Low Ruminal ph Reduces Dietary Fiber Digestion via Reduced Microbial Attachment

200 Asin-Aust. J. Anim. Si. Vol. 20, No. 2 : 200-20 Ferury 200 www.js.info Low Ruminl ph Redues Dietry Fier Digestion vi Redued Miroil Atthment H Guyn Sung, Ysuo Koyshi 1, Jongsoo Chng 2, Ahnul H, Il Hwn Hwng nd J. K. H* Shool of Agriulturl Biotehnology, College of Agriulture nd Life Sienes Seoul Ntionl University, Seoul 11-2, Kore ABSTRACT : In vitro rumen inution studies were onduted to determine effets of initil ph on teril tthment nd fier digestion. Ruminl fluid ph ws djusted to.,.2 nd., nd three mjor firolyti teri tthed to rie strw in the mixed ulture were quntified with rel-time PCR. The numers of tthed nd untthed Firoter suinogenes, Ruminoous flvefiens nd Ruminooous lus were lower (p<0.0) t initil ph of. without signifint differene etween those t higher initil ph. Lowering inution medi ph to. lso inresed teril numers dethed from sustrte regrdless of teril speies. Dry mtter digestiility, gs umultion nd totl VFA prodution were ph-dependent. Unlike teril tthment, mintining n initil ph of. inresed digestion over initil ph of.2. After h in vitro rumen fermenttion, verge inreses in DM digestion, gs umultion, nd totl VFA prodution t initil ph of.2 nd. were 2. nd., 2.0 nd 3.0, nd 1.2 nd 1. times those t initil ph of., respetively. The lg time to reh ove 2% DM digestiility t low initil ph ws tken more times ( h) thn t high nd middle initil ph ( h). Current dt lerly indite tht ruminl ph is one of the importnt determinnts of fier digestion, whih is modulted vi the effet on teril tthment to fier sustrtes. (Key Words : Bteril Atthment, Fier Digestion, ph, Firoter suinogenes, Ruminoous flvefiens, Ruminooous lus). INTRODUCTION The min soure of energy for ruminnts is fier, whih is digested y onsortium of neroi miroorgnisms residing in the rumen. Understnding mehnisms involved in fier digestion y rumen miroorgnisms, therefore, hs een mjor interest of ruminnt nutritionists (Koyshi et l., 200). It hs een well doumented tht teril popultions in the rumen t given time lrgely determine the extent nd rte of fier degrdtion (Akin nd Brton, 13; Miron et l., 2001; Pn et l., 2003; Khmp et l., 200). Also, the tthment of firolyti teri is n oligtory step in fier degrdtion, whih ws proven in previous studies with mjor firolyti teri suh s Firoter suinogenes S (Gong nd Forserg, 1), * Corresponding Author: J. K. H. Tel: +2-2-0-0, Fx: +2-2--, E-mil: jongh@snu..kr 1 Grdute Shool of Agriulture, Hokkido University, Spporo, 00-, Jpn. 2 Deprtment of Agriulturl Siene, Kore Ntionl Open University, Seoul 1-1, Kore. Reeived August, 200; Aepted Otoer 11, 200 Ruminoous lus SY3 (Miron et l., 1) nd Ruminoous flvefiens 00 (Stewrt et l., 10). Rumen ph, together with miroil popultion, nture of sustrtes, environmentl ftors suh s temperture nd existene of tions nd solule rohydrtes hve een suggested s ftors governing teril tthment (Miron et l., 2001). Ruminl ph is one of the most importnt of these ftors, euse firolyti teri re very sensitive dependent on ph hnge. In pure ulture studies, the numer of dhesion ells of F. suinogenes to ellulose deresed when ph ws redued from.0 to. with the numers eing mintined etween ph.0 nd.0, nd flling ruptly ove ph. (Roger et l., 10). In nother study in whih the dhesion of R. lus ws investigted, hnge in ph etween. nd.0 hd little effet ut elow ph.0 there ws mrked derese (Morris, 1). Fier digestion dereses t low rumen ph, espeilly elow ph.0, s oserved previously in studies using ontinuous ulture of mixed ruminl miroorgnisms (Slyter, 1), in vitro rumen ulture (Grnt nd Weidner, 12; Hu et l., 200), in so dispperne (Mould nd Ørskov, 13) nd in vivo tests (Mould et l., 1). It is

Sung et l., (200) Asin-Aust. J. Anim. Si. 20(2):200-20 201 pprent tht low ruminl ph hnges the rumen miroil popultion from firolyti to mylolyti (Tjim et l., 2001). Mourino et l. (2001) reported tht teril dhesion ws severely inhiited y initil low ph (.) ompred to vlues ove ph.0, nd low dhesion of rumen miroorgnisms ws onsidered ustive ftor of redued fier digestion t low ruminl ph, lthough the results were otined from the mesurement N nd P ontent of miroil mss tthed to pure ellulose sustrte. From the literture survey, no diret evidene ppers to hve een reported on the reltionship mong rumen ph, teril tthment nd fier digestion in omplex rumen miroil eosystem. Therefore, in vitro studies with mixed rumen miroorgnisms were onduted to determine effets of initil ph on fier digestion nd firolyti teril tthment y rel-time PCR. MATERIALS AND METHODS Ruminl inoul nd ulture medi Rumen ontents were otined from three rumen fistulted Holstein steers (verge 0 kg) whih ws fed twie dily t 0:00 nd 1:00 with mixture of 0% onentrte nd 0% timothy hy for more thn 2 wk. Rumen ontents whih hd rnge of ph.-. were olleted efore morning feeding, homogenized under neroi onditions, nd strined through four lyers of heeseloth. The strined rumen fluids from three steers were mixed in equl mount nd were used s soure of miroorgnisms. Rie strw ground to pss 2 mm sreen ws used s ron nd energy soure. The sl medium used in this experiment ws on uffer solution of MDougll (1). In vitro fermenttion Bteril tthment to rie strw under different ph onditions ws tested using n in vitro ulture system with some modifition y Sung et l. (200). The rumen fluidssl medium mixture ws prepred y mixing one volume of rumen fluid nd two volumes of the sl medium desried ove. The prepred medium ws djusted to. (high ph),.2 (middle ph) nd. (low ph) using N HCl, nd mde up to the sme volume using distilled wter. Five hundred mg of ground rie strw ws dded to 30 ml of phdjusted inution medium in 0 ml serum ottle nd inuted t 3 C with ontinuous shking t 120 rpm. The triplite ultures were respetively srified to nlysis teril tthment nd fermenttion prmeters t 0, 2,,, 12, 2 nd h of inution. The whole proedure ws rried out under neroi onditions (oxygen-free CO 2 ). Bteril dethment tests were rried out y wshing filter pper (0. 2.0 m) whih ws pre-inuted in rumen fluid t ph. for 12 h. The filter ppers were wshed in uffer solutions (0 ml) hving ph of.,.2 nd. for 30 min in shker t 0 rpm. Quntifition of ellulolyti teri Smple preprtion : The ulture ws entrifuged t g for min to seprte rie strw nd ulture medium. Colleted rie strw ws suspended in 0 ml of 0.% sline solution nd entrifuged three times t g for min to remove esily dethle teri. After entrifugtion, rie strw ws dried using lyophilizer (Ilshin, Kore) nd kept t -0 C until teril mesurement. The olleted ulture medium nd sline solution whih ws used for wshing rie strw were used for nlysis of suspended (untthed) teril popultion. The mixture of olleted ulture medium nd sline solution ws entrifuged t 1,000 g for min. After entrifugtion, the teril pellet ws resuspended with 0.% sline solution nd entrifuged t 1,000 g for min to otin teril pellet. DNA extrtion : Totl DNA ws extrted ording to the method desried y Purdy et l. (1). In detil, 0. g of dried rie strw or entrifuged ulture pellet ws mixed with 0.3 ml of TE uffer ( mm Tris HCl, 1 mm EDTA, ph.0), 0. ml of Tris-uffered phenol nd 0.2 g of sterilized glss ed (0. mm, BioSpe., Produt In., USA.). The tues were shken for 2 min, stood on ie for 2 min nd this step ws repeted three times. After dding 0 µl of % sodium luryl sulfte solution, tues were entrifuged t 13,000 g for 2 min nd superntnt ws olleted. The remining pellet ws resuspended in 20 µl of TE uffer, then entrifuged t 13,000 g for 2 min nd the superntnt ws olleted. Totl DNA ws olleted from pooled superntnt using hydroxyptite hromtogrphy olumn (Hydroxyptite Bio-Gel HTP Gel, Bio-Rd Lortories, In., USA). The RNA ws removed y DNAse-free pnreti RNAse A tretment nd susequent gel filtrtion (MiroSpin S-200 HR Columns, Amershm Biosienes, UK). The Purity nd onentrtion of totl DNA were heked using Biomte spetrophotometer (Thermo Spetroni, USA). PCR primer : Speies-speifi PCR primers for F. suinogenes, R. flvefiens nd R. lus were seleted from the literture (Koike nd Koyshi, 2001). Primers for F. suinogenes, R. flvefiens nd R. lus were: Fs21f ( -GGT ATG GGA TGA GCT TGC-3 ) nd Fsr ( -GCC TGC CCC TGA ACT ATC -3 ); Rf1f ( - TCT GGA AAC GGA TGG TA-3 ) nd Rf2r ( -CCT TTA AGA CAG GAG TTT ACA A-3 ); R121f ( -CCC TAA AAG CAG TCT TAG TTC G-3 ) nd R13r ( -CCT CCT TGC GGT TAG AAC A-3 ), respetively. Amplifition sizes from PCR retions for the three teril speies were, 2 nd 1 p nd nneling

202 Sung et l., (200) Asin-Aust. J. Anim. Si. 20(2):200-20 tempertures were 2, nd C, respetively. Rel-time PCR : Bteril DNA ws mplified nd quntified with n icyler iq rel-time PCR system (Bio- Rd In. USA). The iq Syer Green Supermix (Bio-Rd INC. USA) ws used for PCR mplifition ording to the mnufturer s protool. PCR onditions were: one yle of initil denturtion t C for 3 min, 0 yles of denturtion t C for 30 s, followed y nneling t eh temperture of strins for 30 s nd then n extension t 2 C for 30 s. Therefter, the melting point of PCR produt ws nlyzed to detet speifiity of pplition. The melting urve ws otined y 0.1 C/s inrese of heting temperture from to C with fluoresene detetion t 0.1 C intervls. Bteril popultion ws defined s log opy numer of 1S rdna whih ws lulted from stndrd urve of ontrol plsmid. The ontrol plsmid hd n insert of speifi frgment of 1S rdna mplified with primers speifi to eh speies (F. suinogenes, R. flvefiens nd R. lus). The ontrol plsmid ws onstruted y using the pgem-t nd pgem-t Esy Vetor System (Promeg, USA) ording to the mnul proedure. The stndrd urves were respetively mde y plotting C t vlues for seril dilutions of the eh ontrol plsmid for eh speies. Determintion of DM digestiility nd fermenttion prmeters The DM digestiility ws lulted y the differene etween dry mtter efore nd fter inution. The hnge of ph ws mesured using Mettler Delt 30 ph meter (Mettler Eletronis, UK). The umulted hed gs pressure ws mesured using pressure trnsduer nd reorded using digitl redout voltmeter (Lurel Eletronis, USA). For VFA determintion, 1 ml of ulture superntnt ws treted with 200 µl of met-phosphori id for 30 min t room temperture nd stored t -20 C until individul VFA were nlyzed using HP 0 gs hromtogrph (Hewlett Pkrd, USA) equipped with flme inoniztion detetor. The VFA were seprted with 30 m 0.32 mm 0.2 µm sized HP-FFAP pillry olumn using nitrogen s rrier gs. Sttistil nlysis All experimentl ultures were done in triplite. The dt were nlyzed ording to omplete rndomized design with one-wy nlysis of vrine of ph tretment. The sttistil model ws: Y ij = µ i +T ij, where i ws the numer of tretments nd j ws the numer of replition tues. When the overll tretment effet ws signifint (p<0.0), the differenes etween tretment mens were tested with the LSD test using the SAS progrm (SAS, 1). Log opy No/g DM Log opy No/g DM Log opy No/g DM. 13 12 11 A ph. ph.2 ph. RESULTS Bteril tthment Ative tthment of three mjor firolyti teri to rie strw took ple during erly inution (Figure 1). 12 11 B Inution time (h) 11 C Inution time (h) ph. ph.2 ph. ph. ph.2 ph. Inution time (h) Figure 1. The tthments of F. suinogenes (A), R. flvefiens (B) nd R. lus (C) on rie strw s influened y different ph during in vitro rumen fermenttion (,, Mens with different letters differ t p<0.0).

Sung et l., (200) Asin-Aust. J. Anim. Si. 20(2):200-20 203 Log opy No/ml Log opy No/ml Log opy No/ml 11 3 A ph. ph.2 ph. When it ws omputed from tthed (Figure 1) nd untthed teril popultion (Figure 2), more 0% of totl teri were tthed to rie strw within 2 hr in ll 2 3 2 B Inution time (h) ph. ph.2 ph. 1 Inution time (h) 3 2 C ph. ph.2 ph. 1 Inution time (h) Figure 2. The untthed popultion of F. suinogenes (A), R. flvefiens (B) nd R. lus (C) s influened y different ph during the in vitro rumen fermenttion (,, Mens with different letters differ t p<0.0). Remined teri (Log opy No). ph. ph.2 ph. F. Suinogenes R. Flvefirns R. lus Figure 3. Effets of different ph in wshing solution on the dethment of teri olonized on filter pper (,, Mens with different letters differ t p<0.0). three speies regrdless of initil ph. The tthment of F. suinogenes to rie strw t low (.) initil ph ws signifintly (p<0.0) less thn t middle (.2) nd high (.) initil ph (Figure 1A). However, no signifint differenes in teril tthment were deteted etween high nd middle initil ph. The tthed popultion t ph. ws highest t hr nd then deresed, while tht t higher ph remined inresed until 12 to 2 h. Low initil ph redued (p<0.0) tthment of R. flvefiens ompred to middle nd high initil ph (Figure 1B); pek tthment ws rehed t 12, 2 nd h inution in high, middle nd low ph onditions, respetively. In ommon with the other two teril speies, the tthment of R. lus ws lso ph dependent with signifintly (p<0.0) lower tthment eing hieved t low initil ph thn with either the middle or high initil ph; there ws no signifint differene etween the two higher ph onditions (Figure 1C). Untthed teri There ws tendeny for redued numer of untthed F. suinogenes (those in inution medium) during the erly stge of inution ( h) whih then rehed pek t 2 h inution in ultures t ph. nd.2 (Figure 2A). However, the numer of F. suinogenes in the ph. of ulture deresed up to hrs of inution, nd therefter the numers remined similr during the inution. The numer of untthed F. suinogenes ws higher t high nd middle initil ph ompred to low ph, s ws the se with tthed F. suinogenes. A similr trend ws oserved in untthed R. flvefiens (Figure 2B) nd R. lus (Figure 2C) with numers t the two higher initil ph (.2 nd.) eing muh lrger ompred to low (.) initil ph, lthough the time when effets of ph eme signifint ws different mong the three teril speies.

20 Sung et l., (200) Asin-Aust. J. Anim. Si. 20(2):200-20 ph.0..2...0 Inution time (h) ph. ph.2 ph. Figure. The ph of inution medium s influened y initil ph. Bteril dethment Filter pper ws inuted for 12 h nd wshed with solutions hving three different ph (.,.2 nd.) to see if ph of wshing solution ould influene teril dethment. The numer of remined F. suinogenes on filter pper fter wshing with ph. uffer solution ws signifintly (p<0.0) lower thn with ph. nd.2 (Figure 3). A similr result ws otined in the degree of dethment of R. flvefines nd R. lus. Fermenttion prmeters nd dry mtter digestiility The pttern of medi ph hnge during inution t three different initil phs is presented in Figure. The ph remined ove.0 during h inution when initil ph ws.. However, the ph deresed elow.0 fter 2 h inution when initil ph ws.2. Gs prodution (Figure A) ws dependent on initil ph, nd ws signifintly higher (p<0.0) t higher initil ph. Totl gs prodution fter h inution t middle nd high initil ph ws 2 nd 3 times, respetively, higher thn tht t low initil ph. Inution of rie strw t higher initil ph lso resulted in higher totl VFA prodution (Figure B), with pronouned (p<0.0) effet pprent fter hr inution. The verge inreses in totl VFA prodution t the two higher initil phs were, respetively, 1.2 nd 1. times tht t low initil ph. Figure shows the digestiility of rie strw DM s influened y different initil ph; distintive trend for higher DM digestiility ws oserved t higher initil ph. Rising initil ph from. to.2 nd. improved DM digestiility y 2. nd. times, respetively. The lg time (to reh ove 2% DM digestiility) for rie strw DM digestion t high nd middle initil ph ws h nd tht t low initil ph ws h. Gs prodution (ml) Totl VFA onentrtion (mm) A 1 1 ph. 1 ph.2 12 ph. 2 0 10 130 1 B ph. ph.2 ph. Inution time (h) DISCUSSION Results otined in the present study lerly indite tht initil ruminl ph is one of the importnt ftors influening tthment of mjor firolyti teri to fier sustrtes therey ffeting their digestion. The influene of initil ph ws espeilly pronouned when initil ph ws lower thn.0. The importne of ruminl ph on miroil tthment hs een reported in previous studies, the results of whih, however, seem equivol. For instne some studies reported tht teril tthment ws not hnged in the ph rnge.3 to.0 in ultures of F. suinogens (Gong nd Forserg, 1), ws stle in the ph rnge 3.3 to. in ultures of R. flvefiens (Roger et l., 10), or teril tthment to ellulose ws inresed when ph ws inresed from. to.0 (Mourino et l., 2001). In ddition, the tthment of F. suinogenes to ellulose ws 0 0 0 Inution time (h) Figure. Gs prodution (A) nd totl VFA onentrtion (B) s ffeted y different initil ph (,, Mens with different letters differ t p<0.0).

Sung et l., (200) Asin-Aust. J. Anim. Si. 20(2):200-20 20 DM digestiility (%) 0 3 30 2 20 1 ph. ph.2 ph. inresed when ph ws inresed from. to.0 (Roger et l., 10) nd the tthment of R. lus ws remrkly deresed in the ph rnge from.0 to.. The disrepny mong studies my rise from differenes in ulture ondition, type of sustrtes nd teril quntifition method. The ext mehnism involved in redued tthment t low initil ph is not evident. However, sed on the present study, redued teril growth nd elerted dethment t low initil ph my hve ontriuted to lower tthment. As shown in Figure 2, inuting rie strw t ph. resulted in the lowest onentrtion of three firolyti teri in the medium, inditing inhiited teril growth t low initil ph. A similr result hs een reported y Russell nd Wilson (1), who oserved tht predominnt speies of ruminl ellulolyti teri did not grow t low ph. In the study of Slyter (1) the ellulolyti pility of the ontinuous ulture system ws low when the ph ondition ws mintined elow.0 due to diminished numer of ellulolyti teri. Bht et l. (10) lso reported tht mximum tthment of oth F. suinogenes nd R. flvefiens to rley strw ws hieved t ph.0. The deline of ellulose degrdtion effiieny t lower ph ws ttriuted to the inhiition of ellulolyti teri, sine most ruminl ellulolyti teri re ph-sensitive (Russell nd Wilson, 1). The ph sensitivity n e explined y intrellulr ph regultion of ellulolyti teri. When the extrellulr ph of id-sensitive teri delines, the intrellulr ph is reltively stle, ut the inrese in the trnsmemrne ph grdient use logrithmi umultion of intrellulr fermenttion id nions nd hene leds to nion toxiity nd produt inhiition (Russell nd Wilson, 1; Weimer, 1). Another possile mehnism for redued tthment t 0 Inution time (h) Figure. Rie strw digestiility s influened y different initil ph (,, Mens with different letters differ t p<0.0). low initil ph is dethment of teri from the surfe of sustrte. As indited in Figure 3, when olonized ells on filter pper were wshed with different ph solutions, the ells remining on filter pper were deresed with lower ph of the wshing solution, whih is inditive of inresed teril dethment t low environmentl ph. There ws lmost 0 fold differene in the remining teri on filter pper etween the low nd two higher initil ph onditions, regrdless of teril speies. Binding to lignd using ellulose inding protein is one of the teril tthment mehnisms (Pell nd Shofield, 13), nd the lignd inding might hve een hmpered y low ph nd used teril dethment in this experiment. High initil ph led to n inrese in DM digestiility, gs prodution nd totl VFA prodution in this experiment (Figures nd ), whih ws proly the result of n inrese in tthed teril popultion used y higher initil ph. This study lerly shows tht tthment of firolyti teri is pivotl proess in the sustrt surfe phenomenon for fier digestion nd tht mehnisms of oth tthment nd digestion re pprently influened y ph s n environmentl ftor in the ruminl miroil eosystem. The role of teril popultions tthed to firous sustrt hs een ddressed previously in mirosopi oservtions (Cheng et l., 10; Cheng et l., 1; Be et l., 1). Koike et l. (2003) quntified ell numers of F. suinogenes, R. flvefiens nd R. lus tthed to stem y ompetitive PCR nd showed tht numers of ll three speies inresed grdully with inresed NDF dispperne. Additionl support for the reltionship etween tthment nd susequent fier degrdtion ws given y studies in whih wild isoltes of R. lus strins degrded etter thn mutnt strins lking tthment ility (Morris nd Cole, 1) nd norml type of F. instinlis DR hd higher totl tivities of ellulose-degrding enzymes thn the mutnt (Miron nd Forserg, 1). Bteri ssoited with sustrt hve een mentioned in previous reports (MAllister et l., 1; Miron et l., 2001). The popultions ssoited with feed prtiles re numerilly predominnt nd oupy from 0 to 0% of the totl miroil popultion in the rumen (Crig et l., 1; Forserg nd Lm, 1). These teri rpidly ssoited with nd tthed to reently ingested feed prtiles within min (Bonhomn, 10), nd then primry olonizers formed rih iofilm on the sustrt surfe together with dditionl olonizers. They were responsile for to 1% of ruminl endoglunse nd xylnse tivity (Minto et l., 13). Similrly, in our study the tthed popultion of F. suinogenes, R. flvefiens nd R.. lus rehed >0% of totl numer in eh strins shortly fter inoultion (in 2 h) t ll three initil ph nd the highest digestion rte of rie strw ws hieved therefter. Roger

20 Sung et l., (200) Asin-Aust. J. Anim. Si. 20(2):200-20 Popultion of tthed ter (Log opy No/g DM/h) 12.0 11.0.0.0.0.0.0.0 F. suinogenes R. flvefiens R. lus DM dis..2. ph et l. (10) found tht R. flvefines tthed to ellulose within 1 min of first ontt nd tive tthment of F. suinogenes took ple within 30 min in the in vitro study. Atthment of ruminooi speies to dmged plnt ourred within 1 to min fter their inoultion (Lthm et l., 1). It is ommonly oserved tht low ph<.0 severely inhiits fier digestiility in the rumen (Weimer; 1; Russell nd Ryhlik, 2001), nd this inhiition is strong when low ph is mintined for extended time of periods. The present study lso showed tht digestion of rie strw ws strongly inhiited y low ph with longer lg time for tive digestion (Figure ). Only very wek fermenttion ourred t ph. nd susequently little gs or VFA ws produed (Figures 3 nd ). In similr studies, Grnt nd Weidner (12) reported tht the lg in NDF digestion inresed s ph fell from. to. nd deresing ph of uffer to <.0 drmtilly deresed NDF digestion rte for lflf hy nd orn silge. Hu et l. (200) showed tht ellulose degrdtion did not our t ph<., wheres t ph>.0 there ws inresed ellulose degrdtion. Also, in the sme study, no VFA or reduing sugr were produed t ph<. nd only smll mount of gs ws produed t ph.-.. The present study lerly showed tht strong reltionship exists etween the initil ph, the rte of mjor ruminl teril tthment nd the rte of fier digestion, s shown in Figure. It is pprent tht the rte of fier digestion ws ph dependent, possily modulted vi modified teril tthment to fier sustrtes. Mintining ph>.0 is ritil for effiieny of miroil tthment nd fier digestion in the rumen. ACKNOWLEDGEMENT 1.2 1.0 0. 0. 0. 0.2 0.0 Dispperne of rie strw (g DM/h) Figure. The reltionship etween initil ph, DM digestiility nd teril tthment to rie strw fter 2 h inution. The work ws supported y grnt of Agriulturl R & D Promotion Center (ARPC, 200) nd the Brin Kore 21 Projet, Kore. The rel-time PCR ws helped y the Ntionl Instrumenttion Center for Environmentl Mngement (NICEM) of College of Agriulture & Life Siene in Seoul Ntionl University. Critil review y Dr. J. Steel (CSIRO) is lso knowledged. REFERENCES Akin, D. E. nd F. E. Brton. 13. Rumen miroil tthment nd degrdtion of plnt ell wlls. Fed. Pro. 2:11-121. Be, H. D., T. A. MAllister, E. G. Kokko, F. L. Leggett, L. J. Ymke, K. D. Jkoer, J. K. H, H. T. Shin nd K. J. Cheng. 1. Effet of sili on the oloniztion of rie strw y ruminl teri. Anim. Feed Si. Tehnol. :1-11. Bht, S., R. J. Wlle nd E. R. Orskov. 10. Adhesion of ellulolyti ruminl teri to rley strw. Appl. Environ. Miroiol. :2-203. Bonhomme, A. 10. Rumen ilites: Their metolism nd reltionships with teri nd their hosts. Anim. Feed Si. Tehnol. 30:203-2. Cheng, K. -J., J. P. Fy, R. E. Howrth nd J. W. Costerton. 10. Sequene of events in the digestion of fresh legume leves y rumen teri. Appl. Environ. Miroiol. 0:13-2. Cheng, K. -J., C. S. Stewrt, D. Dinsdle nd J. W. Costerton. 1. Eletron mirosopy of teri involved in the digestion of plnt ell wlls. Anim. Feed Si. Tehnol. :3-120. Crig, W. M., G. A. Broderik nd D. B. Riker. 1. Quntittion of miroorgnisms ssoited with the prtiulte phse of ruminl ingest. J. Nutr. 11:-. Forserg, C. W. nd R. Lm. 1. Use of denosine-- triphosphte s n inditor of the miroil iomss in rumen ontents. Appl. Environ. Miroiol. 33:2-3. Gong, J. nd C. W. Forserg. 1. Ftors ffeting dhesion of Firoter suinogenes S nd dherene defetive mutnts to ellulose. Appl. Environ. Miroiol. :303-30. Grnt, R. J. nd S. J. Weidner. 12. Digestion kinetis of fier: Influene of in vitro uffer ph vried within oserved physiologil rnge. J. Diry Si. :0-. Hu, Z. -H., H. -Q. Tu nd R. -F. Zhu. 200. Influene of prtile size nd ph on neroi degrdtion of ellulose y rumen miroes. Int. Biodeter. Biodegr. :233-23. Khmp, S., M. Wnpt, C. Whirpkorn, N. Nontsol, M. A. Wttiux nd P. Rowlison. 200. Effet of leve;s of sodium DL-mlte supplementtion on ruminl fermenttion effiieny of onentrtes ontining high levels of ssv hip in diry steers. Asin-Aust. J. Anim. Si. 1:3-3. Koyshi, Y., S. Koike, H. Tguhi, H. Itshi, Dong K. Km nd J. K. H. 200. Reent dvnes in gut miroiology nd their possile ontriution to niml helth nd prodution- Review. Asin-Aust. J. Anim. Si. 1:-. Koike, S. nd Y. Koyshi. 2001. Development nd use of ompetitive PCR sys for the rumen ellulolyti teri: Firoter suinogenes, Ruminoous lus nd Ruminoous flvefiens. FEM Miroiol. Letters. 20:31-3. Koike, S., J. Pn, Y. Koyshi nd K. Tnk. 2003. Kinetis of in so fier-tthment of representtive ruminl ellulolyti

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