effects of energy level and leucaena leucocephala leaf meal

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ContentslistsavailableatSciVerseScienceDirect

Animal

Feed

Science

and

Technology

journalhomepage:www.elsevier.com/locate/anifeedsci

Effects

of

energy

level

and

Leucaena

leucocephala

leaf

meal

as

a

protein

source

on

rumen

fermentation

efficiency

and

digestibility

in

swamp

buffalo

S. Kang, M.

Wanapat

∗

_{, P.}

_{Pakdee, R.}

_{Pilajun, A.}

_Cherdthong

TropicalFeedResourcesResearchandDevelopmentCenter(TROFREC),DepartmentofAnimalScience,FacultyofAgriculture,KhonKaenUniversity,Khon Kaen40002,Thailand

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14June2011

Receivedinrevisedform7March2012 Accepted12March2012

Keywords:

Leucaenaleucocephalaleafmeal Heattreatment

Energylevel Digestibility Rumenecology Swampbuffalo

a

b

s

t

r

a

c

t

FourThai–rumenfistulatedmaleswampbuffaloes(Bubalusbubalis),about3yearsoldwith 360±18kgliveweight,wererandomlyassignedaccordingtoa2×2factorialarrangement ina4×4Latinsquaredesigntoreceivefourdietarytreatments.Thetreatmentswereas follows:acassavabasedsupplement(CS)at1g/kgBWandLeucaenaleucocephalaleafmeal (LLM)at300g/d(T1);CSat2g/kgBWwithLLMat300g/d(T2);CSat1g/kgBWandheat treatedLLM(HLLM)at300g/d(T3);andCSat2g/kgBWandHLLMat300g/d.Duringthe experiment,urea–calciumhydroxidetreatedricestrawwasgivenonadlibitumbasis.The resultsrevealedanincreaseinroughageandtotaldrymatter(DM)intake(P<0.05)byCSat 2g/kgBW(T2andT4)ascomparedwithCSat1g/kgBW(T1andT3).Digestioncoefficients ofDM,organicmatter(OM),andcrudeprotein(CP)wereincreasedbyCSat2g/kgBW, whileneutraldetergentfiber(aNDF)andaciddetergentfiber(ADF)weresimilaramong treatments.However,therewasnoeffectofneitherenergylevelnorHLLMonruminal pHandtemperature(P>0.05).Concentrationofruminalammonianitrogen(NH3-N)was

decreasedbyHLLMascomparedwithLLM(P<0.05),whilebloodurea–nitrogenwasnot altered.Therewasanincrease(P<0.05)intotalvolatilefattyacid(TVFA),aceticacid(C2), propionicacid(C3),andbutyricacid(C4)concentrationsandthehighestwerefoundin CSat2g/kgBWwithHLLM(T4),whilethelowestwasinT1andT3.However,nochanges inC2–C3ratiowerefoundinthisstudy.Totalbacterialdirectcountswerefounddifferent (P<0.05),whereasfungalzoosporesandprotozoalpopulationsweresimilaramong treat-ments.Nevertheless,viablebacterialcountswerefoundaffectedbybothconcentratelevel andHLLM.ThetreatmentswithHLLMwerelowerthanthoseinLLMandCSat2g/kgBW werehigherthanthosesupplementedatCSat1g/kgBW(P<0.05).Inaddition,efficiencyof rumenmicrobialCPsynthesistendedtobehigherintreatmentwithhigherlevelofenergy andHLLM.Basedonthisstudy,itcouldbeconcludedthatLLMcouldbeusedasaprotein source,whilethecombinationofHLLMandCSat2g/kgBWcouldenhancethevoluntary feedintake,nutrientdigestibilityandrumenfermentationinswampbuffalofedontreated ricestraw(urea–calciumhydroxidetreatment).

Abbreviations: BW,bodyweight;LLM,Leucaenaleucocephalaleafmeal;HLLM,heattreatedLeucaenaleucocephalaleafmeal;DM,drymatter;OM, organicmatter;CP,crudeprotein;ADF,aciddetergentfiber;aNDF,neutraldetergentfiber;TVFA,totalvolatilefattyacid;C2,aceticacid;C3,propionic acid;C4,butyricacid;NH3-N,ammonianitrogen;CS,acassavabasedsupplement.

∗ Correspondingauthor.Tel.:+6643202368;fax:+6643202368.

E-mailaddress:metha@kku.ac.th(M.Wanapat).

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1. Introduction

Ruminantsraisedinthetropicslargelydependonseasonalfeedresourceswhicharerelativelylowinqualityintermsof lowCPbuthighincrudefiber(CF);hence,themanipulationofrumenefficiencythroughtheusesoflocalfeedresourceswould beanadvantage(Wanapat,2000).FoliagesfromlocallygrownshrubsandtreessuchasLeucaena(Leucaenaleucocephala) havebeensuccessfullyinvestigatedasproteinasupplementforruminants(Sahaetal.,2008).Leucaenaleafmeal,withits richprotein,mineralsandvitamincontent,isalsobecomingapopularingredientinpoultryfeedsinthetropics(D’Mello andTaplin,1978).Itsproteincontentisathighlevelsof292g/kgCPinleafmealand220.3g/kgCPinforage(Garciaetal., 1996).Moreover,itcontainscondensedtannincontentof10.1–10.5g/kgthatcanprotectproteinfromrumenmicrobial degradationandreducemethaneproduction.

Theruminantanimalsderivetheiraminoacidssupplyjointlyfromdietaryproteinwhichescapesrumendegradationand microbialproteinsynthesizedintherumen.Theamountofproteinandaminoacidsthatescapesrumendegradationvaries greatlyamongdifferentfeeds,dependingontheirsolubilityandtherateofpassagetothesmallintestine.Itisoftenthecase insomesituationthatanimal’srequirementsforaminoacidsarenotfullymetfromthenormalsourcesofdietaryprotein. Rapidandextensivedegradationofvaluableproteinsintherumenleadresearchtodeveloptheconceptofproteinprotection fromruminaldegradationwiththeprincipalobjectiveofenhancingthesupplyofessentialaminoacidstotheproductive animal,furtherreducewastefulammoniaproductionintherumenandreductionofnitrogenlossesasureaintheurine (Annison,1981).HeattreatmentoffeedstuffscandecreasedegradationofDMandCPbyblockingreactivesitesformicrobial proteolyticenzymes(BroderickandCraig,1980)andincreasethesupplyofdietaryproteintotheduodenum(Tagarietal., 1986).Severalstudies(FaldetandSatter,1991)onvariousproteinsourceshaveshownacorrelationbetweendecreased ruminaldegradationofproteinandincreasedmilkproduction. Heattreatmenthastheadvantageofbeingsafe,rather inexpensiveandeasilyavailable(notrequestingcomplexequipment).However,theknowledgeontheoptimalconditionof heattreatmentsofL.leucocephalaleafmealisscarce.WhereasdataontheeffectoftheL.leucocephalaleafmealheattreating, itwasyetbeenfoundnodataoftheeffectonfeedintakeandrumenecologyinswampbuffalo.Therefore,theobjectivesof thisstudyweretoinvestigatetheeffectofenergylevelwithheatedandunheatedtreatmentonL.leucocephalaleafmealon feedintake,nutrientdigestibility,rumenfermentationandmicrobialpopulationinswampbuffalo.

2. Materialsandmethods

2.1. Animals,dietsandexperimentaldesign

L.leucocephala(LL)washarvestedfromthetreewithaverageplantingageof4–5yearsandsundried.OnlytheleafofLL wassundriedandground,usedfortheexperiment.Afterthat,theleafmealwaskeptandhalfoftheleafmealwasheated intheovenattemperature100◦_C_for₆₀_min._The_{urea–calcium}_hydroxide_treated_rice_straw_was_prepared_by_adding₂_kg ureaand2kgCa(OH)2(hydratedlime)in100landpouredoverto100kgairdry(910g/kgDM)straw.Therelevantvolume

ofureaandcalciumhydroxidesolutionwassprayedontoastackof5wholestrawbales(approximately20kg)andthen coveredthestackwithaplasticsheetforaminimumof10daysbeforefeedingdirectlytotheanimals(Wanapatetal.,2009). FourThai–rumenfistulatedmaleswampbuffaloes(Bubalusbubalis),about3yearsoldwith360±18kgliveweight,were randomlyassignedaccordingtoa2×2factorialarrangementina4×4Latinsquaredesigntoreceivedietarytreatments.The treatmentswereasfollows:acassavabasedsupplement(CS)at1g/kgBWandL.leucocephalaleafmeal(LLM)at300g/d(T1); CSat2g/kgBWwithLLMat300g/d(T2);CSat1g/kgBWandheattreatedLLM(HLLM)at300g/d(T3);andCSat2g/kgBW andHLLMat300g/d.Eachofthefourperiodslastedfor21days,withthefirst14daysasstrawintakemeasurement,while thelast7daysforsamplecollection.Ingredientcompositionsofconcentratemixture,LLMandroughage(urea–calcium hydroxidetreatedricestraw)areshowninTable1.Allanimalswereindividuallypennedandwaterandmineralblockwere availableatalltimes.Allanimalswerefedonurea–calciumhydroxidetreatedricestrawadlibitum.

2.2. Datacollectionandsamplingprocedures

FeedofferedandrefusalswererecordeddailythroughouttheexperimentalperiodforDMintakecalculationandfeed sampleswererandomlycollectedtwiceaweekforDManalysis.Samplesofconcentratemixture,LLMandtreatedricestraw includingrefusalswerecollecteddailyduringthecollectionperiod.Samplesofricestrawwerecompositedbyperiodaswell assampleofconcentratemixture.LLMandfeedrefusalswerecompositedbyperiodandbyanimalandstoredat−20◦_C_for laterchemicalanalyses.

RumenpH,temperatureandfermentationcharacteristicsweremeasuredatthelastdayofeachperiodpostmorning feeding.Approximately200mlofrumenfluidweretakenfromthemiddlepartoftherumenbyusinga60mlhandsyringeat eachtime.RumenfluidwasmeasuredforpHandtemperatureandthefluidsampleswerethenstrainedthroughfourlayers ofcheeseclothanddividedintothreeparts.Thefirst45mlofrumenfluidsamplewascollectedandkeptinaplasticbottle towhich5mlof1MH2SO4wasaddedtostopfermentationprocessofmicrobeactivityandthencentrifugedat3000×gfor

10min.About20–30mlofsupernatantwascollectedandanalyzedforNH3-NandVFA.Thesecondportionof1mlrumen

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Table1

Feedingredientsandnutritivevaluesusedintheexperiment.

Item Cona _LLMb _HLLMc _{Urea–calcium}_hydroxide_treated_rice_straw

Ingredients,g/kgdrymatter

Cassavachip 750

Ricebran 70

Coconutmeal 70

Palmkernelmeal 50

Molasses 15

Urea 15

Mineralmixture 10

Salt 10

Sulfur 10

Chemicalcomposition

Drymatter,g/kg 923 862 946 542

Organicmatter 907 916 917 862

Crudeprotein 108 273 271 58

Neutraldetergentfiber 182 354 364 765

Aciddetergentfiber 125 163 172 562

a_Concentrate.

b_Leucaena_leucocephala_leaf_meal.

c_Heat_treated_Leucaena_leucocephala_leaf_meal.

formalin)wasaddedandstoredat4◦_C_for_measuring_microbial_population_by_using_total_direction_counts._The_third_portion wasforthetotalviablebacteriacount(cellulolytic,proteolytic,andamylolytic)andtotalviablebacteria.

Abloodsample(about10ml)wasdrawnfromthejugularveinatthesametimeasrumenfluid.Bloodsampleswere immediatelyplacedontheiceandtransportedtothelaboratoryforseparatingplasmafromthewholeblood.Sampleswere refrigeratedfor1handthencentrifugedat3500×gfor20min.Theplasmawereremoved,storedat−20◦_C_and_analyzed_for bloodureanitrogen(BUN)composition.UrinesampleswereanalyzedfortotalNandanalyzedforallantoinconcentration.

2.3. Analyticalprocedure

Thesamplesweredividedintotwoparts,firstpartforDManalyses,whilethesecondpartkeptandpooledattheendof eachperiodforanalysesofAsh,CP,aNDFandADF.Feeds,refusalsandfecalsamplesweredriedat60◦_C_and_ground₍₁_mm screenusingtheCyclotechMill,Tecator,Sweden)andanalyzedusingstandardmethodsofAOAC(1995)forDM(ID967.03) andash(ID942.05).ADFwasdeterminedaccordingtoanAOACmethod(1995;ID973.18)andwasexpressedinclusiveof residualash.aNDFinsampleswasestimatedaccordingtoVanSoestetal.(1991)withadditionof␣-amylasebutwithout sodiumsulphiteandresultsareexpressedwithresidualash.Totalnitrogen(N)wasdeterminedaccordingtoAOAC(1995; ID984.13).

RumenfluidwasimmediatelymeasuredforpHandtemperatureusingaportablepHtemperaturemeter(HANNA Instru-mentsHI8424microcomputer,Singapore)andNH3-NbyKjeltechAuto1030Analyzer(AOAC,1995;ID973.18).VFAwere

analyzedusingHighPressureLiquidChromatography(HPLC,InstrumentsbyWaterandNovapakmodel600E;watermode l484UVdetector;columnnovapakC18;columnsize3.9mm×300mm;mobilephase10mMH2PO4[pH2.5])accordingto Samueletal.(1997).Rumenfluidwasusedfordirectcountsofbacterial,protozoaandfungalzoosporesusingmethods ofGalyean(1989)byhaemocytometer(Boeco,Singapore).Groupsofbacteria(i.e.,cellulolytic,proteolytic,amylolyticand totalviablecountsbacteria)weremeasuredusingtheHungate(1969)roll-tubetechnique.BUNwasmeasuredaccordingto

Crocker(1967).

Urinesamples wereanalyzed for total N(AOAC,1995; ID984.13)and allantoin inurine was determinedby HPLC asdescribedbyChenetal.(1993).Theamountofmicrobialpurinesderivativeabsorptionwascalculatedfrompurine derivative(PD)excretionbasedontherelationshipderivedbytheequationofLiangetal.(1994):Y=0.12X+(0.20BW0.75_).

Thesupply ofmicrobial N(MN)wasestimatedbyurinary excretionofPDaccordingtoChenand Gomes(1995): MN (g/d)=70X/(0.116×0.83×1000)=0.727X;whereXandYare,respectively,absorptionandexcretionofPDinmmol/d. Effi-ciencyofmicrobialNsynthesis(EMNS)wascalculatedusingthefollowingformula:EMNS=microbialN(g/d)/DOMR;where DOMR=digestibleOMapparentlyfermentedintherumen(assumingthatrumendigestionwas650g/kgOMofdigestionin totaltract,DOMR=DOMI×0.65;DOMI=digestibleorganicmatterintake).

2.4. Statisticalanalysis

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Table2

EffectofenergylevelandLLMonvoluntaryfeedintakeandnutrientdigestibility.

Item LLMb _HLLMc _SEM _Interaction

1a ₂a ₁a ₂a _LLMb _Cona _LLMb_×_Cona

Drymatterintake Roughageintake

kg/day 6.1 6.5 5.9 6.4 0.06 ns ** _ns

g/kgBW0.75 _69.9 _73.5 _67.4 _72.8 _0.76 _ns ** _ns

Concentrateintake

kg/day 0.4 0.7 0.4 0.7 0.01 ns *** _ns

g/kgBW0.75 _4.1 _8.2 _4.1 _8.2 _0.01 _ns *** _ns

LLMintake

kg/day 0.26 0.26 0.28 0.28 0.004 ** _ns _ns

g/kgBW0.75 _3.0 _3.0 _3.2 _3.2 _0.06 ** _ns _ns

Totalintake

kg/day 6.7 7.4 6.6 7.3 0.06 ns * _ns

g/kgBW0.75 _77.0 _84.7 _74.7 _84.2 _0.78 _ns * _ns

Apparentdigestibility

Drymatter 0.61 0.70 0.62 0.66 0.02 ns * _ns

Organicmatter 0.64 0.73 0.66 0.69 0.02 ns * _ns

Crudeprotein 0.51 0.60 0.53 0.60 0.006 * * _ns

Neutraldetergentfiber 0.58 0.66 0.60 0.63 0.02 ns ns ns

Aciddetergentfiber 0.54 0.61 0.51 0.55 0.04 ns ns ns

a_Concentrate_(g/kg_BW).

b _Leucaena_leucocephala_leaf_meal.

c _Heat_treated_Leucaena_leucocephala_leaf_meal.

* _P_<_0.05.

** _P_<_0.01.

***_P_<_0.001.

3. Results

3.1. Chemicalcompositionofdiet

ExperimentalfeedandtheirchemicalcompositionsareshowninTable1.Themixtureofconcentrate,consistingof availablelocalfeedresourcessuchasenergysource(cassavachips),proteinsouces(ricebran,coconutmeal,andpalm kernelmeal)andnon-proteinnitrogen(urea),hadahigherqualityintermsofCPandlowinfiber(108,and182g/kgofDM, respectively).ThemeanlevelofCPofLLMandHLLMusedintheexperimentwere273and271g/kgofDM,respectively. Moreover,fibrousfractions,aNDFandADFwerenotdiferentbetweenLLMandHLLM.Ricestrawqualitywasimprovedin CPbythetreatmentwithurea–calciumhydroxide.

3.2. Feedintakeandnutrientdigestibility

Table2presentsdataofdailyfeedintakesandnutrientdigestibility.Feedintakeswereenhanced(P<0.05)byCSat2g/kg BW.Moreover,supplementationatCSat2g/kgBWincreasedricestrawintake,thusresultinginanincreaseintotalintake. ApparentdigestibilityofDM,OMandCPwerealsofoundincreased(P<0.05)inbuffaloesconsumeddietwithCSat2g/kgBW andLLM,thehighestwasinT2(0.70,0.73,and0.60kg/kg,respectively).TheCPdigestibilitywasincreasedbybothfactors CSat2g/kgBWandHLLM.Incontrast,noeffectondigestibilityofaNDFandADFbyenergylevelandLLMsupplementation wasfound(P>0.05).

3.3. Rumenfermentationandbloodmetabolites

Ruminaltemperature,pH,andBUNweresimilaramongtreatmentsandthevalueswerequitestableat39.1-39.3◦_C, pH(6.5–6.7),andBUNat13.6–16.6mg/dl,respectively(Table3).However,BUNinthetreatmentswithLLM(T1=11.9and T2=11.4mg/dl)tendedtobehigherthanthosewithHLLM(T3=10.4andT4=10.0mg/dl).TreatmentswithHLLMwerefound lowerinconcentrationofruminalNH3-NthanwithLLM.BothtreatmentswithHLLMhavelowerconcentrationofNH3-N

(T3=13.6andT4=14.5mg/dl)thaninthetreatmentswithLLM(T1=16.0andT2=16.6mg/dl).Inaddition,theconcentrate levelhasenhancedrumenNH3-Nconcentration.TheavailablerumenNH3-Nwouldbeusedinmicrobialproteinsynthesis

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Table3

EffectofenergylevelandLLMonruminalfermentationandbloodureanitrogen.

RuminalpH 6.7 6.5 6.6 6.5 0.07 ns ns ns

Temperature,◦_C _39.2 _39.2 _39.3 _39.1 _0.11 _ns _ns _ns

NH3-N,mg/dl 16.0 16.6 13.6 14.5 0.45 *** ns ns

Bloodureanitrogen,mg/dl 11.9 11.4 10.4 10.0 1.71 ns ns ns

TotalVFA,mmol/l 82.9 95.6 82.2 99.5 1.22 ns ** _ns

VFA,mol/100mol

Aceticacid(C2) 59.3 67.4 58.7 68.8 0.90 ns ** _ns

Propionicacid(C3) 16.4 18.2 15.3 20.1 1.16 ns * _ns

Butyricacid(C4) 7.2 10.0 8.2 10.6 0.67 ns ** _ns

C2:C3 3.6 3.7 3.8 3.4 0.62 ns ns ns

*_P_<_0.05.

**_P_<_0.01. ***_P_<_0.001.

Table4

EffectofenergylevelandLLMonmicrobialpopulationintherumenofswampbuffaloes.

1a ₂a ₁a ₂a _LLMb _Cona _LLMb

×Cona

Ruminalmicrobes×cell/ml

Bacteria,×109 _3.3 _4.4 _2.9 _3.2 _0.10 ** ** **

Protozoa,×105 _8.1 _7.9 _8.3 _7.9 _0.38 _ns _ns _ns

Fungi,×105 _2.6 _3.9 _2.8 _2.6 _0.35 _ns _ns _ns

Viablebacteria,CFU/ml

Amylolytic,×108 _4.6 _5.1 _4.3 _4.4 _0.75 _ns _ns _ns

Proteolytic,×108 _2.8 _3.1 _2.3 _2.7 _0.15 * * _ns

Cellulolytic,×108 _10.0 _10.5 _8.6 _9.5 _0.49 * * _ns

Total,×109 _4.9 _5.6 _4.0 _4.8 _0.43 * * _ns

*_P_<_0.05.

**_P_<_0.01.

3.4. Rumenmicroorganismpopulation

AsshowninTable4,totalbacteriacountswerefounddifferent(P<0.05).TreatmentswithCSat2g/kgBWandLLMhad thehighestat4.4×109_cell/ml_and_the_lowest_was_in_treatment_with_CS_at₁_g/kg_BW_and_HLLM,_2.9_×₁₀9_cell/ml._Total

countsofbacteriawereaffectedbybothofconcentratelevelandLLM,whileprotozoalandfungalzoosporepopulation werenotdifferent.Totalviablebacteriacounts,cellulolyticbacteria,andproteolyticbacteriacountswerefounddifferent (P<0.05),whileamylolyticbacteriacountswasnotdifferentamongtreatments.ThetreatmentwithCSat2g/kgBWandLLM wasthehighestintotalviablebacteriacounts,cellulolyticbacteria,andproteolyticbacteriacounts(5.6×109_,₁₀

×108_,_and

3.1×108_CFU/ml,_{respectively).}_The_treatments_with_LLM_were_higher_than_those_with_HLLM.

3.5. Nitrogenutilizationandefficiencyofmicrobialproteinsynthesis

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Table5

EffectofenergylevelandLLMonNutilization,purinederivations(PD)andmicrobialcrudeproteinsupply(MCP).

Nutilization,g/day

Nintake 74.4 83.9 73.8 83.8 0.77 ns *** _ns

Nexcretion

Feces 49.4 44.1 45.0 43.9 0.89 * * *

Urine 8.4 8.7 10.6 9.0 1.12 ns ns ns

Total 57.8 52.8 55.6 52.9 1.41 ns ns ns

Nbalance

Absorption 25.0 39.8 28.0 40.0 2.33 ns *** _ns

Retention 16.6 31.1 17.4 30.9 2.56 ns *** _ns

PD,mmol/d

Allantoinexcretion 27.8 31.6 29.0 33.2 3.14 ns ns ns

Allantoinabsorption 89.7 116.2 94.6 120.4 4.94 ns ** _ns

Microbialnitrogensupply,gN/d 65.2 84.5 68.8 87.6 3.59 ns ** _ns

Microbialcrudeprotein,g/d 407.7 528.2 429.9 547.3 7.82 * *** _0.08

EMNSd_,_gN/kg_OMDRe _28.0 _28.6 _30.5 _32.0 _2.16 _ns _ns _ns

b _Leucaena_leucocephala_leaf_meal.

c _Heat_treated_Leucaena_leucocephala_leaf_meal.

d _Efficiency_of_microbial_nitrogen_synthesis.

e_Digestible_OM_apparently_fermented_in_the_rumen.

* _P_<_0.05. ** _P_<_0.01.

***_P_<_0.001.

4. Discussion

4.1. Chemicalcompositionofdiet

Concentrateingredientswerebasedonlocalresources,consistingofcassavachip,ricebran,coconutmealandpalmkernel meal,whichhadahigherqualityintermofCPandlowinfiber.Thisconcentratewaswellconsumedbyanimalsduringthe experimentalperiods.Thenutritivevalueofricestrawhasbeenimprovedbythetreatment.CPcontentofurea–calcium hydroxidetreatedricestrawwas58g/kg.Moreover,ureaandcalciumhydroxidecoulddecreasetheproportionofaNDFand ADFcontentinricestrawto765g/kgand562g/kg,respectively.ThisvaluewassimilartothosevaluesreportedbyWanapat etal.(2009)whousedurea–calciumhydroxidetreatedricestraw.Underthisstudy,therewerenodifferencesbetween chemicalcompositionofHLLMandLLM.ItwasalsoreportedbyFathiNasiretal.(2008)andMahalaandGomaa(2007),who usedheatedwholesoybeanandsesamecake,thattherewasnoeffectonchemicalcompositionbyheating.Itwassimilar tothevalueofYousufetal.(2007)whoreportedthevalues;302,302,173g/kgand247,320,211g/kg,CP,aNDFandADF, respectively.

4.2. Feedintakeandnutrientdigestibility

TheresultsrevealedanincreaseinroughageandtotalDMintake(P<0.05)byCSat2g/kgBW(T2andT4)ascompared withCSat1g/kgBW(T1andT3),butnotbyLLM.RoughageandtotalDMintakesrangedfrom5.9–6.5and6.6–7.4kg/d, respectively,andthehighestwasinCSat2g/kgBWtreatment.However,itwassuggestedthatsupplementationofsmall amountby-passproteintolowqualitydietoftenresultsinahigherintakethanwithout.AsshownbySinghetal.(2009),

Thangetal.(2010),andSahooandWalli(2008),whoreportedthatwhenincreasedlevelofenergyintake,therewasan increaseinDMintake.Moreover,underthisstudy,itwasshownthatlowintakewasfoundintheheatedtreatment.This couldbeexplainedbytheeffectofhighrumenundegradableprotein.AccordingtoSwartzetal.(1991)whofoundthesame effectthattherewasaslightdecreaseinDMintakewhenmoreundegradableproteinwasconsumed.Itwasalsofoundin heatedsoybeanmealwithaslightdecreaseofDMintake(AhrarandSchingoethe,1979).

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ofapparentdigestibilitycoefficientsofOM,grossenergy(GE),aNDFandADFwereobservedinthecattlefedthehighenergy diet(32MJ/day)ascomparedtothelowlevel(25MJ/day).

4.3. Rumenfermentationandbloodmetabolites

TherewerenoeffectofenergylevelandLLMonruminalpHandtemperature(P>0.05).However,ruminalpHand tempera-turewereinnormalrangeat6.5–6.7and39.1–39.3◦_C,_{respectively.}_Ahrar_and_Schingoethe₍₁₉₇₉₎_,_who_used_heated_soybean meal,foundnoeffectonpHbyheattreatment.Moreover,Robinsonetal.(1991)andDuttaetal.(2009)foundthesame resultswhensupplementedwithdifferentenergyratioandrumenundegradableprotein.However,NH3-Nwasaffectedby

energylevelandLLM,butnotforBUN.RuminalNH3-Nconcentrationisacrudepredictorofefficiencyofdietarynitrogen

conversionintomicrobialnitrogen(Firkinsetal.,2007;BroderickandMuck,2009).Inthisstudy,NH3-NinHLLMwaslower

thanLLMtreatment;16.0–16.6and13.6–14.5mg/dl,respectively,andinhighconcentratelevelgroupswerehigherthanin lowerlevel.Thiscouldbeduetoheattreatmentoffeedstuffsinwhichcandecreasecrudeproteindegradationbyblocking reactivesitesformicrobialproteolyticenzymes(BroderickandCraig,1980)and/orincreasedthesupplyofdietaryprotein totheduodenum(Tagarietal.,1986).Robinsonetal.(1991)reportedthatwhenincreasedintakeofrumenundegradable proteinresultedinlowNH3-Nconcentration,similarlytotheresultreportedbyDuttaetal.(2009).Althoughthereisahighly

differenceonNH3-Nconcentrationbyheating,however,noeffectwasfoundonBUNconcentration.However,Ahrarand Schingoethe(1979)foundthatBUNwasaffectedbyheatingsoybeanmeal(HSBM).ThiswasconsistentlywithHudsonetal. (1970),whichindicatedthatconcentrationsofplasmaureafromruminantanimalsfedHSBMremainedbelowthosefedthe unheatedsoybeanmeal(SBM).ThissuggestedthattheproteinintheHSBMwasdegradedataslowerratebytheruminal microorganismsthanproteinfromunheatedmealorthatammonialiberatedfromHSBMwasutilizedmoreefficientlyfor microbialproteinsynthesis.Itmayduetodigestibleproteininthedietofruminantsiseitherdegradedintherumenor escapestotheabomasumandsmallintestinewhereitisdegradedtoaminoacidsandsmallpeptidesthenabsorbedintothe portalbloodsystem.ThatmaybethereasoninthisstudyresultedinlowerBUNintreatmentswithHLLMwhichwaslower degradethanLLMassimilartotheresultofMabjeeshetal.(1998)whoconductedwithheatwholecottonseed(HWCS)in dairycows.

Thereweredifference(P<0.05)inTotalVFA(TVFA),C2,C3andC4whenbuffaloeswerefedwithdifferentlevelofenergy andLLM.Thisincreasewasstronglyrelatedwiththenumberofruminalcellulolyticbacterialspecies.Fibrobactersuccinogenes mainlyproducesprimarilysuccinate,themajorprecursorofpropionateintherumenwhileRuminococcusalbusismainly aspecieswhichproducesacetate.Therefore,withanincreaseinnumberofF.succinogenes,R.albus,bothpropionateand acetateconcentrationwereincreased.Incontrasttothepresentresult,accordingtoDuttaetal.(2009),TVFAconcentration intherumenliquorwasstatisticallysimilaramongtreatmentsgroupswithdifferentratioofenergyandprotein.Moreover, theTVFAconcentrationinruminalfluidwasnotinfluencedbythelevelofenergyinthedietofsheep(Merchenetal.,1986; Carroetal.,2000).Moreover,rumenVFAconcentrationsweresimilarforcowsfedSBMandHSBMrationsreportedbyAhrar andSchingoethe(1979).However,thetreatmentswithHSBMtendedtobehigherthanthosewithSBM.Thiswasexpected sincevaryingaproteinsourceintherationshouldnotaffectrumenVFAgreatlyunlesssuchachangecausesagreatdeficiency innitrogenavailabletotherumenmicroorganisms.AccordingtoMabjeeshetal.(1998),theproportionofpropionatewas thelowestforHWCSdietcomparedtounheatedtreatmentsandtheacetate/propionateratioswerehigheratallsampling timesforthisdiet.ThisisincontrastwiththepresentstudywhichshownthemeanvaluesofTVFA,C2,C3,andC4werethe highestinCSat2g/kgBWwithHLLM(99.5,68.8,20.1,and10.6mmol/l,respectively),whilethelowestwasinT1andT3.

4.4. Rumenmicroorganismpopulation

TotalbacterialdirectcountswerefounddifferentbyconcentratelevelandLLM,whereasfungalzoosporesandprotozoal populationsweresimilaramongtreatments.ThetreatmentwithCSat2g/kgBWwithLLMwasthehighest,whiletheothers threeweresimilar.AccordingtoVerbic(2002),energysupplyisusuallythefirstlimitingfactorformicrobialgrowthinthe rumen.Morethanthat,thiscouldbeexplainedthatNH3-Nisanessentialsourceofnitrogenformicrobialproteinsynthesis.

TherangeofNH3-Nlevelforoptimalrumenecologyhasbeenreportedtobe15.0–30.0mg/dl(Leng,1999).Treatmentswith

CSat2g/kgBWandLLMhadthehighestrangedat4.4×109_cell/ml_and_the_lowest_was_in_treatment_with_CS_at₁_g/kg_BW

andHLLM,2.9×109_cell/ml._This_could_be_explained_by_decreasing_protozoal_population._Van_Soest₍₁₉₉₄₎_suggested_that

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fromDacronbagsincubatedintherumenofsteers(Yangetal.,1993).Makkaretal.(1995)indicatedthattheefficiencyof microbialproteinsynthesiswasgreaterinforagescontainingsaponinandtannins,whichreduceruminalNdegradability.In thisstudy,thetreatmentwithCSat2g/kgBWandLLMwasthehighestintotalviablebacteriacounts,cellulolyticbacteria, andproteolyticbacteriacountsandthelowestwasintreatmentwithCSat1g/kgBWandHLLM(T3).

4.5. Nutilizationandefficiencyofmicrobialproteinsynthesis

EffectofenergylevelandLLMinswampbuffaloesonNutilizationisshowndifferentamongtreatmentsintermsofN intake,Nfeces,Nabsorptionandretention,whilenodifferencewerefoundonNurineandtotalNexcretion(Table5).TotalN intakeandNbalancewerefoundhighestinCSat2g/kgBWsupplementation.Thisindicateshigherproteinavailableforuse bythebuffaloes.However,Nexcretionthroughfeceswashigherinhighenergy–highproteinfedgroup,buturinaryNwas nodifferencebetweenenergyandproteinlevel.Consistencytothepresentresult,AhrarandSchingoethe(1979)reported thattherewerenodifferencesinNbalancehowever;NwasutilizedslightlymoreefficientlybycowsfedHSBM.Nlossesin fecesandurinewereslightlylesswithHSBM.ThisagreedwithGlimpetal.(1967)andLittleetal.(1963)whichindicated thatheattreatmentdecreasedurinaryexcretioninruminantanimals.Resultsofanotherstudy(SherrodandTillman,1962) showedthatasheatingincreasedandsolubilitydecreased,thepercentageofNintakeretainedincreased.

Urinaryexcretionofpurinederivativeisconsideredtobeanindicatorofmicrobialproductioninrumen.Protein degra-dationintherumenisoneofthemainreasonsfortheinefficientutilizationofproteininruminants.Itseemsthatproteins whichhavelowerratesofruminaldegradationtendtoimprovetheefficiencyofmicrobialproteinsynthesis,probably becauseofthebettercaptureofreleasedNbyrumenmicrobes(Russell,2001).Similartothepresentstudy,itwasfound thatthetreatmentwithHLLMhaveahighermicrobialcrudeprotein(MCP)thanLLM.Itmaybeduetoheatedtreatment couldprotectrumendegradationintherumen.Moreimportantthanthat,inthisresult,allantoinabsorptionandMCPwere foundhigherintreatmentwithhigherinenergy.ThiswasreportedbyRussell(2001)thatenergysupplyisusuallythefirst limitingfactorformicrobialgrowthintherumen.Themaximumpotentialofrumenmicrobestoproducemicrobialprotein canbeexploredonlybytheprovisionofhighqualityforage.Inaddition,matchingthereleaseofammonia-Nfromdietary proteinwiththereleaseofusableenergymayimproveNutilization.Inordertoincreasemicrobialyield,itseemsthatthe manipulationofenergyandNfermentationintherumenshouldfirstbeaimedatobtainingthemostevenruminalenergy supplypatternpossiblewithinaparticulardietaryregimen.

5. Conclusions

Basedonthisstudy,itcouldbeconcludedthatLLMcouldbeusedasaproteinsource,whilethecombinationofHLLM andconcentratelevelat2g/kgBWenhancedvoluntaryfeedintake,nutrientdigestibility,rumenfermentationand microor-ganismsinswampbuffalosupplementationwithurea–calciumhydroxidetreatedricestraw.

Acknowledgments

TheauthorswouldliketoexpressourmostsinceregratitudetotheTropicalFeedResourcesResearchandDevelopment Center(TROFREC),DepartmentofAnimalScience,FacultyofAgriculture,KhonKaenUniversity,andtheNorwegian Pro-grammefordevelopment,ResearchandEducation(NUFUProject)fortheirfinancialsupportforthefirstauthor’sstudyat M.Sc.degreelevelandfortheuseoftheresearchfacilities,respectively.

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