterinarianorAnimalCareCommitteeisresponsiblefor
providinginformationaboutbloodsamplingmethodsofthefishspeciesusedfor
scientificstudyintheirrespectiveregions.TheAnimalCareCommitteemay
delegatetrainingoftheseprocedurestoaninstructorwhohasdemonstrated
knowledgeandexperienceintheareasofanatomyandanaesthesiaandhas
demonstratedexcellentsurvivaloffishthathavebeenbloodsampledusingthe
techniquesoutlinedinthistrainingtemplate.Staffmustbetrainedintheproper
bloodsamplingtechniquesforthefishspeciesandsizebeingstudiedpriorto
initiationofanybloodsamplingprocedure(s).
4.4Goalofthistrainingexercise:
1. Learnthemethodstohumanelyremovebloodsamplesfromanaesthetized
fish.
2. Understandtheconsequencesofeachmethodofbloodsamplingonthefish
andthusthepotentialimpactofthemethodselectedandthesamplecollected.
3. Understandthefunction/physiologyofthecirculatorysystem withinafish.
4. Demonstrategentlehandlingtechniquesoffishwhenlivefishareusedinthis
section.
5. Understandproperdisinfectiontechniquestouseaftercompletingthe
procedure(providebiosecuritySOPs).

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4.5TheoreticalTrainingtobecompletedbeforehandsonsession
1. CompletedTheExperimentalFish.
2. CCACguidelines:SectionHExperimentalProcedures6:Collectionofbody
fluids.
3. Completedtheanatomy&physiology,anaesthesiaandeuthanasiatemplates
orhasexperienceortraininginallthreeareas.
4. WHMIStraining.
5. Summarytheorymaterialprovidedwiththistrainingtemplate(AppendixA:
Fishbloodandthecirculatorysystem).

4.6DetailsoftheProcedure:
Introductory:
Methodsofbloodsampling,thatmaybeincludedinthetrainingsession,are:
1. Bloodsamplingbytailablationofsmallfishthathavebeeneuthanized.
2. Bloodsamplingbycaudalvenouspuncture.
3. Bloodsamplingbydorsalaortapuncture.
Advanced:
4. Bloodsamplingbyheartpuncture.
4.6.1. Timeestimateforcompletingthehandsonexercise
Setup:1hour
InstructionandTraining:2hours
4.6.2 EquipmentRequired:
Fish:numberwilldependonthenumberoftraineesinasession
speciesusedwilldependonavailabilityandrequirementsofthe
facility.
TMS(plusNaHCO3 bufferifanaesthetizinginfreshwater)
Dipnet
Containerforanaestheticbath
Airstonesandcompressedairoroxygen
Thermometer
Glovesandsplashglassestobewornwhenhandlingfishor
anaesthesiawater
Disinfectantforcleanupafterbloodsampling
Softspongeormattopreventmucoussloughingduringblood
sampling
Scalpelorsharpknifefortailablation
Papertowelforcontainmentofbloodfromablation
Sterileneedlesorvacutainerneedles(sizewillbedependentonthe
sizeofthefish)
Syringeorvacutainertubeforcollectingbloodsamples(sizewill
dependontheamountofbloodtocollectandthesizeofthefish)
Vacutainerholder
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Tuberacksforbloodcollectiontubes
Hematocrittubes
Critoseal
Sharpscontainer

4.6.3Procedure:
Theinstructorshoulddemonstratealltheprocedurespriortoallowingthe
traineestoattemptthem.Fishsacrificedintheeuthanasiatemplatemake
excellentteachingspecimensforthetraineestopracticetheirtechniques.
Instructorswillneedtousetheirownbestjudgementinchoosingwhich
proceduresareappropriatewiththecommonspeciesusedattheirresearch
facility.Itisrecommendedthatallproceduresbetaughttotraineessothat
theyhavemultipleoptionsattheirdisposalwhenfacedwiththeneedto
samplethebloodoffishofdifferentspecies.
Introductory:
4.3.6.1EquipmentforBloodCollection:
HumanSafetyMeasures
Discusshumansafetymeasureswhilediscussingtheequipmentused
forbloodsampling.
Wearglovesatalltimeswhenhandlingbloodcollectionequipment.
Whilethiswillnotpreventaccidents,itmayreducetheriskof
contamination.
Neverattempttoresheathneedles.Alwaysdiscardthemintoasharps
containerimmediatelyafteruse.
Neveroverfillthesharpscontainer.Adheretotherecommendedfill
lineonthesharpscontainer.
Placethesharpscontainerintheareayouareworkingtoavoid
transportingusedneedlesoutsideofyourworkarea.
Ensuretheareayouareworkingiscleanandalltheequipmentneedto
carryouttheprocedureisreadilyavailable.Allowenoughworkspace
tosafelyobtainthebloodsamplewithoutcontaminatingtheblood
samplingsuppliesshouldthefishexhibitcommonreflexmovements
uponneedleinsertion.Forbloodcollectioninthefielditissuggested
tospreadafinemeshnetundertheentireworkareatopreventlossof
supplies(particularlysharps)thatmayaccidentallydropwhile
sampling.

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EquipmentOptions:
Presentequipmentoptionsavailableforbloodcollection(i.e.needles
ofvariousgaugesandlength,multiplesyringetypesandsizes,
haematocrittubesandcritoseal,vacutainerneedlesandtubes,etc.).
Emphasizetherelationshipbetweenneedlegaugeandneedlesize(e.g.
a20gaugeneedleislargerthana30gaugeneedle).
Discusssafetyequipmentandhowitistobeused(i.e.tuberacksare
forholdingtubesafterfillingwithblood,sharpscontainersarefor
discardingusedneedles,etc.).
Identifytheproperneedlegaugeandlengthrequiredforblood
samplingthefishusedinthistrainingsession.
Discussthecommontypesandsizesofvacutainertubesandchosethe
typeandsizeappropriateforthefishusedinthistrainingsession.
Traineesshouldconsultwiththeprincipalinvestigatortodetermine
theappropriatetubestousewhenbloodsamplesarecollectedfor
researchpurposes.Examplesofvacutainertubesinclude,butarenot
limitedtothefollowing:
Redtop:containsnoadditives.Usedforserumcollection,
bacteriology,andparasitologicalexamination.
Greentop:containstheanticoagulantlithiumorsodiumheparin.
Intendedforclinicalchemistryparametersandplasmacollection.
Purpletop:containstheanticoagulantEDTA.Intendedforwhole
bloodcollectionforhaematology.EDTAwillinterferewith
sometestsandisthereforenotasuitablesampletubeforblood
chemicalanalysis.
Greytop:containstheadditivesodiumfluoride.Intendedfor
glucosemeasurements.
Lightbluetop:containstheadditivesodiumcitrate.Intendedfor
coagulationstudies.
Selecttheappropriatetubeorsyringesizeforthefishusedinthe
trainingsession.Toolargeatubeorsyringecansignificantlyreduce
thebloodreserveinsmallerfishcausingexcessstressand/ordeath.
Paediatrictubesor1mlsyringesshouldbeusedwhenbloodsampling
smallerfish.
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Instructtraineesinthepropermethodtoattachanddetachneedlesto
syringesorvacutainertubes.
Stresstheimportanceoffeelingcomfortablewiththebleeding
apparatuspriortoinsertionintoalivingfish.
Traineesshouldpracticemovingtheplungerupanddowninthe
syringe.Agoodpracticetoolishavingthetraineesinsertasyringe
withattachedneedleintoanorangeandextractasmallamountof
juice.
Traineesshouldpracticeinsertingavacutainerintothevacutainer
holder(withattachedneedle)withoutbreakingthevacuumseal.A
breakinthevacuumsealinairmakesthetubeuselessforblood
collection,asthebloodwillnotaspirateonceintheveinoraortaofthe
fish.
Note:Thefollowingproceduresandphotographshavebeenadaptedwith
permissionoftheauthorfromChapter4.AsepticBacterialExaminationof
Finfishin:FinfishandShellfishBacteriologyManualTechniquesand
Procedures,2004.WhitmanK.A.(author)BlackwellPublishing,IowaState
Press,Iowa,USA.
4.6.3.2.TailAblation:
Fishthataretoosmalltobleedwithasyringeandneedleora
vacutainersystemmaynecessitatelethalsamplingbytailablation.
Euthanizeafish.RefertotheEuthanasiaTemplateforaccepted
methodsofeuthanasia.

Bloodcollectionfrom Carassiusauratus.(PhotobyR.Hebb)
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Severthecaudalpedunclewithascalpelbladeorsharpknife.
Fillahematocrittubewiththebloodasitflowsfromthecaudal
vein.
Plugoneendofthehaematocrittubewithcritoseal.
DisposeofthefishcarcassandwasteasoutlinedinfacilitySOP.
4.6.3.3 CaudalVenousPuncture:

(a)BloodcollectionfromanAmericanshad(Alosasapidissima)usingthe
caudalvein(ventralview).(PhotobyR.Hebb)

(b)Lateral view.(PhotobyR.Hebb)
Insertaneedleattachedtoasyringeoravacutainersystem
undertheskinoftheventralmidlineofthecaudalpeduncleof
ananaesthetizedorfreshlyeuthanizedfish.
Alternatively,alateralapproachcanbeusedbyinsertingthe
needleunderthescalesofthemidportionofthetailjustbelow

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thelaterallineata45o angletothelongaxisofthefishina
cranialdirection.
Easetheneedletowardthevertebralcolumnuntilyoureachthe
baseofthecolumn.
Withdrawtheneedleafractionofamillimetre,andobtainthe
bloodsample.
Removeanddiscardtheneedleinasharpscontainer.
Returnthefishtoarecoverybathoritshometankandmonitor
therecoveryprocess.
Preparethebloodsampleforprocessingasinstructedbythe
instructor.
CleantheworkareaasoutlinedinthefacilitySOP.

4.6.3.4 DorsalAortaPuncture:

BloodcollectionfromanAtlanticsalmon(SalmosalarL.)usingthe
dorsalaorta.(PhotobyR.Hebb).
Inserttheneedleattachedtoasyringeoravacutainersystem,
bevelpointingupward,alongthedorsalmidlineofthemouth,
justpastthejunctureofthesecondgillarchofananaesthetized
orfreshlyeuthanizedfish.
Collectabloodsample.
Removeanddiscardtheneedleinasharpscontainer.

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Returnthefishtoarecoverybathoritshometankandmonitor
therecoveryprocess.
Preparethebloodsampleforprocessingasinstructedbythe
instructor.
CleantheworkareaasoutlinedinthefacilitySOP.
Advanced:
Bloodcollectionusingtheheartpuncturemethodisoftenperformedon
euthanizedfish,thoughitcanbeperformedsuccessfullyonanaesthetized
fishwithpropertraining.
Instructorsshoulddemonstratethisprocedureonseveralfishtoensurethe
traineeiscognitiveofthepotentialrisksassociatedwiththismethod.
Traineesshouldnotbepermittedtoperformthisprocedureonlivefish
withoutafirmgraspoftheanatomyandphysiologyoftheheart.
Itishelpfulfortraineestodissectasacrificedspecimentoobservethe
location(bulbousarteriosus)wheretheneedleistobeinsertedand
determinethepotentialtraumaafishsheartcanundergoafterblood
sampling.
Traineesshouldpracticeonanumberofeuthanizedspecimenstopractice
theirtechniqueandgainconfidencepriortoattemptingalivespecimen.
4.6.3.5 HeartPuncture:

BloodcollectionfromanAmericanshad(A.sapidissima)usingtheheart
puncturetechnique.(PhotobyR.Hebb).

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Holdtheneedleattachedtoasyringeorvacutainersystem
perpendiculartotheskinandinserttheneedleslightlybelow
thetipoftheVshapednotchformedbythegillcoverandthe
isthmusofananaesthetizedorfreshlyeuthanizedfish.
Collectthebloodastheneedleentersthebulbousarteriosus.
Removeanddiscardtheneedleinasharpscontainer.
Returnthefishtoarecoverybathoritshometankandmonitor
therecoveryprocess.
Preparethebloodsampleforprocessingasinstructedbythe
instructor.
Cleanthebloodsampleareaasoutlinedinthefacility SOP.
4.6.4Afterthetrainingsession:
Increasemonitoringfor2 3weeksafterhandlingiflivefishwere
usedforthetraining.
Traineesshouldhaveclearinstructionsforneedle/sharpsdisposaland
carcassdisposal.
Anaesthesiabathsmustbedisposedofinaccordancewithlocalwaste
managementregulations.
Disinfecttheareawherefishwerehandled(providetraineeswithsite
biosecuritySOP).
Traineesmustwashhandswithdisinfectantsoap.
Updateinventoryrecordstoreflectthenumberoffisheuthanizedfor
thissession(ifany).
Updatedruguserecordstoincludeanaestheticuse.

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4.7ACCNotes
Locallysignificantdifferencesrequiredintraining(e.g.species).

Authorizationrequiredtoteach/listofpossibleinstructorsforyourregion.

Anyotherrequirementsforyourregion.

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APPENDIXA:ReviewTheory:FishBloodandtheCirculatorySystem
References:
CCACguidelineson:thecareanduseoffishesinresearch,
teachingandtesting.
Bone,Q.andMarshall,N.B.1982.BiologyofFishes.Blackie&
SonsLtd.,NewYork,USA.
Evans,H.E.1992.Anatomyoftropicalfishesin:Aquariology
MasterVolume,TheScienceofFishHealthManagement.J.B.
GratzekandJ.R.Matthews(Eds.)TetraPressPublicationN.J.,
USA.
Iwama,G.K.andFarrell,A.P.1998.Disordersofthe
CardiovascularandRespiratorySysteminFishDiseasesand
Disorders:Volume2,Noninfectiousdisordersed.J.F.Leatherland
andP.T.K.Woo,OxfordUniversityPress,CABIInternational
Reinert,R.E.1992. Fishphysiologyin:AquariologyMaster
Volume,TheScienceofFishHealthManagement.J.B.Gratzek
andJ.R.Matthews(Eds.)TetraPressPublicationN.J.,USA.

Whitman,K.A.(2004).FinfishandShellfishBacteriologyManual
TechniquesandProcedures. PublishedbyBlackwellScientific,
IowaStatePress,Iowa,USA.
Definitions:
Anticoagulant:Chemicaladditivewhichinhibitsclotting.
Erythrocyte: Red blood cell or RBC, the nucleated (non
mammalian), agranular blood cells containing the oxygen
carrying pigment haemoglobin and responsible forthe red colour
offreshblood.
Haematocrit:Volumetricrelationshipofthecellularelementsof
bloodtothetotalbloodvolumealsoreferredtoaspackedcell
volume.
Haematopoiesis:Formationandgrowthofbloodcells.

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Leukocytes: Collectivetermreferringtoanynonpigmented,
nucleatedbloodcell.Leukocytesinfishbloodinclude440%
granulocytes(subdividedintoneutrophil,acidophil,andbasophil
cellsaccordingtotheirstainingproperties)andgranular
lymphocytes,monocytes,andthrombocytes.
Lymphoid:Resemblingthetissueoflymphglands.
Lymphocyte: Granular leukocyte of the peripheral blood formed
inthelymphatictissue.
Monocyte: Large mononuclear leukocyte with a deeply indented
nucleus,slategrey cytoplasmandazurophilicgranulationthatare
formamacrophageuponmigrationintotissue.
Myeloid:Relatingtothespinalcordorbonemarrow.
Plasma:Thefluidfractionofthebloodcontainingdissolvedsalts
andproteins,asdistinguishedfromcorpuscles.
Serum:Thefluidportionofthebloodthatremainsaftertheblood
isallowedtoclotandthecellsareremoved.
Thrombocyte:Bloodcellthatisinvolvedinbloodclotting.
FishBloodandtheCirculatorySystem
Bloodcellsareformedprimarilyinthekidneyandspleenandtoa
lesserextentintheliver,intestinalsubmucosaandthymus.
Theerythrocytesareovalandnucleated.
Leukocytes are morphologically and functionally diverse and all
arenucleated.Thereareatleast fivedifferentsubgroupsofwhite
bloodcells.
Somearcticfisheslackhaemoglobinandthushaveclearblood.At
lowertemperaturesoxygencanbecarriedinsimplesolutioninthe
plasma.
Lymphatic vessels are present in teleosts, butthere are no lymph
nodes.
Capillary networks or retia are present in the gas bladder for the
introductionorremovalofgas(oxygen,nitrogen).

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Withintheredmusclestrip(aerobicslowcontracting)ofthelateral
body wall, the capillary network functions for gas exchange and
forheatexchangeinlargefishsuchastuna.
In the oviduct and ovarian walls of livebearing fishes, there are
networks for nutrient exchange, which can be considered
placentae.
Fishes have a oneway/single circulatory pathway through the
heart. All the blood leaving the heart passes through at least two
setsofcapillariesthecapillariesofthegillsandthecapillariesof
thebodyorgan.
The blood in the circulatory system transports oxygen to the
tissues,andremovescarbondioxide(CO2)fromthem.Itvariesin
its properties in different fishes depending on the metabolic
demands and the way in which the fish obtains oxygen and
excretesCO2.
Hypobranchial vessels from the gill rather than from the dorsal
aorta supply the heart wall. A few fish species have a coracoid
artery that arises from the dorsal aorta and supplies blood to the
caudalaspectoftheheart.
The pseudobranch also receives a special blood supply from a
branchoftheefferentbranchialarterycalledthemandibularartery.
Allvenousbloodfromtheorgans,bodywall,andthefinsreturns
totheheartviasuperficialanddeepveins.
Thesinusvenosusservesasareceivingchamberforvenousblood
returning from the body and it helps to assure a smooth flow of
blood intotheatrium.The hepatic veinsand anteriorjugular vein
empty directly into the sinus venosus. The anterior and posterior
cardinal veins join to form the common cardinal vein (Cuverian
duct)beforeemptyingintothesinusvenosus.
Blood flows from the atrium to the ventricle, which, with each
contraction (called the systolic period of the heartbeat), ejects
blood into the bulbous arteriosus in teleosts and the conus
arteriosusinotherfishspecies.
Astheventriclerelaxes(thediastolicperiod intheheartbeat),the
high pressure persists in the bulbous arteriosus and serves to
maintainanevenflowofbloodintotheventralaorta.

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Valvesbetweenthebulbousandtheventriclepreventbackflowof
thebloodduringthediastolicperiod.
Fromthebulbousarteriosusthebloodflowsintotheshortventral
aorta(itistheoutflowpathtothegills).Theventralaortagivesoff
pairedafferentbranchialarteriestothebranchialarches.
Fromthe capillaries inthegillstheoxygenatedblood iscollected
by efferent branchial arteries and is emptied into the dorsal aorta
above. These roots join immediately to form the dorsal aorta,
whichisthemajordistributingvesseloffishcirculation.
The dorsal aorta passes caudally beneath the vertebral column,
giving rise along the way to paired segmental intercostal, renal,
and spinal arteries as well as unpaired arteries to the gas bladder
andviscera.
Posterior,wherethepairedribsfusetoformthehaemalarches,the
dorsalaortaentersthehaemalarchandpassesintothe tail.
The caudal vein also lies within the haemal arches beneath the
aorta.
Branchesofthedorsalaortasupplyarterialbloodtothehead,body
musculature,swimbladder,andallinternalorganssuchasthegut,
liver,gonads,andkidney.
BloodCollectionGuidelines:
Nomorethan0.1%ofthefishsbodyweight(i.e.1ml/kg)should
be removed from a fish that will be recovered from blood
collection.
Fish greater than 200 grams can be recovered successfully
following blood collection (dependant on the health status of the
fishpriortobloodsampling).
Fishsmallerthan200gramsmayhavetobesacrificedtoensurean
adequatebloodvolumeiscollected.
Fish must be allowed adequate time to recover and regenerate
bloodvolumeifserialbloodsamplesaretobecollected.
Bloodcollectedforhaematologyandclinicalchemistryparameters
must be taken from living fish. Care must be taken to avoid
contaminationofthesamplewithtissuefluid.
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Fish blood clots very rapidly. The type of anticoagulant used


should be determined in consultation with the principal
investigator. For some parameters an anticoagulant will not be
required.Examplesofanticoagulantsinclude:
EDTA (dipotassium ethylenediamine tetraacetic acid): acidifies
blood, binds divalent cations lowering calcium measurements in
thesample.
Disodiumoxlalate:raisesbloodpHandsodiumlevels.
Trisodiumcitrate:raisesbloodpHandsodiumlevels.
LithiumorSodiumheparin:cancauseerythrocyteclumpingbut
doesnotinterferewithdivalent cation measurementsand has less
effectonbloodpH.Lithiumheparindoesnotinterferewithsodium
levels.
Equipmentusedforbloodcollectionshouldbescaledtothesizeof
thefishandtheexpectedbloodvolumetobecollected.
The length and gauge of a needle used for blood collection will
dependonthesizeandspeciesofthefishbeingsampled.(E.g.a22
gauge, 1 inch needle is most commonly used for salmonids
averaging1kg).
Onlysterileneedlesshouldbeusedforbloodcollection.
Thechoiceofusingavacutainersystemorasyringeandneedleis
basedpurelyonpreference.
BloodSampleHandling:
Blood samples should be processed within 3060 minutes after
collection. The time between blood sampling and laboratory
evaluation of the sample collected is critical. Cells can swell and
rupture, and some parameters may not be stable during the time
betweencollectionandprocessing.
Thehandlingandstorageofsamplesafterprocessingwilldepend
ontheirpurpose.Checkwiththeprincipalinvestigatorto
determinehowtoprocessthesamples.
HERMAJESTYTHEQUEENINRIGHTOFCANADAasrepresentedbythe
MinisterofFisheriesandOceans.
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