Effects of crude glycerin on milk composition, nutrient digestibility and ruminal fermentation of dairy cows fed corn silage-based diets

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ContentslistsavailableatScienceDirect

Animal

Feed

Science

and

Technology

journalhomepage:www.elsevier.com/locate/anifeedsci

Short

communication

Effects

of

crude

glycerin

on

milk

composition,

nutrient

digestibility

and

ruminal

fermentation

of

dairy

cows

fed

corn

silage-based

diets

P.G.

Paiva

a,∗

_,

_T.A.Del

_Valle

b

_,

_E.F.

_Jesus

a

_,

_V.P.

_Bettero

a

_,

_G.F

_Almeida

b

_,

_I.C.S.

Bueno

c

_,

_B.J.

_Bradford

d

_,

_F.P.

_Rennó

b,∗∗

a_Department_of_Animal_Science,_{UNESP—Universidade}_Estadual_Paulista_“Júlio_de_Mesquita_{Filho”/Campus}_Jaboticabal,_Rod._Prof._Paulo

DonatoCastellanekm5,Rural,14884-900Jaboticabal,SP,Brazil

b_Department_of_Animal_Nutrition_and_Production,_School_of_Veterinary_Medicine_and_Animal_Science,_University_of_Sao_Paulo_(USP),_Av.

DuquedeCaxiasNorte,225-CampusdaUSP,13635-900,Pirassununga,SP,Brazil

c_Department_of_Animal_Science,_College_of_Animal_Science_and_Food_Engineering,_University_of_Sao_Paulo,_{Pirassununga,}_SP,_Brazil

d_Department_of_Animal_Sciences_and_Industry,_Kansas_State_University,₆₆₅₀₆_Manhattan,_USA

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2May2015 Receivedinrevisedform 18December2015 Accepted21December2015 Keywords: Biofuels By-product Fiberdigestibility Glycerol Milkcomposition

a

b

s

t

r

a

c

t

Theobjectiveofthecurrentstudywastoevaluatetheeffectsofincreasinglevelsofcrude glycerinondrymatterintake,nutrientdigestibility,ruminalparameters,blood metabo-lites,milkyieldandcompositionofdairycowsfedcornsilage-baseddiets.Twenty-four Holsteincows(16non-cannulatedand8rumen-cannulated;184±50daysinmilk[DIM] and594±39kgofbodyweight[BW])wereassignedtoareplicated4×4Latinsquaredesign experiment,consistingof14daysforadaptationtodietsand7daysforsampling.Cowswere assignedtoreceiveoneofthediets:0(Control),70,140or210gofcrudeglycerin/kgof dietdrymatter(DM).Drymatterintakeandmilkyieldlinearlydecreasedwiththe inclu-sionofcrudeglycerin.However,crudeglycerindidnotaffectmilkcomposition.Total-tract digestibilityofDM,crudeprotein(CP)andetherextract(EE)linearlyincreased,and neu-traldetergentﬁber(NDF)digestibilitylinearlydecreasedaccordingtodietaryinclusionof crudeglycerin.Feedingcrudeglycerinchangedvolatilefattyacidconcentrations:linearly increasedpropionate,quadraticallyaffectedbutyrate,andlinearlydecreasedacetate con-centrations,resultinginloweracetatetopropionateratio.Totalvolatilefattyacidwasnot affectedbycrudeglycerin.RuminalNH3-Nlinearlydecreasedaccordingtocrudeglycerin

dietarylevels.Crudeglycerinlinearlyincreasedbloodglucoseconcentration.Inclusionof highlevelsofcrudeglycerin(210g/kg)indietsofmid-lactatingcows,increaseddry mat-terdigestibilityandruminalpropionateconcentrations,butnegativelyaffecteddrymatter intakeandmilkyield.

Abbreviations: BCFA,branchedchainfattyacids;BUN,bloodureanitrogen;BW,bodyweight;CP,crudeprotein;DIM,daysinmilk;DMI,drymatter

intake;EE,etherextract;aNDF,neutraldetergentﬁberwithresidualash;NFC,non-ﬁbercarbohydrate;VFA,volatilefattyacids.

∗ Correspondingauthor.Fax:+551935654368.

∗∗ Correspondingauthor.

E-mailaddresses:bocazoo@hotmail.com,pagppaiva@gmail.com(P.G.Paiva),francisco.renno@usp.br(F.P.Rennó).

http://dx.doi.org/10.1016/j.anifeedsci.2015.12.016

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1. Introduction

Thegrowthofthebiofuelindustryhasincreasedthedemandforcorn,affectingpricesandthelivestocksector(USDA, 2014).Therefore,newalternativefeedsourcesarerequiredinordertoreduceanimalfeedcosts.Crudeglycerinisa by-productobtainedfromoilprocessinginthebiodieselindustry,whereinforeachliterofbiodieselproduced,0.1lofcrude glycerinisformed(Dasarietal.,2005).Crudeglycerincontains800–880gofglycerol/kg(Donkin,2008)andglycerolor puriﬁedglycerin(>95%glycerol)hasbeenusedtopreventmetabolicproblemsintransitioncows(GoffandHorst,2003; PiantoniandAllen,2015).

Crudeglycerincanbeconvertedintovolatilefattyacid(VFA)inrumen,mainlyintopropionate(Wangetal.,2009),orcan beabsorbedthroughtherumenepitheliumandoxidizedintheliver,increasingtheenergyavailableforanimalutilization (Remondetal.,1993).Severalstudieshavereportedthatreplacingcornwithglycerinincreasedtheruminalmolarproportion ofpropionate(DeFrainetal.,2004;Shinetal.,2015;Boydetal.,2013).

Thereisalackofdataexaminingcrudeglycerinasaprimaryenergysourceingredientinrationsofdairycows,mainly indietarylevelshigherthan100g/kg(drymatter[DM]basis).Thus,theobjectiveofthepresentstudywastoevaluatethe effectsofincreasingdietarylevelsofcrudeglycerin(upto210g/kgdietDM)ondrymatterintake(DMI),nutrientdigestibility, ruminalparameters,bloodmetabolites,milkyieldandcompositionoflactatingdairycowsfedcornsilage-baseddiets.Our hypothesiswasthatcowsfedcrudeglycerincouldmaintainmilkyieldandcompositionwithoutimpairingDMIandnutrient digestibility.

2. Materialsandmethods

ExperimentalprocedureswereapprovedbytheEthicsCommitteeoftheSchoolofVeterinaryMedicineandAnimal ScienceoftheUniversityofSãoPaulo(approvalnumber3058/2013).

2.1. Animals,dietsandexperimentaldesign

Twenty-fourmultiparousHolsteincows(184±50daysinmilk[DIM]and594±39kgbodyweight[BW],mean±SD),16 non-cannulatedand8rumencannulated,wereassignedtoareplicated4×4Latinsquaredesignexperimentwith21day periods(14dayofadaptationand7dayofsampling).Thecowsweregroupedaccordingtomilkyieldandthenassignedto eachsquareaccordingtoBW,exceptfor2squaresthatwereformedbytherumencannulatedcows.Thus,24repetitionsper treatmentwereusedforstatisticalanalysis.Theanimalswereassignedwithineachsquaretoreceiveoneofthefollowing diets:0(Control),70,140or210gofcrudeglycerin/kgofdietDM.DietswereformulatedaccordingtoNRC(2001),and ingredientsandchemicalcompositionaredescribedinTable1.Thecrudeglycerincontained806g/kgofglycerol,63g/kg ofashand124.6g/kgofwater,andwasobtainedfromsoybeanoil(ADM,Rondonopolis,Brazil).Thecrudeglycerinwas mixedintotheconcentratebeforethemorningfeeding.Cowswereindividuallyfedtwicedaily(50%oftotaldietDMin eachfeeding)at0700and1300htosupply105–110%ofexpectedfeedintake(onasfed-basis).Thefeedwasindividually Table1

Ingredientsandchemicalcompositionofexperimentaldiets.

Item,g/kgofdietDM Glycerininclusion,g/kgofDM

0 70 140 210 Cornsilagea _501.4 _500.3 _505.9 _504.0 Groundcorn 300.5 219.2 125.1 45.0 Soybeanmeal 162.1 174.6 192.8 204.8 Crudeglycerinb _0.0 _69.9 _140.2 _210.2 Mineralpremixc _29.8 _29.8 _29.8 _29.8 Urea 5.30 5.30 5.30 5.30 Ammoniumsulphate 0.90 0.90 0.90 0.90 Composition Drymatter 586.4 587.6 585.0 586.6

Neutraldetergentﬁber 301.1 290.5 281.9 271.0

Nonﬁbercarbohydrated _455.7 _464.1 _468.1 _468.1

Crudeprotein 162.3 160.7 161.2 159.5

Etherextract 31.1 31.2 31.0 31.2

Ash 66.3 70.1 74.3 78.0

Netenergye_(Mcal/kg_DM) _1.74 _1.75 _1.75 _1.75

a_Composition:_293.0_of_dry_matter_(DM);_70.5_of_crude_protein_(CP);_556.7_of_neutral_detergent_ﬁber_(NDF);_29.7_of_ether_extract_(EE)_and_38.6_of_ash. b_Composition:₈₀₆_g/kg_of_glycerol,₆₃_g/kg_of_ash_and_124.6_g/kg_of_water,_obtained_from_soybean_oil_(ADM,_{Rondonopolis,}_Brazil).

c_Contained_per_kg:_88.0_g_of_Ca;_42.0_g_of_P;_18.0_g_of_S;_45.0_g_of_Mg;_123.0_g_of_Na;_14.0_mg_of_Co;_500.0_mg_of_Cu;_20.0_mg_of_Cr;_1050.0_mg_of_Fe;_28.0_mg

ofI;1400.0mgofMn;18.0mgofSe;2800.0mgdeZn;80.0mgofBiotin;200.000,00UIvitaminA;40.000,00UIvitaminD;1.200,00UIvitaminE.

d_Non_ﬁber_carbohydrate₌_1000–([CP_-_CP_of_urea₊_urea]₊_NDF₊_EE₊_ash),_Hall₍₂₀₀₀₎_. e_Estimated_using_the_NRC₍₂₀₀₁₎_model.

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weighed,andthesilageandconcentrateofeachcowwereindividuallyhandmixedtoprepareatotalmixedration(TMR). Animalswerehousedinsandedbedfree-stallbarncontainingindividualpens(17.5m2_),_forced_ventilation_and_free_access towater.

2.2. Datacollection,samplingandanalysis

Samplesofingredients(0.5kg)werecollectedonceaweekduringthepreparationofconcentrate.Samples(0.5kg)ofsilage andortsfromeachcowweretakenoncedailyduringthemorningfeedingfromday15untilday21ofeachexperimental period,andcompositesamples(onasfed-basis)ofsilageandortsfromeachcowwereformedandstoredat−20◦Cfor furtherchemicalanalysis.Feedintakewasrecordeddailyasthedifferencebetweenfeedofferedandrefused.Sampleof feedandortsweredriedina55◦Cforced-airovenfor72handgroundtopassthrougha1mmscreen(WileyMill,ArthurH. Thomas,Philadelphia,PA,USA)andthenanalyzedfordrymatter([DM]method930.15;AOAC,2000),crudeprotein([CP]Nx 6.25;method984.13;AOAC,2000),etherextract([EE]method920.39;AOAC,2000),andash(method942.05;AOAC,2000). Neutraldetergentﬁber(aNDF)wasdeterminedasdescribedbyMertensetal.(2002),using␣-amylase,withoutadditionof sodiumsulﬁtetothedetergent(AnkomTech.Corp.,Fairport,NY,USA),andwasexpressedwithresidualash.

Fecalsamples(0.5kgonwetbasis)werecollectedtwicedaily(0630hand1630h)fromday16untilday18ofeach experimentalperiod.Thesixfecalsamplesofeachcowwerecompositedintoasample(1.5kgonwetbasis)andstored at−20◦C.Indigestibleaciddetergentﬁber(iADF)wasusedasaninternalmarkertoestimatefecalexcretionandapparent nutrientdigestibility.Samplesoffeeds,ortsandfecesweredriedat55◦Cinaforced-airovenfor72handgroundtopass througha2mmscreen(Wileymill,ArthurH.Thomas,Philadelphia,PA,USA).Thesesampleswereplacedinbagsof non-woventextile(100g/m2₎_following_the_{recommendation}_of_a_maximum_of₂₀_mg_of_dry_sample_per_cm2₍_Nocek,₁₉₈₈₎_and wereincubatedforaperiodof264hintherumenof2Holsteincows,previouslyadaptedtoahighconcentratediet,according tothetechniquedescribedbyCasalietal.(2008).After264h,bagswereremovedandwashedinrunningwater,driedat 55◦Cinaforced-airovenandsubjectedtoaciddetergenttreatment(Mertensetal.,2002).Totalfecalexcretionwasobtained asfollows:DMfecalexcretion(kg/d)=iADFintake(correctedfororts)/iADFcontentinfeces.

Cowsweremechanicallymilkedtwicedaily(0600hand1600h)andmilkyieldwasrecordedbyanautomaticmilkmeter (Alpro®_,_{DeLaval–Tumba,}_Sweden)._Milk_samples_were_automatic_collected_(Alpro®_,_DeLaval_–_Tumba,_Sweden)_from_day 16untilday18ofeachexperimentalperiod,accordingtomilkyieldofeachcowineachmilking.Sampleswererefrigerated afterthemorningmilkingandmixedwithsamplesoftheafternoonmilkingandanalyzedfreshforfat,crudeproteinand lactoseaccordingtoKaylegianetal.(2006).Milkyieldwascorrectedfor3.5%offataccordingtoSklanetal.(1992).

Bloodsampleswerecollectedond15ofeachperiodfromthecoccygealvesselsbeforethemorningfeeding,centrifuged at3000×gfor10minandplasmawasseparatedandstoredfrozenat−20◦Cuntilanalysis,asdescribedbyColaketal. (2013).Analyseswereperformedusingcolorimetriccommercialkits(Glucose:cat.no.K-082;Urea:cat.no.K-056;allfrom theBioclin,BeloHorizonte,Brazil),andreadingwasperformedwithasemi-automaticspectrophotometer(SBA200,CELM, SãoCaetanodoSul,Brazil).

Onday16and17ofeachexperimentalperiod,spoturinesampleswerecollectedfromeachcow4hafterthemorning feedingandthenﬁlteredtoobtain10mLaliquotswhichwereimmediatelydilutedin40mLof0.036Nsulfuricacid,toavoid bacterialdestructionofthepurinederivativesandprecipitationoftheuricacid,andthenstoredat₋20◦Cforsubsequent analysisofuricacidandallantoinaccordingtoChenandGomes(1992).Apuresampleofurinewasstoredfordetermination oftotalnitrogen(N)andcreatinine.Concentrationsofuricacidandcreatinineweredeterminedusingcommercialkits(uric acidstableliquid:cat.no.k-052;kineticcreatinine:cat.no.K-067;Bioclin).Thedailyurinevolumewasestimatedfromthe dailycreatinineexcretionas24.05mgkg−1_of_body_weight₍_Chizzotti_et_al.,₂₀₀₈_)._The_excretion_of_uric_acid,_allantoin_in urineandmilkwereconsideredasthetotalexcretionofpurinederivatives.Microbialproteinsynthesiswasestimatedas describedbyChenandGomes(1992).

Dataoftwosquarescomposedofcannulatedcowswereusedtoevaluateruminalfermentation,resultingin8repetitions foreachtreatmentpertime.Onday20ofeachexperimentalperiod,ruminalfluidsampleswerecollectedfromeight rumen-cannulatedcows(216±54DIMand596±27kgofBW)before(0h)and2,4,6,8,10and12hafterthemorningfeeding. TheruminalfluidpHvaluesweredeterminedusingapotentiometer(MB-10,MarteCientífica,SantaRitadoSapucaí,Brazil). Aliquotsofsamplesweremixedwithmetaphosphoricacid(0.25mol/LHPO3),centrifugedat7000×g,andsupernatant storedat−20◦CforanalysisofvolatilefattyacidsaccordingtomethoddescribedbyErwinetal.(1961)andadaptedby Getachewetal.(2002).Theremainingaliquotsofsamples(2mL)weremixedwith1mLofsulfuricacid(0.5mol/LH2SO4) andstoredat−20◦Ctodetermineammonianitrogen(NH3-N)byacolorimetricphenol-hypochloritemethod(Broderick andKang,1980).RuminalVFAweremeasuredusingagaschromatograph(GC-2014Shimadzu,Tokyo,Japan)equippedwith acapillarycolumn(Stabilwax,Restek,Bellefonte,PA,USA)at145◦C.Thegasesusedwerehelium(8.01mL/minflow)asthe carrier,hydrogen(pressureof60kPa)asthefuel,andsyntheticair(pressureof40kPa)astheoxidizer.Steamertemperature wassetat220◦C,ionizationdetectorflamesat250◦C,andtheseparationcolumnat145◦Cfor3min,whichwasthenraised 10◦C/minupto200◦C.Externalstandardwaspreparedwithacetic,propionic,isobutyric,butyric,isovalericandvaleric acids(Chemservice,WestChester,PA,USA).ThesoftwareGCsolution(Shimadzu,Japan)wasusedforcalculationofVFA concentrations.

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Table2

Effectsofglycerinlevelsonintake,digestibilityandperformanceoflactatingdairycows.

Item Glycerininclusion,g/kgofDM SEMa _P_-valueb

0 70 140 210 Diet LIN

Drymatterintake,kg/day 21.97 21.97 21.42 21.06 0.32 0.05 0.04

Digestibility

Drymatter 0.696 0.703 0.717 0.724 0.003 <0.01 <0.01

Crudeprotein 0.734 0.736 0.755 0.772 0.003 <0.01 <0.01

Etherextract 0.729 0.761 0.841 0.869 0.008 <0.01 <0.01

Neutraldetergentﬁber 0.599 0.580 0.571 0.543 0.005 <0.01 <0.01

Performance

Milkyield,kg/day 27.74 26.19 26.43 24.64 0.51 <0.01 <0.01

3.5%FCM,kg/dayc _31.74 _30.24 _30.30 _27.89 _0.52 _<0.01 _<0.01 Fat,kg/day 1.21 1.16 1.17 1.06 0.02 <0.01 <0.01 Protein,kg/day 0.87 0.82 0.83 0.78 0.02 <0.01 <0.01 Lactose,kg/day 1.31 1.21 1.24 1.16 0.02 <0.01 <0.01 FCM:DMId _1.49 _1.38 _1.42 _1.33 _0.03 _0.01 _<0.01 Milkcomposition Fat,g/kg 44.07 44.70 44.31 43.60 0.60 0.77 0.59 Protein,g/kg 31.41 31.38 31.40 31.52 0.12 0.84 0.52 Lactose,g/kg 47.10 47.04 47.00 47.17 0.17 0.87 0.75

Milknitrogen,g/day 136.52 126.78 130.15 121.38 2.51 <0.01 <0.01

Nitrogenefﬁciencye _23.69 _22.00 _22.48 _22.08 _0.41 _<0.01 _<0.01

a_Standard_error_of_means.

b_Treatment_effect_(Diet)._Linear_contrast_(LIN)._Quadratic_contrasts_were_tested_but_were_not_signiﬁcant₍_P_>_0.05). c_3.5%_fat_corrected_milk_(FCM)₌_(0.432₊_0.165_×_fat_content)_×_kg_of_milk₍_Sklan_et_al.,₁₉₉₂_).

d_Fat_corrected_milk_to_dry_matter_intake_ratio.

e_Nitrogen_(N)_efﬁciency₌₁₀₀_×_milk_N_(g/day)/N_intake_(g/day).

2.3. Statisticalanalyses

ThedatawereanalyzedwithPROCMIXED(StatisticalAnalysisSystemforWindows9.0-SAS-SASInstituteInc.,Cary, NC,USA),accordingtothefollowingstatisticalmodel:

Yiklm=+Ti+Pk+Sl+Am(S_l)+eijklm

whereY_iklmistheobservedvalueintheanimalm,fromlsquare,inthekthperiod,whichreceivedtheithglycerinlevel;

=overallmean;T_i=fixedeffectofglycerinlevel(3DF);P_k=fixedeffectofexperimentalperiod(3DF);S_l=fixedeffect ofsquare(5DF);A_m(S_l)=randomeffectofanimalwithinsquare(18DF),ande_ijklm=randomresidualerror.Theruminal fermentationvariables(pH,NH3-NandVFA)wereanalyzedasrepeatedmeasuresusingPROCMIXEDofSAS9.0,considering inthestatisticalmodeltheeffectsofanimal,period,square,treatment(glycerinlevel),beyondtheeffectsoftimewith theirinteractionswiththetreatment.Compoundsymmetrywasthebestcovariancestructurebaseduponthesmallest Akaike’sinformationcriterionvalues.Othercovariancestructurestestedincludedheterogeneouscompoundsymmetry, unstructured,autoregressive1andheterogeneousautoregressive1.Resultsarereportedasleastsquaresmeans.Responses toglycerinlevelweretestedwithlinearandquadraticcontrasts,whichweredeclaredsignificantatP<0.05.

3. Results

InclusionofcrudeglycerininthedietslinearlydecreasedtheDMI(P=0.04;Table2).Cowsfeddietscontaining210g/kg ofcrudeglycerinreducedDMIby4.1%or0.910g/dcomparedtocowsfedcontroldiet.TotaltractdigestibilityofDM,CP,and EElinearlyincreased(P<0.01),butneutraldetergentfiber(NDF)digestibilitylinearlydecreased(P<0.01)accordingtothe inclusionofdietarycrudeglycerin.Neutraldetergentfiberdigestibilityreduced5.6%incowsfeddietscontaining210g/kg ofcrudeglycerincomparedtocontrol.Increasingthedietaryinclusionofcrudeglycerinlinearlydecreasedmilkyield, fat-correctedmilk(FCM),milkefficiency(FCM:DMI),fat,proteinandlactose(P<0.01,Table2).However,milkfat,proteinand lactoseproportionweresimilaramongtreatments(P>0.05).DailyNmilkexcretionandNefficiencywerelinearlydecreased (P<0.01)withcrudeglycerininclusioninthediet.

TotalruminalVFAconcentrationwassimilaramongtreatments(P>0.05;Table3).However,theinclusionofcrude glyc-erinindietslinearlydecreasedruminalacetate,acetatetopropionateratio(P<0.01),whereaspropionateandbranchedchain fattyacids(BCFA)linearlyincreased(P<0.01)withcrudeglycerininclusion.Butyratewasquadraticallyaffected(P<0.01)by crudeglycerin,withhighestvalueobservedwiththedietaryinclusionof70g/kgDMofcrudeglycerin.Therewasdiet*time interactionforruminalpH,butyrateproportionandacetatetopropionateratio(P<0.01).RuminalNH3-Nconcentration linearlydecreased(P<0.01)withcrudeglycerininthediet.Microbialproteinsynthesiswasnotaffectedbycrudeglycerin treatments(P>0.05).Bloodconcentrationofglucoselinearlyincreased(P<0.01;Table3)from78.84(control)to86.40mg/dL

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Table3

Effectsofglycerinlevelsonruminalparametersandbloodmetabolitesinlactatingdairycows.

Item Glycerininclusion,g/kgofDM SEMa _P_-valueb

0 70 140 210 Diet Time Diet*Time LIN QUAD

pH 6.31 6.38 6.38 6.32 0.03 0.65 <0.01 <0.01 0.89 0.22

NH3-N,mg/dL 23.74 21.52 18.05 17.54 0.49 <0.01 <0.01 0.19 <0.01 0.39

TotalVFA,mmol/L 119.11 111.40 108.16 113.87 2.26 0.41 <0.01 0.85 0.37 0.16

Acetate,mol/100mol 62.41 52.51 51.13 47.16 0.58 <0.01 <0.01 0.44 <0.01 0.05

Propionate,mol/100mol 20.61 22.47 24.02 29.12 0.32 <0.01 <0.01 0.06 <0.01 0.06

Butyrate,mol/100mol 12.13 19.25 18.93 17.56 0.35 <0.01 <0.01 <0.01 <0.01 <0.01

BCFA,c_mol/100_mol _4.85 _5.77 _5.92 _6.16 _0.07 _<0.01 _<0.01 _0.07 _<0.01 _0.11

C2:C3d _3.05 _2.39 _2.21 _1.73 _0.05 _<0.01 _<0.01 _<0.01 _<0.01 _0.20

MicrobialCP,e_g/day ₂₄₄₆ ₂₄₁₅ ₂₂₅₀ ₂₃₂₃ _67.18 _0.43 _– _– _0.20 _0.57

Bloodmetabolites

Glucose,mg/dL 78.84 83.93 83.96 86.40 0.74 <0.01 – – <0.01 0.30

BUN,f_mg/dL _36.95 _32.50 _32.26 _33.36 _0.76 _0.01 _– _– _0.13 _0.01

a_Standard_error_of_means.

b _Treatment_effect_(Diet)._Time_effect._Diet_and_time_interaction_effect_(Diet*Time)._Linear_contrast_(LIN)._Quadratic_contrasts_(QUAD). c _BCFA:_branched_chain_fatty_acids_(included_valerate,_isovalerate_and_{isobutyrate).}

d _Acetate_to_propionate_ratio. e_Microbial_crude_protein. f _BUN:_blood_urea_nitrogen.

(210g/kgDMofcrudeglycerin);ontheotherhand,bloodureanitrogenconcentrationwasquadraticallyaffectedbycrude glycerin(P=0.01),withlowestvalueobservedwhen140g/kgDMofcrudeglycerinwassupplied.

4. Discussion

Currently,thegrowthofthebiodieselindustryhasresultedingreateravailabilityofcrudeglycerin,aby-productanda potentialreplacertostarchindietsofdairycows.InthecurrentstudyalineardecreaseinDMIwasobservedwhencows werefedupto210g/kgDMofcrudeglycerin.Ezequieletal.(2015)reportedareductionof15%inDMIofdairycowsfed crudeglycerinupto300g/kgofdietDMcomparedtocontrol.Thenegativeeffectsofcrudeglycerinonanimalmetabolism andperformancemightbeattributedtothreefactors:thequalityofcrudeglycerinduetoimpurities,includingmethanol andsalts(ThompsonandHe,2006;Chungetal.,2007);thespeedinwhichglycerolisfermentedinrumen(Remondetal., 1993;Wangetal.,2009;Shinetal.,2012);andtheabilityofrumenepitheliumtoabsorbglyceroltobemetabolizedinliver (Krehbiel,2008).Ezequieletal.(2015)demonstratedthatdietswith300g/kgDMofcrudeglycerinincreased500%ofNaCl dietarycontentanddecreasedfeedintakeofdairycows.Furthermore,highsaltsandsodiumdietarylevelscouldresultinan electrolyteunbalanceinanimalsandaffectacceptabilityofthedietwithcrudeglycerin(Chungetal.,2007;Dasari,2007). Ontheotherhand,crudeglycerinhashighenergycompounds,andcaninﬂuenceoxidationreactionsandincreaseKrebs cycleintermediatesproductionintheliver,stimulatingsatietyandreducingDMI(Bensonetal.,2002;Trabueetal.,2007; Allenetal.,2009).

Inclusionofhighdietarylevelsofcrudeglycerin(210g/kg)indairycowdietsresultedinalargedecreaseofmilkyield (3.1kg/day).Similarly,Boydetal.(2013)foundreducedmilkyieldwhencrudeglycerinwasaddedtothedietofdairycows. However,thisresultcontrastedwithotherpreviousstudiesthatreportednodifferenceinmilkyieldwithglycerinadded tothedietsofmid-lactationdairycows(Donkinetal.,2009;Kassetal.,2012;Shinetal.,2012)whichusedupto150gof glycerinindietDM.Inthecurrentstudy,thedecreaseofmilkyieldcouldberelatedtoDMIanddietcomposition.Despite thedigestibilityofnutrientsincreasedwithcrudeglycerin,dietaryCPcontentandDMIdecreased,andconsequentlyaltered metabolizableproteinavailabletobeabsorbedintheduodenum.Oncethemilkcompositionwasnotaltered,theyieldof solidscomponentsinmilk(fat,proteinandlactose)wouldhavethesameresponseofmilkyield.

Apparenttotal-tractdigestibilityofDM,CPandEEincreasedwhenglycerinwasaddedtothediets.Theseresultsarein agreementwithapreviousstudyinwhichglycerincomprisedupto150g/kgofthediet,resultinginincreaseddigestibility ofDM(Donkinetal.,2009).DespitetheincreaseofDMdigestibilitywhencrudeglycerinwasaddedtodiets,theFCMtoDMI ratiolinearlydecreased.Thelatterreducedbecausethedecreaseofmilkyieldwasproportionallyhigherthanthedecrease ofDMI,mainlywhencowswerefedthehighestlevelofcrudeglycerin(−4.0%vs.−11.1%,respectively).Furthermore,in thecurrentstudy,highcrudeglycerinlevelshadanegativeeffectonNDFdigestibility.Invitrostudiesshowedthatglycerol hasaninhibitoryeffectontheactivityandgrowthofcellulolyticbacteria(Rogeretal.,1992;AboEl-Noretal.,2010),which mayimpairtheNDFdigestibility.OtherinvivostudiesalsoshoweddecreaseofNDFdigestibilitywithdietaryinclusionof glycerinupto150g/kgofDM(Donkinetal.,2009;Shinetal.,2012).Moreover,inthisstudythereducedNDFdigestibility mightbeassociatedwithchangesinpropionateandacetateconcentrations,suggestingthattherewereshiftsintheruminal microorganism;however,thisexplanationshouldbeevaluatedinfurtherstudies.

InclusionofcrudeglycerininthecurrentstudyalteredtheruminalVFAproportions.ThesechangesinVFAconcentrations aresimilartothosereportedinpreviousstudies.Decreaseinacetateandincreaseinpropionate,butyrateandBCFAruminal

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ﬂuidconcentrationswerefoundwhendairycowswerefedglycerin(Carvalhoetal.,2011;Kassetal.,2012;Shinetal.,2012). Theincreaseofruminalpropionateconcentrationisrelatedtothefactthat30–69%ofglycerolconsumedisfermentedin therumen,andmostglycerolisfermentedintopropionate(Remondetal.,1993),suggestingthatruminaldegradationof glycerinwasfasterthangroundcornandsoybeanmealdegradation.Propionateismorehypophagiccomparedtoacetate becauseofitsabilitytostimulateoxidationintheliver(Allen,2000).Furthermore,glycerolisaprecursorofglucoseand mayenterintothegluconeogenicpathwaybeingconvertedtoglucoseintheliver,providingenergyforthecow(Goofand Horst,2003;Allenetal.,2009).Therefore,anotherexplanationforthedecreasedDMIreportedinthecurrentexperimentis relatedtothehepaticoxidationtheory,whichstatesthathepaticfueloxidationincreasesATPconcentrationsinhepatocytes whichsendinhibitorysignalsviavagusnervetonucleustractussolitariesthatinhibithypothalamicsatietycenters(Allen etal.,2009).

ThedecreasedNH3-NruminalconcentrationinthepresentstudymayberelatedtodietcompositionandDMI.TheCP contentofdietsdecreasedandDMIlinearlydecreasedaccordingtotheinclusionofcrudeglycerin;thus,lessproteinwouldbe availableintherumenenvironmenttobedegradedintoNH3whencowswerefeddietswithcrudeglycerin.Indeed,microbial proteinsynthesiswasnotimpairedwhencrudeglycerinwasincludedupto150g/kgofthediet(Donkinetal.,2009;Shin etal.,2012),showingnoeffectsofcrudeglycerinonruminalNsupply.Plasmaglucoseconcentrationincreasedaccordingto dietaryinclusionofcrudeglycerinandthisresultcanberelatedwithincreaseinruminalpropionateconcentrations,which isaglucogenicprecursor.Bloodglucoseconcentrationwasdecreased(Carvalhoetal.,2011),unchanged(Kassetal.,2012; Shinetal.,2012;Boydetal.,2013),orincreased(Donkinetal.,2009)whenglycerinwasaddedupto150g/kgincowdiets. Thedifferencesamongthesestudiescouldberelatedtothepurityofglycerin,amountofglycerinadded,andthetimeof bloodsamplingrelativetomealtime.

5. Conclusion

Inclusionofhighlevelsofcrudeglycerin(210g/kg)inthedietsofmid-lactatingcowsincreaseddrymatterdigestibility andruminalpropionate,butnegativelyaffecteddrymatterintakeandmilkyield.Inaddition,resultssuggestthatcrude glycerinhasthepotentialtopartiallyreplacestarchindietarylevelsbelowof140g/kgDMwithoutlargelyreducedairycow performance.

Conﬂictofinterest

Theauthorsdeclarethatthereisnoconﬂictofinterest.

Acknowledgements

TheauthorsacknowledgetheUniversityofSãoPaulo(USP)andtheDairyCattleResearchLaboratory,forproviding allthephysicalstructureandstaffnecessaryforthisstudy.Inaddition,theauthorsexpressappreciationtotheBrazilian fundingagencyCAPES(‘Coordenac¸ãodeAperfeic¸oamentodePessoaldeNívelSuperior’)forscholarshiptoP.G.Paiva(grant numberBEX3664/14).

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