Effect of increasing levels of glycerin on growth rate, carcass traits and liver gluconeogenesis in young bulls

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ContentslistsavailableatScienceDirect

Animal

Feed

Science

and

Technology

journalhomepage:www.elsevier.com/locate/anifeedsci

Effect

of

increasing

levels

of

glycerin

on

growth

rate,

carcass

traits

and

liver

gluconeogenesis

in

young

bulls

夽

Marcio

M. Ladeira

a,∗

_,

_José

_Rodolfo

_R.

_Carvalho

a

_,

_Mario

_L.

_Chizzotti

b

_,

_Priscilla

_D.

Teixeira

a

_,

_Júlio

_César

_O.

_Dias

a

_,

_Tathyane

_R.S.

_Gionbelli

a

_,

_Aline

_C.

_Rodrigues

a

_,

Dalton

M. Oliveira

c

a_Department_of_Animal_Science,_Universidade_Federal_de_Lavras,_Lavras,_Minas_Gerais,_CEP:_37.200-000,_Brazil

b_Department_of_Animal_Science,_Universidade_Federal_de_Vic¸_osa,_Vic¸_osa,_MG,_36.570-000,_Brazil

c_Universidade_Estadual_de_Mato_Grosso_do_Sul,_Aquidauana,_MS,_79.200-000,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received21December2015

Receivedinrevisedform14June2016

Accepted15June2016 Keywords: Biodiesel Glycerol GK1 Marbling Phosphoenolpyruvatecarboxykinase

a

b

s

t

r

a

c

t

Thisstudyaimedtoevaluateperformance,carcasstraits,glycerolkinase1(GK1)and cyto-plasmaticphosphoenolpyruvatecarboxykinase(PCK1)geneexpression,andglycerolkinase activityinliverofyoungbullsreceivingdifferentlevelsofcrudeglycerin.Forty-four cross-bredyoungbulls(initialbodyweightof368±4.2kg)wereusedinacompletelyrandomized design,withfourtreatmentsand11replicates.Theexperimentperiodlasted84days, pre-cededbyanadaptationperiodof28days.Thebasaldietwascomposedofcornsilage (300g/kg)andconcentrate(700g/kg)containingcornandsoybean.Theexperimental treat-mentswereasfollows:withoutglycerinorincluding60,120or180g/kgofcrudeglycerinin thediet.Bloodsampleswerecollectedonthelastdayoftheexperimentperiodtoevaluate biochemicalparameters.Afterslaughter,carcasstraitsweremeasuredandliversamples werecollectedtoanalyzegeneexpressionandglycerolkinaseactivity.Therewasnoeffect (P=0.21)ofglycerinonglucosebloodconcentrations.However,liverglycerolkinase activ-itywasgreater(P<0.01)andGK1andPCK1genesexpressionsweredownregulate(P<0.01) asglycerinlevelsinthedietincreased.Glycerindidnotaffect(P>0.17)performanceand mostofthecarcasstraits.However,therewasagreater(P=0.02)marblingscoreinthe carcassofanimalsfed120and180g/kgofcrudeglycerin.Inconclusion,theuseofglycerin atalevelofupto180g/kgisrecommendedindietsoffeedlotbeefcattle,anditincreases liverglycerolkinaseactivity,feedefﬁciencyandbeefmarbling.

Abbreviations:ADG,averagedailygain;AQP9,aquaglyceroporin;AST,aspartateaminotransferase;CCW,coldcarcassweight;CK,creatinekinase;CY,

carcassyield;DM,drymatter;DMI,drymatterintake;FBW,ﬁnalbodyweight;GAPDH,glyceraldehyde3-phosphatedehydrogenasegene;GGT,

gamma-glutamylaminotransferase;GKI,glycerolkinase1gene;HCW,hotcarcassweight;LMA,longissimusmusclearea;ME,metabolizableenergy;NFC,

non-ﬁbrouscarbohydrates;PCK1,cytoplasmaticphosphoenolpyruvatecarboxykinasegene;PEPCK-C,cytosolicphosphoenolpyruvatecarboxykinaseenzyme;

PGC-1␣,peroxisomeproliferator-activatedreceptor-␥coactivator1␣;PPAR-gamma,peroxisomeproliferator-activatedreceptor-␥;SFT,subcutaneousfat

thickness.

夽 ResearchfundedbyFAPEMIG.

∗ Correspondingauthor.

E-mailaddress:mladeira@dzo.uﬂa.br(M.M.Ladeira).

http://dx.doi.org/10.1016/j.anifeedsci.2016.06.010

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1. Introduction

Theuseofcoproductstoreplacecommonlyusedingredientssuchascornandsoybeancouldreducefeedingcostand

increaseproﬁtinfeedlots.Amongavailablecoproductsforuseinfeedlots,glycerinisemergingasanoptionduetothe

growthofbiodieselindustryaroundtheworld.Somestudieshaveshownthepotentialofthisbiodieselindustrycoproduct

forruminants’feeding,beingtheﬁrststudiesperformedindairycows(Donkin,2008).Inbeefcattle,severalstudiesevaluated

theeffectofcrudeglycerinonperformanceandbeefquality,butresultswereambiguousandinconclusive.Inotherwords,in

somecasescrudeglycerinincreasedgrowthperformanceoftheanimals(Parsonsetal.,2009),butinothercases,therewas

nopositiveeffect(Egeaetal.,2014;VanCleefetal.,2014),whileinsomestudies,glycerindepressedanimalperformance

andcarcasstraits(Gunnetal.,2010).Inaddition,noneanalyzedperformanceandlivermetabolismatthesametime.

Chemicalcompositionofcrudeglycerinstillisquitevariable,beingimportanttoassesswhethertheuseofthisingredient

mayhavetoxiceffectsinruminants,especiallyregardingmethanolconcentration.AccordingtoElametal.(2008),ithasbeen

hypothesizedthatglycerinsupplementationmaycauseproblemsassociatedwithhepaticlipidmobilizationbyincreasing

theliverabilitytosequesterandre-esterifyfattyacidsfromtissues.Besides,glycerolmaybeasubstrateforgluconeogenesis

intheliver,increasingglycerolkinaseactivityandglucoseturnover,whichcouldimprovebeefmarbling.Itmayoccur

because,accordingtoGilbertetal.(2003),intramuscularadiposetissueusesahighproportionofglucoseforfattyacid

synthesisanditismoresensitivetoinsulin.

Therefore,thehypothesisofthisstudyisthatglycerinwillnotaffectnegativelyfeedlotperformance,whileincreases

livergeneexpressionandactivityofglycerolkinase.Theobjectivewastoevaluateperformance,carcasstraits,expression

ofglycerolkinase1(GK1)andcytoplasmaticphosphoenolpyruvatecarboxykinase(PCK1)genes,andglycerolkinaseactivity

intheliverofyoungbullsfeddifferentlevelsofcrudeglycerin.

2. Materialsandmethods

AnimalcareandhandlingwereapprovedbytheFederalUniversity ofLavrasAnimalCareand UseCommittee.The

experimentwascarriedoutattheAnimalScienceDepartmentoftheFederalUniversityofLavras.

2.1. Animals,dietandslaughter

Fourty-fourcrossbredyoungbulls(¼Angus,¼Nellore,¼Senepol,¼Caracu),withinitialbodyweightof368±4.2kg

wereusedinacompletelyrandomizeddesign,withfourtreatments(0,60,120and180g/kgofcrudeglycerininthediet)

and11replicates.Crudeglycerincompositionusedintheexperimenthadthefollowingcomposition:glycerol:831g/kg,

moisture:111g/kg,ash:60.6g/kg,chlorides36.7g/kg,andmethanol:0.2g/kg.Animalswerehousedinopen-airpenswith

anareaof30m2_per_animal.

This84-dayexperimentwasprecededbyanadaptationperiodof28days,duringwhichanimalsreceivedthesame

experimentaldiet.Atthebeginningoftheadaptationperiod,animalsweretreatedforectoparasitesandendoparasites

(Ivomec,Paulinia,Brazil).Aimingtomeasuretheiraveragedailygain(ADG)usingregressionanalysis,animalswereweighed

atthebeginning,every28daysandattheendofexperimentalperiod,after16hoffasting.

Dietswereformulatedwithcornsilage,andfourdifferentlevelsofcrudeglycerin(Table1)accordingtotheNational

ResearchCouncil–NRC(2000)tomakeitisonitrogenous.Animalswerefedadlibitumat07:30amand03:30pm.Corn

glutenmeal-21wasincludedinthedietswithcrudeglycerintoprovidesimilarlevelsofcrudeproteinandasimilaramino

acidproﬁle.

Samplesofsilageandconcentratewerecollectedevery14days.Thesesamplessuppliedacompositesamplethat,after

pre-dryinginaforced-ventilationovenat65◦Cfor72h,wasgroundinamillwitha1-mmmeshsieve.Chemicalanalyses

ofthedietswereperformedaccordingtoAOAC(2000).Thedrymatter(DM)wasobtainedbyovendryingat100◦Cfor

18h(Method934.01).Theashcontentwasestimatedaftera2-hincinerationprocessina600◦Cmufﬂefurnace(Method

942.05).Thecrudeprotein(CP)contentwasdeterminedbasedontheNcontentmultipliedbythe6.25factor.TheNcontent

wasobtainedbytheKjeldahlprocedure(Method920.87).Evaluationoftheetherextract(EE)contentwasperformedwith

solventusingaSoxhletextractorapparatus(Method920.39).Non-ﬁbrouscarbohydrates(NFC)weredeterminedaccording

toSniffenetal.(1992),andmetabolizableenergy(ME)wascalculatedaccordingtotheNRC(2001)andMachetal.(2009).

BloodsampleswerecollectedfromthecoccygealveinusingVacutainer®_tubes_(BD,_Juiz_de_Fora,_Brazil)_at_the_end_of_the

experimentafter16hoffasting.Thesesampleswereplacedinacoolerwithiceandtransporteddirectlytothelaboratory,

whereanalysesofcreatinineandaspartateaminotransferase(AST),gamma-glutamylaminotransferase(GGT),andcreatine

kinase(CK)enzymeswereperformedusingcommercialkits(LabtestDiagnóstica,LagoaSanta,Brazil).Bloodsamplesfor

glucoseanalysiswerecollectedinanotherVacutainer®_tube_(BD,_Juiz_de_Fora,_Brazil)_containing_a_ﬂuoride_{anticoagulant.}

Animalswereslaughteredatacommercialabattoirbycaptiveboltandexsanguination,followedbyhideremovaland

evisceration,withoutelectricalstimulus.Thecarcasseswerewashed,dividedintotwoequalhalvesandweighedtoobtain

thehotcarcassweight(HCW)andcarcassyield(CY).Thesubcutaneousfatthickness(SFT)andlongissimusmusclearea

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Table1

Compositionofingredientsandnutrientscontentsoftheexperimentaldiets(g/kg).

Ingredients Glycerinlevels(g/kg)

0 60 120 180

Cornsilage 300 300 300 300

Groundcorn 560 480 370 260

Soybeanmeal 120 120 120 120

Cornglutenmeal-21 – 20 70 120

Crudeglycerin1 _– ₆₀ ₁₂₀ ₁₈₀ Mineralmixture2 ₂₀ ₂₀ ₂₀ ₂₀ Nutrients Drymatter3 ₅₉₂ ₅₈₈ ₅₈₄ ₅₈₀ Crudeprotein4 ₁₃₁ ₁₂₉ ₁₃₁ ₁₃₃ NDF4 ₂₂₂ ₂₂₄ ₂₃₆ ₂₄₈ NFC4 ₅₆₆ ₅₆₆ ₅₅₀ ₅₃₃ Glycerol4 _– ₅₆ ₁₁₂ ₁₆₈ Etherextract4 ₃₄ ₃₄ ₃₃ ₃₂ ME2_(MJ/kg_MS) _11.7 _11.7 _11.6 _11.6

1_Guaranteed_levels_per_kilogram_of_product:_Glycerol:_831,_Moisture:_111,_Ash:_60.6,_Chlorides_36.7,_Methanol:_0.2.

2_Guaranteed_levels_per_kilogram_of_product:_Ca:₁₇₀_g;_P₃₁_g;_Na:₁₅₅_g;_Zn:₂_mg;_Cu:₃₉₆_mg;_Mn:₅₁₅_mg:_Co:₁₅_mg;_I:₂₉_mg;_Se:_5.4_mg;_Vit._A:

111.000UI;Vit.D3:22.000UI;Vit.E:265UI.

3_As_fed_basis.

4_Dry_matter_basis.

Table2

Targetprimersusedintherelativequantiﬁcationbyq-PCR.

Gene Primer Sequence Accesscode

(GenBank)

Bpa _R2 _Efﬁciency

GK Glycerolkinase F:TCTTCTGTAGTCTGCCCTTGG BC122692.1 87 0.936 99.4

R:TTTACGGCATACCTGAGAGG

PEPCKC Phosphoenolpyruvate

carboxykinase

F:CCCCCAGAGATCAAGAATCA AY145503.1 86 0.998 98.3

R:ATTGGAGGTGGACAGTCAGG

ˇ-actin ˇ-actin F:GTCCACCTTCCAGCAGATGT BC142413.1 90 0.996 100.0

R:CAGTCCGCCTAGAAGCATTT

GAPDH Glyceraldehyde3-phosphate

dehydrogenase

F:CGACTTCAACAGCGACACTC NM001034034.1 96 0.998 101.4

R:TTGTCGTACCAGGAAATGAGC

a_Amplicon_size_in_base_pairs.

refrigerationinacoldchamber(1◦C)for24h.Aftercooling,carcasseswerereweighedtoobtainthecoldcarcassweight

(CCW).MarblingwasanalyzedaccordingtoDowetal.(2011)equation,usingether-extractablefatofthelongissimusmuscle.

2.2. Geneexpressionandenzymeactivity

Immediatelyafterslaughter,liversampleswerecollected,frozeninliquidnitrogenandstoredin−80◦_C_to_analyze_GK1

andPCK1genesexpressionthatencodeglycerolkinaseandcytoplasmaticphosphoenolpyruvatecarboxykinaseenzymes,

respectively.Inaddition,samesampleswereusedtomeasureglycerolkinaseactivity.

TargetandreferenceprimersweredesignedusingsequencesdepositedandpublishedintheGenBankpublicdatabase,

aNCBI(NationalCenterforBiotechnologyInformation)platform.ORFs(OpenReadingFrames)oftheselectedsequences

wereobtainedbyusingNCBIORFFindertool,andtheencodedproteinsequenceswereobtainedfromtheTranslatetoolof

ExPASyproteindatabase.TheprimersequencesweredesignedusingtheOligoPerfectTM_Designer_software;_sequences_are

showninTable2.

TotalRNAwasextractedfromliversampleswithQIAzolreagent(QIAgen,Valencia,CA,USA)andtreatedwithDNA-free

DNase(Ambion,Austin,TX,USA)accordingtothemanufacturer’sprotocol.TotalRNAwassubjectedtoelectrophoresisin

10.0g/kgagarosegel(w/v)stainedwithGelRedNucleicAcidGelStainandvisualizedinaUVITECFireReaderXSD-77Ls-20M

foranalysisofthe28Sand18SribosomalRNA(rRNA)bands.Sampleswerequantiﬁedbyspectrophotometry(Nanodrop

ND-1000Spectrophotometer)atA260nmtodeterminetheamount(ng/uL)andquality(260/280and260/230)oftheRNA.

ThecDNAsynthesiswasperformedwiththeHigh-CapacitycDNAReverseTranscriptionkit(AppliedBiosystems,FosterCity,

CA,USA)accordingtothemanufacturer’sprotocol.Afterthereaction,sampleswerestoredat−20◦_C.

ForquantitativegeneexpressionanalysisbyRT-qPCR,ABIPRISM7500RealTimePCRmachine(AppliedBiosystems)

wasusedwithSYBRGreendetectionsystem(AppliedBiosystems,FosterCity,CA,USA).Thereactionparameterswereas

follows:2minat50◦C,10minat95◦Cand40cyclesof15minat95◦C,1minat60◦Cand15sat95◦C.Datawerecollected

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cDNA,0.3␮Lofeachprimer(forwardandreverse)and5.0␮LofSYBRGreenMasterMixinaﬁnalvolumeof10.0␮L/sample

inaMicroAmpOptical96-wellreactionplate(AppliedBiosystems,FosterCity,CA,USA).TheRT-qPCRanalysesforeachgene

studiedwereperformedusingcDNAsfrom11experimentalunits(animals),withthreetechnicalreplicatesforeachcDNA.

ResultswerenormalizedusingCycleThreshold(CT)obtainedfromreferencegenesˇ-actinandglyceraldehyde3-phosphate

dehydrogenase(GAPDH)expression.Thechoiceofthesereferencegeneswasbasedonapre-studyusingGAPDH,Actin,

UbiquitinandCycloPilina,inwhichˇ-actinandglyceraldehyde-3-phosphatedehydrogenase(GAPDH)hadthebestefﬁciency.

TheCTwasdeterminedfromthenumberofcyclesusingthecomparativeCTmethod.Asonerequisiteofthismethod,a

validationassaywasperformedtodemonstratethattheampliﬁcationefﬁcienciesofthetargetandreferencegeneswere

approximatelythesame(Table2).Standardcurvesweregeneratedforthestudiedgeneswiththefollowingdilutions:1:5,

1:25,1:125,1:625,and1:3125.ThisprocedureallowstheusertodeﬁnethecDNAconcentrationsusedineachreaction(in

thiscase,10ng/␮L).Theconcentrationofeachprimerwas1.5␮M.

TherelativeexpressionlevelswerecalculatedaccordingtothemethoddescribedbyPfafﬂ(2001),whichisbasedonCt

valuescorrectedforeachprimerpairampliﬁcationefﬁciency.

Forglycerolkinase(EC2.7.1.30)extraction,1.5goffrozenliverwasused.Thetissuewashomogenizedin3mLbuffer

(Tris-HCl0.05MandpH8.0)usingapolytron(Ultra-turraxT25basic-IKA,Wilmington,USA).Aftertissuehomogenization,

sampleswerecentrifugedat15,000gfor20minat5◦Candthesupernatantscollectedandstoredat4◦C(Bernardinoetal.,

2013).TheenzymaticactivityinthesupernatantswasquantiﬁedaccordingtothemethoddescribedbyKiharaetal.(2009).

Initially,glycerolkinasepresentinthesamplesphosphorylatesglyceroltoglycerol-3-phosphateandADP,inthepresence

ofATP.Aftertwosequentialreactionscatalyzedbyglycerol-3-phosphateoxidase(EC1.1.3.21)andperoxidase(EC1.11.1.7),

glycerol-3-phosphateisconvertedtoquinoneimine,thatcanbeanalyzedusingacolorimeter.

Thekineticassaywasconductedin96-wellmicroplateswith880␮LofTris-HClbuffer(50mM,pH8.0),10␮Lof400mM

ATP(pH7.0,Amresco,code:0220-25G),10␮Lof200mMMgCl2,10␮Lofglycerol-3-phosphateoxidasefromPediococcus

sp.(100Uof enzyme/500␮Lofbuffer;Sigma-Aldrich,code:G9637-100UN),10␮Lofhorseradishperoxidase(450Uof

enzyme/mlofbuffer;Amresco,code:0343-25,000U),10␮Lof150mM4-aminoantipyrine(Sigma-Aldrich,code:

A4382-100G),10␮Lof150mMphenoland50␮Lofthesupernatant.Tostartthebatteryofreactions,10␮Lof1Mglycerolwere

addedandtheplateswerethenincubatedat25◦Cinanautomaticmicroplatereader(MultiskanGO,ThermoScientiﬁc,

Vantaa,Finland)programmedtoreadtheabsorptionat500nmevery20sforamaximumperiodof15min.Theblank

sampleswereincludedformeasuringtheabsorbanceofthereagentsusedinthekineticassay.Subsequently,datafromthe

enzymaticassaywereusedtogeneratealinearcurve.

Proteincontentinthesamples(lgofprotein/mlofhepaticextract)wasmeasuredaccordingtoBradfordmethod(1976)

usingbovineserumalbuminasastandard,tocalculatespeciﬁcactivityoftheglycerolkinaseasfollows:

Speciﬁcactivity(U/␮gofprotein)= volumetricactivity/proteincontentinthesample.

Therefore,theglycerolkinaseactivitywasexpressedinU,deﬁnedasthequantityofenzymethatgenerates1␮molof

glycerolperminuteofreactioninasolutionwithpH=8.0and25◦C.

2.3. Statisticalanalyses

TheShapiro-Wilktestwasusedtoanalyzethedatadistribution.Ifthedatawerenotnormallydistributed,transformation

wasperformedusingthePROCRANKofSAS9.3(SASInst.Inc.,Cary,NC,USA).Datawereanalyzedasacompletelyrandomized

designusingtheMIXEDprocedure(SASInst.Inc.,Cary,NC)withdietasﬁxedeffectandaccordingtothefollowingmodel:

Yi,j=+Ti+e;with:Yi,jistheobservationonjexperimentalunitreceivingtreatmenti;istheoveralltreatmentmean;

Tiistheeffectofhavingtreatmentleveli;andeistherandomerror.Animalsweretheexperimentalunit,andorthogonal

contrastswereusedtoevaluatethelinearandquadraticeffectsofglycerinandwithoutglycerinvs.glycerintreatment.

TreatmentmeanswerecomputedwiththeLSMEANSoption.EffectswereconsideredsigniﬁcantatP-valueof≤0.05,with

tendenciesdeclaredatP-valuesbetween0.05and0.10.

3. Results

Bloodcreatinine,AST,GGTandCKwerenotaffectedbythelevelsofglycerininthediet(Table3).However,bloodglucose

tended(P=0.08)todecreaselinearlywiththelevelofglycerininthediets.Liverglycerolkinaseactivityincreasedlinearly

followingglycerininclusion(Fig.1A).Ontheotherhand,oppositeresultweredetectedonGK1andPCK1expressions(Fig.1B

andC).ExpressionofGK1intheliverwas1.61,3.34and5.45timeslower,whentheanimalsfed60,120and180g/kgofcrude

glycerin,respectively,thanintheliverofanimalsfeddietwithoutglycerin.ExpressionofPCK1alsopresentedaquadratic

effectbeingthemaximumvalueinthecurvewhenglycerincontentwas59.1g/kgofDM.

Despitethelackofglycerinversusnoglycerindietseffect(P=0.41)ondrymatterintake(DMI),linearandquadraticeffects

weredetected(Table4).Analyzingthemeansandthemaximumvalueinthequadraticcurve,61.2g/kgofglycerininthe

diet,itispossibletoconcludethatthenegativeeffectofglycerinonDMIstartedafterthispointanditwasmorepronounced

whenanimalswerefed180g/kgofglycerininthediet.Therewasnoeffect(P>0.18)ofcrudeglycerinonanimalsADGduring

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Table3

Biochemicalproﬁleofthebloodofyoungbullsfeddifferentlevelsofcrudeglycerin.

Variables Glycerinlevels(g/kg) SEM Gly1 _L2 _Q3

0 60 120 180 Glucose,mg/dl 83.2 73.1 85.1 71.9 5.40 0.30 0.08 0.95 Creatinine,mg/dl 1.53 1.41 1.47 1.48 0.09 0.49 0.40 0.92 AST4_,_U/L _93.0 _95.4 _85.8 _84.9 _6.80 _0.59 _0.83 _0.20 GGT5_,_U/L _12.5 _12.2 _15.7 _17.5 _2.36 _0.34 _0.94 _0.08 CK6_,_U/L ₃₄₆ ₅₀₄ ₃₇₆ ₃₃₆ _60.2 _0.39 _0.11 _0.25 1_Gly₌₀_vs._Glycerin. 2_L₌_Linear. 3_Q₌_Quadratic.

4_AST₌_Aspartate_{aminotransferase.}

5_GGT₌_{gamma-glutamyl}_{aminotransferase.}

6_CK₌_creatine_kinase.

Fig.1. Glycerolkinaseactivity(1A),relativeexpressionofGK1(1B)andrelativeexpressionofPCK1(1C)intheliverofyoungbullsfedwithdifferentcrude

glycerinconcentrations.

Crudeglycerindidnotaffect(P>0.36)ﬁnalbodyweight(FBW),HCW,CCWandSFToftheanimals(Table5).

Neverthe-less,quadraticeffects(P<0.05)wereobservedincarcassyield,longissimusmuscleareaandmarblingwiththeusecrude

glycerininthediets.Maximumvaluesinthecurvesforcarcassyieldandmarblingwere212and244g/kgofcrudeglycerin,

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Table4

Drymatterintake(DMI),averagedailygain(ADG)andfeedefﬁciency(G:F)inyoungbullsfeddifferentlevelsofcrudeglycerin.

Variable Glycerinlevels(g/kg) SEM Gly1 _L2 _Q3

0 60 120 180 DMI,kg/d 11.8 11.9 11.8 11.0 0.178 0.41 <0.01 <0.01 ADG0–28,kg/d 1.84 1.85 2.11 1.95 0.132 0.40 0.32 0.54 ADG28–56,kg/d 1.82 2.11 2.08 1.95 0.149 0.20 0.57 0.18 ADG56–84,kg/d 1.60 1.79 1.45 1.49 0.116 0.87 0.20 0.49 ADG0–84,kg/d 1.75 1.92 1.88 1.80 0.007 0.32 0.80 0.21 G:F4 _0.148 _0.160 _0.158 _0.172 _0.007 _0.08 _0.04 _0.91 1 _Gly₌₀_vs._Glycerin. 2 _L₌_Linear. 3 _Q₌_Quadratic. 4 _Gain:feed_ratio. Table5

FinalBodyweight(FBW),hotcarcassweight(HCW),coldcarcassweight(CCW),longissimusdorsimusclearea(LMA),LMAper100kgcoldcarcass

(LMA/100kg),carcassyield(CY),subcutaneousfatthickness(SFT),andmarblingscoreofyoungbullsfeddifferentlevelsofcrudeglycerin.

Variables Glycerinlevels(g/kg) SEM Gly1 _L2 _Q3

0 60 120 180 FBW,kg 513 522 527 516 14.9 0.61 0.82 0.77 HCW,kg 284 289 299 291 9.2 0.39 0.85 0.36 CCW,kg 278 283 287 279 8.5 0.62 0.89 0.73 CY,g/kg 554 555 568 565 4.3 0.08 0.99 <0.01 LMA,cm2 _83.1 _78.5 _85.4 _87.8 _2.69 _0.79 _0.34 _0.04 LMA,cm2_/100_kg _30.0 _28.0 _29.8 _31.6 _0.87 _0.84 _0.28 _0.05 SFT,mm 3.18 3.27 3.18 3.18 0.433 0.95 0.89 0.91 Marblingscore 336 342 540 534 62.3 0.07 0.97 <0.01 1 _Gly₌₀_vs._Glycerin. 2 _L₌_Linear. 3 _Q₌_Quadratic. 4. Discussion

Theconcentrationsofplasmacreatininearewithinthenormalrange(1–2mg/dL)reportedbyKanekoetal.(1997).This

indicatesthatthekidneyglomerularﬁltrationrateforthismetabolitewasadequate,withnomethanolinterferenceofthe

crudeglycerin.Theglycerinusedinthepresentstudycomprised200mg/kgofmethanol,whichisabovetherecommended

valuebyUnitedStatesDepartmentofAgriculture(USDA)andBrazilMinistryofAgriculture(MinistériodaAgricultura,Pecuária

eAbastecimento)of150ppm.

TheCKvalues,although notaffectedbythediet, werewellabovethoserecommended byKanekoetal. (1997)of

4.8–12.1U/L.Thisresultcanbeexplainedbytheexertedphysicaleffortoftheanimalsduringhandlingforweighing;since

CKisasensitiveandspeciﬁcindicatorofmuscleinjury.AccordingtoHodgson(1994),increasesofupto500U/Larenormal

inmoderatephysicaleffortsituations.Injointanalysis,increasesinbloodASTandCKmaybesuggestiveofmyonecrosis

(Hodgson,1994),whatdidnothappenwhenanimalsfedglycerin.TheGGTenzymefoundinplasmaisindicativeof

cholesta-sis,whichisahepaticfailureintheexcretionandsecretionofpigments,bileacidsandsaltsinruminants(Santosetal.,2008).

Similartotheotherhepaticenzymes,therewasnoeffectoftheglycerinlevelsinthedietonGGTconcentrations,which

werealsowithinthenormalrangeofupto140U/L(BarrosFilho,1995).Therefore,glycerinaddedtothedietsdidnotaffect

bloodparameters,indicatinganormalhepaticmetabolismandnohepatocytedestruction.

Thedecliningtrendinbloodglucosewhileusingglycerincanindicatethattissuesweremoresensitivetoinsulin-mediated

glucoseuptake,sincegreaterglycerolkinaseactivityoccurredintheliverofthesebulls,resultinginapossiblegreaterliver

gluconeogenesis.Thisenzymeisabletoinducegluconeogenesisfromtheglycerolabsorbedintherumenandtransported

totheliver(Aschenbachetal.,2010).Inthiscase,liverglucoseproductionmayhaveincreasedpancreasinsulinsecretion

andthen,glucoseuptakebytissuesasmuscle.AccordingtoMajdoubetal.(2003),gluconeogenesisprecursorsstimulate

insulinpancreaticsecretion.Thepositiveeffectofthecrudeglycerinonmarblingscorealsosupportsthistheory.According

toChungetal.(2007),glucoseplaysarelevantroleascarbondonorindenovofattyacidsbiosynthesis.

Theliverglycerolabsorptionoccursduetothepresenceofmembraneproteins,speciﬁcally,aquaglyceroporin9(AQP

9)(Frogeretal.,2001;Hibuseetal.,2005).AccordingtoKrehbiel(2008),glycerolhasthreefatesintherumen:escape

intothepostrumen(130g/kg),fermentationintherumen(440g/kg)andabsorptionbytheruminalepithelium(430g/kg).

Therefore,duetotheresultsintheglycerolkinaseactivity,alargeramountofglycerolandlesspropionatewasprobably

absorbedintherumenasglycerinlevelsinthedietincreased.Besides,thelowerexpressionofPCK1,whichencodescytosolic

phosphoenolpyruvatecarboxykinaseenzyme(PEPCK-C),intheliverofanimalsfedhighlevelsofglycerin,supportsthislast

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feed-forwardmechanismofsubstratecontrolforhepaticgluconeogenesisthatislinkedtotheﬁnalproductsofrumen

fer-mentation.Consequently,probably,ruminalpropionateproductionincreasedwhencrudeglycerinwasusedupto59.1g/kg

ofthediet(maximumvalueforPCK1expressioncurve)andthenitdecreased,reducingPCK1expression.Inaddition,ina

studyperformedbyHalesetal.(2015),methaneproductionincreasedbetween100and150g/kgofglycerin,anditiswell

knownthatmethaneandpropionateproductionareantagonistic,supportingthehypothesisoflowpropionate

produc-tionwhenanimalsfed180g/kgofglycerindietinthepresentstudy.Boydetal.(2013)observedthatmolarproportionsof

butyrateweregreaterwhenanimalswerefeddietswithglycerolinclusionand,accordingtotheseauthors;ruminalbutyrate

wouldimprovethegrowthofruminalepithelialtissueandpossiblyincreasenutrientsabsorptionfromtherumen,aswell

asglycerol.

Althoughglycerolkinaseactivityincreasedwiththeglycerinlevelinthediet,expressionofGK1,thegeneresponsible

toencodethisenzyme,wasinhibitedwithincreasinglevelsofglycerin.Therefore,despitethepositiveeffectofpropionate

inPCK1expression,glycerolseemedtoexertanegativefeedbackinGK1expression.Inthissense,studiesevaluatingwhich

transcriptionfactorsregulateexpressionofgenesinvolvedinlivergluconeogenesisinruminantsarenecessary.Yoonetal.

(2001)showedthatthePeroxisomeProliferator-ActivatedReceptor-␥Coactivator1␣(PGC-1␣)isakeymodulatorofhepatic

gluconeogenesisinmice,activatingthePCKgene.Inaddition,PeroxisomeProliferator-ActivatedReceptor-␥(PPAR-gamma)

activationalsoinducestheexpressionofPCKinmice(Perryetal.,2014;Schultzetal.,2015).Probably,theimportantrole

ofthesetranscriptionfactorsinliverofnon-ruminantsisapathwhereresearchinruminantsmustfollow.

ThelowerDMI whenanimalsfedglycerin(Table4)canbeexplainedbyametabolicregulationand otherglycerin

components.Intheﬁrstsituation,thegreaterinsulinsecretion,asglycerinlevelsinthedietincreased,mayhaveaffected

negativelyDMI.AccordingtoPlumetal.(2005)andPorteetal.(2005),insulinenterthebrainandreactswithspeciﬁc

insulinreceptorsonneurons,reducingfoodintake.Besidesthemetaboliceffect,accordingtoHalesetal.(2015),highNa

concentrationsincattledietmayhaveinhibitoryeffectsonDMI.Inthepresentstudy,glycerinhad36.7g/kgofchlorides.Hales

etal.(2015)alsodescribedthatDMIreductionincattle,asglycerinconcentrationisincreased,seemsconsistentthroughout

literature.ThelinearnegativeeffectonDMIandthelackofeffectonADGexplainedwhyfeedefﬁciencyincreasedlinearlyas

glycerinincreasedinthediet.Therefore,animalperformanceobservedinthisstudyindicatesthatupto180g/kgofglycerol

inclusioninDMhasnonegativeimpact,consideringthetypeofdietused.

Similarly,Machetal.(2009)foundnoeffectonanimalsADGwhenassessingupto120g/kgofcrudeglycerininclusion

inthediet.However,Parsonsetal.(2009),workingwithdietswithupto160g/kgofcrudeglycerin,observedaquadratic

effectonanimalsADG,andthemaximumconcentrationrecommendedbytheauthorsforglycerinwas80g/kgonDMbasis.

ThehighestcrudeglycerinlevelanalyzedforbeefcattlesofarwasreportedbyVanCleefetal.(2014),whoevaluatedthe

inclusionofupto300g/kgintheDM.TheauthorsdescribedthattherewerenodifferencesamongtreatmentsforADGand

feedefﬁciency;however,theyreportedanegativeeffectofthetreatmentwith300g/kgofglycerinonﬁberdigestibility.

Therefore,therecommendedlevelofcrudeglycerinisinﬂuencedbytheglycerinquality,forage:concentrateratio,protein

source,cornprocessingetc.

Oneplausibleexplanationfortheobservedeffectincarcassyieldisapossiblelowergastrointestinalcontentweightinthe

animalsfedcrudeglycerin.Evaluatingglycerolsupplyeffectonstress,associatedwithlongtransportperiodsinZebucattle,

Parkeretal.(2007)observednodifferencesintheanimalsbodyweightafteratripof24h,similartotheresultsobtainedin

thepresentstudy.However,bodywatercontentincreasedinanimalsfedglycerol,possiblyduetotheactionofglycerolin

enhancingintestinalﬂow,andthusincreasingthegastrointestinalcontentloss.AccordingtoWythesetal.(1980),animal

weightlossthattakesplaceduringfastingandextendedtransportationisaconsequenceofintestinalﬁlling,urinaryand

defecationrates.Anotherexplanationfortheobservedlinearincreaseincarcassyieldisthatglycerolhaspositiveeffectson

aminoacidsandnitrogenretentioninthebody,leadinganeconomyingluconeogenicaminoacidsusage.Theincreasein

glycerolkinaseactivitysupportsthisstatement.

5. Conclusion

Theuseofglycerinincreasedfeedefﬁciencyand beefmarblingand,therefore,atalevelofupto180g/kgofDMis

recommendedforfeedlotdiets.GlycerolkinaseactivityandexpressionofGK1andPCK1geneswereaffectedwiththeuse

ofcrudeglycerin,whichinﬂuencelivergluconeogenesis.

Conﬂictofinterest

Theauthorsdeclarethattherearenoconﬂictsofinterest.

Acknowledgement

ThisstudywasfundedbyFundac¸ãodeAmparoàPesquisadoEstadodeMinasGerais–Fapemig(BeloHorizonte,MG,Brazil).

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