Effect of invasive slug populations (Arion vulgaris) on grass silage I. Fermentation quality, in-silo losses and aerobic stability

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ContentslistsavailableatScienceDirect

Animal

Feed

Science

and

Technology

journalhomepage:www.elsevier.com/locate/anifeedsci

Effect

of

invasive

slug

populations

(Arion

vulgaris)

on

grass

silage

I.

Fermentation

quality,

in-silo

losses

and

aerobic

stability

Åshild

Taksdal

Randby

a,∗

,

Kristine

Gismervik

b

,

Arild

Andersen

c

,

Ida

Skaar

d

aNorwegianUniversityofLifeSciences,POB5003,N-1432Ås,Norway bNorwegianVeterinaryInstitute,Pb5695Sluppen,7485Trondheim,Norway

cNorwegianInstituteforAgriculturalandEnvironmentalResearch,Fr.A.Dahlsvei20,1430Ås,Norway dNorwegianVeterinaryInstitute,Pb750Sentrum,0106Oslo,Norway

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received30June2014 Receivedinrevisedform 25September2014 Accepted27September2014 Keywords: Sluginvasion Grasssilage Arionvulgaris Silageadditives Arionlusitanicus Silagequality

a

b

s

t

r

a

c

t

ThisstudyaimedtoexplorehowinvasiveslugpopulationsofArionvulgarisinfluence fer-mentationquality,in-silolossesandaerobicstabilityofgrasssilage,andtheefficiencyof silageadditivesandwiltingtoimprovethequalityofsilagesfromslugcontaminatedcrops. Theeffectoffourlevels,includingcontrol,ofaslugcontaminatedgrasscrop,wasevaluated inlaboratoryscale.Thecropusedwaswiltedtotwodrymatter(DM)levels:low(253g DM/kg)andhigh(372gDM/kg).AdultslugswereappliedtothelowDMcrop correspond-ingto5(lowlevel),10(medium)and20(highlevel)7-gsizedA.vulgarisperm2inan

assumedharvestedregrowthyieldof2.5tonDMperha.ForthehighDMcrop,theapplied sluglevelscorrespondedto6(lowlevel),12(medium)and24(highlevel)slugsperm2.

AtlowDMlevel,theeffectoffouradditivetreatments,control(C),inoculationwith Lactobacillusplantarum(LP),aformic,propionicandbenzoicacidmixture(ACID)anda chemicaladditivecontainingbenzoicacid,NaNO2,hexamethylenetetramineandpropionic

acid(CHEM)weretested.Increasingslugcontaminationgaveincreasingquality reduc-tionsbothinsilagescontaining253and372gDM/kg.Comparedwithuntreatedsilage, LP-treatmentdidnotimprovesilagefermentationqualityofcontaminatedcrops.Treatment withACIDandCHEM,however,considerablyimprovedthequalityofheavilycontaminated silages.Themuchhighercrudeproteinconcentrationinslugscomparedtograsscropmade slugsamore“difficult-to-ensile”material.Wiltingoftheharvestedcropto372gDM/kgwas notsufficienttocontrolsilagefermentationofslugcontaminatedcrop.Withcontamination levelsfrom138to553gfreshslugweight/kgcropDM,efficientsilageadditiveswereable toensureacceptablefermentationqualityofgrasssilages.

©2014TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/3.0/).

Abbreviations:ACID,formic,propionicandbenzoicacidmixture;AA,aceticacid;BA,butyricacid;BC,bufferingcapacity;C,control;CHEM,chemical additivewithbenzoicacid,NaNO2hexamethylenetetramineandpropionicacid;CP,crudeprotein;DM,drymatter;FA,formicacid;FC,Fermentability

coefficient;LA,lacticacid;LAB,lacticacidbacteria;LP,Lactobacillusplantaruminoculation;N,nitrogen;NADPH,Nicotinamideadeninedinucleotide phosphate;PA,propionicacid;WSC,watersolublecarbohydrates.

∗ Correspondingauthor.Tel.:+4767232689;fax:+4764965101.

E-mail addresses: ashild.randby@nmbu.no (Å.T. Randby), stine.gismervik@vetinst.no (K. Gismervik), arild.andersen@nmbu.no (A. Andersen),

ida.skaar@vetinst.no(I.Skaar).

http://dx.doi.org/10.1016/j.anifeedsci.2014.09.026

0377-8401/©2014TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons. org/licenses/by-nc-nd/3.0/).

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1. Introduction

ExtensiveinvasionsbytheslugsArionvulgaris(innameconfusionsalsoreferredtoasArionlusitanicus)ontofarmland

haverecentlybeenreportedinnorthernEuropeduringwetsummers(SpörndlyandHaaga,2010).Theslugsoriginatefrom

arelativelydryclimateinSouthFrance,whereitoperatesinlowdensities,buthavebeenwidespreadinEuropethelast

decades.Adultslugsweigharound10ginAugust,withvariationfrom3to27gwithinandbetweenlocations(Brinerand

Frank,1998a).Densepopulationsofmorethan50slugspersquaremeterhavebeenreportedfromwildflowerstripsand

meadows(BrinerandFrank,1998b).Asaconsequence,highamountsofslugsmightcontaminategrasssilageandcausea

potentialthreattoanimalfeedquality.

SilageisthemainformofpreservedgrassandotherforagesofferedcattleandsheepinEuropeandNorthAmerica.

AnaerobicconditionsandrapidloweringofpHbylacticacidfermentationareessentialtoobtainhighqualitysilage.Forage

composition(drymatter(DM),solublesugarsandnitrate,bufferingcapacityandlacticacidbacteria(LAB)content)together

withthesilage-makingtechniquedeterminesthesuccessoftheconservationprocessandtherebythefeedquality(Driehuis

andOudeElferink,2000).Inordertocontrolthesilagefermentationprocess,alargenumberofadditives(generallyassigned

eitherfermentationstimulantsorinhibitors)arecommerciallyavailable.Still,theuseofsilageadditivesvariesalotamong

countries.InNorway,forinstance,approximately3/4ofsilagesareadditivetreated(WilkinsonandToivonen,2003),tower

andbunkersilagestoagreaterextentthanroundbalesilages.Theensilingprocessisverycomplexandmaybehardto

controlespeciallywithlowDMgrassharvestedundersuboptimalconditionslikerainyweather.Undersuchconditionsare

alsoslugsbeingactive,andSwedishfarmersexperiencedunspecificsymptomsincattleofferedslugcontaminatedsilage

harvestedduringthewetsummerin2007.Addressingthisproblem,SpörndlyandHaaga(2010)studiedslugcontaminated

silage,untreatedortreatedwithanacidicadditive.Theyusedjuvenileslugsof0.64gintheprimarygrowth,inrelevant

contaminationlevelsforearlysummer,andconcludedthatsilagequalitywasnotseverelyaffected,possiblyduetohigh

levelsofLABdetectedintheslugs.Slugsincreasetheirweightconsiderablyfromearlytolatesummer.Itwastherefore

necessarytoinvestigateslugcontaminatedsilagealsofromtheregrowthharvest.Thisstudyaimedtoexplorehowinvasive

slugpopulationsofA.vulgarisinfluencefermentationquality,in-silolossesandaerobicstabilityofgrasssilageandthe

efficiencyofsilageadditivesandwiltingtocontrolsilagefermentationofslugcontaminatedcrops.Microbiologicalaspects

ofthesamesilagesarereportedanddiscussedelsewhere(Gismerviketal.,2014).

2. Materialsandmethods

2.1. Experimentaldesign

Theeffectonsilagequalityoffourlevels,includingcontrol,ofaslugcontaminatedgrasscrop,wasevaluatedinalaboratory

scaleensilingstudy.ThecropusedwaswiltedtotwodifferentDMlevels:low(253gDM/kg)andhigh(372gDM/kg).At

lowDMlevel,theeffectoffouradditivetreatments,includingcontrol,wastestedina4×4factorialarrangementwithslug

contaminationusingthreereplicates(altogether48silages).AthighDMlevel,thefourlevelsofslugcontaminationwere

testedwiththreereplicateswithoutanyadditivetreatment(12silages).

2.2. Cropforensiling

TheharvestedcropwasfromthefirstregrowthofafifthyearorganicmeadowattheNorwegianUniversityofLife

Sciences,Ås,Norway.Theswardwasinitiallysownwith15kg/haoftimothy(Phleumpratense)and7kg/haofredclover

(Trifoliumpratense),butcontainedadditionallyavarietyofherbs,legumesandweedswhenharvested.In2012thesward

wasfertilizedwith30tonperhaofdilutedcattlemanureon2May,and,followingtheprimarygrowthharvest,with15ton

perhaon15June.Itstartedrainingon16June,andtotalprecipitationuntiltheregrowthharvestwas245mm.Thecrop

wasmown13August2012at12h,andwiltedinthefieldfor2h.Cropwasfilledintonettingbagsthatallowedairtopass

throughforfurtherwilting.Thebagswerestoredindoorsat12–15◦Covernight,becauseshowerswereexpected.LowDM

cropwasensiledon14August.CropforhighDMsilagewasfurtherwiltedoutdoorsinthesun,spreadonatrailer.Thetrailer

wasmovedindoorsovernight.HighDMcropwasensiledon15August.Cropswerechoppedtoapproximately30–50mm

length.Thegrasscropwasnotnaturallycontaminatedbyslugs.

2.3. Slugsforensiling

A.vulgariswerecollectedonfourlocationsinSouth-EastNorway(Hortenmunicipality).Allslugswerecollectedbynight

atabout15◦C,672slugson12–13and13–14ofAugust(usedforlowDMsilages)and343slugson14–15ofAugust(used

forhighDMsilages).Theslugswerekeptinplasticboxes(36×27×17cm),53–61slugsfromthesamelocationineach

box.Averageweightoftheslugsperboxwascalculatedbydividinggramsbynumber,andoverallmeanweightforslugs

collectedwere7.0g(lowDM)and6.1g(highDMsilages).Topreventtheslugsfromemptyingtheirintestine,theywere

offeredfreshplantleaves(Taraxacumofficinale,Dactylisglomerata,Rumexlongifolius,TrifoliumrepensandT.pratense)from

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Foreachreplicateofsilages,thecorrespondingnumberofslugstobeaddedwascollectedevenlyfromeachstoragebox.

Halfoftheslugswerecutbyscissors,tosimulatetheharvestingprocesswhereslugspartlygetmashedorcut,andtheslug

masswasmixedbyhand.TheamountofslugsappliedtothelowDMcropwere138,277and553gfreshslugweightperkg

cropDMcorrespondingto5(lowlevel),10(medium)and20(highlevel)7-gsizedmatureA.vulgarisperm2inanassumed

harvestedregrowthyieldof2.5tonDMperha.ForthehighDMcrop,theappliedslugweightwas167,333,and669gper

kgcropDMcorrespondingto6(lowlevel),12(medium)and24(highlevel)7-gsizedslugsperm2withthesameharvested

yield.ThehighersluglevelsaddedtohighthantolowDMcrop,wasduetoslightlylowerDMlevelinwiltedcropthan

assumedduringpreparationofthesilages.

2.4. Silageadditives

Fouradditivetreatments,appliedat4ml/kgofthefreshcropandslugmixture,wereusedforlowDMsilage:1.Control

(C;noadditiveapplied),2.InoculationwithtwostrainsofLactobacillusplantarum(LP;KofasilLife,Addcon,EuropeGmbH,

Bonn,Germany).Thefreeze-driedpremixtureofLPplusgrowthmediumwasactivatedwithwaterat20◦Cfor48hand

appliedgiving,accordingtomanufacturer,aminimumof4×105CFU/gofthefreshcropandslugmixture.3.Acid-treatment

(ACID);57.8%formicacid,12.0%propionicacidand1.5%benzoicacid(GrasAATPlus,AddconNordicAS,Porsgrunn,Norway).

4.Chemicaltreatment(CHEM);11.0%benzoicacid,10.4%NaNO2,6.9%hexamethylenetetramineand3.3%propionicacid

(KofasilUltra,AddconEuropeGmbH,Bonn,Germany).

2.5. Ensiling

Portionsofcropsforsilageswereweighed(1000gforeachportionoflowDMsilageand900gforeachportionofhighDM

silage)andplacedonplasticsheetsofapproximately1m2.Slugswereaddedtoeachcropportionaccordingtoplan.Crop

andslugswerethereaftermixedbyhandandspreadoutinathinlayeronthesheetforapplicationofsilageadditivesusing

micropipette.Themasswasthenthoroughlymixedbyhand,filledintoplasticbagsandsealedusingMagicVacMaxima

vacuumsystem(FlaemNuovaS.p.A.,Brescia,Italy).Silageswereweighedindividually.ForLowDMsilages,replicateone

wasmadeat10:15–13:00,replicatetwoat13:00–15:10andreplicatethreeat15:00–17:15.ThethreereplicatesofhighDM

silagesweremadeat9:35–10:50,10:45–11:50and12:30–14:00thefollowingday.Indoortemperatureduringtheensiling

workwasapproximately25–28◦C.Silageswerefurtherstoredat15–20◦C.

2.6. Samplesoffreshcropandslugs

Duringensiling,acropsamplefromeachreplicatewithineachcropDMlevelwasfrozen(−20◦C)forchemicalanalyses.

Theslugmassaddedtoeachreplicatewasalsosampled,keptat4–6◦Cin24handfrozen(−70◦C)forchemicalanalyses.

2.7. Silageopeningandtestofaerobicstability

SilageswereopenedinNovember,after84,90and97daysofstorageforreplicate1,2and3oflowDMsilage,respectively,

andafter103daysforhighDMsilage.Forunknownreason,fivesilagesoflowDMandtwosilagesofhighDMwereobserved

withslightdamageoftheplastic,whichhadcausedsmallspotsofmoldsinthesilage.Thesesilageswereomittedfromall

results.Nomoldswereobservedinsilageswithundamagedplastic.Silageswereweighedindividually.Silagesamplesfor

chemicalanalyseswereimmediatelyfrozen(−20◦C).

SamplesfromeachlowDMsilage(500g)andhighDMsilage(400g)wereplacedinperforatedplasticbagsinperforated

polystyreneboxesat20◦C.Thetemperatureofthesilagesandtheambienttemperaturewereloggedevery4hfor14days.

Silageswereregardedstableuntilsilagetemperaturereached2◦Caboveambienttemperature.

2.8. Chemicalanalysesandcalculations

FreshcropsampleswereanalyzedforbufferingcapacityaccordingtoPlayneandMcDonald(1966).Slugsampleswere

freezedriedandequilibratedtoroomhumidityovernight,andmilledtopassthrougha1mmscreen(RetschGmbHcutting

mill,Haan,Germany).Thedriedslugsampleswerefurtherdriedat103◦Cfor4hforDMdetermination,analyzedforash

(550◦Cfor4h),fatwithAcceleratedSolventExtraction(ASE)200(Dionex,USA)usingpetroleumether(Applicationnote

345),andfortotalNasdescribedbelowforcropsamples.

Cropandsilagesampleswereoven-driedat60◦Ctostableweight,atleast48h,andweighedwarmforDMdetermination.

AllanalyseswereperformedonundriedsamplesthatwerehomogenizedinfrozenconditionusinganIKAA11basicchopper

(IKA–WerkeGmbh&CoKG,StaufenimBreisgau,Germany).Cropandsilagesampleswereanalyzedfortotalnitrogen(N)for

determinationofcrudeprotein(CP;totalN×6.25)andwater-solublecarbohydrates(WSC).Silagesampleswereadditionally

analyzedfororganicacids,2,3-butandiol,ethanol,pHandammonia-N.TotalNwasdeterminedbytheKjeldahlmethodon

Kjeltec2400(TecatorAB,Höganäs,Sweden)usingaCucatalyst.FordeterminationofWSC,sampleswereextractedin0.05M

Na-acetatebufferatroomtemperaturefor18handfilteredthroughpaper.Sucroseandfructansinthefilteredextractwere

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Table1

Effectofadditivetreatmentandlevelofslugcontaminationonchemicalcompositionoflowdrymattergrasssilage.

Additive Sluglevel N DM,g/kg g/kgDM NH3-value pH

CP WSC LA FA AA PA BA Eth. C None 3 251 123 17 104 0.0 22 0.0 0.8 11.5 120 4.07 Low 3 263 121 17 87 0.0 16 0.0 6.0 9.2 137 4.19 Med. 2 251 139 20 70 0.7 17 0.0 14.3 9.0 169 4.44 High 2 248 166 10 67 1.2 30 1.2 21.7 10.3 216 4.74 LP None 2 262 116 20 102 0.0 20 0.0 0.7 9.3 121 4.03 Low 2 280 118 23 90 0.0 17 0.0 5.9 8.6 143 4.15 Med. 3 279 127 26 83 0.4 17 0.0 10.0 8.2 165 4.29 High 3 249 147 19 80 1.6 17 1.2 30.0 9.6 210 4.57 ACID None 3 273 117 54 64 11.5 12 1.3 0.4 3.7 75 4.08 Low 3 279 121 56 68 11.5 14 1.8 1.1 4.3 107 4.11 Med. 3 251 150 49 80 12.5 17 2.1 2.8 4.8 124 4.19 High 3 262 144 24 83 10.4 21 2.7 7.0 4.6 168 4.28 CHEM None 3 275 117 34 72 0.0 19 0.0 0.0 2.9 80 4.20 Low 2 273 127 31 65 0.0 16 0.0 0.0 2.9 105 4.25 Med. 3 263 145 29 88 0.0 22 0.0 0.0 3.8 103 4.28 High 3 264 144 27 89 0.0 22 0.1 0.4 3.0 137 4.37 SEMe 13.3 9.6 7.6 7.0 0.87 3.1 0.26 1.90 0.54 8.3 0.040 C 10 253 137 16a 82ab 0.5a 21 0.3a 10.7a 10.0a 161a 4.36a LP 10 268 127 22ab 89a 0.5a 18 0.3a 11.6a 8.9b 160a 4.26b ACID 12 266 133 46c 73b 11.5b 16 2.0b 2.8b 4.3c 118b 4.17c CHEM 11 269 133 30b 78ab 0.0a 20 0.1a 0.1c 3.1d 106b 4.28b SEMf 6.6 4.8 3.8 3.5 0.43 1.5 0.13 0.95 0.27 4.2 0.020 None 11 265 118a 31 85 2.9 18ab 0.3a 0.5a 6.9 99a 4.09a Low 10 274 122a 31 77 2.9 16a 0.5a 3.2a 6.2 123b 4.17b Med. 11 261 140b 31 80 3.4 18ab 0.5a 6.8b 6.4 140c 4.30c High 11 255 150b 20 80 3.3 23b 1.3b 14.7c 6.9 183d 4.49d SEMf 6.6 4.8 3.8 3.5 0.43 1.5 0.13 0.95 0.27 4.2 0.020 P Additive 0.42 0.59 <0.001 0.05 <0.001 0.16 <0.001 <0.001 <0.001 <0.001 <0.001 P Slugs 0.36 <0.001 0.13 0.52 0.79 0.05 <0.001 <0.001 0.33 <0.001 <0.001 P A×S 0.92 0.84 0.70 0.007 0.80 0.58 0.23 <0.001 0.08 0.35 <0.001

C=control;LP=twostrainsofL.plantarum;ACID=formic,propionicandbenzoicacid;CHEM=benzoicacid,NaNO2,hexamethylenetetramine,

propio-nicacid;DM=drymatter;CP=crudeprotein;WSC=water-solublecarbohydrates;LA=lacticacid;FA=formicacid;AA=aceticacid;PA=propionicacid; BA=butyricacid;Eth.=ethanol;NH3–value=NH3–N,g/kgtotalNcorrectedforadditive-derivedNH3(fromCHEM).

a,b,c,dMeanswithdifferentlettersdifferatP<0.05.

eSEMmustbemultipliedwith1.016forrowswhereN=2.

fSEMmustbemultipliedwith1.061forrowswhereN=11andwith1.118forrowswhereN=10.

fructose-6-phospatebyanenzymaticmethodusingkitK-FRUGL(Megazyme,Wicklow,Ireland).Theconcentrationswere

determinedspectrophotometricallybytheincreaseinabsorptionofNADPHat340nm.Silagesamplesweredilutedwith

distilledwaterandstoredfrozen,thenthawedandfiltratedbeforeanalyzinglacticacid(LA),formicacid(FA),aceticacid

(AA),propionicacid(PA),butyricacid(BA),2,3butanediolandethanolusingaWatersAlliance2795HPLCsystem(Separation

ModulewithatemperaturecontrolmoduleII,range40–70◦C)andWaters2414RIDetector(WatersCorp.,Milford,MA,USA).

SeparationcolumnwasReproGelH,9␮m300×8mm(Dr.A.Maisch,Ammerbuch,Germany)andpre-columnReproGel

H,9␮m30×8mm(Dr.A.Maisch,Ammerbuch,Germany).Mobilephasewas0.005MH2SO4 at0.8ml/min,andcolumn

temperature60◦C.pHwasanalyzedusingaTermoOrion420A+pH-meterwithOrion9107BNelectrode(ThermoScientific,

Beverly,MA,USA)andammonia-NwithMAN-TECHPC-titrate(Guelph,ON,Canada)usinganOrionionanalyzer901.

OvenDMcontentsofthesilageswerecorrectedforvolatilelossaccordingtotheNordicfeedevaluationsystemNorFor

(Åkerlindetal.,2011).Ammonia-NinsilagederivedfromNaNO2,andhexamethylenetetramineinCHEMadditive(46gNH3

perkg)wassubtractedfromtotalNandNH3–Nanalyses,inordertoexpresstheamountofNH3–Noriginatingfromcrop

proteolysisinthepresentedNH3-values(Table1).Forthe11CHEM-treatedsilages(oneofthe12silageswasomitteddue

toplasticdamage),themeancorrectedNH3-valuewas106,versus135fortheuncorrectedvalue.

2.9. Statisticalanalyses

ThechemicalcompositionandaerobicstabilityoflowDMsilageswereanalyzedusingtheGLMprocedureofSAS(release

9.3,2002–2010;SASInstituteinc.,Cary,NC,USA),bythefollowingmodel:Yijk=+Ai+Sj+Ai×Sj+Rk+eijk,where=general

mean,Ai=effectofadditivetreatmenti,Sj=effectofsluglevelj,Ai×Sj=effectofinteractionbetweenadditiveiandslug

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andAi×Sjcomponentswereomittedfromthemodel.TreatmentmeanswereseparatedusingthePDIFFstatementinSAS.

Resultsaregiveninleastsquare(LS)means.

3. Results

3.1. Freshcropcomposition

ThecropusedforlowDMsilagecontained(mean±SD)253±24gDM/kg,andperkgDM:120±11gCP,46±4gWSC,

andhadabufferingcapacity(BC)of28±4mEqNaOH/100gDM.ThecropusedforhighDMsilagecontained372±26g

DM/kg,andperkgDM:112±6gCP,74±12gWSC,andhadBCof25±2mEqNaOH/100gDM.

3.2. Slugcomposition

TheslugsusedforlowDMsilagecontained(mean±SD)173±2.1gDM/kg,andperkgDM:842±15.5gorganicmatter,

552±4.9gCPand74±4.5gfat.TheslugsusedforhighDMsilagecontained170±9.2gDM/kg,andperkgDM:840±5.6g

organicmatter,594±3.1gCPand72±5.5gfat.OnDMbasis,thesluginclusionconstituted0.026(lowsluglevel),0.050

(mediumsluglevel)and0.096(highsluglevel)oftheensiledlowDMmixtureoffreshcropplusslugs,and0.029,0.057and

0.107,respectively,ofthehighDMensiledmixture.DuetothehighCPconcentrationinslugDMrelativetocropCP,slugCP

constituted0.104(lowsluglevel),0.188(medium)and0.317(highsluglevel)oftotalCPintheensiledlowDMmixtureof

freshcropplusslugs,and0.142,0.248and0.397,respectively,ofthehighDMensiledmixture.

3.3. Silagefermentationquality

ChemicalcompositionoflowDMsilageispresentedinTable1.TreatmentwithLPreducedpH(P=0.005)andethanol

fermentation(P=0.02),butsilagecompositiondidotherwisenotdifferfromCsilage.ACID-treatmentreducedBA(P<0.001),

ethanol(P<0.001)andNH3–N(P<0.001)concentrations,increasedWSCconcentrations(P<0.001),andreducedpHcompared

withC(P<0.001)andLP-treated(P=0.005)silage,andreducedLAconcentrationcomparedwithLP-treatment(P=0.008).

CHEM-treatmentreducedBA(P=0.005)andethanol(P=0.005)concentrationscomparedwithallothertreatments,reduced

NH3–NconcentrationscomparedwithC(P<0.001)andLP-treatment(P<0.001),andreducedpH(P=0.02)andincreased

WSC(P=0.02)concentrationcomparedwithC-treatment.Increasinglevelsofslugcontaminationincreasedconcentrations

ofCP,BAandNH3–N,andincreasedpH.ThehighestslugcontaminationincreasedPAconcentrations(P<0.001),tendedto

increaseAAconcentrations(P=0.05)andtendedtoreduceWSCconcentrations(P=0.05)comparedwithC-treatment.No

silagesamplescontained2,3-butandiol.

SignificantadditivebysluginteractionswerefoundforLAandBAconcentrationsandpH.WhereasC-andLP-treated

silageshoweddeceasingLAconcentrationswithincreasingslugcontamination,ACID-andCHEM-treatedsilageshowed

increasingLAconcentrationswithincreasingslugcontamination.And,whereasC-andLP-treatedsilagedisplayeddramatic

increasesinBAconcentrationswithincreasingslugcontamination,thecorrespondingBAincreasewasmoderatein

ACID-treatedsilageandabsentinCHEM-treatedsilage.Withincreasingslugcontamination,pHincreaseddramaticallyinC-and

LP-treatedsilage,butmoderatelyinACID-andCHEM-treatedsilage.

InhighDMsilage,increasingslugcontaminationdecreasedDMconcentrationsandincreasedconcentrationsofCPand

NH3–N,andpH(Table2).ThehighestslugcontaminationdecreasedLAconcentration(P=0.009)andincreasedPA(P<0.001)

andBAconcentrations(P=0.02).

Table2

Effectoflevelofslugcontaminationonchemicalcompositionofuntreatedhighdrymattergrasssilage.

Slug DM,g/kg g/kgDM NH3 -value level N CP WSC LA FA AA PA BA Eth. pH None 3 394a 106a 21 72a 0.0 14 0.0a 0.0 7.9 114a 4.20a Low 3 376ab 123b 17 74a 0.0 15 0.0a 5.0 7.7 156b 4.36ab Med. 2 362b 132c 14 79a 0.0 15 0.0a 9.2 8.3 170b 4.42b High 2 333c 161d 16 50b 0.5 32 0.9b 20.8 8.1 206c 4.81c SEMe 6.3 2.2 4.7 3.7 0.15 5.7 0.00 4.10 1.25 7.2 0.048 P 0.005 <0.001 0.82 0.01 0.29 0.26 <0.001 0.09 0.99 0.001 0.001

DM=drymatter;CP=crudeprotein;WSC=water-solublecarbohydrates;LA=lacticacid;FA=formicacid;AA=aceticacid;PA=propionicacid;BA=butyric acid;Eth.=ethanol;NH3-value=NH3–N,g/kgtotalN.

a,b,c,dMeanswithdifferentlettersdifferatP<0.05.

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Table3

Effectofadditivetreatmentandlevelofslugcontaminationoflowdrymattergrasssilageonweightlossduringtheanaerobicphaseandonthesubsequent aerobicstability.

Silage additive

Sluglevel N Weight lossf,g/kg

Aerobicg

stabilityofallsilages

Silagesthat neverheatedh Silagesthat heated Aerobicstabilityof heatedsilages Hoursh N N Hours C 10 17.5a 327 6 4 180 LP 10 17.2a 321 5 5 263 ACID 12 14.2b 318 6 6 236 CHEM 11 13.3b 342 7 4 227 SEMe 0.41 26.5 34.1i None 11 13.8a 226a 2 9 194 Low 10 15.2b 342b 6 4 235 Med. 11 15.5b 378b 8 3 311 High 11 17.7c 362b 8 3 247 SEMe 0.41 26.5 25.5j P Additive <0.001 NS 0.53 P Slugs <0.001 0.003 0.18 P A×S 0.09 NS k

C=control;LP=twostrainsofL.plantarum;ACID=formic,propionicandbenzoicacid;CHEM=benzoicacid,NaNO2,hexamethylenetetramine,propionic

acid.

a,b,c:MeanswithdifferentlettersdifferatP<0.05.

eSEMmustbemultipliedwith1.061forrowswhereN=11andwith1.118forrowswhereN=10. fWeightlossincludedappliedadditivesandslugs.

gAerobicstabilitywasmeasuredashourstoreach2Caboveambienttemperature.

hSeveralsilagesdidnotreach2Caboveambienttemperatureduringthe14-dtestperiod.Forthosesilages,thetimetoreach2Caboveambient

temperaturewassetto400h(≈17d).

iSEMmustbemultipliedwith1.094forrowswhereN=5andwith1.223forrowswhereN=4. jSEMmustbemultipliedwith1.502forrowswhereN=4andwith1.733forrowswhereN=3. kAdditive×Sluginteractioncouldnotbeestimated.

3.4. Weightloss

TheweightlossoflowDMsilageduringtheanaerobicphasewashigherforCandLP-treatedsilagecomparedwith

ACID-orCHEM-treatedsilage(Table3).Weightlossesincreasedwithincreasingslugcontaminationforbothlow(Table3)and

highDMsilage(Table4).

3.5. Aerobicstabilityofsilage

Severalsilagesdidnotreach2◦Caboveambienttemperatureduringthe14-dayaerobicstabilitytest.Forthosesilages,

thetimetoreach2◦Caboveambienttemperaturewassetto400h(≈17d).Thereafter,statisticalanalysisofhourstoreach

2◦Caboveambientwasdoneforallsilages,andaloneforthesilagesthatactuallyheated(Tables3and4).Additivetreatment

didnotaffecttheaerobicstabilityoflowDMsilage.Asmallerproportionoftheslugcontaminatedsilages(10of32silages)

thanofthesilageswithoutslugs(9of11silages)heatedwithin14days,andthetimetakentoreach2◦Caboveambient

temperaturewaslongerforcontaminatedthanfornotcontaminatedsilages.ThiswasthecasebothwhenalllowDMsilages

Table4

Effectoflevelofslugcontaminationofhighdrymattergrasssilageonweightlossduringtheanaerobicphaseandonthesubsequentaerobicstability.

Slug level N Weight lossd Aerobic stabilitye ofallsilages Silagesthat never heatedf Silagesthat heated Aerobic stability ofheated silages g/kg Hoursf N N Hours None 3 20.1a 246a 0 3 246 Low 3 21.0a 400b 3 0 Med. 2 23.4b 400b 2 0 High 2 26.9c 400b 2 0 SEMe 0.48 27.5 P <0.001 0.02

a,b,c:MeanswithdifferentlettersdifferatP<0.05. dWeightlossincludedslugs.

eAerobicstabilitywasmeasuredashourstoreach2Caboveambienttemperature.

fTheslugcontaminatedsilagesdidnotreach2Caboveambienttemperatureduringthe14-dtestperiod.Forthosesilages,thetimetoreach2Cabove

ambienttemperaturewassetto400h(≈17d).

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wereconsidered(P<0.001),andfortheheatedsilagesalone(P=0.07).ThethreehighDMsilageswithoutslugsheatedafter

246hofaeration,whereasnoneofthesevenslugcontaminatedsilagesheated.Whentimetoreach2◦Caboveambient

temperaturewassetto400hforthose,theyweremorestablethanthethreenotcontaminatedsilages.

4. Discussion

4.1. Cropforensiling

Theharvestedcrophadlowbufferingcapacity,andfairlyhighDMandWSCconcentrations.ThehigherWSCconcentration

inthehigh,comparedwiththelow,DMcropwasprobablyduetodegradationofhemicellulosebyplantenzymesduring

theprolongedwilting(McDonaldetal.,1991).Calculatedfermentabilitycoefficients(FC)(WeissbachandHonig,1996)were

38forthelowand61forhighDMcrop.ThisindicatedthatonlywellfermentedsilagewasexpectedfromthehighDMcrop

(FCabove45).FromthelowDMcrop,wellfermentedsilagewasexpectedifLABcountexceeded105CFU/gfreshweightor

ifNO3contentexceeded0.5g/kgDM(FCabove35).

4.2. Levelofslugcontamination

Kozlowski(2007)revealedhighdifferentiationsinslugincidenceatdifferenttypesoflocationsduring1995–2005in

Poland,rangingfroma fewto100slugs/m2 dependingonthelocality.On average12slugs/m2 werecountedinbalks,

fieldridgesandfieldswithhighweeddensityandbarrenlands.Slugdensitiesof100slugs/m2wererecordedfromtypical

gatheringsiteswithfreshplantdebris,compost,deepditchesandshadedslopesofwatercourses.Inanotherstudyfrom

Poland,anaveragenumberof16.8slugs/m2wereobservedinJulyinaclovercultivation(KozlowskiandKozlowski,2011).

InasimilarlaboratoryscalestudyperformedinSweden,SpörndlyandHaagaapplied,perkgcropDM,approximately0,

26,57and118gofjuvenileA.vulgaris,withanaverageweightof0.64g,toaprimarygrowthharvestinlateMay(Spörndly

andHaaga,2010).ComparedwiththeSwedishstudy,theslugcontaminationinthepresentstudywasroughlyahalfon

thenumberofslugbasis,butfivetimesashighonagslugperkgcropDMbasis,becauseeachslugwasapproximately10

timesbigger.Thetwostudiesrepresentrelevantcontaminationlevelsthatareobservedinthetwocountriesinearlyand

latesummer,respectively.

OnaslugweightperkgcropDMbasis,allcontaminationlevelsintheSwedishstudywerebelowthelowestcontamination

levelinthepresentNorwegianstudy.Using asilagecropwiltedto370gDM/kg, SpörndlyandHaaga(2010)observed

increasedLAconcentrationsandreducedpHinalllevelsofslugcontaminatedsilage,comparedwithcontrolsilage.They

attributedthistotheobservationthattheslugscontainedahigherLABconcentration(5.5logCFU/g)thantheharvested

crop(2.8logCFU/g),andconcludedthataddingjuvenileslugstoacropforsilagetosomeextentresembledtheeffectof

addingsilageinoculants(SpörndlyandHaaga,2010).Nosucheffectofslugcontaminationonthechemicalcompositionof

silagewasobservedinthepresentstudy.

4.3. Silagefermentationquality

AccordingtoNorwegianrecommendations(Eurofins,2010),maximumvaluesoforganicacidsinwell-fermentedsilage

withDMconcentrationsupto250g/kg,are(g/kgDM):3BA,30AAand80LA.SilageswithhigherDMshouldcontainlower

levels.TheconcentrationsofFAandPAshouldreflecttheamountsappliedwithsilageadditives,orotherwisebebelow

2g/kgDM.Theconcentrationoftotalacidsshouldbebelow100g/kgDM.Ethanolshouldbeatamaximumof7g/kgDM.

Theammoniavalue(NH3–N,g/kgtotalN)shouldbeatamaximumof80,andforsilageswithDMconcentrationsbelow

250g/kg,pHshouldbebelow4.2.

Accordingtotheserecommendations,onlyACID-andCHEM-treatedlowDMsilagewithoutslugcontaminationcouldbe

regardedaswellfermented(Table1).Allothersilagesexceededthemaximumammoniavalue.AllC-andLP-treatedslug

contaminatedsilages,andACID-treatedsilagewiththehighestslugcontamination,additionallyexceededtheupperlimit

forBAconcentration.CHEM-treatmenteffectivelyinhibitedBAfermentationevenatthehighestlevelofslugcontamination.

Thesefindingsaresupportedbymicrobialenumerationsfromsilagesinthesamestudy(Gismerviketal.,2014),wherethe

BAproducingClostridiumtyrobutyricumincreasedwithincreasingslugcontaminationandonlysilageswithoutslugsand

silagestreatedwithCHEMhadnon-detectablelevels.CHEMcontainsnitrite,knownforefficientclostridiainhibition,and

alsohexaminethatcombinedwithnitriteisfoundtoinhibitclostridia(Woolford,1975;Spoelstra,1985;Knick ´yandLingvall,

2001).EthanolconcentrationswereabovetherecommendedlevelforallC-andLP-treatedsilages,butwithinrecommended

levelsforallACID-andCHEM-treatedsilages.

AlthoughLP-treatment,comparedwithCtreatment,slightlyreducedethanolandpHvalues,andnumericallyobtained

somewhathigherWSCandLAconcentrationsinslugcontaminatedsilage,itwasnotareliablesilageadditiveforusein

highlyslugcontaminatedcrops.ACID-andCHEM-treatment,however,considerablyimprovedfermentationqualityofslug

contaminatedsilage.AtthehighestcontaminationlevelCHEMwasevenmoreeffectivethanACID,possiblyduetoitsstrong

inhibitoryeffectagainstClostridiumspp.thatrestrictedBAfermentationinsilage.TheincreasedsilageCPconcentrations

withincreasingslugcontaminationfollowedcloselythecalculatedCPconcentrationsinthefreshcropplusslugmixtures

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none,low,mediumandhigh,respectively.Withincreasingslugcontamination,increasingproportionsfrom0to0.32of

theinitialensiledCPwerederivedfromslugs.ThemuchhigherCPconcentrationmakesslugsamore“difficult-to-ensile”

materialthangrasscrops.Thismayhavecontributedtoanevenstrongerincreaseinsilageammoniavalues,thaninCP

values,indicatingincreasedproteolysisofcropand/orslugproteinwithincreasingcontaminationlevel.Thiswasinline

withtheincreasingsilagepHandBAfermentationwithincreasingsluglevels.SpörndlyandHaaga(2010)alsoobserved

increasedammoniavaluesinslugcontaminatedsilage.

ThedecreasingsilageDMconcentrationswithincreasingslugcontaminationinhighDMsilages(Table2)followeda

calculateddecliningDMconcentrationintheensiledcropplusslugmixture.ThefermentationqualitiesobtainedinhighDM

silagesweresimilartothoseobtainedinC-treatedlowDMsilage.ThecalculatedFC(WeissbachandHonig,1996)suggested

thatthehighDMcropwaseasytoensilewithoutadditivetreatment.Apartfromamoderateexceedofthelimitforammonia

value,theuncontaminatedhighDMsilagewaswellfermented.Similarly,forlowDMcrop,CandLPtreateduncontaminated

silageswereofacceptablequalityinspiteoftheirammoniavalues,inlinewiththecalculatedFC.Thereducedextentof

fermentationoftenobservedduetolowerwateractivityinhighDMcropwasnotsufficienttopreserveslugcontaminated

silages,whereupto0.4ofCPintheensiledcropandslugmixturewasderivedfromslugs.Ademandforeffectivesilage

additiveswassurelyapparentalsoforhighDMcrop.

InlinewithMcDonaldetal.(1991)andRandby(2000)ACID-treatmentrestrictedLAfermentation intherelatively

“easy-to-ensile”uncontaminatedcropbutencouragedLAfermentationinthe“difficulttoensile”crop(mediumorhigh

slugcontamination).Restrictedfermentationgenerallyimprovessilageintakebydairycows,butwhencropsaredifficult

toensile,avoidanceofbutyricacidfermentationismoreimportant.LowlevelsofPAandFAdetectedinsilagesthatarenot

treatedwithacidsindicatetheirproductionduringthecourseoffermentation.Inlinewithpreviousresearch(Randby,2000),

thisoccurredonlyinpoorlyfermentedsilageswithhighpH,aswasthecaseforsilagescontaminatedwiththemediumor

highsluglevels.PropionicacidbacteriagrowathighpHonly(McDonaldetal.,1991).Theobservedweightlossesduring

thesilagefermentationarecausedbygas,primarilyCO2,producedduringfermentationandescapingthroughtheplastic

coverofthesilages(Tables3and4).Thoselosseswerecloselyrelatedtotheextentofpoorfermentation.Volatilefattyacids

andammoniahavegreaterdepressiveeffectsonintakethanLA(Huhtanenetal.,2002).ACID-andCHEM-treatmentwould

thereforebeexpectedtoimprovesilageintake.

4.4. Expectedsilageintake

Attemptingtoevaluatedifferencesinexpectedsilageintake,therelative silageDMintakeindex(SDMI-index)was

calculatedaccordingtoHuhtanenetal.(2002).TherevisedSDMI-index(Huhtanenetal.,2007)wasnotchosenbecause

itwasdevelopedusingfewpoorlyfermentedsilages,anddonotcontainaclearmeasureofsecondaryfermentation(BA

orammonia).Therevisedindexmaythereforeoverestimatetheintakeofverypoorlyfermentedsilages(Huhtanenetal.,

2007).EvaluatedaccordingtoHuhtanenetal.(2002),allsilagesinthepresentstudyapartfromACID-andCHEM-treated

uncontaminatedlowDMsilagewereclassifiedaspoorly,orsecondarily,fermented.

TheoriginalSDMI-indexwascalculatedbasedonsilagedigestibleorganicmatterperkgDM(DOMD)andconcentrations

oftotalacids(LA+AA+PA+BA)andNH3–NintotalN(Huhtanenetal.,2002).Anassumed,lowvalue,fixedforallsilages,

600gDOMDwasused.Thisreflectedthehighmaturityofthecropthatwasharvested2monthsfollowingtheprimary

growthharvest,however,thechosenleveldoesnotinfluencetherelativedifferencesbetweensilages.Thehighest

SDMI-index,84,wasfoundforACID-treateduncontaminated,lowDMsilage,andthelowestindex,74,forLP-treatedlowDMsilage

contaminatedwiththehighestsluglevel.Onaverageoverslugcontaminationlevels,C-,LP-,ACID-andCHEM-treatment

producedSDMI-indexesof77,77,81and80(SEM=0.53,P<0.001),suggestingthatcowswoulddepressintakeby5%when

offeredC-orLP-treatedsilage,andwith2%whenofferedCHEM-treatedsilage,comparedwithACID-treatedsilage.On

averageoveradditivetreatments,theslugcontaminationlevelsnone,low,mediumandhighproducedSDMI-indexesof80,

80,79and76(SEM=0.53,P<0.001),suggestingthatthehighestslugcontaminationwoulddepressintakewith5%compared

withuncontaminatedsilage.ForhighDMuntreatedcrop,SDMI-indexesofthefourincreasingsluglevelswere82,80,78

and77(SEM=0.40,P=0.001),suggestingthatthehighestslugcontaminationwoulddepressintakewith6%comparedwith

uncontaminatedcrop.TheseestimatesdonottakeintoconsiderationthepossibilitythatBAfermentationmayreducesilage

DOMDconcentrations(McDonaldetal.,1991),neitherthepossibleeffectsontheimmediatesmell,tasteorpalatabilityof

thesilagescausedbytheslugstherein.FarmersinbothSwedenandNorwayhavereportedthatcattlewerereluctantto

consumefreshlyharvestedgrassandsilagewithahighdegreeofslugcontamination(SpörndlyandHaaga,2010),implying

thatthismaybeaproblem.

4.5. Aerobicstabilityofsilages

Thehigheraerobicstabilityofslugcontaminated,comparedwithuncontaminated,silagewasprobablyduetotheir

highercontentofBAandNH3–N,andlowerWSCconcentration.Extensivelyfermentedsilageswithhighconcentrations

offermentationproducts,particularlyBA,andlimitedconcentrationsofresidualWSCareknowntohavehigheraerobic

stabilitythanwellfermentedsilage(McDonaldetal.,1991).Whentheaerobictestwascompletedafter14days,andsilages

wereremovedfromthepolystyreneboxes,mostslugcontaminatedsilagesstilllookedastheydidatthebeginningofthe

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minorlevelsofbutyricacidarefound,ACID-andCHEM-treatedsilagesarenormallymorestablethanuntreatedorLP-treated

silage,inspiteoftheirhigherresidualsugarconcentration(Randby,2010).Silagesinoculatedwithhomofermentativestrains

ofL.plantarum,asinthepresentstudy,areoftenfoundtobelessstablethanuntreatedsilage(Muck,2012).Therelativelyhigh

numericalvalueforaerobicstabilityfoundforLPtreatedsilageinthisstudymightbeduemainlytoitshighconcentrations

ofBA.Thelackofsignificantdifferencesinaerobicstabilitybetweensilage-additivetreatmentsinthisstudymaybedueto

thefactorialarrangementwithslugcontaminatedpoorlyfermentedsilages,andthelownumberofreplicatesilagesthat

actuallyheated.

4.6. Onfarmconsiderations

Heavilysluginvadedgrassmeadowsmightoftenbewet(<25%DM).Basedonthisstudy,wiltingaftermowingwould

notbesufficienttocontroltheensilingprocesseswithslugsofhighmoistureandproteincontent.Althoughpreventive

measuresthatcouldreducethesluginvasionmightbetheidealeffort,theuseofadditivesbasedonacidsorotherchemicals

mayensuresilageofacceptablefermentationquality.

5. Conclusion

SluginvasionbyadultA.vulgarisintoregrowthsilagecropsmayseverelydisturbsilagefermentation.Increasingslug

contaminationgaveincreasingqualityreductionsbothinsilagescontaining253and372gDM/kg.Comparedwithuntreated

silage,LP-treatmentdidnotimprovefermentationqualityofcontaminatedcrop.TreatmentwithACIDandCHEM,however,

considerablyimprovedthequality,evenofheavilycontaminatedsilages.

Conflictofinterest

Wewishtoconfirmthattherearenoknownconflictsofinterestassociatedwiththispublicationandtherehasbeenno

significantfinancialsupportforthisworkthatcouldhaveinfluenceditsoutcome.

Weconfirmthatthemanuscripthasbeenreadandapprovedbyallnamedauthorsandthattherearenootherpersonswho

satisfiedthecriteriaforauthorshipbutarenotlisted.Wefurtherconfirmthattheorderofauthorslistedinthemanuscript

hasbeenapprovedbyallofus.

Acknowledgments

TheResearchCouncilofNorway;FoundationforResearchLevyonAgriculturalProductsandAgriculturalAgreement

ResearchFoundandTineareacknowledgedforfundingthisproject(199401)andthePh.D.studentshipsofK.G.Theauthors

wouldliketothankHallvardGjøsteinforhelpwithsilageproductionandtheaerobicstabilitytest,andAddconforproviding

silageadditives.

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