Comparative Serum Fatty Acid Profiles of Captive and Free-Ranging Cheetahs

14 Pages·2016·1 MB·English

RESEARCHARTICLE
Comparative Serum Fatty Acid Profiles of
Captive and Free-Ranging Cheetahs (Acinonyx
jubatus) in Namibia
AdrianS.W.Tordiffe1,2,3*,BettinaWachter4,SonjaK.Heinrich4,FredReyers5,Lodewyk
J.Mienie3
1 DepartmentofParaclinicalSciences,FacultyofVeterinaryScience,UniversityofPretoria,Onderstepoort,
SouthAfrica,2 DepartmentofResearchandScientificServices,NationalZoologicalGardensofSouth
Africa,Pretoria,SouthAfrica,3 CentreforHumanMetabonomics,FacultyofNaturalSciences,North-West
a11111 University,Potchefstroom,SouthAfrica,4 DepartmentofEvolutionaryEcology,LeibnizInstituteforZooand
WildlifeResearch,Berlin,Germany,5 IdexxLaboratories(Pty)Ltd,WoodmeadWillowOfficePark,
Johannesburg,SouthAfrica
*[email protected]
Abstract
OPENACCESS
Cheetahs(Acinonyxjubatus)arehighlyspecialisedlargefelids,currentlylistedasvulnera-
Citation:TordiffeASW,WachterB,HeinrichSK,
ReyersF,MienieLJ(2016)ComparativeSerum bleontheIUCNreddatalist.Incaptivity,theyareknowntosufferfromarangeofchronic
FattyAcidProfilesofCaptiveandFree-Ranging non-infectiousdiseases.Althoughlowheterozygosityandthestressofcaptivityhavebeen
Cheetahs(Acinonyxjubatus)inNamibia.PLoS
suggestedaspossiblecausalfactors,recentstudieshavestartedtofocusonthecontribu-
ONE11(12):e0167608.doi:10.1371/journal.
tionofpotentialdietaryfactorsinthepathogenesisofthesediseases.Fattyacidsarean
pone.0167608
importantcomponentofthediet,notonlyprovidingasourceofmetabolisableenergy,but
Editor:GovindhaswamyUmapathy,Centrefor
servingotherimportantfunctionsinhormoneproduction,cellularsignallingaswellaspro-
CellularandMolecularBiology,INDIA
vidingstructuralcomponentsinbiologicalmembranes.Todevelopabetterunderstanding
Received:June10,2016
oflipidmetabolismincheetahs,wecomparedthetotalserumfattyacidprofilesof35captive
Accepted:October17,2016
cheetahstothoseof43free-rangingindividualsinNamibiausinggaschromatography-
Published:December19,2016 massspectrometry.Theunsaturatedfattyacidconcentrationsdifferedmostremarkably
Copyright:©2016Tordiffeetal.Thisisanopen betweenthegroups,withallofthepolyunsaturatedandmonounsaturatedfattyacids,
accessarticledistributedunderthetermsofthe exceptarachidonicacidandhypogeicacid,detectedatsignificantlylowerconcentrationsin
CreativeCommonsAttributionLicense,which
theserumofthefree-ranginganimals.Theinfluenceofageandsexontheindividualfatty
permitsunrestricteduse,distribution,and
acidconcentrationswaslessnotable.Thisstudyrepresentsthefirstevaluationoftheserum
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. fattyacidsoffree-rangingcheetahs,providingcriticalinformationonthenormalfattyacid
profilesoffree-living,healthyindividualsofthisspecies.Theresultsraiseseveralimportant
DataAvailabilityStatement:Allcheetahserum
fattyaciddataareavailablefromUniversityof questionsaboutthepotentialimpactofdietaryfattyacidcompositiononthehealthofchee-
Pretoria’sresearchdatarepository(http://hdl. tahsincaptivity.
handle.net/2263/58345).
Funding:FundingwasreceivedbyBWandSKH
fromtheMesserliFoundation(Switzerland)and
LeibnizInstituteforZooandWildlifeResearch
(Germany)forthesamplingofthefree-ranging
cheetahs.Otheraspectsofthestudywere Introduction
supportedbyNorthWestUniversity,theAfriCat
FoundationandtheNationalZoologicalGardensof Cheetahs(Acinonyxjubatus)arekeptinmanyfacilitiesworldwide.Despiteobviousimprove-
SouthAfrica.FRisemployedandreceivesasalary mentsinhusbandrysincecheetahswerefirstkeptincaptivity,theystillsufferfromarangeof
PLOSONE|DOI:10.1371/journal.pone.0167608 December19,2016 1/14
SerumFattyAcidProfilesinCheetahs
fromIdexxLaboratories(Pty)Ltd,acommercial unusualdiseasesnottypicallyseeninotherlargecaptivefelids.Theseincludeglomerulosclero-
laboratory.Allfundersprovidedsupportintheform sis[1–4],renalamyloidosis[4],lympho-plasmacyticgastritis[2,5,6],veno-occlusivedisease
ofsalariesand/orresearchmaterialsforauthors
[2,7],splenicmyelolipomas,cardiacfibrosis[2,4],adrenalcorticalhyperplasia[1,2,4,8]with
(ASWT,BW,SKH,FRandLJM),butdidnothave
lymphocyticdepletionofthespleen[2],pancreaticatrophy[2]aswellasseveralill-defineddis-
anyroleinthestudydesign,datacollectionand
ordersoftheneurologicalsystem[2,9].Someofthesechronicdegenerativediseaseseventually
analysis,decisiontopublish,orpreparationofthe
manuscript.Thespecificrolesoftheseauthorsare affectthemajorityofcheetahsincaptivityandareconsideredtobetheprimarycauseofmor-
specifiedinthe‘authorcontribution’section. bidityandmortalityinadultanimals[2,10].Incontrast,theincidenceofsimilarconditionsin
free-rangingcheetahswasfoundtobeverylow[10,11].Althoughlowheterozygosityandthe
CompetingInterests:Oneoftheauthors(FR)is
currentlyemployedbyIdexxLaboratories(Pty) stressofcaptivityhavebeensuggestedascausalfactors[8,12],recentstudieshavestartedto
Ltd,acommercialveterinarylaboratory.Besides focusonthecontributionofpotentialdietaryfactorsinthepathogenesisofthesediseases[13–
providingsupportintheformasalary,Idexx 15]
Laboratoriesplayednoroleinanyaspectofthis
Thereisincreasingevidenceofthecriticalroleofdietaryandcirculatingfattyacids(FA)in
studyandthisdoesnotalterouradherenceto
healthanddiseaseinvariousspecies[16–19].Besidesprovidingavaluablesourceofenergy,
PLOSONEpoliciesonsharingdataandmaterials.
Theauthorsfurthermoredeclarethatnocompeting FAsalsoperformothervitalfunctionsinthebody,includinghormoneproduction,cellular
interestexist. signallingaswellasprovidingstructuralcomponentsofbiologicalmembranes.Long-chain
polyunsaturatedfattyacids(PUFAs),with20carbonatoms,aretheprecursorsofeicosanoids,
suchasprostaglandins,leukotrienesandthromboxanes,whichhaveawiderangeofregula-
tory,autocrineandparacrineeffects.PUFAswith20to22carbonatomsarealsotheprecur-
sorsofautacoids,includingresolvins,lipoxinsandneuroprotectins,whichareinvolvedinthe
activeresolutionofinflammation[20].Furthermore,FAsareknowntoplayaroleasmodula-
torsofgenetranscription[21,22].
Giventheirbiologicalimportance,dietaryFAsarelikelytoplayasignificantroleinthe
healthofcaptivecheetahs.SerumFAshavebeenshowntoprovideagoodapproximationof
dietaryintake[23–26].Todatefewstudieshavefocusedonthemetabolismoflipidsinthis
speciesandnoclearlinkshavebeenestablishedbetweentheFAintakeandthediseasessuf-
feredbycheetahsincaptivity.SuspectedessentialFAdeficiencieshavebeendocumentedina
fewcheetahcasereports.[27,28].Currently,serumreferencevaluesexistforphospholipid
fractionsofaselectednumberofFAsin28captivecheetahsfromtwofacilitiesintheUnited
States[29].However,thephospholipidFAfractiongenerallymakesuponlyaround30%ofthe
totalserumorplasmaFAsandisnotnecessarilyreflectiveoftotalserumorplasmaFAcompo-
sition[26]Furthermore,thesecheetahswerefedacommercialdietthatisonlyreadilyavail-
ableinNorthAmerica.Giventherangeofdietsfedtocaptivecheetahs,thesevaluesare
unlikelytoprovidereliableserumreferencevaluesforcheetahsinothercaptivefacilities.
Wehypothesizedthatthedifferencesindiet,feedingintervalandenergyexpenditure
betweencaptiveandfree-rangingcheetahsresultindifferencesintheirserumFAprofiles.
CaptivecheetahsinsouthernAfricaareoftenfeddietsthatconsistprimarilyofleanmuscle
meatfromcattle,horsesordonkeys,supplementedwithamultivitamin/mineralpowder.Most
facilitiesadheretoguidelinesthatrecommendfeedingmealsofbetween1.2and1.4kgperday
forsixdaysoftheweek[30].Incontrast,thefree-rangingcheetahshavebeenshowntocon-
sumeavarietyofgamespecies,consistingprimarilyofsmalltomediumsizedantelopeand
smallerquantitiesofwarthogs,haresandbirds[31].Free-rangingcheetahsalsogenerallyfeed
lessfrequently,withsuccessfulkillsonlybeingrecordedevery2.6to7days[32,33].Thehigh
proportionofantelopeinthefree-rangingcheetahdietisexpectedtoresultinalowerpropor-
tionalintakeofunsaturatedfat,sincetheunsaturatedtosaturatedfatratioinruminanttissues
issubstantiallylowerthaninmonogastricanimals.Ruminantmuscletissuetypicallyhasan
unsaturatedtosaturatedFAratioofbetween0.11and0.15,whiletheratioindonkeymeatis
around1.45[34–36].Thespecificcomponentsandorgansofpreyanimalsconsumedbyfree-
rangingcheetahsshouldalsoleadtolowerserumPUFAtoSFAratios,astheintra-abdominal
fatdepots,frequentlyconsumedbyfree-rangingcheetahs,alsohaveahigherproportionof
PLOSONE|DOI:10.1371/journal.pone.0167608 December19,2016 2/14
SerumFattyAcidProfilesinCheetahs
saturatedfatthaneitherintramuscularorsubcutaneousadiposetissue[37].MostAfrican
ungulateshavesubstantialintra-abdominalfatreserves,including,mesenteric,omental,peri-
renalandchannelfat.Consumptionofwholepreycarcassesisthereforelikelytoresultinan
increasedintakeofdietaryfatcomparedtocaptivedietsthataremostlylimitedtomusclemeat
andbone[38].
Theincreasedproportionalintakeofdietaryfat,decreaseinfeedingfrequencyand
increasedphysicalactivityinfree-rangingcomparedtocaptivecheetahsareallpredictedto
resultinenhancedmitochondrialFAoxidationthroughtheloweringofcirculatingglucose
concentrationsandinsulin:glucagonratios[39].Duringfasting/refeedingcyclesandincreased
levelsofexercise,tissuePUFAconcentrationshavebeenshowntodepleterapidlyinboth
humansandrats[40,41].ThesestudiesshowthatmostPUFAs,includingα-linolenicacid
(ALA)andlinoleicacid(LA),arepreferentiallyoxidizedinperiodsofexerciseorfasting.Dur-
ingrefeeding,SFAsandmonounsaturatedfattyacids(MUFAs),suchaspalmiticacidand
oleicacid,arealsomorerapidlyreplacedthananyofthePUFAs.Similarly,theconcentrations
ofmostplasmaPUFAsandMUFAshavebeenshowntobesignificantlylowerinratsfeda
highfatketogenicdietthanincontrols[42].ThepredictedincreaseinFAoxidationinfree-
rangingcheetahsisthereforelikelytoalsoskewtheirserumFAprofilestowardlowerpropor-
tionalserumconcentrationsofPUFAsandMUFAsrelativetoSFA.
InthisstudywecomparedthetotalserumFAprofiles(combinedfreeandboundFAs)of
captivecheetahsfedameat-baseddiettothoseoffree-rangingcheetahsfeedingonnatural
prey.Ouraimwastoinvestigateourhypothesisthatthedifferentfeedinghabitsoffree-rang-
ingandcaptivecheetahsleadtodifferentserumFAprofilesandtotesttwopredictionsderived
fromthishypothesis.Weexpectedthatfree-rangingcheetahshave;1)lowerPUFA:SFAratios
and2)lowerMUFA:SFAratiosthanthecaptivecheetahs.Theprofilesobtainedalsoprovide
valuablebaselineinformationwithwhichtoevaluatesuspectedFAdeficienciesincaptive
cheetahsandadvanceourunderstandingoflipidmetabolismanditsimpactonthehealthof
thisspecies.
MaterialsandMethods
Ethicalandpermitconsiderations
TheprojectwasapprovedbytheNationalZoologicalGardensofSouthAfrica’sResearchand
EthicsCommittee(Projectno.P11/07).Research/collectingpermits(1846/2013)and(1689/
2012)wereobtainedfromtheNamibianMinistryofEnvironmentandTourismandthesam-
pleswereimportedintoSouthAfricawiththeCITESexport(no.0042838)andimport(no.
137670)permits,aswellasaveterinaryimportpermit(no.13/1/1/30/2/10/6-2013/11/002397).
OnceinSouthAfrica,thesamplesweretransportedandstoredwiththenationalThreatened
orProtectedSpecies(TOPS)ordinarypermit(no.05238).
Samples
Bloodsampleswerecollectedfrom35adultcaptivecheetahs(14femalesand21males)during
theirannualhealthassessmentsinJuly2013attheAfriCatFoundationnearOtjiwarongoin
Namibia(Table1).Thecheetahswerefastedfor24to36hoursandimmobilizedviaremote
injectiondart(Daninject,Denmark)with0.03mg/kgmedetomidinehydrochloride(Medeto-
midine20mg/ml,KyronLaboratories,SouthAfrica)incombinationwith1.2mg/kgzolaze-
pam/teletamine(Zoletil1,Virbac,SouthAfrica).Theseanimalswerefedadietconsisting
mostlyofmusclemeatfromevisceratedandexsanguinateddonkeys,supplementedwitha
multivitaminandmineralpowder(PredatorPowder1,Healthtech,SouthAfrica).
PLOSONE|DOI:10.1371/journal.pone.0167608 December19,2016 3/14
SerumFattyAcidProfilesinCheetahs
Table1. Numbersofcheetahsinthecategoriesage,sexandcaptivitystatus.
Sex Agecategory Captive Free-ranging Total
Males Sub-adults 0 14 14
Youngadults 6 9 15
Matureadults 8 11 19
Oldadults 7 0 7
Total 21 34 55
Females Sub-adults 0 2 2
Youngadults 4 3 7
Matureadults 3 4 7
Oldadults 7 0 7
Total 14 9 23
doi:10.1371/journal.pone.0167608.t001
SampleswerealsocollectedbetweenJuly2012andMay2013from43free-rangingsub-
adultandadultcheetahs(34malesand9females)livingoncommercialfarmlandintheKho-
masandOmahekedistrictsincentralNamibia.Theywerecapturedinnon-baitedboxtraps,
placedatknowncheetahmarkingtrees.Oncesecuredintheboxtraps,theywereimmobilised
byremoteintramuscularinjectionusingacombinationof0.06mg/kgmedetomidinehydro-
chloride(Medetomidine10mg/ml,KyronLaboratories,SouthAfrica)and3.2mg/kgketamine
(Ketamine1G,KyronLaboratories,SouthAfrica).Theagesofthefree-rangingcheetahswere
estimatedbyusingthekeyforbodysizeestablishedby[43],usingshoulderheight,appearance
ofthemaneandphysicallesionssuchaselbowcalluses,scarsaswellasdentalwear.
Within15to35minutesofimmobilisation,bloodwascollectedasepticallybydifferent
operators,usingslightlydifferentmethods.Inthecaptiveanimals,bloodwascollectedfroma
jugularveinwitha20mlsyringeand18gaugeneedleandthentransferredinto6mlBDVacu-
tainer1tubes(Becton,DickinsonandCompany,SouthAfrica),whileinthefree-rangingchee-
tahsthebloodwascollectedfromthecephalicveindirectlyintothe6mlBDVacutainer1tubes
througha21gaugeVacutainer1needle.Allthebloodsampleswereallowedtoclotoniceina
coolerbox.Sampleswerecentrifugedwithin4hoursateither1500gfor10minutes(captive
cheetahs)orwithin4to12hoursat400gfor15minutes(free-rangingcheetahs),afterwhich
theserumwaspipettedoffandimmediatelyfrozenat-20˚C.Thesampleswerethentransported
ondryicetothelaboratoryinNovember2013andkeptat-80˚CuntilanalysisinJanuary2014.
Afterthesamplecollectionandcompletionofthehealthassessments,thecaptivecheetahs
wereplacedinwoodenrecoverycratesand0.075mg/kgatipamezole(Antisedan1,Zoetis,
SouthAfrica)wasadministeredintramuscularly,whereasthefree-rangingcheetahswere
placedinapaddedboxand0.11mg/kgatipamezole(Antisedan1,Pfizer,SouthAfrica)was
administeredintramuscularly.Oncetheanimalshadrecoveredsufficientlyfromtheeffectsof
theanaesthesiainthecratesorpaddedboxes,respectively,thecaptivecheetahswerereleased
backintotheirenclosuresandmonitoredintermittentlyfor24hours,whereasthefree-ranging
cheetahswerereleasedatthespotofcaptureandmonitoreduntiltheywalkedawayandoutof
sight.Mostofthefree-rangingcheetahswerecollaredwithaGPScollarandtheirmovements
afterreleasewereconfirmedtobenormal.Inallcasestheyrecoveredwithoutincident.
Samplepreparationandreagents
Afterthawingthesamplesonice,theyweresubjecttobothacidicandalkalinehydrolysisand
thenextractedintohexaneasdescribedby[44]fortheanalysisofverylong-chainfattyacids
(VLCFA).Theprotocolwas,however,modifiedfortheadditionalquantificationoflong-chain
PLOSONE|DOI:10.1371/journal.pone.0167608 December19,2016 4/14
SerumFattyAcidProfilesinCheetahs
fattyacids(LCFA)byaddinganadditionalstableisotopestandard(eicosanoicacid-d )tothe
39
internalstandardsolutionataconcentrationof50μmol/l.Derivatisationwasachievedwith
N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide(MTBSTFA)asdescribedbythe
sameauthors.
Eicosanoic-d acidwasobtainedfromSigma-AldrichPtyLTD(KemptonPark,South
39
Africa).TheVLCFAstableisotopestandards:C26:0-d ,C24:0-d ,C22:0-d ,pristanicacid-d
4 4 4 3
andphytanicacid-d wereobtainedfromDr.HermantenBrink(VUMedicalCentre,Amster-
3
dam,TheNetherlands)(http://www.vumc.nl/metabool/index.html).Otherreagentswere
obtainedfromLarodan(KarolinskaInstitutetSciencePark,Retziusva¨g8,SE-17165Solna,
Sweden),MerckPtyLTD(Modderfontein,SouthAfrica)andRegisTechnologies,Inc(Morton
Grove,IL,USA).
Gaschromatography-massspectrometry
TheGC-MSanalysisoftheserumLCFAsandVLCFAs,wasconductedonaHewlettPackard
HP6890seriesgaschromatography(GC)systemwithanAgilent5973NMassselectivedetec-
torfittedwithanelectronionization(EI)ionsource,anAgilentTechnologies7683autosam-
pleranda7683Binjector.TheGCsystemwasfittedwithaPhenomenexGCFocusLinerliner
forHP,split/splitless,w/wool,singletaper,4mmIDx78.5mmLx6.3mmOD(partnumber
AG04680)andaCPSil19capillarycolumn(25m×0.25mm×0.20μm;Varian).Theinjection
systemwasusedinthesplitlessmodeandkeptat300˚C.Theinterfacetothemassselective
detectorwassetat290˚C.
EachsamplewasinjectedinitiallyforthedetectionofVLCFAsintheselected-ionmonitor-
ing(SIM)modeandasecondtimeforthedetectionoftheLCFAsinscanmode.Forthedetec-
tionofVLCFAs,theGCseparationoftheanalyteswasachievedusingthefollowingcolumn
temperatureprogram:initialtemperature60˚Cfor1min,increaseto240˚Catarateof30˚C/
min,furtherincreaseto270˚Catarateof10˚C/min,finalincreaseto300˚Cat4˚C/minand5
minisothermalatthelatterlevel.Themassspectrometerwasoperatedat70eVintheSIM
mode.Adwelltimeof100msandarelativeEMvoltageof400Vhigherthanthatinthescan-
ningmodewerechosenforeachionmonitored.
ForthedetectionofLCFAs,theGCseparationoftheanalyteswasachievedusingthefol-
lowingcolumntemperatureprogram:initialtemperature50˚Cfor1min,increaseto270˚Cat
arateof30˚C/min,furtherincreaseto320˚Catarateof4˚C/minand5minisothermalatthe
latterlevel.Themassspectrometerwasoperatedat70eVintheSCANmode(scanrangeof
50–650amu).Heliumwasusedasthecarriergasataconstantflowrateof1.0ml/minforboth
VLCFAanalysisandtotalFAanalysis.MSconditionswereasfollows:EImode,ionsource
temperature,200˚C,multipliervoltage,1.182V,solventdelay5min.
TheFAs,werequantifiedwithAgilentMSDChemStationE02.00software.Alinearregres-
sionwasusedtocalibratetheidentificationandquantificationoftheFAs.Thelinearity,lower
detectionlimitandmaximumdetectionlimitweredeterminedforalltheFAs.Aqualitycon-
trol(QC)standardmixtureofhumancontrolserumwasrunwitheachbatch.Thehumancon-
trolwaspreparedbyaliquoting100μlportionsofanormalrangepooledserumintoscrew-
capvials.ThemeanandstandarddeviationforeachQCsamplewascalculatedwithamini-
mumof20between-runvalues.Controlvaluesthatfellwithinstandarddeviationsofthemean
wereconsideredacceptable.
Statisticalanalysis
Three-wayANOVAswereconductedtocomparethemainandinteractiveeffectsofage,sex
andcaptivityoneachoftheFAconcentrations.Cheetahswereclassifiedassub-adults(<2
PLOSONE|DOI:10.1371/journal.pone.0167608 December19,2016 5/14
SerumFattyAcidProfilesinCheetahs
yearsofage),youngadults(2to4yearsofage),matureadults(5to9yearsofage)oroldchee-
tahs(>10yearsofage).DatawithineachsubsetwastestedfornormalityusingtheShapiro-
Wilk’stest(p>0.05).Theconcentrationsofpalmiticacid,stearicacid,nonadecanoicacic,ara-
chidicacid,behenicacid,lignocericacid,pristanicacid,phytanicacid,totalSFA,LA,γ-linone-
nicacid,arachidonicacid(AA),eicosadienoicacid,eicosapentanoicacid,totalω-6,total
PUFAandtotalFAconcentrationswerenormallyornear-normallydistributed.Theconcen-
trationsofmyristicacid,heptadecanoicacid,ceroticacid,hypogeicacid,palmitoleicacid,oleic
acid,totalMUFA,SFA:PUFA,ω-6:ω-3andthedesaturaseindexhadtobelogarithmically
transformedtomeetnormalityrequirementsfortheANOVAanalyses.Allanalyseswereper-
formedwithSPSSversion23.0forWindows(IBMCorporation).Duetothecomparisonof
multipleFAs,aBonferroniadjustmentwasappliedtothemaineffectsintheANOVAs,taking
intoaccountthenumberofanalyses.Thisresultedinanadjustedstatisticalsignificancelevel
of0.0018(0.05/28).Forthetwo-wayandthree-wayinteractions,thestatisticalsignificancewas
keptat0.05.AllsimplepairwisecomparisonswererunwithaBonferroniadjustment.
Results
AnalysisofthemaineffectofcaptivitystatusonthevariousFAconcentrationsintheANO-
VAsresultedinanumberofsignificantdifferences,assummarisedinTable2.Alloftheindi-
vidualserumMUFAsandPUFAsaswellasthetotalMUFAandPUFAconcentrations
differedbetweenthefree-rangingandcaptivecheetahs(p<0.0005),withfree-rangingchee-
tahshavinglowervaluesformostFAs.Free-rangingcheetahsalsohadlowertotalFAconcen-
trationsthanthecaptivecheetahs(p=0.001),whereastotalSFAconcentrationsdidnotdiffer.
BoththeSFA:PUFAandSFA:MUFAratioswereonaveragemorethanthree-foldhigherin
free-rangingcheetahsthanincaptivecheetahs(p<0.0005).Eicosapentaenoicacidwasthe
onlyω-3PUFAdetectedinserumofbothfree-rangingandcaptivecheetahs.Otherω-3
PUFAs,suchasALA,docosahexaenoicacid(DHA)andeicosatetraenoicacid,wereeither
absentorbelowthelimitsofdetection.Whiletotalω-3andω-6concentrationswerelowerin
thefree-rangingcheetahs(p<0.0005),theω-6:ω-3ratiowasnotsignificantlydifferent
(p=0.198).
Otherthanforcaptivitystatus,maineffectsforagecategoryweresignificantforγ-linolenic
acidF =3.92,p=0.01(TableC2inS1File),eicosadienoicacidF =5.82,p=0.001
3,64 3,64
(TableC5inS1File),andarachidonicacidF =3.10,p=0.03(TableC3inS1File).Theγ-
3,66
linolenicacidconcentrationswerehigherincaptiveyoungadultcheetahs(131.48μmol/L)
thanincaptivematureadults(58.94μmol/L,p<0.0005)andcaptiveoldadults(74.9μmol/L,
p=0.001),buttheγ-linolenicacidconcentrationsmeasuredinthevariousagecategoriesof
free-rangingcheetahsdidnotdiffer.Arachidonicacidconcentrationswerelowerinsub-adult
free-rangingcheetahs(39.87μmol/L)thaninmatureadultfree-rangingcheetahs(59.50μmol/
L,p=0.008),butnotsignificantlydifferentfromthoseofyoungadultfree-rangingcheetahs
(51.72μmol/L).Arachidonicacidconcentrationsdidnotdifferbetweenanyofthecaptive
cheetahagecategories.Eisosadienoicacidconcentrationsdecreasedwithageinthecaptive
cheetahswithmeanvaluesof13.62μmol/Linyoungadults,10.16μmol/Linmatureadults
and9.34μmol/Linoldadults.Theconcentrationsdifferedonlybetweenyoungandmature
adults(p=0.003)aswellasyoungandoldadults(p<0.0005).Incontrast,meaneicosadienoic
acidconcentrationsdidnotdifferwithageinthefree-rangingcheetahs.
IntheANOVAstherewerenosignificantthree-wayinteractions(age,sexandcaptivitysta-
tus)foranyoftheserumFAsmeasured(TablesA1–D4inS1File).Therewasonlyonesignifi-
canttwo-waysex(cid:3)captivitystatusinteractionwhichwasforstearicacidF =5.09,p=0.03
1,66
(TableA4inS1File).Thesimplemaineffectofcaptivitystatusonstearicacidconcentrations
PLOSONE|DOI:10.1371/journal.pone.0167608 December19,2016 6/14
SerumFattyAcidProfilesinCheetahs
Table2. Totalserumfattyacidconcentrationsasmeansandstandarddeviations(SD)inμmol/Loffree-ranging(n=43)andcaptive(n=35)chee-
tahs. Theresultsofmaineffectofcaptivitystatusinthethree-wayANOVAsareshown,comparingtheserumfattyacidconcentrationsoffree-rangingand
captivecheetahs.ForstatisticaldetailsseesupplementarytablesA1-D4inS1File.SFA=saturatedfattyacids,MUFA=monounsaturatedfattyacids,
PUFA=polyunsaturatedfattyacids.Desaturaseindex=stearicacid/oleicacid.ND=notdetected.
Fattyacid(FA) Abbreviatedchemicalstructure Free-ranging Captive F p-value
Mean(SD) Mean(SD)
Myristicacid C14:0 32.88(17.20) 31.16(16.19) 0.046 0.83
Palmiticacid C16:0 1385.2(366.0) 1182.3(242.7) 3.387 0.07
Heptadecanoicacid C17:0 84.67(42.8) 45.10(9.9) 21.020 <0.0005*
Stearicacid C18:0 2127.4(520.3) 2028.9(451.4) 0.121 0.729
Nonadecanoicacid C19:0 0.03(0.02) 0.05(0.02) 3.147 0.081
Arachidicacid C20:0 30.93(7.5) 41.05(9.1) 17.106 <0.0005*
Behenicacid C22:0 40.41(12.6) 29.77(4.7) 12.928 0.001*
Lignocericacid C24:0 56.48(16.8) 36.48(6.2) 30.367 <0.0005*
Ceroticacid C26:0 0.83(0.4) 0.91(0.4) 2.582 0.113
Pristanicacid 0.71(0.8) 0.089(0.05) 6.883 0.011
Phytanicacid 13.41(8.9) 2.30(0.7) 23.746 <0.0005*
TotalSFA 3727(832) 3398(676) 1.464 0.231
Hypogeicacid C16:1ω9 24.14(16.8) 5.87(6.7) 20.632 <0.0005*
Palmitoleicacid C16:1ω7 18.35(17.4) 46.97(19.3) 26.443 <0.0005*
Oleicacid C18:1ω9 178.67(213.3) 821.44(362.4) 59.628 <0.0005*
TotalMUFA 221.1(214.9) 874.3(374.7) 55.081 <0.0005*
Linoleicacid C18:2ω6 294.2(125.9) 1049.79(329.3) 107.985 <0.0005*
γ-Linolenicacid C18:3ω6 8.83(6.6) 86.58(58.1) 52.124 <0.0005*
Eicosadienoicacid C20:2ω6 0.83(0.9) 10.90(3.4) 247.177 <0.0005*
Dihomo-γ-linolenicacid C20:3ω6 ND ND - -
Arachidonicacid C20:4ω6 52.19(17.8) 35.91(8.7) 19.845 <0.0005*
Totalω6 357(137) 1183(355) 114.912 <0.0005*
α-Linolenicacid C18:3ω3 ND ND - -
Eicosatetraenoicacid C20:4ω3 ND ND - -
Eicosapentaenoicacid C20:5ω3 5.54(4.7) 14.69(4.6) 36.860 <0.0005*
Docosahexaenoicacid C22:6ω3 ND ND - -
Totalω3 5.54(4.7) 14.69(4.6) 36.860 <0.0005*
TotalPUFA 363(140) 1183(355) 114.695 <0.0005*
ω6:ω3 281(462) 84.0(24.2) 1.692 0.198
SFA:PUFA 11.15(2.9) 3.01(0.8) 190.34 <0.0005*
SFA:MUFA 22.07(6.93) 5.78(5.1) 106.66 <0.0005*
Desaturaseindex 0.09(0.10) 0.41(0.17) 62.181 <0.0005*
TotalFA 4314(984) 5470(1253) 13.062 0.001*
*p<0.0018,Bonferroniadjustment0.05/28tests.
doi:10.1371/journal.pone.0167608.t002
wasneithersignificantformales(F =2.09,p=0.15),norforfemales(F =1.82,p=0.18),
1,66 1,66
butstearicacidconcentrationstendedtobelowerinfree-rangingfemales(1843.42μmol/L)
thaninfree-rangingmales(2198.43μmol/L,F =3.90,p=0.053,(datanotshown).
1,66
Significanttwo-waycaptivitystatus(cid:3)ageinteractionsweredetectedforoleicacidF =
1,66
5.75,p=0.019(TableB3inS1File),γ-linolenicacidF =9.72,p=0.003(TableC2inS1
1,64
File),eicosadienoicacidF =6.80,p=0.011(TableC5inS1File),SFA:MUFAF =4.89,
1,64 1,66
p=0.03(TableD1inS1File)andthedesaturaseindexF =5.31,p=0.024(TableD3inS1
1,66
File).Oleicacidconcentrations,eicosadienoicacidconcentrationsandthedesaturaseindexes
PLOSONE|DOI:10.1371/journal.pone.0167608 December19,2016 7/14
SerumFattyAcidProfilesinCheetahs
didnotdifferbetweenthedifferentagecategoriesofeithercaptiveorfree-rangingcheetahs
(datanotshown).
Discussion
Inthisstudy,thetotalserumFAprofilesoffree-rangingcheetahscontrastedmarkedlywith
thoseinthecaptivecheetahs,reflectingsignificantdifferencesindietaryFAcompositionand/
orFAmetabolism.Differencesindietaryfatintake,dietaryFAcomposition,energyexpendi-
ture,andfeedingfrequencycouldallpotentiallycontributetothesecontrastinglipidprofiles.
TheunsaturatedFAconcentrationsdifferedmostremarkablybetweenthegroups,withmost
ofthePUFAsandMUFAshavinglowerserumconcentrationsinfree-rangingcheetahsand
onlyarachidonicacidandhypogeicacidhavinglowerserumconcentrationincaptivechee-
tahs.Aspredicted,boththeSFA:PUFAandSFA:MUFAratiosweresignificantlyhigherinthe
free-ranginganimals.TheinfluenceofageandsexontheindividualFAconcentrationswas
lessnotable.
Thehigherpalmitoleicacidandoleicacidserumconcentrationsincaptivecheetahsmaybe
areflectionofdietaryintakeoftheseFAs,becausethesetwoMUFAsmakeupapproximately
34%oftheFAsindonkeymeat[35],whereastheircombinedproportionofmuscularFAsin
mostgamespeciesisbelow20%[45].However,boththeseMUFAsarealsosynthesisedinthe
liverbystearoylcoenzyme-Adesaturase(SCD)withpalmiticandstearicacidassubstrates.
TheregulationofSCDhasbeenshowntohaveconsiderablephysiologicimportance,and
alterationsinSCDexpressionandregulationhavebeenimplicatedinseveralmetabolicdis-
easesinhumansandlaboratorymice[46].TheplasmaratioofC18:0(stearicacid)toC18:1
(oleicacid)hasbeenusedasa“desaturaseindex”tomeasureSCDactivityinhumans[47].The
desaturaseindexwasmorethanfour-foldhigherinthecaptivecheetahsthaninthefree-rang-
ingindividuals.InSCD1knockoutmice,tissuelevelsofoleicandpalmitoleicacidarereduced,
whilestearicacidandpalmiticacidareincreased.Otherthangeneticmutations,elevatedcir-
culatinginsulinandglucoselevelsareknowntoincreaseSCDexpressionintheliver[48].
Seruminsulinandglucoseconcentrationshavenotbeenadequatelyevaluatedinnon-anaes-
thetisedcaptiveorfree-rangingcheetahs,butthehigherdesaturaseindexincaptivecheetahsis
consistentwithourhypothesisthattheseanimalspotentiallyhavehighercirculatingglucose
andinsulinlevelsduetomoreregularfeedingandahigherdietaryproteinintake.
ThedifferencesinserumPUFAconcentrationsbetweenfree-rangingandcaptivecheetahs
arealsoconsistentwithourhypothesis,becausetheseunsaturatedFAsoccuratlowerserum
concentrationsinthefree-rangingthanincaptivecheetahs,primarilyduetolowerdietary
intake.WesuggestthatthepreferentialoxidationofPUFAsinperiodsofincreasedexerciseor
fastinginthefree-rangingindividualscouldalsopotentiallycontributetowardsthesediffer-
ences.ArachidonicacidwastheonlyPUFAforwhichconcentrationswerehigherinthefree-
rangingcheetahs.Thisagainmayreflectdifferencesindietaryintake,asAAmakesupalarger
proportion(7.63%to9.3%)oftheintramuscularFAsinwildgamespeciessuchasthespring-
bok(Antidorcasmarsupialis)[49]thanindonkeymusclemeat(1.65%to2.09%)[36].Agreater
consumptionoforganmeatbyfree-rangingcomparedtocaptivecheetahsmayalsoresultina
higherproportionalintakeofAA[50].ArachidonicacidcanbesynthesisedfromLA,however,
similartodomesticcats,theactivityoftheΔ-6desaturaseenzyme,responsibleforcatalysing
thefirststepofthispathway,islikelytobeverylowincheetahs[29,51].PUFAsareparticularly
pronetooxidativerancidityandtheperoxidationofAAinstoredcarcassmeatcouldalso
potentiallycontributetolowerserumconcentrationsincaptivecheetahs.Domesticcats,feda
diethighinLA,withadequatelevelsofAAandantioxidants,werefoundtohavesignificantly
lowerplasmaAAlevelsatday140ofthetrial,thancatsfedadietcontaininglowerlevelsofLA
PLOSONE|DOI:10.1371/journal.pone.0167608 December19,2016 8/14
SerumFattyAcidProfilesinCheetahs
[52].Generallythecarcassmeatisnotstoredfor140daysatmostcaptivecheetahfacilities,but
itispossiblethatsomeperoxidationofthelong-chainFAstakesplaceinstorageasnoantioxi-
dantsareaddedtothemeatuntiljustbeforeitisfedtotheanimals.
Theserumconcentrationsofω-3FAswerelowinboththecaptiveandfree-rangingchee-
tahs,makinguponly0.13%and0.27%ofthetotalserumFAs,respectively(calculatedfrom
Table2).Omega-3FAs,suchasALA,DHAandeicosatetraenoicacid,reportedinserumor
plasmaofcaptivecheetahs[29]anddomesticcats[53],wereeitherabsentorbelowthelimits
ofdetection.ThereasonfortheabsenceofDHAintheplasmaofbothcaptiveandfree-rang-
ingcheetahsispotentiallyshowninanstudybyPawloskyetalinwhichtheydemonstrated
thatdeuteriumlabelledLAandALAadministeredtocatsresultsintheirliversproducingdeu-
teriumlabelledlong-chainedFAsuptoadrenicacid(22:4ω-6)anddocosapentaenoicacid
(22:5ω-3),butnotDHA[54].DeuteriumlabelledDHAandseveralotherlong-chainedFAs
were,however,detectedinthebraintissueofthecats,suggestingthatthedocosapentaenoic
acidistransportedfromthelivertothebrain,andprobablytheretina,whereitisthenfurther
metabolisedtoDHAandwhereitiscriticallyrequiredfornormalfunction[55].Theverylow
concentrationsorabsenceoftheseessentialω-3FAsintheserumofhealthyfree-livingchee-
tahsisthusaclearindicationthattheircirculatingconcentrationsshouldnotbeusedtoevalu-
atedietarydeficienciesinthisspecies.
Heptadecanoicacid,alsoknownasmargaricacid,isaSFAwithanunevennumberofcar-
bonatomsandisnormallyfoundatlowconcentrationsintheadiposetissueandmilkfatof
ruminants.ThemeanserumconcentrationofthisSFAwashigherinfree-rangingcompared
tocaptivecheetahs.Recently,theimportanceofthisFAwasdemonstratedinanothercaptive
hypercarnivore,thebottlenosedolphin(Tursiops truncates)[56].Lowserumheptadecanoic
acidconcentrationswereassociatedwithelevatedserumconcentrationsofknownmarkersof
metabolicsyndrome,includingserumferritin,glucose,insulinandtriglyceridesinthedol-
phins.Achangeinthefishspeciesprovidedinthedolphindiet,withhigherheptadecanoic
acidcontent,resultedinanormalizationofthesemarkers.TwootherFAsthatcorrelatednega-
tivelywiththemarkersofmetabolicsyndromeindolphinswereAAandbehenicacid.Bothof
theseFAswerealsodetectedatsignificantlylowerserumconcentrationsincaptivecheetahs.
Captivecheetahsarenotknowntosufferfrommetabolicsyndrome,althoughithasbeensug-
gestedthattheglomerulosclerosislesionscommonlyfoundintheseanimalsmaybecausedby
mildbutchronicelevationsinbloodglucoseconcentrations[1].Itis,nevertheless,interesting
thatthisSFA,presumedtobenon-essentialinthediet,hadsuchadramaticmetaboliceffectin
anotherobligatorycarnivore.
Thehigherserumconcentrationsofpristanicandphytanicacidsinthefree-rangingchee-
tahsarelikelytoresultfromthegreaterconsumptionofruminantprey.Phytanicacid
(3,7,11,15-teramethylhexadecanoicacid)isproducedfromphytol,thesidechainofchloro-
phyll,bythemicro-organismspresentintheruminantgastrointestinaltract[57].Mostsatu-
ratedVLCFAsarecomponentsofsphingolipids.TheC24sphingomyelin,whichincorporates
lignocericacid,isparticularlyabundantinliverandkidneytissue[58].Therefore,higher
serumconcentrationsoflignocericandbehenicacidinthefree-rangingcheetahsmayreflect
theirhigherdietaryintakeoftheseorganmeats.
ThereasonsfortheagedifferencesinAAinfree-rangingcheetahs,andγ-linolenicand
eicosadienoicacidincaptivecheetahs,isnotclear.Thecaptivecheetahsallreceivedthesame
diet,regardlessofage,andthereforethesedifferencearemorelikelytoinvolvesmallage
relatedchangesinendogenoussynthesisand/oroxidationoftheseFAratherthandifferences
indietaryintakeinthisgroup.Similardifferenceswerenotobservedinthefree-rangingchee-
tahsandthereforeotherfactorsrelatedtocaptivityarepotentiallyinvolved.Despiteapparent
differencesinthepreypreferencesoffree-rangingmaleandfemalecheetahs[31],noneofthe
PLOSONE|DOI:10.1371/journal.pone.0167608 December19,2016 9/14
SerumFattyAcidProfilesinCheetahs
serumFAsdifferedsignificantlybetweenthesexes,suggestingthatthedietaryfattyacidintake
isreasonablyuniforminfree-ranginganimals.ItisthereforeunlikelythatthelowerserumAA
concentrationsinfree-rangingsub-adultsaretheresultoflowerdietaryintakeofthisFA.The
absenceofanysub-adultcheetahsinthecaptivecheetahgroupleavesagapinourdataandit
ispossiblethatfuturestudiesthatincludecaptivecheetahsinthisagecategorymayfindlower
AAserumconcentrationsthaninoldercaptiveanimals.
Inthisstudy,wedocumentedthetotalserumFAprofilesof43apparentlyhealthyfree-
rangingcheetahsinNamibia.Theunusualdiseasessufferedbycaptivecheetahsarerarely
reportedinfree-rangingcheetahs[3,10,11].Thus,theFAprofilesoffree-rangingcheetahspro-
videareasonablesetofreferencevaluesagainstwhichtheprofilesofcaptiveindividualscanbe
assessed.ThepotentiallinksbetweenthediseasessufferedbycaptivecheetahsandtheFA
compositionoftheirdiet,however,stillremainunclear.Futurestudieswillneedtofocuson
evaluatingtheassociationsbetweentheincidenceand/orseverityofdiseaseandthevarious
serumFAs,aswellastheclinicaleffectsofdietarymanipulationsand/orFAsupplementation.
TherelativeeffectsofextendedfeedingintervalsandincreasedphysicalactivityonserumFAs
incaptivecheetahswillalsoneedtobeassessed.Free-rangingcheetahsrarelyscavengeand
consumemostoftheirpreywithinafewhoursafterakill.Fatsconsumedunderthesecondi-
tionshavelittletimetobecomerancidandthehigherintakeoftheoxidativelymorestable
SFAswouldresultinareducedneedforextensiveantioxidantmechanismstodealwithperox-
idisedFAsinthisspecies.TheextentandimpactofdietaryFAperoxidationshouldtherefore
alsobeassessedinfuturestudies.
Alimitationofourstudywasthelackofcontroloverthefastingperiodpriortosamplingin
thefree-rangingcheetahs.Althoughnobaitwasusedintheircapturetraps,itispossiblethat
someofthecheetahsmayhavefedwithinafewhoursbeforeimmobilizationandsampling.
Duringthepostprandialperiod,thenon-esterifiedfattyacids(NEFAs)generallydeclinein
responsetorisinginsulinlevels,whilethetriacylglycerollipidfractionsincreaseslightlyor
remainstableinhealthyindividuals[59].SincetheNEFAfractionmakesuponlyapproxi-
mately6%ofthetotalserumFAs[26],thepostprandialvariationintotalserumFAsislikelyto
besmall.Nevertheless,itwouldbevaluabletoassessthechangesinserumFAsduringthepost-
prandialperiodinhealthycaptivecheetahsfedondifferentdiets.
Anadditionalpotentiallimitationinourstudywasthedifferencesinserumseparationand
samplestoragetimespriortoanalysisinthecaptiveversusthefree-rangingcheetahs.Although
circulatingNEFAfractionspotentiallyincreaseinbloodandplasmaafter48hoursat4˚C[60],
storageofvariousplasmalipidclasses,includingtriglyceride,cholesterolester,phospholipid
andNEFAfractionsat-20˚Cforayear,wasshowntohaveminimaleffectonstabilityeven
withouttheuseofnitrogenstorage[61].Inarecentreview,MetherelandStarkconcludedthat
plasma/serumstorageat4˚Cforlessthan6days,-20˚Cforonetothreeyearsand-80˚Cforup
to10yearswouldnotlikelyresultinsignificantchangesinPUFAconcentrations.Theeffects
ofdelayedbloodcellseparationontotalserumorplasmaFAconcentrationshavenottoour
knowledgebeenevaluated.However,adelayof24hoursbeforeseparationinsampleskeptat
4˚C,resultedinanincreaseinNEFAconcentrationsofbetween0%and8%inplasma[62,63]
and10%inserum[62].Theseincreasesaremostlikeduetotheactionoflipolyticenzymes
whichresultinaFAshiftfromtheboundfractionstofreeFApool.Suchchangesarenotlikely
toaffectthecombined/totalplasmaorserumFAconcentrationsandthereforethedifferences
insamplehandling,priortostorage,wouldbeexpectedtohaveaminimaleffectonourresults.
TheimpactofdifferentsamplehandlingproceduresontotalserumorplasmaFAsunderfield
conditionsshouldneverthelessbeinvestigatedinfuturestudies.
Inconclusion,wefoundmarkeddifferencesintheserumFAsofcaptiveversusfree-ranging
Namibiancheetahs.Thesedifferencesarelikelytolargelyreflectdifferencesindietaryfat
PLOSONE|DOI:10.1371/journal.pone.0167608 December19,2016 10/14