Probiotic supplementation affects the glycan composition of mucins secreted by Brunner's glands of the pig duodenum.

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ContentslistsavailableatScienceDirect

Annals

of

Anatomy

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / a a n a t

Probiotic

supplementation

affects

the

glycan

composition

of

mucins

secreted

by

Brunner’s

glands

of

the

pig

duodenum

夽

Gianluca

Accogli

a

_,

_Alberto

_Maria

_Crovace

b

_,

_Maria

_Mastrodonato

c

_,

_Giacomo

_Rossi

d

_,

Edda

G. Francioso

a

_,

_Salvatore

_Desantis

a,∗

a_Section_of_Veterinary_Clinics_and_Animal_Productions,_Department_of_Emergency_and_Organ_{Transplantation}_(DETO),_University_of_Bari_Aldo_Moro,_S.P.

CasamassimaKm3,70010Valenzano,Bari,Italy

b_Dottorato_di_Ricerca_in_Sanità_e_Scienze_Sperimentali_Veterinarie,_University_of_Perugia,_Perugia,_Italy c_Department_of_Biology,_University_of_Bari_“Aldo_Moro”,_Via_E._Orabona_4,₇₀₁₂₄_Bari,_Italy

d_School_of_Biosciences_and_Veterinary_Medicine,_University_of_Camerino,_Via_{Circonvallazione}_93/95,₆₂₀₂₄_Matelica,_MC,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24October2017

Receivedinrevisedform21March2018 Accepted29March2018 Keywords: Mucins Glycohistochemistry Lectins Probiotics Diet Swine

a

b

s

t

r

a

c

t

Theeffectofadietaryprobioticblendonthecarbohydratecompositionofmucinssecretedbythe Brun-ner’sglandsintheduodenumofgrowing-finishingpigswasinvestigatedbymeansofconventional (periodicacid-Schiff,AlcianBluepH2.5,highirondiaminestaining)andlectin(15lectins) histochem-istry.Pigswereassignedtotwodietarytreatments:acontrolbasaldietwithouttheprobioticblend (No-Pro)andatestdietthatincludedtheprobioticblend(Pro).Duodenaltissuefragmentswerefixedin 4%phosphate-buffered-saline-bufferedparaformaldehyde,dehydratedthroughagradedalcoholseries, andembeddedinparaffinwax.ThesecretorycellsoftheBrunner’sglandsfromNo-Propigsprimarily producedneutralglycoproteinsandasmallamountofacidicnon-sulphatedmucins.Thisglycan pat-ternwasoppositethatoftheBrunner’sglandsfromProanimals.Acomparisonoflectin-bindingprofiles ofthesecretorycellsofBrunner’sglandsinthesetwogroupsshowedthatinPropigs,therewas(i)a decreaseinN-linkedglycanscontaining␣1,2-linkedfucose(ConA,UEAI);(ii)alossofcomplextypesof N-glycans(PHA-L,PHA-E)terminatingwithlactosamine(RCA120),␣1,6-and␣1,3-linkedfucose(LTA),and

␣-galactose(GSAI-B4),aswellasofO-glycanswithterminalGal␤1,3GalNAc(PNA);and(iii)anincrease

inO-glycanscontainingGalNAcHPA.No-ProandProsamplesshowednochangeintheexpressionof␣2,6 sialoglycansandterminalGlcNAcresiduesandnoafﬁnityforMALII,DBA,andSBA.Theseresults indi-catethatprobioticsupplementationaffectstheglycancompositionofmucinsproducedintheBrunner’s glandsofgrowing-ﬁnishingpigs.Thesechangescouldeffectivelyactonthegastrointestinalfunctionand healthstatusoftheseanimalsbecausetheprobioticblendinducedhighergrowthperformanceandmeat qualityinthetestprobioticgroupthanitdidinthecontrolbasaldietgroup(Tufarellietal.,2017).

1. Introduction

Brunner’s glands, or duodenal glands, are speciﬁc to

mam-mals and are located in the submucosa of the duodenalwall,

althoughtheycanbefound,evenifdiscontinuously,inthejejunum

ofbigherbivores and pigs (Krause, 2000).Brunner’s glands are

mucin-secretingtubulo-alveolarglands.Mucinsconstitutea

fam-ilyofdenselyglycosylatedproteins,thehighglycosylationgiving

themgel-likepropertiesandtheabilitytoresistproteolysisand

holdwater(Perez-VilarandHill,1999).Thehighlyviscousmucus

夽 ThispaperbelongstothespecialissueAnimalAnatomyIII. ∗ Correspondingauthor.

E-mailaddress:salvatore.desantis@uniba.it(S.Desantis).

secretedbyBrunner’sglandsprotectstheunderlyingmucosafrom

mechanicalinsults,neutralizestheacidityofgastricjuice(Florey

andHarding,1933),modulatesabsorptionofingesta,inhibitsattack

bypathogens,andmaintainsbacterialmicroﬂora(Krause,2000;

FlemstromandIsenberg,2001).

Theimportanceoftheintestinalmicrobiotaforgastrointestinal

functionandhealthhasbeenshowninmanystudies(Heinritzetal.,

2013;BüsingandZeyner,2015;Huetal.,2015;Liuetal.,2017).In

particular,probioticshaveastimulatingeffectonthedigestive

pro-cessesandimmunityofanimals(Fuller,2006;Mengetal.,2010).

Therefore,theiruseissuggestedasanalternativetoantibioticsor

anti-inﬂammatorydrugs.However,themodeofactionofprobiotics

ispoorlyunderstoodandthereportedmechanismsofactionare

oftentheresultsofinvitroexperiments.Theseresultsshould

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forebeconﬁrmedbyinvivostudies(Oelschlaeger, 2010).More

recently,theuseofaprobioticcomplex(SivoyTM_,_SLAB51)_has_been

showntoenhancethegrowthperformanceandmeatqualityof

growing-ﬁnishingpigsandtoreducepollutionfromanimalexcreta

(Tufarellietal.,2017).

Despitethephysiologicalimportanceofthemucinssecretedby

Brunner’sglands, todate nothingis knownabouttheeffects of

microbiotaonthecompositionofthemucinssecretedbythese

glands. Previous studies have, however, demonstrated that the

compositionofmucinsfromBrunner’sglandscouldbeaffectedby

dietarychange(seeKrause,2000,forreference).

Theaimofthepresentstudywastoexaminetheinsitueffect

of a dietary probiotic complex (SivoyTM_, _SLAB51) _on _the

gly-cancompositionofmucinsproducedin theBrunner’s glandsof

growing-ﬁnishingpigs.Weusedbothconventionalandlectin

his-tochemistry.The conventional technicalapproach discriminates

neutral and acidic classes of glycoconjugates, whereas lectins

allowanalysisofthecarbohydratecompositionofcomplexglycans

(Spicer and Schulte, 1992; Sharon and Lis, 2004). The

inves-tigation was carried out in pigs. Because of their anatomical,

physiological,andgeneticcomparabilitytohumans,they

repre-sentapromisinganimalmodeltodeterminequestionsofbasic,

applied,andtranslationalbiomedicalresearch(Aigneretal.,2010;

Stramandinoli-Zanicottietal.,2014),includingstudiesofhuman

nutritionandhealthproblems(Guilloteauetal.,2010;Vermaetal.,

2011;Pratheretal.,2013;Gonzalezetal.,2015).

2. Materialsandmethods

2.1. Probioticsources

The probiotic preparation used in the present trial was

obtainedfromacommercialcompany(SLAB51,MendesSA,Lugano,

Switzerland).TheprobioticSLAB51iscomposedofablendofthe

followingstrains:StreptococcusthermophilesDSM32245,amixture

ofthetwostrainsBiﬁdobacteriumanimalisssp.lactisDSM32246and

DSM32247,LactobacillusacidophilusDSM32241,Lactobacillus

hel-veticusDSM32242,LactobacillusparacaseiDSM32243,Lactobacillus

plantarumDSM32244,andLactobacillusbrevisDSM27961.

2.2. Animals

ThetrialreceivedethicalapprovalfromtheItalianMinistryof

Health(n.597/2015-PRdel23/06/2015)andwasconductedinstrict

accordancewiththerecommendationsoftheGuidefortheCare

andUseofLaboratoryAnimalsoftheNationalInstitutesofHealth

(Art.18D.L.4March2014,no.26).

Twentypigs[(Landrace×Yorkshire)×Talent]withanaverage

initialbodyweight(BW)of22.80±0.95kg(SE)wereusedina

12-weekexperiment.Pigswereassignedtotwodietarytreatments:

thecontrolbasaldietwithouttheprobioticblend(No-Pro)andthe

experimentaldietthatincludedtheprobioticblend(Pro).The

pro-bioticmixturewasusedasadietarysupplementforthepigsduring

theentirefeedingperiodatadoseof100mg/kgofBW.Thebasal

dietwasformulatedtomeetorexceedthenutrientrequirements

ofpigsaccordingtotheNRC(1998).Pigswerehousedinan

envi-ronmentallycontrolledroomwithaconcreteﬂoorandwerefedad

libitum.

2.3. Samplingandhistologyprocessing

Attheendofthetrial,pigswereslaughteredandspecimensof

duodenaltissuewereimmediatelyremovedfrom5cmofthecaudal

partofthepyloricregionandﬁxedin4%(v/v)

phosphate-buffered-saline-buffered paraformaldehydefor 24hat 4◦C. Thesamples

werethendehydratedthroughagradedalcoholseriesand

embed-ded inparafﬁnwax.Serialsections(4-␮mthick) werecutand,

afterbeingde-waxedwithxyleneandhydratedinanethanolseries

ofdescendingconcentrations,stainedwithhematoxylin-eosinfor

morphologicalandmorphometricstudiesandbyconventional

his-tochemicalproceduresorlectinhistochemistryforglycoconjugate

characterization.

2.4. Conventionalhistochemistry

Sectionsweretreatedwith(1)periodicacid-Schiff(PAS)

reac-tionforneutralglycoconjugates(McManus,1948);(2)AlcianBlue

pH2.5(AB2.5)forsulphateestersandcarboxylgroupsin

glycocon-jugates(Pearse,1968);and(3)combinedhighirondiamine-Alcian

BluepH2.5(HID-AB2.5)forsimultaneousstainingofsulphated

(brown-black) and non-sulphated (blue) acidic glycans (Spicer,

1965).Torevealcellularcombinationsofbothacidicandneutral

glycoconjugates,weperformedAB2.5/PASandHID/AB2.5staining

sequences.

2.5. Lectinhistochemistry

Thebindingof15lectinswastestedtoinvestigatethe

compo-sitionanddistributionofoligosaccharidicchainsintheBrunner’s

glandsofthepigs(Table1).AlllectinswerepurchasedfromVector

LaboratoriesInc.(Burlingame,CA,USA).

Tissue sectionsstained with ﬂuorescent lectins were rinsed

in0.05MTris–HCl-bufferedsaline(TBS)pH7.4andincubatedin

appropriatedilutionsofeachlectindilutedinTBS(Table1)for1h

atRTinthedark.AfterthreerinsesinTBS,slidesweremountedin

Vectashieldmountingmedium(VectorLaboratories,Burlingame,

CA,USA).Tissuesectionsstainedwithbiotinylated MALIIwere

immersedin3%v/vsolutionofH2O2inmethanolfor10minto

sup-presstheendogenousperoxidaseactivity,rinsedinTBSpH7.4,and

incubatedinalectinsolution(25␮g/mlfor1hatRTinthedark).

AfterthreerinsesinTBS,thesectionsweretreatedwith

strepta-vidin/peroxidasecomplexfor30minandsubsequentlywith0.05%

(w/v)3,3-diaminobenzidine(VectorLaboratories,Burlingame,CA,

USA)plus 0.003%(v/v)H2O2 in0.05MTBS (pH7.6)for10min.

SectionsweredehydratedandmountedusingEukitt.

Eachexperimentwasrepeatedtwiceforeachsample.Controls

forlectinstainingincluded(1)substitutionofthesubstratemedium

withbufferwithoutlectinand(2)incubationwitheachlectinin

thepresenceofitshaptensugar(0.5MinTBS).Allcontrol

experi-mentsgavenegativereactions.Slideswereobservedwiththelight

photomicroscopeEclipseNi-U(Nikon,Japan)at20×magniﬁcation

and photographedwitha digital camera(DS-U3, Nikon,Japan).

Theimageswereanalyzedbytheimage-analyzing programNIS

ElementsBR(Version4.20)(Nikon,Japan).

2.6. Morphometryandstatisticalanalysis

ThediameterofBrunner’sglandadenomeresfrombothNo-Pro

and Prosampleswasmeasuredon15microphotographicﬁelds

casuallydetectedandphotographedwithadigitalcamera(DS-U3,

Nikon,Japan)connectedtothelightphotomicroscopeEclipse

Ni-U(Nikon,Japan),usinga20_×lens.Imageswereanalyzedbythe

image-analyzingprogramNISElementsBR(Vers.4.30)(Nikon,JP).

Eachﬁeldsurfacewas140,000␮m2_._We_measured_the_diameter

of450transversallycutadenomeresofBrunner’sglandfromthe

No-ProandProsamples.Valueswereexpressedasmeans±SD.The

resultswereevaluatedforstatisticalsigniﬁcancebyStudent’sttest

andthecompareddatawereconsideredstatisticallysigniﬁcantat

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Table1

Lectinsused,theirsugarspeciﬁcities,andtheinhibitorysugarsusedincontrolexperiments.

Lectinabbreviation Sourceoflectin(␮g/ml) Sugarspeciﬁcity Inhibitorysugar

MALIIa _Maackia_amurensis ₂₅ _{NeuNAc˛2,3Gal␤1,3(±NeuNAc␣2,6)GalNAc} _NeuNAc

SNA Sambucusnigra 15 Neu5Ac˛2,6Gal/GalNAc NeuNAc

PNAb _Arachis_hypogaea ₂₅ _{Gal␤1,3GalNAc} _␤-D-Gal

RCA120 Ricinuscommunis 20 Gal␤1,4GlcNAc Gal

GSAI-B4 Griffoniasimplicifolia 20 ␣Gal Gal

DBA Dolichosbiﬂorus 25 GalNAc␣1,3(LFuc␣1,2)Gal␤1,3/4GlcNAc␤1 D-GalNAc

SBAb _Glycine_max ₂₀ _{␣/␤GalNAc} _D-GalNAc

HPA Helixpomatia 20 ␣GalNAc D-GalNAc

ConA Canavaliaensiformis 15 ␣Man>␣Glc Man

PHA-E Phaseolusvulgaris 20 Gal␤l,4GlcNAc␤1,2Man␣1,6 Man

PHA-L Phaseolusvulgaris 20 GlcNAc␤l,2Man,triantennarycomplexoligosaccharides Man

succWGAb _Triticum_vulgaris ₁₅ _␤GlcNAc _D-GlcNAc

GSAII Griffoniasimplicifolia 20 D-GlcNAc D-GlcNAc

UEAI Ulexeuropaeus 20 L-Fuc␣1,2Gal␤1,4GlcNAc␤ ␣-L-Fuc

LTA Lotus

tetragonolobus

25 L-Fuc␣1,6GlcNAc

L-Fuc␣1,2Gal␤1,4[L-Fuc1,3]GlcNAc␤1,6R ␣-L-Fuc Fuc:fucose,Gal:galactose,GalNAc:N-acetylgalactosamine,Glc:glucose,GlcNAc:N-acetylglucosamine,Man:mannose,NeuNAc:N-acetylneuraminic(sialic)acid,succ: succinylatedWGA.

a_{Biotin-labeled}_lectin.

b _{Rhodamine-labeled}_lectin._Non-marked_lectins_are_ﬂuorescein_{isothiocyanate-labeled}_lectins.

Fig.1. LightmicrographofpigduodenumshowingBrunner’sglands(Bg)and duodenalvilli(dv).Notetheduct(arrowhead)ofaBrunner’sglandpiercingthe muscularismucosae(asterisk)andenteringtheoverlyingduodenalmucosa.Inset displaysthedetailsofanacinousadenomere(a)andduct(d).Hematoxylin-eosin staining.

3. Results

3.1. Morphology

SwineBrunner’sglandsweretubulo-acinouswithamain

excre-toryduct opening at the base of the duodenal crypts (Fig. 1).

MorphologicalanalysisofBrunner’sglandsdidnotreveal

signif-icantdifferencesbetweentheNo-ProandProsamples.Therewas

alsonosigniﬁcantdifferenceinadenomerediameter,which

mea-sured42.66±7.38␮mintheNo-Proand44.3±6.32␮minthePro

specimens.

3.2. Glycohistochemistry

Theresultsofconventionalandlectinstainingpatternsof

Brun-ner’sglandsofbothNo-ProandPropigsaresummarizedinTable2.

ThecombinationAB2.5/PASprocedurerevealedthewidespread

presenceofPAS-positive(magenta)cellsandafewAB2.5-positive

(blue)cellsintheBrunner’sglandsfromNo-Prosamples(Fig.2A).

Thisstaining pattern was reversed in the Pro group Brunner’s

Table2

ConventionalandlectinhistochemistrystainingpatternoftheduodenalBrunner’s glandsofno-probioticandprobiotic-fedpigs.

No-probiotic Probiotic PAS + * AlcianBluepH2.5 * ++ HID − − SNA +/++lc +/++lc PHA-L +/++lc −/++lc PHA-E ±/++lc −/±lc succinylWGA −/+lc −/+lc GSAII ++ ++ ConA ++ + UEAI + ± GSAI-B4 ++ − LTA + − PNA + − RCA120 ± − HPA + ++ MALII − − DBA − − SBA − −

Lc:luminalcontent,*:fewreactivecells,−:negativereaction,±:faintlyvisible reaction,+:weakpositivereaction,++:intensepositivereaction.

glands(Fig.2B).ThecombinationHID/AB2.5methodsrevealedthe

absenceofHIDreactivity(brown)inbothProandNo-ProBrunner’s

glands(Fig.2C,D).

Comparisonof lectin reactivity in No-Pro and Pro Brunner’s

glandsshowedseveraldifferentbindingpatterns.ExceptforMALII,

DBA,andSBA,whichwereunreactivewithBrunner’sglandsfrom

bothNo-ProandPropigs,theotherlectinsboundtheglandularcells

and/ortheluminalcontent(SNA,PHA-L,PHA-E,succinylWGA)or

onlytheadenomericcells(GSAII,ConA,UEAI,GSAI-B4,LTA,PNA,

RCA120,HPA).TheresultsaresummarizedinTable2.

Inparticular,SNAdisplayednochangeinthestainingintensity

ofthesecretorycellsandtheadenomericluminalcontentof

Brun-ner’sglandsfrombothNo-ProandProanimals(Fig.3A,B);PHA-L

reactedwiththeadenomericcellsandtheluminalcontentof

No-ProBrunner’sglands(Fig.3C)and withtheadenomericluminal

contentoftreatedanimals(Fig.3D);PHA-Eboundweakly with

theadenomeresandstronglywiththeirluminalcontentinNo-Pro

specimens(Fig.3E),whereasitdidnotbindtheadenomeresbut

weaklystainedtheluminalcontentoftreatedpigs(Fig.3F);and

succinylWGAshowednoreactionwiththesecretorycells,whereas

itstainedtheluminalcontentinallinvestigatedBrunner’sglands

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Fig.2.Duodenumfromno-probiotic(A,C)andprobiotic-fedpigs(B,D),stainedwithPAS/AlcianBlue(AB)2.5(A,B)andHID/AB2.5(C,D)procedures.(A)No-probiotic Brunner’sglandsexhibitbroadPASpositivity(magenta)andafewAB2.5-positive(blue)cells.(B)Brunner’sglandsofprobiotic-fedpigsshowbroadAB2.5positivityand somePAS-positivecells.(A)and(B)demonstrateAB2.5-positivegobletcellsinduodenalvilli(dv).(C,D)HID/AB2.5methodsrevealedtheabsenceofHID(brown)reactivity intheBrunner’sglandsofbothno-probioticandprobiotic-fedanimals.ThereliabilityofHIDstainingwasdemonstratedbytheHIDpositivityofthegobletcells(arrow)in duodenalvilli(dv).Bg,Brunner’sgland.(Forinterpretationofthereferencestocolorinthisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.)

Concerningthelectinsthatboundonlytheadenomericcells,

GSAIIexhibitednodifferenceinthestainingintensitybetween

No-ProandProsamples.ConA(Fig.4A,B)andUEAI(Fig.4C,D)showed

strongerafﬁnityintheNo-Pro samplesthanintheProsamples.

GSAI-B4,LTA,RCA120,andPNAboundtheadenomericcellsofthe

Brunner’sglandsfromNo-Propigs,whereasitdidnotreactwiththe

secretoryepitheliumofProBrunner’sglands(Fig.4E,F).HPAwas

thesolelectinshowinga decreaseinreactivityintheBrunner’s

glandsofNo-PropigscomparedwiththatintheBrunner’sglands

ofPropigs(Fig.4G,H).

4. Discussion

Thisis theﬁrststudy todemonstratethat feedingof a

pro-biotic complex affects the glycosylation pattern of the mucins

secretedbypigBrunner’sglands.Thisinvestigationfollowsarecent

reportdemonstratingthattheuseofadietaryprobioticcomplex

(SivoyTM_,_SLAB51)_enhances_growth_performance_and_meat_quality

ingrowing-ﬁnishingpigs(Tufarellietal.,2017).

ConventionalhistochemistryrevealedthatBrunner’sglandsof

no-probiotic-fedpigsproduceprimarilyneutralglycoproteins(PAS

reaction),asmallamountofacidiccarboxylatedmucins(AB2.5

positivity), and no sulfoglycans (HID negativity). These results

are consistentwith those of previousstudies onthe Brunner’s

glandsofseveralmammals(Krause,2000;Verdiglioneetal.,2002;

Schumacheretal.,2004;ScillitaniandMentino,2015),including

youngadultpigs(TakehanaandAbe,1986).

Using15differentlectins,wecharacterizedthecarbohydrate

compositionofthemucinsfromBrunner’sglandsmorethoroughly.

Thesecretory epithelium ofBrunner’s glands from No-Pro pigs

showedthepresenceofbothN-andO-linkedglycans.N-Glycans

werehighmannose(ConA)andcomplextypes(PHA-L,PHA-E).

O-GlycanscontainedtheterminaldisaccharideGal␤1,3GalNA(PNA)

andthesimplestmucinO-glycanmadebyasingleGalNAclinkedto

serineorthreonine(HPA).Moreover,weobservedglycans

termi-natingwithfucose(LTA,UEAI),GlcNAc(GSAII),galactose(GSA

I-B4), lactosamine (RCA120), and Neu5Ac␣2,6Gal/GalNAc (SNA).

AlthoughourresultsagreewithpreviousreportsonpigBrunner’s

glands(TakehanaandAbe,1986;Gelbergetal.,1992),ourstudy

providesamorein-depthcharacterizationoftheoligosaccharide

chainsofBrunner’sglandglycoproteinsbecauseSNA,PHA-L,

PHA-E,LTA,andHPAhavenotbeenusedpreviously.Theluminalcontent

accumulatedhighmannoseandcomplextypesofN-glycansaswell

asglycanswithinternalGlcNAc(succinylWGA)andterminal

␣2,6-linkedsialicacid(SNA).Theroleofsugarresiduesinthesecretory

glycoproteins ofBrunner’s glands is not wellknown. However,

oligosaccharidechainscontaininglactosamineinhigh

mannosy-latedN-glycanshavebeenobservedinthehumanmucinMUC6

(Toribaraetal.,1997).Notably,thegeneencodingthelattermucin

isalsoexpressedinhumanBrunner’sglands(Bartmanetal.,1998).

Concerningsialic acid,thenegativechargeof thismoleculehas

beenshowntohavearoleinthetransportofchloride,

bicarbon-ate,water,andprotonsinBrunner’sglands(Collacoetal.,2013).

Insecretedmucus,sialicacidcancontributetotheviscosityand

protectionoftheunderlyingepitheliumfromlysisbygastricjuice

andbacteria(Schauer,2004)andcanactasaligandforseveral

symbioticandpathogenicmicroorganisms(Lehmannetal.,2006).

GalNAcandGalresidualscaninhibitthecellbindingofthe

tropho-zoitesofEntamoebahistolytica,thecausativeagentofamoebiasis

(RalstonandPetri,2011).Regardingfucosylatedresiduals,theycan

beimportantinboththemaintenanceofthebacterialﬂorainvolved

inthedegradationoffucosefromfood(BeckerandLowe,2003)and

inincreasingtheviscosityofmucus(Liquorietal.,2012).O-Glycans

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nat-Fig.3.LectinbindingpatternofBrunner’sglandsfromno-probioticandprobiotic-fedpigs.NotethedecreasedreactivityofPHA-LandPHA-EinBrunner’sglandadenomeres ofprobiotic-fedanimals.Bg,Brunner’sgland;dv,duodenalvilliwithpositivegobletcells;arrow,Brunner’sglandluminalcontent;arrowhead,basementmembrane.(A–F): FITC-conjugatedlectins;(G,H)rhodamine-labeledsuccinylatedWGA.

uralantibioticandasatumorsuppressorfordifferentiated-type

adenocarcinoma(Nakayama,2014).

Theprobioticblendinduceda greatchangeinthe

glycosyla-tionpathwayoftheepitheliumofBrunner’sglands.Conventional

histochemistryshowedadrasticreductioninneutralmucinsand

theprimarypresenceofcarboxylacidicglycoproteins.Inaddition,

lectinhistochemistryrevealedareductioninhighmannose(Con

A)and␣1,2-linkedfucosylatedglycans(UEAI)andthe

disappear-anceofcomplextypesofN-glycans(PHA-L,PHA-E)terminating

withlactosamine(RCA120)andfucosylatedoligomersbindingLTA,

aswellasO-linkedglycansterminatingwithGal␤1,3GalNAc(PNA)

and terminal galactose (GSA I-B4). Moreover, theluminal

con-tentofBrunner’sglandsexhibitedareducedamountofbisecting

GlcNAc-and Gal-bearingglycoproteins(PHA-E)whencompared

withthatfromprobiotic-fedpigs.However,thedietaryprobiotic

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Fig.4. LectinbindingpatternofBrunner’sglandsfromno-probioticandprobiotic-fedpigs.NoteConA,UEAI,andPNA(B,D,F)decreasedreactivity,aswellasHPA(H)increased afﬁnityintheBrunner’sglandsofprobioticfedpigs.Bg,Brunner’sgland;dv,duodenalvilli;it,interstitialtissue.(A–D,G,H)FITC-conjugatedlectins;(E,F)rhodamine-labeled PNA.

O-linkedglycansterminatingwithGalNAc(HPA).Theseﬁndings

areinlinewiththeevidencethatcomponentsofthedietandthe

gutmicroﬂoraareincontactwithintheintestinaltractandthat

dietarycomposition may inﬂuence thecarbohydrate structures

ofthemucosaland mucinglycoconjugateswithmarked

conse-quencesfortheadherenceofmicroﬂoraandforthegutitself(Kelly

etal.,1992).Somestudiesdemonstratedthatpre-treatmentwith

probioticsincreasedtheexpressionofMUC2,MUC5AC,andMUC6

in ratstomach(Caballero-Franco etal., 2007;Lam et al.,2007;

Gomietal.,2013).Moreover,experimentalevidencesupportsthe

hypothesisoftheadaptabilityofBrunner’sglandmucinstodietary

changes(Krause,2000;Desantisetal.,2011).

Inconclusion,this studydemonstratesthatprobiotic

supple-mentationaffectstheglycancompositionofthemucinsproduced

intheBrunner’sglandsofgrowing-ﬁnishingpigs.Sinceprobiotics

haveastimulatingeffectondigestiveprocessesandtheimmune

systemofpigs(Mengetal.,2010;Huetal.,2015;Liuetal.,2017)

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meatqualitythandidthecontrolbasaldiet(Tufarellietal.,2017),

webelievethattheobservedchangesinthecompositionofthe

secretorymucinsfromBrunner’sglands couldacteffectivelyon

thegastrointestinalfunctionandhealthstatusofanimals.However,

furtherstudiesarenecessarytounderstandthemechanismof

pro-bioticactionontheglycosylationpathwayofthemucinssecreted

byBrunner’sglands.

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