Maternal Plane of Nutrition during Late Gestation and Weaning Age Alter Angus × Simmental

20 Pages·2015·0.56 MB·English

RESEARCHARTICLE
Maternal Plane of Nutrition during Late
Gestation and Weaning Age Alter Angus ×
Simmental Offspring Longissimus Muscle
Transcriptome and Intramuscular Fat
SoniaJ.Moisá1,DanielW.Shike2,LindsayShoup2,SandraL.Rodriguez-Zas2,3,Juan
J.Loor1,2,4*
1 MammalianNutriPhysioGenomics,DepartmentofAnimalSciences,UniversityofIllinois,Urbana,Illinois,
UnitedStatesofAmerica,2 DepartmentofAnimalSciences,UniversityofIllinois,Urbana,Illinois,United
StatesofAmerica,3 TheInstituteforGenomicBiology,UniversityofIllinois,Urbana,Illinois,UnitedStatesof
America,4 DivisionofNutritionalSciences,IllinoisInformaticsInstitute,UniversityofIllinois,Urbana,Illinois,
UnitedStatesofAmerica
* [email protected]
Abstract
OPENACCESS
Citation:MoisáSJ,ShikeDW,ShoupL,Rodriguez- Inmodelorganismsboththenutritionofthemotherandtheyoungoffspringcouldinduce
ZasSL,LoorJJ(2015)MaternalPlaneofNutrition long-lastingtranscriptionalchangesintissues.Inlivestock,suchchangescouldhaveimpor-
duringLateGestationandWeaningAgeAlterAngus
tantrolesindeterminingnutrientuseandmeatquality.Themainobjectivewastoevaluateif
×SimmentalOffspringLongissimusMuscle
planeofmaternalnutritionduringlate-gestationandweaningagealtertheoffspring’sLong-
TranscriptomeandIntramuscularFat.PLoSONE
10(7):e0131478.doi:10.1371/journal.pone.0131478 issimusmuscle(LM)transcriptome,animalperformance,andmetabolichormones.Whole-
transcriptomemicroarrayanalysiswasperformedonLMsamplesofearly(EW)andnormal
Editor:PascaleChavatte-Palmer,INRA,FRANCE
weaned(NW)Angus×Simmentalcalvesborntograzingcowsreceivingnosupplement
Received:March26,2015
[lowplaneofnutrition(LPN)]or2.3kghigh-grainmix/day[mediumplaneofnutrition(MPN)]
Accepted:June1,2015
duringthelast105daysofgestation.BiopsiesofLMwereharvestedat78(EW),187(NW)
Published:July8,2015 and354(beforeslaughter)daysofage.DespitegreaterfeedintakeinMPNoffspring,blood
Copyright:©2015Moisáetal.Thisisanopen insulinwasgreaterinLPNoffspring.CarcassintramuscularfatcontentwasgreaterinEW
accessarticledistributedunderthetermsofthe offspring.Bioinformaticsanalysisofthetranscriptomehighlightedamodestoverall
CreativeCommonsAttributionLicense,whichpermits
responsetomaternalplaneofnutrition,resultinginonly35differentiallyexpressedgenes
unrestricteduse,distribution,andreproductioninany
(DEG).However,weaningageandahigh-graindiet(EW)stronglyimpactedthetranscrip-
medium,providedtheoriginalauthorandsourceare
credited. tome(DEG=167),especiallycausingalipogenicprogramactivation.Inaddition,between
78and187daysofage,EWsteershadanactivationoftheinnateimmunesystemduepre-
DataAvailabilityStatement:Allmicroarraydata
filesareavailablefromtheNCBIGEOdatabase sumablytomacrophageinfiltrationofintramuscularfat.Between187and354daysofage
(accessionnumberGSE65560). (the“finishing”phase),NWsteershadanactivationofthelipogenictranscriptomemachin-
Funding:ThisstudywasfundedbytheDivisionof ery,whileEWsteershadaclearinhibitionthroughtheepigeneticcontrolofhistoneacety-
NutritionalSciencesVision20/20”(Universityof lases.Resultsunderscoredtheneedtoconductfurtherstudiestounderstandbetterthe
Illinois,Urbana-Champaign)interdisciplinarynutrition-
functionaloutcomeoftranscriptomechangesinducedintheoffspringbypre-andpost-natal
relatedresearchprogram,andproject#ILLU-971–
nutrition.Additionalknowledgeonmolecularandfunctionaloutcomeswouldhelpproduce
352(NationalInstituteofFoodandAgriculture,USA).
Thefundershadnoroleinstudydesign,data moreefficientbeefcattle.
collectionandanalysis,decisiontopublish,or
preparationofthemanuscript.
PLOSONE|DOI:10.1371/journal.pone.0131478 July8,2015 1/20
NeonatalNutritionAltersOffspringMuscleTranscriptome
CompetingInterests:Theauthorshavedeclared Introduction
thatnocompetinginterestsexist.
Theprenatalperiodsduringwhichtheorganismissusceptibletoenvironmentalstimulilead-
ingtofetalprogrammingaretheembryonicphase,themid-gestationperiod(organogenesis),
andlategestation(rapidgrowth).Theeffectsofmaternalnutritiononfetalgrowthandits
carry-overeffectsonoffspringgrowthanddevelopmentwerereviewedrecently.Asanexam-
ple,extremeintrauterinegrowthretardationcanresultinslowergrowththroughoutpostnatal
life[1].Maternalnutritionseemstoelicitdifferentoutcomesintheoffspringdependingonthe
gestationalstageduringwhichtreatmentsbegin.Forexample,proteinsupplementationof
grazingcowsduringlategestationinthewinterenhancedfeedlotperformanceandcarcass
qualityoftheoffspring.
Inareviewonfetalprogrammingandskeletalmuscledevelopmentintheruminant[2],it
wasconcludedthatadipogenesisisinitiatedduringmid-gestation.Atthistime,apoolofundif-
ferentiatedmesenchymalstemcellsispresent.Fromthispool,eithermyocytesoradipocytes
areabletodifferentiatefromcommittedmesenchymalcellstobecomeskeletalmuscleoradi-
posetissue[3].Theincreaseinnumberofstemcellsthroughoutmiddle-to-lategestationledto
thehypothesisthatnutritionalmanagementhasthepotentialtobemoreeffectiveduringthe
prenatalperiodratherthanthepostnatalportionofananimal’slife[2].Insheep,astudy
revealedthatdifferencesinmaternalnutritionduringmid-to-lategestationcanimpactthepro-
gramingoffetalmuscleandfattissues[4].However,itremainstobedeterminedifplaneof
nutritionduringlate-pregnancy(last90days)canelicitcarryovereffectsacquiredthroughpro-
gramminginbeefcattle.
Thereareseveralmaternal-nutritionstudiesutilizingreal-timeRT-PCRtoevaluatespecific
targetgenesinadiposetissueorlongissimusmuscle(LM)ofbeef[5],lamb[6]andsheep[7]
offspring.Despitetheseefforts,tothebestofourknowledge,therearenopublishedstudiesof
whole-transcriptomeprofilesinLMofoffspringfrommothersfedhighorlowplanesofnutri-
tionduringlate-pregnancyinbeefcattle.
Ourhypothesiswasthatahigh-planeofnutritionofthecowduringlategestationwould
resultinearlyactivationofgenesassociatedwithmyogenesis,adipogenesis,lipogenesisandthe
synthesisofadipokinesintheoffspring’sskeletalmuscle.Furthermore,changesinnutritionof
thepregnantmotheralsowouldelicitalterationsassociatedwithepigeneticregulationofgene
expression.Theobjectivesofthisstudyweretoassesstheeffectofmaternalplaneofnutrition
andearlyweaningtoahigh-graindietontheskeletalmuscletranscriptomeoftheoffspring.
MaterialsandMethods
AnimaluseinthisstudywasapprovedbytheInstitutionalAnimalCareandUseCommittee
(IACUC)oftheUniversityofIllinois.Asubsetof20Angus×Simmentalbeefcowsfromthe
UniversityofIllinoisDixonSpringsAgricultureCenterinSimpson,IL(USA),wereselected
fromagroupofanimalsutilizedinaparallelstudy[8].Maineffectsevaluatedwerematernal
planeofnutritionduringlategestationandpostnatalmanagementoftheoffspring.Three
monthspriortotheprojectedparturitiondatecowswereassignedtotreatments(lowor
mediumplaneofnutrition)inasplit-plotdesign.Lowplaneofnutrition(LPN)wasachieved
bygrazingendophyte-infectedtallfescue/redcloverpasturesduringJuly,August,andSeptem-
berwithnosupplement.Mediumplaneofnutritioncowdiet(MPN)wasachievedbygrazing
endophyte-infectedtallfescue/redcloverpasturessupplementeddailywith2.3kgofdrieddis-
tiller’sgrainswithsolublesandsoyhulls(70%DDGS/30%soyhulls).Cowsupplementationwas
initiatedat103±11daysprepartumwhileonpastureanditwashaltedatthemidpointofpar-
turition(2±11dayspostpartum).Moreinformationaboutcowsupplementationisreported
elsewhere[8].
PLOSONE|DOI:10.1371/journal.pone.0131478 July8,2015 2/20
NeonatalNutritionAltersOffspringMuscleTranscriptome
Compositionofthediet[drymatter(DM)basis]fedtoearlyweaned(EW)steersupon
arrivaltothefeedlotandpriortonormalweaning(NW),andthefeedlotdietfedtoEWand
NWsteersafternormalweaningarereportedinTable1.Angus×Simmentalsteercalveswere
randomlyassignedtoearlyornormalweaning(EWorNW)treatmentswithineachgestational
treatment.At78±2dayspostpartum,EWoffspringwereweaned,transportedtoUniversityof
IllinoisBeefandSheepFieldLaboratory(Urbana,IL,USA),andadaptedtoahigh-graindiet
untiltheyhadadlibitumconsumption.At187±2dayspostpartum,NWoffspringwere
weanedandtransportedtoUniversityofIllinoisBeefandSheepFieldLaboratory.Alloffspring
wereco-mingledamongtreatments.
Bloodwascollectedfromthejugularveinat78,187and296daysofagetoisolateserumfor
insulin(BovineInsulinELISAkit,CatNo.10–1201–01,MercodiaAB,Uppsala,Sweden),glu-
cose(HexokinaseG-6-PDHmethodusingaBeckmanCoulter,Fullerton,CA,USA;Diagnos-
ticsLaboratory,CollegeofVeterinaryMedicine,UniversityofIllinois,Urbana,USA)and
adiponectin(Millipore,LA,USA).ThelatterwasdeterminedusingaliquidRIA(Millipore,
LA,USA)followingaprotocolpreviouslydescribed[9].Afternormalweaning,alloffspring
wereplacedonacommon,grain-basedfinishingdietthatistypicalofindustrymanagement
[crudeprotein(CP)%,18.1,neutraldetergentfiber(NDF)%,25.3,aciddetergentfiber(ADF)
Table1. Compositionofdiet(DMbasis)fedtoearlywean(EW)steersuponarrivaltofeedlotandprior
tonormalweaning1andfeedlotdietfedtoEWandnormalwean(NW)steersafternormalweaning.
Inclusion,%DM
Item EWdiet Feedlotdiet
Ingredient,%
MWDGS2 45 45
DryWholeCorn 25 25
HighMoistureCorn — —
CornHusklage 20 20
Groundcorn 7.3 7.3
Limestone 2.5 2.5
Tracemineralsalt3 0.1 0.1
Rumensin904 0.018 0.018
Tylosin405 0.012 0.012
Soybeanoil 0.076 0.076
Analyzednutrientcontent,%
Crudeprotein 17.3 18.1
Neutraldetergentfiber 23.9 25.3
Aciddetergentfiber 14.1 14.3
Crudefat 5.3 5.1
1Ageatweaning:EW=78±11daysofage;NW=186±11daysofage.
2MWDGS=ModifiedWetDistillersGrainswithSolubles.
3TraceMineralSalt=8.5%Ca(asCaCO3),5%Mg(asMgOandMgSO4),7.6%K(asKCl2),6.7%Cl(as
KCl2)10%S(asS8,prilled),0.5%Cu(asCuSO4andAvaila-4(ZinproPerformanceMinerals;Zinpro
Corp,EdenPrairie,MN)),2%Fe(asFeSO4),3%Mn(asMnSO4andAvaila-4),3%Zn(asZnSO4and
Availa-4),278ppmCo(asAvaila-4),250ppmI(asCa(IO3)2),150Se(Na2SeO3),2,205KIU/kgVitA(as
retinylacetate),662.5KIU/kgVitD(ascholecalciferol),22,047.5IU/kgVitE(asDL-α-tocopherylacetate),
andlessthan1%CP,fat,crudefiber,salt.
4Rumensin90(198gmonensin/kgRumensin90;ElancoAnimalHealth,Greenfield,IN,USA).
5Tylosin40(88gtylan/kgTylosin40;ElancoAnimalHealth,Greenfield,IN,USA).
doi:10.1371/journal.pone.0131478.t001
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NeonatalNutritionAltersOffspringMuscleTranscriptome
%,14.3,crudefat%,5.1](Table1).Alltheoffspringinthestudywereharvestedatacommer-
cialpackingplantwhentheyreachedtheselectedendpointtargetbackfatthicknessof1.1cm.
Reportedfinalbodyweight(BW)wascalculatedfromhotcarcassweightusinga62%dressing
percentage.
LMbiopsieswereharvestedfromasubsetof5animalspergestational×postnataltreatment
fromthemainherdat~78daysofage,~187days,andduringthelastweekpriortoharvest
(~354days).Selectionofsteerprogenyforbiopsywasperformedbasedon2criteria:first,off-
springforbiopsywereselectedbasedontheirdam’sperformance.Onlyoffspringfromcows
whoseBWandBWchangeduringlategestationwaswithin½ofastandarddeviationoneither
sideofthemean(LPNorMPN)wereconsideredforbiopsy.SelectingbasedoncowBWand
BWchangeensuredthatonlycalvesfromcowsthatwererepresentativeoftheirtreatment
wereutilizedfortranscriptomics.Thefinalselectionofsteersforbiopsywasbasedonsteer
BW.OnlysteerswhoseBWwaswithin½ofastandarddeviationoneithersideofthemean
wereutilized.Thisselectioncriterionstrategyminimizedtheeffectsofvariationindam’smilk
production,whichwasnotsignificantlydifferentbetweenLPNandMPNtreatments[8].
Transcriptomicswasperformedwithatranscriptome-widebovinemicroarray(Agilent-
015354BovineOligoMicroarray-4x44K)thatcontains21,475uniquegenesandtranscriptsof
BosTaurus,withtwoprobespergene.Themethodsusedforhybridizationandscanningwere
accordingtomanufacturer’sprotocolsandLooretal[10].Themicroarraydataweredeposited
intheNationalCenterforBiotechnologyInformation(NCBI)GeneExpressionOmnibus
(GEO)database(http://www.ncbi.nlm.nih.gov/gds)withaccessionnumberGSE65560.
Datamining
TheentiremicroarraydatasetwithassociatedstatisticalP-valueswereimportedintoIngenuity
PathwaysAnalysis(IPA,www.ingenuity.com)inordertoexaminethenumberofactivated
andinhibiteddifferentiallyexpressedgenes(DEG).EntrezGeneIDswereusedtoidentifyindi-
vidualsequences.
Statisticalanalysis
Datafromthemicroarrayanalysiswerenormalizedfordyeandmicroarrayeffects(i.e.,Lowess
normalizationandarraycentering)andusedforstatisticalanalysis.TheMIXEDprocedureof
SAS(SASInstitute,Inc.,Cary,NC,USA)wasusedforstatisticalanalysis.Fixedeffectswere
treatment(EW,NW),diet(LPN,MPN),time(78,187,and354daysofage),first,secondand
thirdorderinteractionsbetweendiet,timeandtreatment,anddye(Cy3,Cy5)andrandom
effectsincludedsteerandmicroarray.RawPvalueswereadjustedusingBenjaminiandHoch-
berg’sfalsediscoveryrate(FDR).
Thestatisticalmodelusedwas:Yijklm=μ+T +D +W +S +(T×D) +(D×W) +
i j k l ij jk
(T×W) +(D×T×W) +ε ;where,Y isthebackground-adjustednormalizedfold
ik ijk ijklm ijklm
changevalue;μistheoverallmean;T isthefixedeffectoftime(3levels);D isthefixedeffect
i j
ofcowplaneofnutrition(2levels);W isthefixedeffectofweaningage(2levels);S istheran-
k l
domeffectofsteernestedwithintreatment;T×D,D×W,T×Waretheinteractionsoftime
bydiet,dietbyweanandtimebywean,respectively;D×T×Wistheinteractionofthird
orderforthemaineffects;andε istherandomerror(0,σ 2)associatedwithY .All
ijklm e ijklm
meanswerecomparedusingthePDIFFstatementofSAS(SASInstitute,Inc.,Cary,NC,USA).
StatisticalsignificancewasdeclaredatP(cid:1)0.05andFDR(cid:1)0.10.Animalfeedlotperformance,
carcassqualityparameters,ultrasoundandblooddatawerealsoanalyzedusingtheMIXED
procedureofSAS,andtreatmentwasthefixedeffectinthestatisticalmodel.Therandomeffect
inallmodelswassteerwithintreatment.
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NeonatalNutritionAltersOffspringMuscleTranscriptome
Dynamicimpactapproach(DIA)
BioinformaticsanalysisofmicroarraydatawasperformedusingDIA[11]andinformation
fromthefreely-availableonlinedatabasesKyotoEncyclopediaofGenesandGenomes(KEGG)
andDatabaseforAnnotation,Visualization,andIntegratedDiscovery(DAVID)v6.7data-
bases.Alistofgeneidentifiers(EntrezGeneIDs)wasuploadedallatoncetoextractandsum-
marizefunctionalannotationsassociatedwithgroupsofgenesorwitheachindividualgene.
Thesignificancevalueassociatedwithbiologicalprocessesandpathwaysisameasureofthe
likelihoodthatthedistributionofDEGinthesepathwaysandbiologicalprocessesisdueto
chance.ThesignificanceisexpressedasaP-value,whichiscalculatedusingtheright-tailed
Fisher'sExactTestandadjustedusingFDR.DetailsoftheDIAapproachanditsvalidation
havebeenreportedpreviously[11].Theinterpretationofthebioinformaticsanalysiswasper-
formedfollowingthesameapproachasourpreviousstudy[12].
Results
Animalperformance
Performancedatafortheentiregroupofanimalsonstudyarereportedelsewhere[8].Feedlot
performanceofEWandNWsteersusedfortranscriptomicsispresentedinTable2.Onlyini-
tialBWatthetimesteersenteredthefeedlotwassignificant(P<0.01)withEWsteersbeing
heavierascomparedwithNWsteers.Drymatterintake(DMI)washigherforMPNas
Table2. Feedlotperformance,ultrasoundmeasurementsandcarcassqualityparametersforAngus×Simmentalsteers(n=20)fromcowsthat
receivedalow(LPN)oramedium(MPN)planeofnutrition(D)duringthelategestationperiod. Weaningtimes(W)areearlyweaning(EW)andnormal
weaning(NW).
Treatments
EW NW P-value1
Item LPN MPN LPN MPN SEM D W D*W
FeedlotPerformance
InitialBW 258 278 212 204 18.25 0.73 <0.01 0.42
FinalBW2 548 581 524 533 19.78 0.26 0.07 0.52
ADG(kg/d) 1.74 1.66 1.81 1.69 0.15 0.40 0.56 0.85
DMI(kg/d) 7.84 9.33 8.18 8.48 0.41 0.03 0.52 0.14
Ultrasoundmeasurements
BFatEW(cm) 0.31 0.35 0.33 0.34 0.02 0.13 0.71 0.52
BFatNW(cm) 0.33 0.33 0.31 0.33 0.02 0.54 0.57 0.74
MarblingatEW 427 388 434 419 27.80 0.30 0.48 0.65
MarblingatNW 330 409 376 418 40.41 0.13 0.47 0.63
Carcassqualityparameters
HCW(kg) 340 360 325 331 12.27 0.27 0.07 0.51
CalculatedYG 3.0 3.0 2.5 2.3 0.27 1.00 0.03 1.00
LMarea(cm2) 77.3 82.5 78.6 79.4 3.27 0.34 0.76 0.48
Marbling 418 573 480 482 43.13 0.07 0.72 0.07
Backfatthickness(cm) 1.24 1.37 1.30 1.12 0.13 0.83 0.41 0.22
KPH(%) 2.1 2.1 2.3 2.1 0.12 0.29 0.28 0.70
Daystoharvest 367 377 392 412 11.10 0.16 0.01 0.59
1D=cowplaneofnutritioneffect(diet),W=weaningageeffect,D×W=diet×weaninginteraction.
2Basedon62%dressingpercentage.
doi:10.1371/journal.pone.0131478.t002
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NeonatalNutritionAltersOffspringMuscleTranscriptome
comparedwithLPNsteers(P=0.03).UltrasoundatthetimeofEW(78daysofage)revealed
nosignificantdifferences(P>0.05)betweentreatments(i.e.allpossiblecombinationsbetween
weaningageandcowplaneofnutrition)formarblingandbackfatthickness(Table2).
Hotcarcassweightdidnotdiffer(P>0.10)(Table2).Calculatedyieldgradehadaweaning
effect(P=0.03)withlowervaluesforNWascomparedwithEWsteers.Marblingscorewas
greater(P=0.07)forEW-MPNsteers.Lastly,therewasaweaningeffect(P=0.01)associated
withEWsteersreachingtheback-fatthicknesstargetend-pointearlierthanNWsteers
(Table2).
Serumglucoseconcentrationhadasignificanttimeeffect(P<0.05)withlowervaluesat
296daysofage(Fig1).Adiponectinconcentrationhadasignificantcowplaneofnutrition
(diet),time,anddiet×timeinteraction(P<0.05)withincreasingconcentrationsbetween78
and187daysofageandaswitchtodecreasingconcentrationsafter187daysofageforNW
steers,butserumadiponectindidnotchangeforEWsteers(Fig1).Insulinconcentrationwas
affectedbyweaningage,time,andweaning×time(P<0.05)witharelativelyconstantcon-
centrationbetween78and187daysofageforalltreatmentsandanoticeableincreaseincon-
centrationbetween187and296daysofage(Fig1).
Microarrayanalysis
AtanFDR<0.10(uncorrectedPvalue<0.05),atotalof35DEGwereaffectedoverallbythe
cowplaneofnutrition(Table3),145DEGwereaffectedbyweaningtimeand7,639DEGwere
affectedbytime.Inaddition,therewere13DEGforthetime×wean×dietinteraction
(Table4),43DEGforthetime×diet,31DEGforthewean×dietinteraction,and167DEG
forthewean×timeinteraction.The167DEGforthewean×timeinteractionwereanalyzed
separatelyforEWandNWsteers:between78and187daysofage,187and354daysofageand
between87and354daysofage.ForthisanalysisweutilizedacutoffPvalue<0.05forany
givencomparison(Fig2).
Whenfocusingonthe167DEGduetotime×treatmentinteraction,thehighestnumberof
DEG,91down-regulatedand44upregulated(uncorrectedPvalue<0.05;FDR<0.10),were
detectedinEWsteersbetween78and187daysofage(Fig2).Duringthesametime-frame,
NWsteershadthelowestDEGwith53downregulatedand4upregulatedgenes(uncorrectedP
value<0.05;FDR<0.10).ItisnoteworthythatthenumberofDEGinNWsteersincreased
markedlybetween187and354daysofage.Thus,whencomparingtheresponsesbetween78
and354dayofage,EWandNWsteershadasimilarnumberofDEG(Fig2).
TheDIAanalysiswasperformedwithDEG(n=167)atanuncorrectedPvalue=0.05and
anFDR=0.10.Fig3containsthesummaryofKEGGpathwaysresultsfromDIA.Thetop-
threemostimpactedcanonicalpathwaysarereportedinFig4andthegeneswithhighestacti-
vationinFig5.Forbiologicalprocesses,onlythosewithanimpactvaluehigherthan50%of
themaximumtotalimpactvalueforeachtimecomparisonforEWandNWsteersaredis-
cussed(Fig6).
TheDIAanalysisrevealedthatfattyacidbiosyntheticprocess,biosynthesisofunsaturated
fattyacids,andinsulinsignalingwerehighly-activatedinEWsteersbetween78and187days
ofage(Fig4).Analysesfurtherrevealedthatactivationofthesepathwayswasnamelydueto
upregulationofFASN(lipogenicenzyme),SCD(monounsaturatedfattyacidsynthesis)and
PCK2(involvedinglyceroneogenesis)(Fig5).IncontrasttoEWsteers,between78and187
daysofageNWsteershadahigherimpactwithnoapparentdirectionofthecytochrome
P450-relatedpathwaysandGlutathioneMetabolism(Fig4).
Between187and354daysofageinEWsteerstherewasasignificantnegativeimpacton
Pyrimidinemetabolism(NucleotideMetabolismSubcategorywithintheMetabolismKEGG
PLOSONE|DOI:10.1371/journal.pone.0131478 July8,2015 6/20
NeonatalNutritionAltersOffspringMuscleTranscriptome
Fig1.Glucose,adiponectinandinsulinserumconcentrationsinAngus×Simmentalsteersfrom
cowsthatreceivedalow(LPN)ormedium(MPN)planeofnutritionduringthelategestationperiod.
Weaningtimesareearlywean(EW)andnormalwean(NW).*Weaning,**Diet,***Time,#weaning×diet,
##weaning×time,###diet×timeand$time×weaning×dietinteractioneffects(P<0.05).
doi:10.1371/journal.pone.0131478.g001
Category),RNApolymerase(TranscriptionSubcategorywithintheGeneticInformationPro-
cessingKEGGCategory)andCytosolicDNAsensingpathway(ImmuneSystemSubcategory
withintheOrganismalSystemKEGGCategory)(Fig4).Polymerase(RNA)III(DNAdirected)
polypeptideA,155kDa(POLR3A)wastheonlyDEGthatcouldexplaintheinhibitionofthese
pathways(Fig5).InthecaseofNWsteersbetween187and354daysofage,Biosynthesisof
unsaturatedfattyacids,FattyacidbiosynthesisandGlycerophospholipidmetabolism(all
withintheKEGGLipidMetabolismSubcategory)werethemost-impactedandactivatedpath-
ways(Fig4).Between78and354daysofage,FattyacidbiosynthesisandBiosynthesisof
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NeonatalNutritionAltersOffspringMuscleTranscriptome
Table3. Symbol,entrezgeneID,logratioexpressionvalue[lowplaneofnutrition(LPN)vs.mediumplaneofnutrition(MPN],typeofmoleculeand
localizationinthecellforthe35differentiallyexpressedgenesaffectedbycowplaneofnutrition.
Symbol EntrezGeneName LogRatio p-value Location1 Type(s)2
ABHD11 abhydrolasedomaincontaining11 0.30 8.74E-06 Cytoplasm enzyme
ACLY ATPcitratelyase 0.35 7.74E-05 Cytoplasm enzyme
AGPAT6 1-acylglycerol-3-phosphateO-acyltransferase6 -0.22 5.90E-05 Cytoplasm enzyme
ARAP1 ArfGAPwithRhoGAPdomain,ankyrinrepeatandPHdomain1 -0.44 1.53E-04 Cytoplasm other
ART3 ADP-ribosyltransferase3 -0.78 4.64E-05 PlasmaMemb. enzyme
ATP5S ATPsynthase,H+transporting,mitochondrialFocomplex,subunits 0.33 1.09E-04 Cytoplasm transporter
C15orf40 chromosome15openreadingframe40 -0.21 1.72E-04 Other other
C8orf48 chromosome8openreadingframe48 -0.72 2.18E-05 Other other
CARD14 caspaserecruitmentdomainfamily,member14 0.96 4.31E-05 Cytoplasm other
CHST12 carbohydrate(chondroitin4)sulfotransferase12 0.78 5.32E-05 Cytoplasm enzyme
DEXI Dexihomolog(mouse) 0.29 1.50E-04 Other other
DHDH dihydrodioldehydrogenase(dimeric) 0.40 8.49E-08 Other enzyme
DYNLL1 dynein,lightchain,LC8-type1 0.47 2.29E-05 Cytoplasm other
EDC3 enhancerofmRNAdecapping3 0.55 2.31E-05 Cytoplasm other
EHD4 EH-domaincontaining4 0.60 1.39E-04 PlasmaMemb. enzyme
ENTPD2 ectonucleosidetriphosphatediphosphohydrolase2 0.53 7.24E-05 Cytoplasm enzyme
EPC1 enhancerofpolycombhomolog1(Drosophila) -0.28 9.13E-05 Nucleus TR
FCAR FcfragmentofIgA,receptorfor -0.52 6.40E-05 PlasmaMemb. other
GSTA4 glutathioneS-transferase,alpha4 -1.74 1.37E-05 Other enzyme
HLF hepaticleukemiafactor 0.72 1.69E-04 Nucleus TR
IQGAP1 IQmotifcontainingGTPaseactivatingprotein1 -0.55 5.28E-05 Cytoplasm other
KRT74 keratin74 -1.01 7.04E-05 Cytoplasm other
LOC789391 tRNAmethyltransferasecatalyticsubunitTRMT61Alike -0.76 1.07E-04 Nucleus enzyme
MARCH7 membrane-associatedringfinger7,E3ubiquitinproteinligase -0.62 7.57E-09 Extracell.Space other
MSRB1 methioninesulfoxidereductaseB1 -0.49 3.86E-05 Other other
NR2C2 nuclearreceptorsubfamily2,groupC,member2 -0.27 7.21E-05 Nucleus LDNR
NSMCE4A non-SMCelement4homologA(S.cerevisiae) 0.34 1.67E-04 Nucleus other
PNMAL1 paraneoplasticMaantigenfamily-like1 0.89 1.31E-04 Other other
PSPH phosphoserinephosphatase 0.74 1.62E-04 Cytoplasm phosphatase
PYCR1 pyrroline-5-carboxylatereductase1 0.47 8.97E-05 Cytoplasm enzyme
SCUBE1 signalpeptide,CUBdomain,EGF-like1 1.05 1.03E-04 PlasmaMemb. transm.receptor
SERF1A smallEDRK-richfactor1A(telomeric) 0.41 1.48E-04 Other other
SRSF5 serine/arginine-richsplicingfactor5 -0.53 9.98E-05 Nucleus other
TAGLN3 transgelin3 0.34 1.17E-04 ExtracellSpace other
TOMM34 translocaseofoutermitochondrialmembrane34 0.36 1.14E-04 Cytoplasm other
1Extracell.Space=extracellularspace;PlasmaMemb.=plasmamembrane;Greceptor=Gproteincoupledreceptor.
2TR=transcriptionregulator;LDNR—ligand-dependentnuclearreceptor;Transm.Receptor=transmembranereceptor.
doi:10.1371/journal.pone.0131478.t003
unsaturatedfattyacidswerethemost-impactedandactivatedpathwaysinbothEWandNW,
buttheactivationofthesepathwaystookplaceatdifferenttimepoints(Fig4).
Whenwefocusonthebiologicalprocesses(BP),between78and187daysofageinEW
steerstherewasaclearactivationoftheBPrelatedtoadipogenesisandlipogenesis(Fig6).Bio-
syntheticprocessandfattyacidbiosyntheticprocesshadthehighestimpact,withalower
impactdetectedforfattyacidelongationofsaturatedfattyacids,long-chainfattyacidbiosyn-
theticprocessandregulationoftriglyceridebiosyntheticprocess(Fig6).Between78and187
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NeonatalNutritionAltersOffspringMuscleTranscriptome
Table4. Symbol,entrezgeneID,logratioexpressionvalue,typeofmoleculeandlocalizationinthecellforthe13differentiallyexpressedgenes
affectedbytheweaning×diet×timeinteraction.
Symbol EntrezGeneName LogRatio p-value Location1 Type(s)2
ARID1A ATrichinteractivedomain1A(SWI-like) 2.885 2.04E-04 Nucleus TR
BTG1 B-celltranslocationgene1,anti-proliferative -1.262 2.07E-03 Nucleus TR
EPAS1 endothelialPASdomainprotein1 -1.797 2.08E-01 Nucleus TR
HSBP1 heatshockfactorbindingprotein1 1.064 2.38E-02 Nucleus TR
IMPG1 interphotoreceptormatrixproteoglycan1 1.577 2.22E-05 Extracell.Space other
KIAA0232 KIAA0232 1.889 3.33E-04 Extracell.Space other
KRTDAP keratinocytedifferentiation-associatedprotein -12.672 8.49E-01 Extracell.Space other
MARK4 MAP/microtubuleaffinity-regulatingkinase4 -1.247 2.99E-02 Cytoplasm kinase
NME1 NME/NM23nucleosidediphosphatekinase1 -5.373 1.17E-01 Cytoplasm kinase
OR51F1 olfactoryreceptor,family51,subfamilyF,member1 -1.393 4.66E-04 PlasmaMemb. Greceptor
OTX1 orthodenticlehomeobox1 -3.683 2.31E-01 Nucleus TR
PLAGL1 pleiomorphicadenomagene-like1 1.313 5.06E-03 Nucleus TR
SLC25A5 solutecarrierfamily25,member5 -4.401 2.36E-01 Cytoplasm transporter
1Extracell.Space=extracellularspace;PlasmaMemb.=plasmamembrane.
2TR=trascriptionregulator;Greceptor=Gproteincoupledreceptor.
doi:10.1371/journal.pone.0131478.t004
Fig2.Differentiallyexpressedgenes(DEG;FDR<0.10anduncorrectedPvalue<0.05)inearlywean
(EW)andnormalwean(NW)steersduringthegrowing(78to187daysofage),finishing(187to354
daysofage)andgrowingandfinishingphases(78to354daysofage).NumberofDEGforeach
comparisonarethosewithoverallFDR<0.10,uncorrectedPvalue<0.05,andP<0.05betweenthespecific
timecomparisons.
doi:10.1371/journal.pone.0131478.g002
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NeonatalNutritionAltersOffspringMuscleTranscriptome
Fig3.DynamicImpactApproach(DIA)ofdifferentiallyexpressedgenes(Pvalue<0.05;FDR<0.10)
ontheKyotoEncyclopediaofGenesandGenomes(KEGG)Pathwaysdatabase.Fluxrepresentsthe
directionofeachcategoryandthecorrespondingsubcategory(greencolor=inhibition,yellowcolor=stable,
redcolor=activationwithdifferentcolorintensitiesaccordingwiththelevelofup-regulationordown-
regulation).Bluebarsdenotetheimpactofeachcategoryandthecorrespondingsubcategories.
doi:10.1371/journal.pone.0131478.g003
Fig4.Resultsofthe3mostimpactedpathwayswithindifferentiallyexpressedgenesaffectedduringthegrowingphase(187vs.78d),finishing
phase(354vs.187d)andthewholeexperiment(354vs.78d).AnalysisperformedusingtheDynamicImpactApproach(DIA)basedontheKyoto
EncyclopediaofGenesandGenomes(KEGG)Pathwaysdatabase.Fluxrepresentsthedirectionofeachcategoryandthecorrespondingsubcategory
(greencolor=inhibition,yellowcolor=stable,redcolor=activationwithdifferentcolorintensitiesaccordingwiththelevelofup-regulationordown-
regulation).Bluebarsdenotetheimpactofeachbiologicalprocess.
doi:10.1371/journal.pone.0131478.g004
PLOSONE|DOI:10.1371/journal.pone.0131478 July8,2015 10/20