Jouany 2009 Levaduras equinos

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J.-P. Jouany, B. Medina, G. Bertin and V. Julliand

high-starch diettheir polysaccharidase and glycoside hydrolase activities in horses fed a high-fiber orEffect of live yeast culture supplementation on hindgut microbial communities and

doi:10.2527/jas.2008-1602 originally published online May 22, 2009;2009.87:2844-2852.J Anim Sci

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ABSTRACT: Four cecum an right ventra coon-fistuate horses were assigne in a 4 4 Latin squareesign an fe a high-fiber (HF) or a high-starch (HS)iet with or without 10 g ofSaccharomyces cerevisiae(SC; CBS 493.94) containing 4.5 109 cfu/g. The HFan HS iets consiste of peete fees an ong wheatstraw (18.0 an 3.5 g of DMkg1 of BW1, respec-tivey) given in 2 equa meas to provie an NDF:starchratio of 3.5 an 1.0, respectivey. After a 21- aapta-tion perio intestina contents were coecte 4 h afterthe morning mea on 23 an 25 to etermine bacte-ria an SC concentrations. Poysacchariase activities(CMCase, xyanase, amyase) an activities of gycosiehyroases (-l-arabinosiase, -d-ceobiosiase, -d-gucosiase, -d-xyosiase) were etermine in iqui-associate bacteria (LAB) an soi-aherent bacteria(SAB) isoate from both compartments. Lactobaciiwere increase in the cecum (P = 0.012) an coon(P= 0.086) when starch intake increase, whereas to-

ta anaerobes, ceuoytics, an streptococci i notchange in either compartment. In yeast-suppementehorses, SC concentrations were greater in cecum (4.4 106 cfu/mL) than in right-ventra coon (5.6 104cfu/mL) an i no change with iet. Concentrations

of actobacii an actic-aci utiizers were greater (P= 0.099 an 0.067, respectivey) in the cecum but re-maine simiar in the coon of SC-suppemente horses.The CMCase activities of SAB were not affecte byiet. Coonic xyanase activities of SAB were reuce(P = 0.046) by starch aition, but no change wasseen in the cecum. A SAB gucosie hyroase activi-ties in the cecum an coon, except -d-xyosiase inthe cecum, were ecrease when starch intake was in-crease. The LAB CMCase (P= 0.049 in the coon)an xyanase (P= 0.021 in the cecum; P< 0.001 inthe coon) activities ecrease with starch intake. Noeffect of starch on LAB or SAB amyase activity wasobserve. Aition of SC improve SAB CMCase inthe cecum (P= 0.019) an coon (P= 0.037) as weas -d-ceobiosiase (P= 0.002) an -d-gucosiase(P = 0.041) in the cecum. Ony xyanase in the ce-cum (P= 0.015) an -d-xyosiase in the cecum (P= 0.028) were improve with SC, whereas coonic LAB

-amyase activity was significanty ecrease (P =0.046). Most enzymes invove in pant ce wa iges-tion were increase after SC aition. This fact maycontribute to expain a better igestion of fiber that hasbeen previousy reporte in SC-suppemente horses.

Key words: ceuoytic, enzymatic, fiber igestion, horse, arge intestine, Saccharomyces cerevisiae

2009 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2009. 87:28442852oi:10.2527/jas.2008-1602

INTRODUCTION

Horses have eveope a speciaize igestive systemthat aows them to utiize ceuosic an hemiceuosic

materias via microbia egraation that occurs in thehingut an provies them with energy via the pro-uction of VFA. Athough the microbia breakown ofpant materia occurs at the beginning of the igestivetract of ruminants an at the en of the igestive tractof horses, the mechanisms invove in the igestion ofceuosic materia have great simiarities in their basicapproach in both types of animas. Thus, in horses, ikeruminants, the efficiency of microbia igestion of ceu-osic fee is greaty epenent on feeing practices. Forexampe, the aition of grain concentrates to the ietcan ater the microbia popuation an its enzymatic

Effect of live yeast culture supplementation on hindgut microbialcommunities and their polysaccharidase and glycoside hydrolase

activities in horses fed a high-fiber or high-starch diet1

J.-P. Jouany,* B. Medina, G. Bertin, and V. Julliand2

*Institut Nationa e a Recherche Agronomique (INRA), UR1213 Herbivores, Site e Theix,

63122 Saint Genes Champanee; Atech-France, 14 Pace Marie-Jeanne Bassot, 92300 Levaois-Perret;an Etabissement Nationa Enseignement Suprieur Agronomique e Dijon (ENESAD),

26 Bouevar Dr Petitjean, BP 87999, 21079 Dijon Ceex, France.

1The authors thank D. Juniper (University of Reaing, UK) forhis support on reviewing the raft of this manuscript an J. Wi-iams (Atech-France) an J. Gobert (ENESAD) for their contribu-tion.

2Corresponing author: [email protected] November 3, 2008.Accepte May 18, 2009.

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activities in ifferent ways epening on the botani-ca origin of cereas, fee processing, or the amount ofstarch that is ingeste. In horses, a eve of starch in-take greater than 2% BWmea1 enhances the quantityof starch reaching the hingut, which can have a nega-tive effect on the microbia fibroytic activity an affectfiber egraation (Juian et a., 2006).

Because the aition of ive yeasts has now been rec-

ognize as a way to improve the igestive efficiency ofrumen microbes (Jouany et a., 1998a,b; Chaucheyras-Duran et a., 2007) attempts have been mae to eter-mine a simiar effect on the igestive efficacy of the mi-crobia ecosystem of horses offere the same probiotic,especiay when animas are fe high-starch (HS) iets.Previous stuies in equines inicate that ive yeastscan increase the concentration of tota anaerobic bacte-ria, change the pattern of hingut fermentation (Mei-na et a., 2002), an improve the igestibiity of ietaryceuose (Gae, 1991a,b; Jouany et a., 2008). In rumi-nants fe a high concentrate iet, the effect of ive yeaston fiber egraation has been associate with increase

poysaccharie-egraing activities of the soi-associ-ate bacteria fraction of rumina contents (Jouany eta., 1998a; Chaucheyras-Duran an Fonty, 2006). Suchan impact on microbia fibroytic activities in the ce-cum or coon of equines remains ignore. Therefore,the objective of the present work was to evauate theeffect of suppementing the iet of horses with a yeastcuture Saccharomyces cerevisiaeCBS 493.94 (SC) onceca an coonic concentrations of soi-aherent bac-teria (SAB) an iqui-associate bacteria (LAB) inthe igestive content of the equine hingut an theirpoysacchariic an gycosie hyroase activities whenanimas were fe a high-fiber (HF) or HS iet.

MATERIALS AND METHODS

The experiment was conucte in the anima re-search unit of ENESAD (Etabissement NationaEnseignement Suprieur Agronomique e Dijon) un-er a icense eivere by the Heath an Anima We-fare Department of the French Veterinary Authority.

Animals

Four crossbre mae horses (12 5 yr) with an aver-age BW of 387 19.1 kg at the start of the experimentwere use. Animas were fitte with cecum an right-ventra coon poyviny chorie cannuae (i.. 30 mm)at east 6 mo before the beginning of the experiment.

Horses were worme 2 wk before the experimentstarte with a oube ose of pyrante (Strongi, Labo-ratoire Pfizer, Orsay, France) foowe 1 wk ater by asinge ose of ivermectin (Eqvaan, Laboratoire Meria,Lyon, France). Animas were kept insie the barn ur-ing the entire experimenta perio an were given ac-cess to a sany paock for 10 h per wk uring the ietaaptation perio (21 ) preceing each experimentaperio. Inoor housing consiste of concrete-foore in-

iviua boxes (2.0 2.5 m) bee with fax shavings(ECOLIT, Croissanvie, France).

Diets

The physica form an composition of iets were cho-sen to mimic norma French feeing practices use inhorse riing schoos. Animas were fe the 2 foowingiets: a peete HF iet or a peete HS iet (Tabe

1); both peets were offere in a mixture of ong wheatstraw as a coarse ingreient. The HF an HS iets werepresse into 3-mm iameter peets after grining theingreients to a partice size of 1.5 mm. The 2 iets weresuppemente (HF+SC; HS+SC) or not (HF+0;HS+0) with a yophiize cuture of Saccharomycescerevisiaestrain CBS 493.94, pus the growth meium(ot 22700, Yea-Sacc, Atech Inc., Lexington, KY). Theive yeast cuture suppement containe 4.5 109 cfu/gan was given at a ose of 10 g1horse1.

The minimum aiy vountary intake of wheat straw(3.5 g of DMkg of BW11), which was etermineon a horses uring a pre-experimenta perio usingthe same HF an HS peets, was then given for theentire uration of the experiment. The 4 iets were is-tribute at the same eve 21.5 g of DMkg1 of BW1(i.e., 18.0 g of peete fees + 3.5 g of ong wheatstraw) to minimize its impact on the rate of passage ofigesta (Drogou et a., 2001; Pearson et a., 2001) anminimize its impact on microbia activities. This intakeeve provie 100 an 130% of energy requirements forHF an HS fe animas, respectivey (Martin-Rosset eta., 1994).

The HF an HS iets provie a range of NDF:starchratios that were arge enough (3.5 an 1.0 for the HF

Table 1. Composition of the high-fiber (HF) an high-starch (HS) iets

Item HF iet1 HS iet2

DM, % of fresh matter 88.7 88.0OM, % of DM 88.2 90.3CP, % of DM 12.6 12.7NDF, % of DM 41.0 30.7ADF, % of DM 25.8 16.1

ADF-ADL,3

% of DM 20.5 13.1NDF-ADF,4 % of DM 15.2 14.6Starch, % of DM 11.6 30.1DE,5 Mca/kg of DM 2.63 3.19

1The HF an HS iets ha the foowing composition, respectivey,on a DM basis (%): ong wheat straw (16.3 an 16.3), ehyrateafafa (46.0 an 8.3), wheat bran (21.3 an 18.8), barey (10.9 an44.8), soybean mea (0.0 an 6.4), CaCO3 (2.12 an 2.98), sugar canemoasses (1.63 an 0.80), NaC (0.81 an 0.80), an mixture of vita-mins an trace eements (5.4 an 5.4).

2Suppie per kiogram of mixture: vitamin A, 15,000 IU; vitaminD3, 2,500 IU; vitamin E, 135 IU; biotin, 0.2 mg; an Cu, 25 mg.

3The ifference ADF-ADL was use to estimate the ceuose con-tent.

4The ifference NDF-ADF was use to estimate the hemiceuosecontent.

5Digestibe energy was cacuate from equations reporte by Fon-nesbeck (1981).

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an HS iets, respectivey) to assess possibe interac-tions between iet composition an yeast content. Dai-y rations were offere as 2 equa meas in iniviuatroughs at 0800 an 1700 h. Mean ingestion of starchwas equa to 2.7 an 1.0 gkg1 of BWmea1 with HSan HF iets, respectivey. Peete iets were initiayistribute with or without the SC cuture top-resse(5 g/mea), an the wheat straw fraction of the iet

was provie 30 min ater.Horses were weighe on 2 consecutive ays beforeeach aaptation perio to ajust fee aowances to ac-coring to BW. Animas were given iniviua free ac-cess to cean potabe rinking water an a ick bockof trace-minera sat (composition per kg of mixture:vitamin A, 15,000 IU; vitamin D3, 2,500 IU; vitamin E,135 IU; biotin, 0.2 mg; an Cu, 25 mg).

Experimental Design

The 4 horses were istribute in a 4 4 Latin squareesign [4 animas, 4 perios, 2 iets (HF an HS) an

2 treatments within each iet (with an without SC)].Animas were aapte to iet for a perio of 21 pre-ceing each perio.

Collection of Digesta Samples

Approximatey 200 mL of igesta were sampe on 23 an 25 of each experimenta perio 4 h after themorning mea through the ceca an coonic cannuaefor microbia an enzymatic anaysis, respectivey. Thefraction of igesta trappe insie the cannuae wascarefuy remove; then the fui fraction of the igestawas coecte in a sterie botte saturate with CO2 an

maintaine at 39C.

Enzyme Extraction from SAB and LAB

Bottes containing ceca an coonic igesta werequicky transferre to an anaerobic chamber kept unerCO2 where the foowing proceures were unertaken.Sampes of igesta were homogenize by han agita-tion of the bottes an then straine through a 100-mnyon fiter (Bute, SAATI Inc., Saiy Saiise, France)to separate the soi phase from the iqui phase. Thefitrate cae Fitrate 1 was pace uner anaerobicconitions into a fask for further treatment.

Exacty 15 g of soi phase was washe in 200 mL ofanaerobic buffer prewarme at 39C to remove nona-herent bacteria. The SAB were recovere by fitration(100 m). The fitrate cae Fitrate 2 was pace intoa fask for further treatment. A fitrations were maeuner anaerobic conitions.

Five grams of the previousy washe igesta contain-ing the SAB was suspene at 4C into 20 mL of aMESDTT soution consisting of an anaerobic MES buf-fer soution mae of 0.025 Mof sufonic aci 2-(N-mor-phoin) ethane at pH 6.5 containing 1 mMithiothreito(DTT) an store at 80C before enzyme extraction.

Two successive cyces of freezing to 20C an thawingwere appie to the microbia suspension accoring tothe methos of Noziere an Michaet-Doreau (1994) toreease the attache bacteria. The suspension was thenpace in ice an sonicate uner anaerobic conitions(4 cyces for 30 s with 30-s intervas) to break microbiaces. Finay, the suspension was centrifuge at 15,000 gfor 15 min at 4C to remove unbroken ce materia,

an anayses of enzymatic activities of SAB were maeon supernatants. As inicate by Noziere an Michaet-Doreau (1994), the enzymes of fee origin were consi-ere as negigibe because they represent ess than 5%of tota nitrogen of enzymes in the supernatants.

Fitrates 1 an 2 were combine an centrifuge at15,000 gfor 15 min at 4C. The peets that containethe LAB were subjecte to the same treatments as theSAB fraction. They were suspene into a MESDTTsoution, then frozen an thawe, sonicate, an finaycentrifuge. A steps from fitrates unti enzyme prep-aration were performe uner anaerobic conitions.Anayses of enzymatic activities of LAB were mae on

supernatants.

Determination of Enzyme Activities

Poysacchariase activities invove in the iges-tion of pant ce was (CMCase, xyanase) an starch(amyase) were etermine accoring to the methosof Martin an Michaet-Doreau (1995). The amountsof reucing sugars iberate from purifie poymers(Birchwoo-xyan, Sigma X-0502; carboxymethyce-uose, Sigma C-5678; potato starch, Sigma S-2002;Sigma-Arich Chemie, Lyon, France) after incubationwith ceuar extracts, were use to cacuate enzyme

activities. The voumes of LAB an SAB extracts, theamounts of substrates, an the incubation times aregiven in Tabe 2. The reaction was stoppe by heatingat 100C for 5 min. Reucing sugars were quantifiespectrophotometricay at 410 nm (Lever, 1977).

Activities of gycosie hyroases (-l-arabinosiase,-d-ceobiosiase, -d-gucosiase, -d-xyosiase)were estimate through the reease of para-nitrophe-no from para-nitropheno erivate substrates (para-nitropheno--l-arabinofuranosie, Sigma N-3641;para-nitropheno--d-ceobiosie, Sigma N-5759;para-nitropheno--d-gucopyranosie, Sigma N-7006;para-nitropheno--d-xyopyranosie, Sigma N-1232;Wiiams et a., 1984). Incubations were carrie out asescribe by Wiiams et a. (1984; 45 min at 39C uneragitation in a water bath). The reaction was stoppeby increasing pH with a 0.4 Msoution of gycine/soa(1/1 vo/vo). The reease paranitropheno was oseby spectrophotometry at 420 nm. Banks were mae forenzymes an substrates to assess the rea activities ofextracte enzymes.

Protein content of enzyme preparations was eter-mine accoring to the metho of Pierce an Sueter(1977) using BSA as the stanar. Enzyme activitieswere expresse as specific activities [i.e., as the quan-

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tity of reucing sugar (for poysacchariases) or parani-tropheno (for gycosiases) reease per mg of proteinper unit of time].

Concentrations of Bacterialand Yeast Populations

Total Viable Anaerobic Bacteria. Bacteria werecutivate for 96 h at 38C in ro tubes prepare witha moifie compete agar meium (Leee an Hespe,1980; Juian et a., 1999). Four repicate tubes were

use for each 106, 107, an 108 iution of intestinaigesta.

Cellulolytic Bacteria. Ceuoytic bacteria werecutivate with a broth meium (Haiwe an Bry-ant, 1963; Juian et a., 1999) for 15 at 38C. Theconcentrations were cacuate by the most probabenumber metho on 4 tubes inocuate with 105, 106,an 107 iutions of each intestina igesta.

Lactic Acid-Utilizing Bacteria. Lactic aci uti-izing bacteria were seectivey cutivate for 96 h at38C on the meium use by Mackie an Heath (1979).Four repicate ro tubes were prepare for each 105,106, an 107 iution of intestina igesta.

Streptococci spp. An overay metho with bie es-cuin azie agar meium (BK158HA, Biokar iagnos-tics, Beauvais, France) was use to enumerate Strep-tococcus. Three repicate Petri pates prepare with105, 106, an 107 iutions of intestina igesta werecounte after 48 h of incubation at 38C.

Lactobacilli spp. The overay metho with a Ro-gosa agar meium (BK158HA, Biokar iagnostics,Beauvais, France) was appie to 3 repicate Petripates prepare from 105, 106, an 107 iutions ofintestina igesta an incubate for 48 h at 38C.

Saccharomyces cerevisiae. Thirty miiiters of

igesta were iute with 270 mL of sterie water anhomogenize for 3 min in a stomacher (Stomacher 400Lab Bener, Sewar Meica, Lonon, UK). Yeastswere numerate in a Sabourau meium (BK025HA,Biokar iagnostics, Beauvais, France) with an ethanosoution of choramphenico (0.17%). Three repicatePetri pates were counte for each iution from 103to 107 of igestive content after 48 h of incubation at35C. The genetic profie of ive yeasts were systemati-cay checke accoring to the PCR metho with spe-cific 1 primers (Ness et a., 1993) an was comparewith the profie of the CBS 493.94 strain of the prouctYea-Sacc1026.

Calculations and Statistical Analyses

Data were anayze using the MIXED proceure(SAS Institute Inc., Cary, NC) for each igestive com-partment. The moe use, Yij = + Ai + Pj + (Dk+ SC) + Dk SC + Eijk, incue the anima (A),perio (P), iet (D), yeast (SC) as singe effects, the D SC interaction, an E as resiua error.

Ony iet an yeast effects, as we as their interac-tion, wi be iscusse in the present paper. Logarithmictransformations were performe on microbia counts for

statistica anayses. Means were compare by FishersLSD when a significant overa treatment F-vaue wasobserve. Two eves of significance (P< 0.10 an P 0.10) by iet(HS or HF; Tabe 3).

Concentration of Total Anaerobic Bacteriaand Cellulolytic Bacteria

The concentrations of tota anaerobic bacteria or ce-uoytic bacteria in the cecum an the coon were notaffecte (P> 0.01) by SC aition or by ietary coni-

tions (Tabe 4).

Concentration of Bacteria Involved in LacticAcid Metabolism

There was no effect of iet on the concentration ofactic aci-utiizing bacteria present in the ceca an thecoonic content, whereas actobacii were more numer-ous in the cecum (P= 0.012) an coon (P= 0.086) ofhorses fe the HS iet. Saccharomyces cerevisiaeCBS493.94 aition increase concentrations of actic aci-utiizing bacteria an actobacii (P= 0.067 an 0.099,respectivey) in the cecum but ha no effect (P> 0.10)

Table 2. Protoco use for the etermination of enzymatic poysacchariase activitiesin equine intestina content

Enzyme

Voume of soution, mLIncubationtime, min

Stanar soution, range of sugarconcentrations (number of pointson the stanar curve)Bacteria extract Substrate

Amyase 0.2 2.0 80 0 to 30 g of gucose/mL (6)CMCase 0.5 2.0 70 0 to 30 g of gucose/mL (6)Xyanase 0.2 2.0 60 0 to 30 g of xyose/mL (6)




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on bacteria in the coon. Concentrations of streptococciwere not affecte (P> 0.10) by iet or SC aition forceca an coonic igestive contents (Tabe 4).

Polysaccharidase Activities of SAB

No ietary or yeast effects were observe on amyaseactivity of SAB isoate from the coon or cecum of

horses. The CMCase activities of SAB were improvewith SC suppementation in the cecum (P= 0.019) ancoon (P= 0.037) but were not affecte (P> 0.10) byiet in either igestive compartment.

Xyanase activities of SAB isoate from the coonwere greater (P= 0.046) with HF iet than HS iet, buti not change with SC suppementation (P> 0.10).Xyanase activities of ceca SAB were not affecte (P>0.10) by iet or yeast suppementation (Tabe 5).

Glucoside Hydrolase Activities of SAB

Vaues of -d-ceobiosiase activities of SAB were

greater in both igestive compartments (P< 0.001) inthe coon (P= 0.072 in the cecum) when HF iets werecompare with HS. A significant an positive effect ofSC on -d-ceobiosiase activities was observe in thececum (P= 0.002) but not in the coon (P= 0.954).

The -d-gucosiase activities of SAB were greater inhorses fe the HF iet than the HS iet (P= 0.001 inthe cecum; P< 0.001 in the coon). A significant anpositive effect of SC aition (P= 0.041) was observeon -d-gucosiase activities of SAB isoate from thececum but not in those isoate from the coon (P>0.10).

The -l-arabinosiase activities of SAB from the co-

on an cecum ha greater vaues (P

< 0.001) in HFthan HS iet. The aition of SC improve arabinosi-ase activities of SAB in the coons of horses fe theHF iet (P= 0.056) but ha no effect on arabinosiaseactivities of SAB extracte from the cecum regaressof iet.

The -d-xyosiase activities of SAB ha greatervaues (P = 0.015) in the coon when animas werefe HF iet than HS. Saccharomyces cerevisiae CBS493.94 stimuate bacteria -d-xyosiase activities inthe coon an cecum (P< 0.05) of horses fe the HSiet, which reache the vaues observe with HF iet(Tabe 5).

Polysaccharidase Activities of LAB

Amyase activities of LAB isoate from the cecuman coon were not affecte (P> 0.10) by iet. Sac-

Table 3. Concentrations (og cfu/mL) of ive Saccharomyces cerevisiaeCBS 493.94 strain in the cecum an coonof fistuate horses (n = 4/treatment) fe a high-fiber (HF) or a high-starch iet (HS) with (+SC) or without (+0)ive yeast suppementation

Digestivecompartment

Diet

SEM

P-vaue

HF + 0 HF + SC HS + 0 HS + SC Diet Yeast Diet yeast

Cecum NDa 6.4b NDa 6.8b 0.7 >0.10 0.10Coon NDa 4.9b NDa 4.5b 0.5 >0.10 0.10

a,bMeans with ifferent superscripts on the same ine are ifferent (P< 0.05); ND: ces ofS. cerevisiae(strain CBS 493.94) were not etect-e.

Table 4. Ceca an coonic bacteria concentrations in fistuate horses (n = 4/treatment) fe a high-fiber (HF)or a high-starch iet (HS) with (+SC) or without (+0) ive yeast suppementation

ItemDigestivecompartment

Diet

SEM

P-vaue


Tota anaerobic bacteria, og10 cfu/mL Cecum 7.4 7.7 7.9 8.5 0.34 0.105 0.210 0.693Coon 7.6 7.8 8.2 7.9 0.20 0.139 0.722 0.206

Ceuoytic bacteria, og10 MPN1/mL Cecum 4.3 5.3 4.7 4.2 0.40 0.410 0.568 0.137

Coon 4.7 5.1 5.4 4.7 0.44 0.803 0.728 0.271Lactic-aci utiizing bacteria, og10 cfu/mL Cecum 6.5 7.0 6.9 7.5 0.25 0.118 0.067 0.792

Coon 6.5 6.5 7.2 6.7 0.28 0.170 0.465 0.391Lactobacillusspp., og10 cfu/mL Cecum 6.5

b 6.6b 6.8ab,B 7.2a,A 0.13 0.012 0.099 0.337Coon 6.1B 6.2B 6.7A 6.8A 0.29 0.086 0.884 0.983

Streptococcusspp., og10 cfu/mL Cecum 6.9 6.7 6.5 6.9 0.18 0.633 0.711 0.123Coon 6.4 6.5 6.8 6.8 0.21 0.166 0.870 0.682

a,bMeans with ifferent superscripts within a row are ifferent (P< 0.05).A,BMeans with ifferent superscripts within a row are ifferent (P< 0.10).1MPN = most probabe number.

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charomyces cerevisiaeCBS 493.94 aition reuce (P= 0.046) amyase activity in the coon of animas fethe HS iet.

The CMCase activities of LAB isoate from the ce-cum were not affecte by iet (P= 0.940). Those iso-ate from the coon ha greater vaues when horseswere fe the HF iet rather than the HS iet (P =0.049). Saccharomyces cerevisiaeCBS 493.94 aitionha no significant effect on CMCase activities in thecoon (P= 0.554) or in the cecum (P= 0.551) irrespec-

tive of iet.

Xyanase activities of LAB were greater with the fi-ber-rich iet than in the iet rich in starch (P= 0.021in the cecum; P< 0.001 in the coon). The aitionof SC increase (P< 0.05) xyanase activities of LABin the coon of horses fe HF iet an in the cecum ofthose fe HS iet (Tabe 6).

Glucoside Hydrolase Activities of LAB

No effects of iet or treatment on -d-ceobiosiase

activities of LAB isoate from the coon an cecum (P

Table 5. Enzymatic activities of soi-aherent bacteria (SAB) in the coon an cecum of fistuate horses (n =4/treatment) fe a high-fiber (HF) or a high-starch iet (HS) with (+SC) or without (+0) ive yeast suppementa-tion

ItemDigestivecompartment

Diet

SEM

P-vaue


-Amyase1 Cecum 513.5A 381.1A 329.7B 529.0A 73.2 0.808 0.652 0.033Coon 410.7 366.0 398.8 517.3 112.0 0.540 0.745 0.474

CMCase1 Cecum 136.6b 411.7a 118.0b 192.7b 69.4 0.101 0.019 0.162Coon 232.9AB 357.3AB 193.2B 422.4A 80.0 0.874 0.037 0.518

Xyanase1 Cecum 371.6 484.7 458.7 539.1 66.4 0.298 0.159 0.807Coon 813.6ab 1,015.0a 547.2b 670.5ab 144.6 0.046 0.273 0.789

-d-Ceobiosiase2 Cecum 715.9b 1,381.3a 486.1b,B 974.1ab,A 170.4 0.072 0.002 0.598Coon 1,112.7a 1,018.5a 546.8b 627.8b 112.6


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> 0.05) were observe. The -d-gucosiase activitiesof LAB in the coon, which were not ifferent betweenthe 2 iets (P= 0.136), were improve by SC suppe-mentation (P= 0.072). In the cecum, -d-gucosiaseactivities of LAB were not affecte by iet or yeast a-ition (P> 0.10).

The -l-arabinosiase activities of LAB were not af-fecte (P> 0.10) by iet or SC aition irrespective of

the igestive compartment. The -d-xyosiase activi-ties of LAB increase with SC suppementation (P=0.028) in the cecum. Those obtaine from the cooni not change (P> 0.10) regaress of iet or yeastsuppementation (Tabe 6).

DISCUSSION

The resuts of this stuy confirm that a arge partof ingeste ive SC can survive uring transit throughthe igestive tract of horses reaching the cecum ancoon (Newman an Spring, 1993; Moore et a., 1994).Gobert et a. (2006) reporte that ive SC fe at simi-

ar concentrations to those of the present stuy ap-peare in the coonic content of horses 3 h after theiringestion. In the present stuy, ive SC concentrationfoun in the cecum (106 cfu/mL) is equivaent to theconcentration usuay foun in the rumen (Fiems et a.,1993; Duran-Chaucheyras et a., 1998) or in the ieum(Newbo et a., 1990) of ruminants fe approximateythe same ora ose. In agreement with previous ata(Gobert et a., 2006), the concentration of ive SC inthe coon was ess, an beow the thresho of efficacy(105 cfu/mL) areay set for the rumen (Brossar et a.,2006). This may expain why changes ue to ive SCsuppementation were observe more frequenty within

ceca contents than coonic contents.For the first time, the technique of separation of LAB

an SAB escribe for rumen bacteria (Martin et a.,1993) has been appie to ceca an coonic igestivecontents of horses, with each type of bacteria being ana-yze in terms of bacteria concentration an enzymaticactivities. Like the rumen of cows an sheep (Martin eta., 1993; Eugne et a., 2004) the activity of poysac-chariases invove in ce-wa egraation originatesmainy from bacteria boun to partices (SAB fraction)isoate from coon or cecum content. Amyase activi-ties were aso greater in SAB than in LAB.

Simiary, gucosie hyroases showe a esser spe-cific activity in the iqui fraction when compare withthe soi fraction, but the ifferences between the 2bacteria fractions varie accoring to the enzyme be-ing consiere. For exampe, -d-ceobiosiase activi-ties of LAB an SAB were comparabe, whereas -d-gucosiase, -d-xyosiase, an -l-arabinosiase weremuch greater in SAB than LAB. The vaues foun herefor -d-xyosiase in the iqui fraction of ceca con-tents are simiar to those previousy pubishe for hors-es (Bonhomme-Forentin, 1988).

Comparison of poysacchariase activities in the ce-cum an coon of horses were cose to an agree with

the concentrations of ceuoytic bacteria, which wereaso very simiar in both igestive compartments. Thisresut agrees with previous numeration of ceuoyticbacteria etermine simutaneousy in the ceca anthe coonic contents, which showe that the microbiaensities were not significanty ifferent between the 2igestive compartments (Moore et a., 1994; Juianet a., 2001; Meina et a., 2002; e Fombee et a.,

2003). However, the proportion of cutivabe ceuoyt-ics among tota anaerobes appeare to be greater inthe cecum than in the ower parts of the hingut anconfirme that this bin pocket was probaby the mostpropitious igestive compartment for ceuoysis (eFombee et a., 2003).

The profies an activities of the intestina micro-fora in horses have been reporte to be moifie bythe NDF:starch ratio of the iet (Meina et a., 2002).Activity of most poysaccharie epoymerases angycosie hyroases reating to fiber egraation weregreater in HF than in HS iet, whereas the numberof ceuoytic bacteria remaine unchange regaress

of iet. This resut with enzymes impies that morefiber was igeste in the HF iet. In a previous stuyconucte with the same experimenta esign an simi-ar iets, the igestibiity of ADF was greater in HFiet than HS iet, which corroborates the present ata(Jouany et a., 2008). These ata aso agree with thegreater acetate concentration reporte by Meina eta., (2002) who observe that the concentration of ce-uoytic bacteria remaine unchange between the 2iets. This suggests that the iet affecte the activityof ceuoytic (an hemiceuoytic) bacteria withoutaffecting their tota concentration when measure withconventiona cutura techniques. A simiar stuy con-

ucte in the rumen of cows showe that the aitionof a arge amount of starch to a hay-base iet simi-ary ecrease the fibroytic activity of the 3 main ce-uoytic bacteria (Ruminococcus albus, R. flavefaciens,an Fibrobacter succinogenes) without moifying theirnumber (Martin et a., 2001).

Amyase activities of LAB an SAB isoate from thehingut of horses were not affecte by iet athoughthe amount of ietary starch was significanty ifferentbetween HF an HS iets. Such a resut is ikey ex-paine by the igestion of starch in the sma intestine,which was probaby compete for the HF iet (intake= 1.0 g of starchkg1 of BWmea1) but aowe asma amount of starch to reach the hingut for the HSiet (intake = 2.7 g of starchkg1 of BWmea1). Theincrease in starch-igesting bacteria concentration suchas actobacii confirms this hypothesis. However, thesuppy of starch to the arge intestine of HS-fe horseswas not sufficient enough to significanty moify bacte-ria amyase activities.

Like the rumen, starch is converte in the hingut ofhorses by amyoytic bacteria into actic aci (Garneret a., 1978; Rowe et a., 1994; Meina et a., 2002),which is then metaboize into propionate by actic ac-i-utiizing bacteria. Using simiar iets, Juian et a.

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(2001) an Meina et a. (2002) note that actic acian propionate i not accumuate in the hingut ofhorses, which confirms that the amount of starch escap-ing the igestion in the sma intestine an reaching thehingut was probaby sma, even for the HS iet.

Saccharomyces cerevisiae CBS 493.94 suppemen-tation ha no effect on the concentrations of a theteste bacteria communities in the coon of horses.

In contrast, the popuations of actobacii an acticaci-utiizing bacteria in the cecum increase when SCwas present. This specific resut on bacteria invove inthe metaboism of actic aci is ikey expaine by thegreater concentrations of ive yeasts foun in the cecumthan in the coon of animas (4.4 106 cfu/mL vs.5.6 104 cfu/mL) as previousy mentione. The samereason cou expain why SC improve more CMCas-es, ceobiosiases, -d-gucosiases, an xyosiases ofSAB in the cecum than in the coon.

These ata unerine that arge simiarities exist be-tween the rumen an the hingut of horses with regarof the effects of ive yeasts on the igestive microbia

ecosystem an its activities (Waace an Newbo,1992; Chaucheyras et a., 1996; Doreau an Jouany,1998; Chaucheyras-Duran an Fonty, 2006). There-fore, ive yeasts can be use in horses to baance anstabiize the igestive microbia ecosystem as they arein ruminants. Their main positive effect is ue to in-creases in the enzymatic activities of bacteria invovein the igestion of ceuosic materia rather than as airect effect on bacteria biomass. This effect is ikeymagnifie in the case of a high suppy of fermentativecarbohyrates (sugars or starch) in the igestive tract.Athough prececa igestion, which occurs in horses,tens to imit the risk of aciosis in the hingut, an

overoa of grain in the iet can affect the coonic eco-system an generate microbia an igestive isorerseaing to ecrease igestion of ce wa carbohyrates(e Fombee et a., 2001; Juian et a., 2001), whichcan be correcte by aition of ive yeasts to the ietof animas.

The increase in the major enzymes invove in pant cewa igestion (e.g., CMCases, -d-ceobiosiases, -d-xyosiases, -d-gucosiases, an -l-arabinosiases inthe cecum or coon of horses after SC aition to HSiet) partiay expain why the fiber fraction is betterigeste by SC-treate horses fe with a high-cereaiet (see Jouany et a., 2008). The same mechanism asescribe in the rumen by Caaway an Martin (1997)who inicate that yeasts stimuate growth of bacte-ria that utiize actate an igest ceuose an can beappie to horses fe an HS iet [because SC tene(P= 0.067) to increase the popuation of actic-aciutiizing bacteria]. In aition, our experiment showesignificant an positive effects of SC suppementationon CMCases, -l-arabinosiases, -d-ceobiosiases inhorses fe an HF iet. Athough no expanation coube given for such evoution, it can be concue thatyeasts have positive effects on the utiization of fiber inhorses regaress of iet.

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