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RESEARCHARTICLEOpenAccessExpressionofgenesinvolvedinhepaticcarnitinesynthesisanduptakeindairycowsinthetransitionperiodandatdifferentstagesoflactationGloriaSchlegel1,JanineKeller1,FrankHirche2,StefanieGeiler2,FriederJSchwarz3,RobertRingseis1,GabrieleIStangl2andKlausEder1*AbstractBackground:Inrodentsandpigs,ithasshownthatcarnitinesynthesisanduptakeofcarnitineintocellsareregulatedbyperoxisomeproliferator-activatedreceptora(PPARA),atranscriptionfactorwhichisphysiologicallyactivatedduringfastingorenergydeprivation.
Dairycowsaretypicallyinanegativeenergybalanceduringearlylactation.
Weinvestigatedthehypothesisthatgenesofcarnitinesynthesisanduptakeindairycowsareenhancedduringearlylactation.
Results:mRNAabundancesofPPARAandsomeofitsclassicaltargetgenesandgenesinvolvedincarnitinebiosynthesis[trimethyllysinedioxygenase(TMLHE),4-N-trimethylaminobutyraldehydedehydrogenase(ALDH9A1),g-butyrobetainedioxygenase(BBOX1)]anduptakeofcarnitine[novelorganiccationtransporter2(SLC22A5)]aswellascarnitineconcentrationsinliverbiopsysamplesof20dairycowsinlatepregnancy(3wkprepartum)andearlylactation(1wk,5wk,14wkpostpartum)weredetermined.
From3wkprepartumto1wkpostpartum,mRNAabundancesofPPARΑandseveralPPARΑtargetgenesinvolvedinfattyaciduptake,fattyacidoxidationandketogenesisintheliverwerestronglyincreased.
Simultaneously,mRNAabundancesofenzymesofcarnitinesynthesis(TMLHE:10-fold;ALDH9A1:6-fold;BBOX1:1.
8-fold)andcarnitineuptake(SLC22A5:13-fold)andtheconcentrationofcarnitineintheliverwereincreasedfrom3wkprepartumto1wkpostpartum(P<0.
05).
From1wkto5and14wkpostpartum,mRNAabundancesofthesegenesandhepaticcarnitineconcentrationsweredeclining(P<0.
05).
Thereweremoreoverpositivecorrelationsbetweenplasmaconcentrationsofnon-esterifiedfattyacids(NEFA)andhepaticcarnitineconcentrationsat1wk,5wkand14wkpostpartum(P<0.
05).
Conclusions:Theresultsofthisstudyshowforthefirsttimethattheexpressionofhepaticgenesofcarnitinesynthesisandcellularuptakeofcarnitineisenhancedindairycowsduringearlylactation.
Thesechangesmightprovideanexplanationforincreasedhepaticcarnitineconcentrationsobservedin1wkpostpartumandmightberegardedasaphysiologicmeanstoprovidelivercellswithsufficientcarnitinerequiredfortransportofexcessiveamountsofNEFAduringanegativeenergybalance.
BackgroundCarnitine(3-hydroxy-4-N,N,N-trimethylaminobutyricacid)isanessentialmetabolitethathasanumberofindispensablefunctionsinintermediarymetabolism.
Themostimportantfunctionliesinitsroleinthetransportofactivatedlong-chainfattyacids(acylgroups)fromthecytosolintothemitochondrialmatrixwhereb-oxidationtakesplace[1].
Carnitineisderivedfromdietarysourcesandsynthesizedendogenouslyfromtrimethyllysine(TML),whichisreleaseduponproteindegradation.
ThereleasedTMLisfurtheroxidizedtog-butyrobetaine(BB)bytheactionoftrimethyllysinedioxygenase(TMLHE),3-hydroxy-N-TMLaldolaseand4-N-tri-methylaminobutyraldehydedehydrogenase(ALDH9A1).
Inthefinalbiosyntheticstep,BBishydroxylatedbyg-butyrobetainedioxygenase(BBOX1)toformcarnitine.
Incattlethislaststepoccursonlyinliverandkidney[2].
Tissueswhicharenotcapableofproducing*Correspondence:klaus.
eder@ernaehrung.
uni-giessen.
de1InstituteofAnimalNutritionandNutritionPhysiology,Justus-Liebig-UniversittGiessen,Heinrich-Buff-Ring26-32,D-35392Giessen,GermanyFulllistofauthorinformationisavailableattheendofthearticleSchlegeletal.
BMCVeterinaryResearch2012,8:28http://www.
biomedcentral.
com/1746-6148/8/282012Schlegeletal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
carnitinedependontheuptakeofcarnitinefrombloodbynovelorganiccationtransporters(OCTN),particu-larlynovelorganiccationtransporter2(SLC22A5)whichisthephysiologicallymostimportantcarnitinetransporter[3,4].
Studiesinrodentsandpigsdemon-stratedthatcarnitinebiosynthesisanduptakeofcarni-tinefrombloodintocellsbySLC22A5aredirectlyregulatedbyperoxisomeproliferator-activatedreceptora(PPARA),atranscriptionfactorwhichplaysacentralroleintheadaptationofmetabolismtoenergydefi-ciency[5].
Inthesespecies,activationofPPARAsuchasinducedbyfastingortreatmentwithsyntheticagonistsledtoincreasedtissuecarnitineconcentrationsduetoanincreasedrateofbiosynthesisandanincreaseduptakeofcarnitinefrombloodintotissues[6-10].
Indairycows,thetransitionfromlatepregnancytoearlylactationisassociatedwithseveremetabolicadap-tations.
Productionofmilkleadstoastrongincreaseoftheenergyrequirement,whichhowevercannotbemetasthefoodintakecapacityislimited.
Thus,duringearlylactation,dairycowsaretypicallyinanegativeenergybalancewhichiscompensatedbythemobilizationofnon-esterifiedfattyacids(NEFA)fromadiposetissue.
NEFAaretransportedbybindingwithserumalbuminandaretakenupbyfattyacidtransportersintotissues,mainlytheliver[11].
StudiesinrodentshaveclearlyestablishedthatNEFAtakenupintotheliverareabletobindtoandactivatePPARA[12,13].
Incontrasttothelargebodyofliteratureinnon-ruminants,verylittleworkhasbeenconductedtodefinethespecificeffectsormechanismsofPPARAincattleliversofar.
However,arecentstudyusingclofibrateasasyntheticagonistinweanedcalvesshowedthatPPARAisfunctionalincat-tleliver[14].
Moreover,ithasbeenshownthatlongchainfattyacidsareabletoactivatePPARAalsoinbovinecells[15].
Inaccordancewiththisfinding,anegativeenergybalanceindairycattle,eitheroccurringphysiologicallyduringearlylactationorinducedbyfeedrestriction,wasassociatedwithanup-regulationofsev-eralPPARAtargetgenesinvolvedinfattyacidoxidationorketosisintheliver,indicativeofanactivationofPPARA[16,17].
Toourknowledge,theregulationofcarnitinehome-ostasisindairycattlehasbeenlessinvestigated.
How-ever,ithasbeenfoundthathepaticcarnitineconcentrationindairycowsisincreasingduringthetransitionfromlatepregnancytoearlylactation[18,19].
ThatfindingandtheassumptionthatgenesinvolvedincarnitinehomeostasisindairycowsmightberegulatedbyPPARAsuchasinotherspecies,promptedustothehypothesisthathepaticgenesofcarnitinesynthesisanduptakeofcarnitineareup-regulatedduringearlylacta-tionindairycows.
Toinvestigatethishypothesis,wedeterminedmRNAabundancesoftherelevantgenesinvolvedincarnitinesynthesisaswellascarnitinecon-centrationsinliverbiopsysamplesofdairycowsinlatepregnancyandearlylactation.
MethodsTheanimalexperimentwasconductedattheAgricul-turalExperimentalStationHirschauoftheTechnicalUniversityofMunich,Germany.
ItwasapprovedbytheBavarianstateanimalcareandusecommittee.
AnimalsandfeedingThisstudyincludedtwentyHolsteincows(fourprimi-andsixteenmultiparous,2.
7±0.
3parities,mean±SE)asexperimentalanimalswithanexperimentalperiodfrom3wkprepartumuntil14wkpostpartum.
Theani-malswerehousedinaplaypen.
Feedingwascomposedofapartialmixedration(PMR)foradlibitumintakeofbasicfeedandseparatelyallocatedconcentrates[supple-mentalconcentrate(SUPP),0.
63kgDM/dforeachcow;individualconcentrate(CONC),individualaccess].
PMRconsisted(drymatter,DM,basis)of33.
7%grasssilage,44.
9%maizesilage,6.
4%hayand14.
9%concentratewhileSUPPcontained(DMbasis)24.
4%soybeanmeal,48.
3%grainmaizeand27.
3%rumen-protectedfatsup-plement.
Withanassumeddrymatterintakeof16kgofPMR/dandtheallotedamountofSUPP,thecalculatednutrientsupplycoveredtheenergyandproteinrequire-mentsfor23kgofmilk/d.
CONCwasindividuallyallo-catedatfourcomputer-operatedfeedingstationswithanautomaticfeedingprogram(DeLavalAlpro,Glinde,Germany).
CONCwascomposedof24.
8%grainmaize,21.
8%wheat,20.
1%soybeanmeal,15.
2%driedsugarbeetpulpwithmolasses,14.
9%barleyand3.
2%vitamin-mineralpremixincludinglimestone(DMbasis).
TheallocationofCONCwasincreasedfrom1.
2to8.
0kgofDM/dduringthefirst42doflactation,andthereafter,itwasdependentonthemilkperformanceoftheindivi-dualcow.
DailyintakesofPMRandCONCwererecordedforeachindividualcow.
Thecowsweregener-allyinagoodhealthcondition,althoughfourcowshadslightmetabolicdiseases(subclinicalketosis,subclinicalacidosis)andninecowssufferedtemporarilyfrommas-titis.
Allusedfeedcomponentsweresampledandana-lyzedforDMcontent,forcrudenutrients,crudeash,crudefibreandcrudefataccordingto[20],andcrudeproteinbyDumasmethod.
AccordingtotheGermanSocietyofNutritionPhysiology[21],thenetenergycon-tent(MJNEL)andtheavailableCPattheduodenumwerecalculated.
NutrientconcentrationsandenergycontentofallfeedcomponentsareshowninTable1.
SamplecollectionMilkingoflactatingcowsoccuredtwicedaily(0500and1500h)ina2*6herringbonemilkingparlor(DeLaval).
Schlegeletal.
BMCVeterinaryResearch2012,8:28http://www.
biomedcentral.
com/1746-6148/8/28Page2of12Milkyieldsofeachcowwererecordedautomaticallyandstoredindatafiles.
Representativemilksamples(50mL)fromeveryindividualcowcomprisedtwoconsecu-tivemilkingprocedures(oneeveningandnextmorningmilking)andwerecollectedtwiceweekly.
Milksamplingat1,5and14wkpostpartumoccurredatdays5.
1±1.
6,29.
7±1.
9,and92.
7±1.
9(means±SE),respec-tively.
At3wkprepartum(21.
1±6.
0dprepartum)and1,5and14wkpostpartum(3.
7±1.
5,30.
9±1.
9and94.
2±2.
6dpostpartum),bloodsamplesofthedammedvenajugularisweredrawnusingsterile20Gcanulasandlithiumheparintubes(Greinerbio-one,Kremsmun-ster,Austria).
Bloodsamplinghappenedbeforemorningfeedingbetween0730and0900h.
Tubeswerekeptoniceuntilsubsequentcentrifugation(2000*g;15min).
Then,plasmawastransferredinto1.
5mLtubes(Grei-nerbio-one,Frickenhausen,Germany)andstoredinali-quotsat-20°Cuntilanalysis.
Furthermore,liverbiopsiesweretakenat3wkprepartum(20.
4±5.
8dprepartum),and1,5and14wkpostpartum(3.
8±1.
4,31.
5±2.
1and94.
9±2.
9dpostpartum).
Forthispurpose,cowswereseparatedandfixedaftermorningmilkingbeforefeedingbetween0700and0900h.
Theliverbiopsysiteontherightsideofthecowbetweenthe11thand12thribsonalinebetweentheolecranonandthetubercoxaewasshavedanddisinfectedbeforealocalsubcuta-neousanesthesiawith5mLIsocaine2%(Procainhy-drochloride/Epinephrin,Selectavet,Weyarn/Holzolling,Germany)wasperformed.
Thenanautoclavedcanulawasintroducedasaductforthesterile14Gbiopsyneedle(DispomedWittoHG,Gelnhausen,Germany)andabout50mgoflivertissuewereremovedandimmediatelysnap-frozeninliquidnitrogen.
Sampleswerestoredat-80°Cuntilfurtheranalysis.
Thebiopsysitewastreatedwithwoundsprayandanimalswerekeptseparatedforoneday.
SampleanalysisMilkproteinandmilkfatcontentswereanalyzedbyinfraredspectrophotometry(MilkoScan-FT-6000,FossAnalyticalA/S,Hillerod,Denmark)atthelaboratoryofMilchprüfringBayerne.
V.
,Wolnzach,Germany.
NEFAandBHBAweredeterminedinthethawedplasmasamplesusingcommercialavailablekits[NEFA-HR(2)andAutokit3-HB,obtainedfromWakoChemicalsGmbH,Neuss,Germany].
Lipidsfromliverbiopsysam-pleswereextractedwithamixtureofn-hexaneandiso-propanol(3:2,vol/vol)[22].
Analiquotoftheextractscontaining25-50nmolesofTAGwaspipettedintoaglassvial(1.
5ml),andthesolventwasevaporatedbyvacuum.
Thelipidswereresolvedina20μlportionofa1:1-mixtureofchloroformandTritonX-100[23],andagainthesolventwasevaporated.
Then1mlofcom-merciallyavailableenzymaticTAGkitreagent(FluitestTG,AnalyticonBiotechnologiesAG,Lichtenfels,Ger-many)wasadded,andafterincubation-accordingtotheinstructionofthemanufacturer-theTAGcontentwasdeterminedbycolorimetry.
EnergybalanceForcalculationoftheaveragedailyenergybalanceofeveryindividualcow,energyintakewascalculatedfromthemeandailyintakeofPMR,SUPPandCONCandthecorrespondingenergycontents(MJNEL).
Bodyweights(BW)ofthecowswereautomaticallyrecordeddailybyelectronicscalesinstalledinthefeedingsta-tions.
UsingtheweeklymeanBWofthecows,energyrequirementsformaintenancewerecalculatedaccordingtotheGermanSocietyofNutritionPhysiology[21].
Thoseformilkproductionwerecalculatedonthebasisofweeklymeansofdailymilkyield,milkproteincon-tentandmilkfatcontent[21].
Changesinbodycompo-sitionwerenotconsideredinenergybalanceevaluation.
CarnitineanalysisConcentrationsoftotalcarnitine,freecarnitine,acetyl-carnitineandpropionylcarnitineinplasma,milkandlivertissueweredeterminedbytandemmassspectro-metry[24,25].
Inbrief,freezedriedtissuesampleswereextractedwithmethanol:water(2:1v/v)byhomogeniza-tion(TissueLyser,Qiagen,Hilden,Germany),followedbysonificationfor20minandincubationat50°Cfor30mininashaker.
Aftercentrifugation(13000*g,10min)20μLofthesupernatantwereaddedwith100μLmethanolcontainingtheinternalstandards,mixed,incu-batedfor10min,andcentrifuged(13000*g,10min).
Plasmaandmilksampleswerehandledat4°Cinthesamemannerasthesupernatantaftertissueextraction.
Thefinalsupernatantswereusedforquantificationofthecompoundsbya1100seriesHPLC(AgilentTech-nologies,Waldbronn,Germany)equippedwithaKro-masil100column(125mm*2mm,5μmparticlesize,CS-ChromatographieServiceLangerwehe,Germany)andanAPI2000LC-MS/MS-System(AppliedBiosys-tems,Darmstadt,Germany).
Aseluents,methanolandamethanol:water:ACN:aceticacidmixture(100:90:9:1v/v/v/v)wereused.
Table1NutrientvaluesofexperimentalfeedstuffPMRCONCSUPPEnergy*(MJNEL/kgofDM)6.
458.
0012.
8Crudefibre(g/kgofDM)2146769Crudeash(g/kgofDM)817249Crudefat(g/kgofDM)3220303CP(g/kgofDM)129184140AvailableCP(g/kgofDM)*142187151*calculatedvaluesSchlegeletal.
BMCVeterinaryResearch2012,8:28http://www.
biomedcentral.
com/1746-6148/8/28Page3of12RNAisolationandquantitativereal-timePCR(qPCR)TotalRNAwasisolatedfromliverbiopsiesusingTrizolreagent(Invitrogen,Karlsruhe,Germany)accordingtothemanufacturer'sprotocol.
RNAfrom10mgofeachsamplewasisolatedwithinoneweekafterfinishingthetrial.
IsolatedRNAwaspreservedat-80°Cuntiluse.
ToestimateRNAconcentrationandpurity,theopticalden-sityat260and280nm,respectively,wasdeterminedusinganInfinite200MmicroplatereaderandaNano-QuantPlate(bothfromTecan,Mannedorf,Switzerland).
TheA260/A280ratioswere1.
96±0.
05.
Inaddition,theopticaldensityat230nmwasdeterminedandtheA260/A230ratioswerecalculatedtocontrolforthepre-senceofcontaminationssuchasguanidinethiocyanate.
AlthoughtheA260/A230ratioofsomesampleswasbelow2.
0indicatingthepresenceofguanidinethiocya-nate,ithasbeenshownthatguanidinethiocyanatehasnomeasurableeffectondownstreamapplicationssuchasRT-qPCRuntilconcentrationsofmorethan100mM[26]Moreover,RNAqualitywasassessedby1%agarosegelelectrophoresis.
RNAwasjudgedassuitableforonlyifthesamplesexhibitedintactbandscorrespondingtothe18Sand28SribosomalRNAsubunits.
cDNAwassynthesizedafterRNAextractionfrom1.
2μgoftotalRNAusing100pmoldT18primer(EurofinsMWGOperon,Ebersberg,Germany),1.
25μL10mmol/LdNTPmix(GeneCraft,Ludinghausen,Germany),5μLbuffer(Fermentas,St.
Leon-Rot,Deutschland),and60unitsM-MuLVReverseTranscriptase(MBIFermentas,St.
Leon-Rot,Germany)at42°Cfor60min,andafinalinactivatingstepat70°Cfor10mininBiometraTher-malCycler(WhatmanBiometraR,Gttingen,Germany).
Subsequently,cDNAwasstoredinaliquotsat-20°C.
ForthestandardcurveacDNApoolofallsampleswasmade.
qPCRwasperformedusing2μLcDNAcombinedwith18μLofamixturecomposedof10μLKAPASYBRFASTqPCRUniversalMastermix(Peqlab,Erlan-gen,Germany),0.
4μLeachof10μMforwardandreverseprimersand7.
2μLDNase/RNasefreewaterin0.
1mLtubes(LtfLabortechnik,Wasserburg,Germany).
Gene-specificprimerpairsobtainedfromEurofinsMWGOperon(Ebersberg,Germany)weredesignedusingPrimer3andBLAST.
FeaturesofprimerpairsarelistedinTable2.
Allprimerpairsweredesignedtohaveannealingtemperaturesofabout60°C,and,ifpossible,bothprimersofaprimerpairweredesignedtobelocatedindifferentexons.
qPCRrunswereperformedwithaRotorgene2000system(CorbettResearch,Mor-tlake,Australia),andincludedallsamplesanda5pointrelativestandardcurveplusthenon-templatecontrol(NTC).
TheqPCRprotocolwasasfollows:3minat95°C,followedby40cyclesofatwo-stepPCRconsistingof5secat95°C(denaturation)and20secat60°C(anneal-ingandextension).
Subsequently,meltingcurveanalysiswasperformedfrom50°Cto95°CtoverifythepresenceofasinglePCRproduct.
Inaddition,theamplificationofasingleproductoftheexpectedsizewasconfirmedusing2%agarosegelelectrophoresisstainedwithGel-RedTMnucleicacidgelstain(Biotium,California,USA).
DataonqPCRperformanceforeachgenesmea-suredarealsoshowninTable2.
ReferencegenestabilitywasdeterminedbyperformingGeNormanalysis[27]whichisbasedoncalculationofareferencegene-stabi-litymeasureM.
Outofsixtestedpotentialreferencegenes,thethreereferencegeneswiththelowestMvalueshavethemoststableexpressionandareusedtocalculatetheGeNormnormalizationfactor.
Therefore,allCtvaluesweretransformedintorelativequantifica-tiondatabyusingthe2-ΔΔCtequation[28].
UsingtheGeNormnormalizationfactor,relativeexpressionlevelswerecalculated,andfromnormalizedexpressiondata,meansandSEwerecomputedforsamplesofthesamelactationweek.
Themeanof3wkprepartumwassetto1andrelativeexpressionratiosof1,5and14wkpost-partumareexpressedasfoldchangescomparedto3wkprepartum.
StatisticsDatawerestatisticallyevaluatedbyageneralizedlinearmodel,includingthefactorstimepointofsampling,ani-mal,paritynumberandtheinteractionsbetweenthesefactors,usingtheMinitabStatisticalSoftwareRelease13.
0(Minitab,StateCollege,PA,USA).
Priortostatisti-calanalysis,alldatawerecheckedfornormalityandoutliersbeforestatisticalanalysis.
Astherewasnosig-nificanteffectofanimalandparitynumberonthepara-metersinvestigated,onlytheeffectsoftimepointofsamplingarereportedintheresultssection.
Linearregressionmodelsforrelationshipsofcarnitineconcen-trationsinlivertissuewithmetabolicparametersatthedifferenttimepointsweresubjectedtoanalysisbyfittedlineplots.
ThesignificancesofdifferencesbetweenthegroupsovertimewereanalyzedbyFisher'smultiplerangetest.
DifferenceswereregardedassignificantforP<0.
05.
ResultsDrymatterintake,performanceandenergybalanceindairycowsinthetransitionperiodandatdifferentstagesoflactationDrymatterintakeofthecows1wkpostpartumwassimilartothat3wkprepartumandincreasedthereaftertowards5and14wkpostpartum(Table3).
Theonsetoflactationledtoastrongnegativeenergybalance(Table3).
Energybalancethatwasstrongestnegativein1wkpostpartumwasthenimproving.
At14wkpost-partum,thecowsreturnedtoaslightpositiveenergybalance(Table3).
Milkyieldwasincreasingfrom1to5Schlegeletal.
BMCVeterinaryResearch2012,8:28http://www.
biomedcentral.
com/1746-6148/8/28Page4of12wkpostpartumandwasthereafterdecliningtowards14wkpostpartum(Table3).
Milkfatandmilkproteincon-tentswerehighestat1wkpostpartumandwerethere-afterdeclining(Table3).
RelativemRNAabundancesofPPARAandgenesinvolvedinfattyaciduptake,fattyacidoxidationandketogenesisintheliverofdairycowsinthetransitionperiodandatdifferentstagesoflactationTherelativemRNAabundanceofPPARAintheliverwasincreasedfrom3wkprepartumto1wkpostpartum(P<0.
05)andwasthereafterdeclining,reachingvaluesat5wkand14wkpostpartumsimilarwiththatof3wkprepartum(Figure1).
InaccordancewiththeexpressionpatternofPPARA,targetgenesinvolvedinfattyaciduptake(SLC27A1,CD36),mitochondrialandperoxiso-malb-oxidation(ACOX1,CPT1A,ACADM)andketo-genesis(HMGCS2)wererisingfrom3wkprepartumto1wkpostpartum(P<0.
05,Figure1).
From1to5and14wkpostpartum,mRNAabundancesofthesegenes,withtheonlyexceptionofCD36,weredeclining(P<0.
05,Figure1).
mRNAabundancesofACOX1,SLC27A1andHMGCS2remainedatahigherlevelat5and14wkpostpartumthanat3wkprepartum(P300.
991.
990.
039CACCTTCACCGTTCCAGTTTSDHAGCAGAACCTGATGCTTTGTG185NM_174178-0.
240.
991.
740.
048CGTAGGAGAGCGTGTGCTTATP5BGGACTCAGCCCTTCAGCGCC229NM_175796.
2-0.
160.
991.
440.
039GCCTGGTCTCCCTGCCTTGCRPS9GTGAGGTCTGGAGGGTCAAA108BC148016-0.
310.
992.
040.
040GGGCATTACCTTCGAACAGAPPIAGGCAAATGCTGGCCCCAACACA87NM_178320.
2-0.
340.
992.
130.
034AGTACCACGTGCTTGCCATCCARPL12CACCAGCCGCCTCCACCATG84NM_205797.
1-0.
350.
992.
250.
036CGACTTCCCCACCGGTGCACTargetgenesACADMGCGAGTACCCTGTCCCATTA243NM_001075235-0.
290.
991.
93CCTCAGTCATTCTCCCCAAAACOX1CCATTGCCGTCCGATACAGT99BC102761-0.
270.
961.
88GTTTATATTGCTGGGTTTGATAATCCAALDH9A1CAGGATTCGGCAGAGAGAAC229NM_001046423-0.
280.
991.
90TGAGCCATGAAGAGCATCACBBOX1TCCAGCTGCCTACTCTGGAT292BC149884.
1-0.
280.
991.
91AGCTGAACCTTACCCCAGGTCD36GCATTCTGAAAGTGCGTTGA179BC103112-0.
280.
981.
91CGGGTCTGATGAAAGTGGTTCPT1ACAAAACCATGTTGTACAGCTTCCA111FJ415874-0.
320.
992.
09GCTTCCTTCATCAGAGGCTTCAHMGCS2GCCCAATATGTGGACCAAAC209NM_001045883-0.
290.
991.
96ATGGTCTCAGTGCCCACTTCPPARAGGTGGAGAGTTTGGCAGAACCAGA168BT020756.
1-0.
230.
991.
70TCCCACTGCCCAGCTCCGATCSLC22A5CACAGTGGTCAGGAACATGG181BC105377-0.
280.
991.
89AATGGTGTCTGGGAGTGGAGSLC27A1CTGAAGGAGACCTCCACAGC208NM_001033625.
2-0.
300.
991.
99GTGGTACAGGGGCAGACAGTTMLHETGGCAGGACACTGCTAGTTG222NM_001076064.
1-0.
310.
992.
05GACAGCCCGGTCATAGTTGTSchlegeletal.
BMCVeterinaryResearch2012,8:28http://www.
biomedcentral.
com/1746-6148/8/28Page5of120.
05,Figure1).
Incontrast,mRNAabundancesofCPT1AandACADM,twoenzymesofmitochondrialb-oxidation,returnedtolevelssimilartothoseat3wkprepartum(Figure1).
RelativemRNAabundancesofgenesinvolvedincarnitinesynthesisandcarnitineuptakeintheliverofdairycowsinthetransitionperiodandatdifferentstagesoflactationmRNAabundancesofthetwogenesinvolvedinthefor-mationofg-butyrobetaine-theprecursorofcarnitine-TMLHEandALDH9A1,werestrongly(10-,and6-fold,resp.
)increasedfrom3wkprepartumto1wkpostpar-tum(P<0.
05,Figure2).
mRNAabundanceofBBOX1,theenzymewhichconvertsg-butyrobetaineintocarni-tine,wasmoderately(1.
8-fold)increasedfrom3wkpre-partumto1wkpostpartum(P<0.
05,Figure2).
mRNAabundanceofSLC22A5,themostimportantcarnitinetransporter,wasstrongly(13-fold)increasedfrom3wkprepartumto1wkpostpartum(P<0.
05,Figure2).
WiththeexceptionofBBOX1,mRNAabundancesofallthesegenesinvolvedincarnitinesynthesispathwayandcarnitineuptakeweredecliningfrom1wkto5and14wkpostpartum.
Nevertheless,mRNAabundancesofthesegenesin5and14wkpostpartumremainedatlevelshigherthanthoseat3wkprepartum(P<0.
05,Figure2).
Concentrationsofcarnitineinliver,plasmaandmilkofdairycowsinthetransitionperiodandatdifferentstagesoflactationIntheliver,freecarnitinewasnearlytheexclusiveformofcarnitinewhereasconcentrationsofcarnitineesters(<1nmol/g)wereonlyslightlyabovethedetectionlimitandarethereforenotreported.
Liverfreecarnitinecon-centrationwasrisingfrom3wkprepartumto1wkpostpartum(P<0.
05)andwasthereafterfallingtolevelsbelowthoseobservedat3wkprepartum(Table4).
Inplasma,theconcentrationoffreecarnitinewasstronglydecreasingfrom3wkprepartumto1wkpostpartumandwasthereafterrisingtovalueswhichremainedhow-everbelowthoseobserved3wkprepartum(P<0.
05,Table4).
Incontrast,theconcentrationofcarnitineestersinplasmawasincreasingfrom3wkprepartumto1wkpostpartum(P<0.
05)andwasthereafterdecreas-ing;plasmaconcentrationofcarnitineestersat14wkpostpartumwasevenlowerthanthat3wkprepartum(P<0.
05,Table4).
Plasmaconcentrationoftotalcarni-tinedecreasedfrom3wkprepartumto1wkpostpar-tum(P<0.
05)andremainedthereafterataconstantlevel(Table4).
Concentrationsoffree,esterifiedandtotalcarnitineinmilkwerehighestat1wkpostpartum;concentrationswerethereafterdecreasingandweresimilarat5wkand14wkpostpartum(Table4).
Correlationsbetweenlivercarnitineconcentrationandphenotypicmeasures(plasmaNEFAandBHBA,hepaticconcentrationofTAG)InordertoassesswhetherplasmaNEFAconcentrationsinfluencelivercarnitineconcentrations,wecalculatedalinearregressionbetweenplasmaNEFAandlivercarni-tineconcentrations.
Asexpected,plasmaNEFAconcen-trationswerestronglyincreasingfrom3wkprepartumto1wkpostpartumandwerethereafterdeclining(Table5).
Atallthethreetimepointsconsideredduringlactation(1wk,5wk,14wkpostpartum),asignificantpositivecorrelationbetweenplasmaNEFAconcentra-tionandlivercarnitineconcentrationwasobserved(P<0.
05,Table6).
Incontrast,aninversecorrelationbetweenplasmaNEFAconcentrationandlivercarnitineconcentrationwasobservedat3wkprepartum.
InordertoelucidatewhetherlivercarnitinestatusinfluenceshepaticTAGaccumulationorketonebodyformation,wecalculatedlinearregressionsbetweenlivercarnitineconcentrationsandliverTAGconcentrationsorplasmaBHBAconcentrations.
Asexpected,liverTAGTable3PerformanceofdairycowsinthetransitionperiodandatdifferentstagesoflactationVariable3wkprepartum1wkpostpartum5wkpostpartum14wkpostpartumwks1to14postpartumP-valueDrymatterintake(kg/d)13.
4c±0.
3313.
4c±0.
4818.
4b±0.
6220.
4a±0.
5318.
5±0.
49<0.
001Milkyield(kg/d)-28.
6c±0.
7637.
5a±0.
9032.
0b±0.
8132.
7±0.
67<0.
001FCM*(kg/d)-39.
2a±1.
3940.
9a±1.
5133.
0b±1.
0337.
7±0.
87<0.
001Milkfat(%)-6.
40a±0.
254.
51b±0.
204.
11b±0.
135.
00±0.
17<0.
001Milkprotein(%)-4.
09a±0.
092.
85c±0.
053.
13b±0.
063.
35±0.
08<0.
001Energybalance(MJNEL/d)46.
9a±2.
42-64.
9d±5.
15-30.
4c±3.
053.
89b±2.
11-12.
5±5.
52<0.
001Energyintake(%ofrequirement)215.
1±7.
9258.
9±2.
4582.
3±2.
22102.
1±2.
1081.
1±2.
63<0.
001Valuesaremean±SE(n=20)a,b,c,dMeanswithdifferentsuperscriptsdiffersignificantly(P<0.
05)*Correctedfor4%milkfatcontentCalculatedvalueSchlegeletal.
BMCVeterinaryResearch2012,8:28http://www.
biomedcentral.
com/1746-6148/8/28Page6of12ACADMPPARACPT1ASLC27A1HMGCS2Figure1CD36ACOX13wkprepartum1wkpostpartum5wkpostpartum14wkpostpartum3wkprepartum1wkpostpartum5wkpostpartum14wkpostpartum3wkprepartum1wkpostpartum5wkpostpartum14wkpostpartum3wkprepartum1wkpostpartum5wkpostpartum14wkpostpartum3wkprepartum1wkpostpartum5wkpostpartum14wkpostpartum3wkprepartum1wkpostpartum5wkpostpartum14wkpostpartum3wkprepartum1wkpostpartum5wkpostpartum14wkpostpartumrelativemRNAabundancerelativemRNAabundancerelativemRNAabundancerelativemRNAabundanceFigure1RelativemRNAabundancesofPPARAandgenesinvolvedinfattyaciduptake,fattyacidoxidationandketogenesis.
RelativemRNAabundancesofPPARAandgenesinvolvedinfattyaciduptake(SLC27A1,CD36),fattyacidoxidation(ACOX1,CPT1A,ACADM)andketogenesis(HMGCS2)intheliverofdairycowsinthetransitionperiodandatdifferentstagesoflactation;barsrepresentmeans±SE(n=20)andareexpressedrelativetothemRNAabundanceat3wkprepartum.
a,b,cBarswithdifferentsuperscriptsdiffersignificantly(P<0.
05).
Schlegeletal.
BMCVeterinaryResearch2012,8:28http://www.
biomedcentral.
com/1746-6148/8/28Page7of12TMLHEALDH9A1SLC22A5BBOX1Figure23wkprepartum1wkpostpartum5wkpostpartum14wkpostpartumrelativemRNAabundancerelativemRNAabundance3wkprepartum1wkpostpartum5wkpostpartum14wkpostpartum3wkprepartum1wkpostpartum5wkpostpartum14wkpostpartum3wkprepartum1wkpostpartum5wkpostpartum14wkpostpartumFigure2RelativemRNAabundancesofgenesinvolvedincarnitinesynthesisandcarnitineuptake.
RelativemRNAabundancesofgenesinvolvedincarnitinesynthesis(TMLHE,ALDH9A1,BBOX1)andcarnitineuptake(SLC22A5)intheliverofdairycowsinthetransitionperiodandatdifferentstagesoflactation;barsrepresentmeans±SE(n=20)andareexpressedrelativetothemRNAabundanceat3wkprepartum.
a,b,cBarswithdifferentsuperscriptsdiffersignificantly(P<0.
05).
Table4Concentrationsofcarnitineinliverbiopsy,plasmaandmilksamplesofdairycowsinthetransitionperiodandatdifferentstagesoflactation3wkprepartum1wkpostpartum5wkpostpartum14wkpostpartumP-valueLivertissuefreecarnitine,nmol/gwetweight37.
4b±4.
5355.
6a±4.
0926.
0c±1.
7618.
2c±1.
26<0.
001Plasmafreecarnitine,μmol/L3.
82a±0.
251.
40c±0.
102.
18b±0.
202.
35b±0.
17<0.
001carnitineesters*,μmol/L2.
07b±0.
102.
54a±0.
122.
45ab±0.
181.
64c±0.
11<0.
001totalcarnitine,μmol/L5.
89a±0.
333.
94b±0.
174.
64b±0.
323.
99b±0.
26<0.
001Milkfreecarnitine,μmol/L-84.
5a±5.
9250.
3b±4.
3460.
2b±2.
65<0.
001carnitineesters*,μmol/L-131.
1a±12.
165.
9b±6.
2341.
3c±2.
60<0.
001totalcarnitine,μmol/L-215.
5a±16.
6116.
2b±7.
92101.
5b±3.
93<0.
001Valuesaremean±SE(n=20)a,b,cMeanswithdifferentsuperscriptsdiffersignificantly(P<0.
05)*Sumofacetyl-andpropionylcarnitine,SumoffreecarnitineandcarnitineestersSchlegeletal.
BMCVeterinaryResearch2012,8:28http://www.
biomedcentral.
com/1746-6148/8/28Page8of12concentrationwashighestat5wkpostpartumandBHBAconcentrationswerehighestat1wkand5wkpostpartum(Table5).
However,nosignificantcorrela-tionsbetweenlivercarnitineconcentrationsandliverTAGorplasmaBHBAconcentrationsemerged,bothpre-andpostpartum(Table6).
DiscussionThisstudywasperformedtoinvestigatethehypothesisthattheonsetoflactationindairycowsleadstoanup-regulationofgenesinvolvedinhepaticcarnitinesynth-esisanduptakeofcarnitine.
Asexpected,thetransitionfromlatepregnancytoearlylactationwasassociatedwithastrongnegativeenergybalanceresultinginincreasedconcentrationsofNEFAandBHBAinplasmaandanincreaseinhepaticTAGconcentration.
Similarmetabolicchangesduringtheperiparturientperiodincowshavebeenobservedinmanyotherstudies[e.
g.
[16,18,29]].
Inagreementwitharecentstudy[16],weobservedthatthenegativeenergybalanceoccurringatearlylactationwasassociatedwithanincreasedexpres-sionofseveralPPARAtargetgenesinvolvedinfattyaciduptake,mitochondrialandperoxisomalfattyacidoxidationandketogenesis.
AlthoughwewerenotabletogivedirectproofofPPARAactivationduetosmallliversampleamountavailable,anup-regulationofvar-iousPPARAtargetgenesisindicativeofanactivationofthattranscriptionfactorintheliver.
WhilethereislessresearchaboutactivationofPPARAincattle,studiesinotherspeciessuchasrodentsorpigshaveclearlyshownthatincreasedplasmaNEFAconcentrations,inducedbyenergydeprivation,areleadingtoanactivationofPPARAinliverandothertissues[12,13,30].
Aslong-chainfattyacidsarealsoactingasagonistsofPPARAinbovinecells[15],itseemsjustifiedtospeculatethathighplasmaNEFAconcentrationsindairycowsduringearlylactationwerecausinganactivationofPPARAintheliver.
Itshouldbenoted,however,thatinoppositetoourstudyandthestudyofLooretal.
[16],therearealsostudieswhichdidnotobserveanup-regulationofPPARAandPPARAtargetgenesintheliverofdairycattleduringearlylactation,particularlywhencowshadprepartumacaloricintakeinexcessof100%oftheirenergyrequirement[31,32].
Thelackofup-regulationofPPARAduringearlylactationinthesestudieshasbeenexplainedbyahepaticinflammatoryresponse,inducedbyanexcessiveprepartumcaloricintake,whichdecreasedpre-andpostpartumhepaticexpressionofexpressionofPPARA[32].
Inaccordancewiththehypothesisofthisstudy,weobservedforthefirsttimethatthetransitionfromlatepregnancytoearlylactationleadstoanup-regulationofvariousgenesinvolvedincarnitinesynthesis(ALDH9A1,TMLHE,BBOX1)andcarnitineuptake(SLC22A5)intheliverofcowsat1wkpostpartum.
AsallthesegenesarePPARAtargetgeneswithfunctionalPPREsidentifiedintheirpromotersorfirstintrons[32-35],weassumethattheup-regulationofthesegenesintheliverofdairycowsat1wkpostpartumwascausedbyapotentialactivationofPPARA.
ThefindingofpositivecorrelationsbetweenplasmaconcentrationsofNEFA,whichmightberegardedasnaturalagonists,andhepaticcarnitineconcentrationsduringlactationsupportsaroleofPPARAintheregulationofgenesofcarnitinesynthesisanduptake.
Thepresentstudyconfirmspreviousstudiesinshow-ingthatlivercarnitineconcentrationisincreasingdur-ingthetransitionfromlatepregnancytolactationandisthereaftercontinuouslydecreasingtovaluessimilarorevenbelowthoseobservedinpregnancy[18,19].
InthestudyofCarlsonetal.
[19],hepatictotalcarnitinecon-centrationswerearound1.
6-foldhigheron2doflacta-tioncomparedto3wkprepartum,whilevaluesatd28wereevenlowerthanthose3wkprepartum.
InthestudyofGrumetal.
[18],concentrationsofacid-solublecarnitine(freecarnitineplusshort-chainacylcarnitine)sharplyincreasedfrom3wkprepartumto1dpostpar-tumandreturnedtoprepartumvaluesatd21postpar-tum.
Ascarnitineissynthesizedfromtrimethyllysinereleasedprimarilyfromturnoverofskeletalmusclepro-teins,Grumetal.
[18]suggestedthatanincreasedhepa-ticcarnitineconcentrationat1dpostpartummightbeduetoanenhancedcatabolismofmuscleproteininthisearlystageoflactation.
Althoughwehavenotdirectlymeasuredcarnitinesynthesisanduptakeintocells,thefindingsofincreasedmRNAexpressionofgenesofcar-nitinesynthesisanduptakesuggestthatincreasedhepa-ticcarnitineconcentrationsatearlylactationcouldalsoTable5Metabolicparametersinliverbiopsyandplasmasamplesofdairycowsinthetransitionperiodandatdifferentstagesoflactation3wkprepartum1wkpostpartum5wkpostpartum14wkpostpartumP-valuePlasmaNEFA(μmol/L)103d±10864a±47276b±16171c±11<0.
001Livertriglyceride(mg/gwetweight)4.
56c±0.
4523.
5b±2.
664.
3a±10.
33.
18c±0.
28<0.
001PlasmaBHBA(μmol/L)484b±23707a±42724a±46521b±24<0.
001Valuesaremean±SE(n=20).
a,b,c,dMeanswithdifferentsuperscriptsdiffersignificantly(P<0.
05)Schlegeletal.
BMCVeterinaryResearch2012,8:28http://www.
biomedcentral.
com/1746-6148/8/28Page9of12bedue,atleastinpart,toanincreasedcarnitinesynth-esisintheliverandanincreaseduptakeofcarnitinefrombloodintotheliver.
Thissuggestionissupportedbystudiesinrodentsandpigswhichfoundthatanup-regulationofenzymesofcarnitinesynthesisintheliver,causedeitherbytreatmentwithPPARAagonistsorbyenergydeprivation,leadstoanincreasedhepaticcarni-tineconcentrationwithoutchangingtheconcentrationsofBBorTML,theprecursorsofcarnitinesynthesis[7,8,36,37].
Thefindingthattheconcentrationoffreecarnitineinplasmaisstronglydecreasingfrom3wkprepartumto1wkpostpartumfitsintothissuggestion.
Studiesinrodentshaveshownthatanup-regulationofSLC22A5bytreatmentwithPPARAagonistsorbyenergydeprivationleadstoareductionofplasmacarni-tineconcentration,duetoanincreasedtransportofcar-nitinefromplasmaintotissues[6,8,36].
Thedecreaseinplasmacarnitineconcentrationsduringearlylactationmightbeinpartexplainedbythetransferofcarnitinefromplasmaintothemilkinmammarygland.
Never-theless,withrespecttothefindingsinrodents,weassumethatreducedplasmacarnitineconcentrationsinearlylactationcouldalsobecausedbyanincreaseduptakeoffreecarnitinefromplasmaintotissues,includ-ingtheliver.
Inaccordancewithpresentstudy,Carlsonetal.
[19]alsoobservedastrongreductionofplasmacarnitineconcentrationfrom21dprepartumto9or27dpostpartum.
Thoseauthors[19]alsofoundthatcarni-tineconcentrationinskeletalmuscleisnotchangingsignificantlyduringtransitionfrompregnancyintolacta-tion.
Thisfindingalsoagreeswithstudiesinrodentswhichshowthatenergydeprivationdoesnotinfluencemusclecarnitineconcentrations[7,36].
Furthermore,weobservedthatmilkcarnitineconcen-trationishighestat1wkpostpartumandisthereafterdecreasingto5wkand14wkpostpartum.
ThisfindingagreeswiththestudyofCarlsonetal.
[19]whichfoundastrongreductionofmilkcarnitineconcentrationfrom2wkto6wkpostpartum.
Itispossiblethatthehighcarnitineconcentrationinmilkatwk1postpartumisduetothestrongnegativeenergybalanceofthecows.
Carlsonetal.
[38]foundthatenergyrestrictionofcowsincreasedmilkcarnitineconcentrations.
Inthemam-marygland,carnitineissecretedintothemilkbyseveraltransporters(SLC22A4,SLC22A5,OCTN3,SLC6A14,SLC6A10)[39].
Possibly,oneormoreofthesetranspor-tersareup-regulatedinastateofanegativeenergybal-ance.
Studiesinratsalsofoundareductionofmilkcarnitineconcentrationfromearlytolaterstageoflacta-tion,duetoadown-regulationofSLC22A5,OCTN3andSLC6A14[39].
Ketosisandfattyliveraretwodiseasesindairycowsduringearlylactationwhicharelinkedtohepaticfattyacidoxidation[40].
Ascarnitineisinvolvedinb-oxida-tionduetoitsroleinthetransportoflongchainfattyacidsintothemitochondrion,itwasinterestingtoexplorewhetherplasmaketonebodyconcentrationsorhepaticTAGconcentrationsarecorrelatedwithhepaticcarnitineconcentrations.
Theobservationthattherewerenocorrelationsbetweenhepaticcarnitineconcen-trationandboth,plasmaBHBAandhepaticTAGcon-centrations,at1,5and14wkpostpartumsuggeststhattheavailabilityofcarnitineintheliverhadnoinfluenceonketogenesisandTAGaccumulationintheliver.
TheobservationthathepaticcarnitineconcentrationdoesnotcorrelatewithBHBAconcentrationisinaccordancewithastudyshowingthatcarnitinesupplementationdoesnotinfluenceplasmaBHBAconcentrationinearlylactatingdairycattle[41].
ThefindingthattheactivityofCPT1Adoesnotplayaprimaryroleintheetiologyofketosis[42]isanotherindicationthattheavailabilityofcarnitine,whichactsasacofactorofthatenzyme,isTable6Linearregressionparametersfortherelationshipofconcentrationoffreecarnitineinliverbiopsysamples(nmol/gwetweight)aspredictorvariablewithdifferentmetabolicparametersofdairycowsinthetransitionperiodandatdifferentstagesoflactationasresponsevariablesResponsevariable3wkprepartum1wkpostpartum5wkpostpartum14wkpostpartumPlasmaNEFA(μmol/L)Intercept152.
7285.
7115.
781.
4Slope-1.
1710.
25.
864.
66R20.
290.
640.
380.
26P-value0.
015<0.
0010.
0080.
035Livertriglyceride(mg/gwetweight)Intercept3.
0730.
544.
13.
81Slope0.
04-0.
140.
65-0.
05R20.
150.
090.
010.
09P-value0.
1120.
2910.
6730.
305PlasmaBHBA(μmol/L)Intercept562.
8605.
3603.
7503.
3Slope-1.
841.
434.
560.
60R20.
130.
020.
030.
00P-value0.
1130.
6210.
5050.
907Schlegeletal.
BMCVeterinaryResearch2012,8:28http://www.
biomedcentral.
com/1746-6148/8/28Page10of12notakeyfactorintheproductionofketonebodies.
AccumulationofTAGintheliverisexplainedbytheobservationthatthecapacityofbovinelivertissuetoconvertfattyacidstoesterifiedproductsisstronglyincreasedduringtheearlypostnatalperiod,whereasfattyacidoxidationisonlyslightlyincreased,meaningthatNEFAfrommobilizationaredirectedtowardscon-versiontoTAG[43].
Thefindingthattherewasnocor-relationbetweenhepaticcarnitineconcentrationandhepaticTAGconcentrationthusindicatesthatahigherhepaticcarnitineconcentrationdidnotstimulatehepaticfattyacidoxidation.
Thisindicationis,however,incon-tradictiontosomeinvitroandinvivostudiesindairycows.
Invitrostudiesusingbovineliversliceshaveshownthatadditionofcarnitineenhancestheoxidationofpalmitate[44,45].
Moreover,postruminalinfusionofcarnitineenhancedpalmitateoxidationanddecreasedliverlipidaccumulationincowswithexperimentallyinducednegativeenergybalance[41].
Thesestudiessug-gestedthatcarnitinemightbetherate-limitingfactorofhepaticb-oxidationindairycowsduringtheperiparturi-entperiodandthatcarnitinesupplementationmightpreventthedevelopmentofafattyliver.
ConclusionsThepresentstudyshowsforthefirsttimethathepaticmRNAabundancesofgenesinvolvedincarnitinesynth-esisandcellularuptakeofcarnitineindairycowsareincreasedduringthetransitionfromlatepregnancytolactation.
Anup-regulationofgenesinvolvedincarni-tinebiosynthesisanduptakecouldcontributetoele-vatedhepaticcarnitineconcentrationinearlylactationobservedinthisandpreviousstudies.
AcknowledgementsGloriaSchlegelwassupportedbyH.
WilhelmSchaumann-Stiftung(Hamburg,Germany).
Authordetails1InstituteofAnimalNutritionandNutritionPhysiology,Justus-Liebig-UniversittGiessen,Heinrich-Buff-Ring26-32,D-35392Giessen,Germany.
2InstituteofAgriculturalandNutritionalSciences,Martin-Luther-UniversittHalle,Von-Danckelmann-Platz2,D-06120Halle,Saale,Germany.
3AnimalNutrition,TechnischeUniversittMünchen,Liesel-Beckmann-Strasse6,D-85354Freising,Germany.
Authors'contributionsGS:conductedtheanimalexperiment,performedthestatisticalanalysesandwrotethemanuscript.
JK:performedthePCRanalyses.
FHandSG:performedthecarnitineanalysesandhelpedtodraftthemanuscript;FJS:participatedinthedesignofthestudyandsupervisedtheanimalexperiment;RR:supervisedPCRanalyses.
GIS:supervisedthecarnitineanalyses.
KE:conceivedofthestudy,andparticipatedinitsdesignandcoordinationandhelpedtodraftthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
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doi:10.
1186/1746-6148-8-28Citethisarticleas:Schlegeletal.
:Expressionofgenesinvolvedinhepaticcarnitinesynthesisanduptakeindairycowsinthetransitionperiodandatdifferentstagesoflactation.
BMCVeterinaryResearch20128:28.
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