Saponin rich tropical fruits affect fermentation and methanogenesis in faunated and defaunated rumen fluid

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1 Animl Feed Science nd Technology 109 (2003) Sponin rich tropicl fruits ffect fermenttion nd methnogenesis in funted nd defunted rumen fluid H.D. Hess, M. Kreuzer,, T.E. Díz b, C.E. Lscno c, J.E. Crull d, Crl R. Soliv, Andre Mchmüller Institute of Animl Sciences, Animl Nutrition, Swiss Federl Institute of Technology (ETH) Zurich, CH-8092 Zurich, Switzerlnd b Ntionl Progrm of Animl Physiology nd Nutrition, Corpoic, Bogotá, Colombi c Tropicl Grss nd Legume Project, CIAT, Cli, Colombi d Deprtment of Animl Production, Ntionl University of Colombi, Bogotá, Colombi Received 4 October 2002; received in revised form 14 My 2003; ccepted 28 My 2003 Abstrct A comprison of the effects on rumen fermenttion of three sponin rich tropicl fruits supplemented to forge-bsed diets ws completed using rumen simultion technique (Rusitec). The diets contined either no tropicl fruit or 100 mg/g of Spindus sponri (crude sponins, 120 mg/g), 200 mg/g of Enterolobium cyclocrpum (crude sponins, 19 mg/g) or 200 mg/g of Pithecellobium smn (crude sponins, 17 mg/g). The four diets were evluted with funted nd defunted rumen fluid obtined from single donor cow. Compred to the control diet, P. smn decresed (P <0.05) mmoni concentrtion of rumen fluid, nd E. cyclocrpum nd P. smn incresed (P <0.05) n-butyrte proportion of totl voltile ftty cids. Defuntion enhnced (P <0.05) propionte proportion with corresponding reductions of cette nd n-butyrte. Orgnic mtter degrdtion of the S. sponri diet did not differ from tht of the control diet but ws higher (P <0.05) with P. smn nd E. cyclocrpum. Only one of the sponin rich fruits evluted, S. sponri, decresed (P <0.05) protozol count (by 54%) nd dily methne relese (by 20%) reltive to control, but without ffecting the methnogen count. Defuntion suppressed methnogenesis by 43% over ll diets (P <0.05), nd the effect of S. sponri on methne ws more pronounced in defunted (29%) versus funted rumen fluid (14%). When relted to orgnic mtter pprently fermented, differences reltive to the control diet persisted (P <0.05), but methne relese per unit of fibre degrded did not differ between the S. sponri diet nd the control diet. This study demonstrted Abbrevitions: CP, crude protein; DM, dry mtter; NDF, neutrl detergent fibre; OM, orgnic mtter; Rusitec, rumen simultion technique; S.E.M., stndrd error of men; VFA, voltile ftty cids Corresponding uthor. Tel.: ; fx: E-mil ddress: michel.kreuzer@inw.grl.ethz.ch (M. Kreuzer) /$ see front mtter 2003 Elsevier B.V. All rights reserved. doi: /s (03)

2 80 H.D. Hess et l. / Animl Feed Science nd Technology 109 (2003) tht supplementtion with S. sponri is effective ginst ruminl methnogenesis, but tht this ws not exclusively n effect of the ssocited depression in protozol count Elsevier B.V. All rights reserved. Keywords: Spindus sponri; Enterolobium cyclocrpum; Pithecellobium smn; Sponins; Methne; Protozo 1. Introduction Livestock re one of the lrgest single sources of methne emission with million tonnes per yer, equivlent to 15 20% of totl nthropogenic methne (IPCC, 2001). The globl cttle popultion is responsible for 73% of methne emissions of ll livestock. It is estimted tht t lest hlf of this popultion is locted in tropicl regions (McCrbb nd Hunter, 1999). Grsslnds in the tropics constitute lrge nturl feed resource which is minly suited for grzing by ruminnts. Tropicl grsses re of low to moderte digestibility (on verge 13% lower dry mtter (DM) digestibility thn temperte grsses) nd re often deficient in criticl nutrients such s protein nd phosphorus (Minson, 1990). Under such conditions, methne produced during ruminl fermenttion represents loss of 10 11% of gross energy intke (McCrbb nd Hunter, 1999). Selective suppression of the rumen protozo hs been suggested to be promising pproch to reduce methne relese (Moss et l., 2000) s shown by the results of defuntion (e.g. Whitelw et l., 1984; Dohme et l., 1999). Finly et l. (1994) described symbiosis of protozo with methnogenic Arche. Other studies hve shown tht 9 25% of totl methnogens re ssocited with the protozo (Newbold et l., 1995). Considering the difficulties of on-frm defuntion (Moss et l., 2000) reduction, rther thn the totl elimintion, of rumen protozol popultion, which ws shown to hve similr effects (Veir et l., 1983), hs been suggested s wy to improve productivity on tropicl diets (Dominguez Bello nd Escobr, 1997). Folige (Rosles et l., 1989; Nvs-Cmcho et l., 1994; Newbold et l., 1997) s well s fruits (Díz et l., 1993; Nvs-Cmcho et l., 1994) of severl tropicl nd sub-tropicl multipurpose shrubs nd trees hve been reported to suppress rumen protozo. The compounds ssumed to be responsible for these nti-protozol effects re sponins or sponin-like substnces. Among the promising tree species tht produce fruits with moderte to high sponin contents re Enterolobium cyclocrpum, Pithecellobium smn nd Spindus sponri (Nvs-Cmcho et l., 1994; Restrepo nd Jiménez, 1999). Sheep consuming the seed pericrp of S. sponri hd reduced rumen protozol count, which ws ssocited with n increse in live weight gin (Nvs-Cmcho et l., 2001). However, in none of these studies ws the effect of these sponin rich feeds on methne relese from ruminl fermenttion mesured. Results of Dohme et l. (1999) illustrted tht the methne suppressing effects of n ctive feed component (i.e. lipids) re not necessrily medited through the decline in protozol counts, even when simultneous suppression of protozo tkes plce. The objective of this in vitro study ws to compre the utility of three sponin rich fruits, included t high proportions in low qulity forge-bsed diet, to ffect methne relese s

3 H.D. Hess et l. / Animl Feed Science nd Technology 109 (2003) well s methnogen nd protozol counts in the rumen. In order to be ble to distinguish between direct nd protozo-medited effects, funted nd defunted rumen fluid ws used. 2. Mterils nd methods 2.1. In vitro technique A rumen-simultion technique pprtus (Rusitec; Czerkwski nd Breckenridge, 1977) s modified by Mchmüller et l. (2002) nd equipped with eight fermenter vessels ws used. Rumen fluid ws collected in glss flsk from one rumen-fistulted Brown Swiss cow in lte lcttion for immedite use in the fermenters. The cow ws housed ccording to Swiss guidelines for niml welfre nd ws fed hy (d libitum) nd diry cttle concentrte (1 kg per dy). Rumen fluid ws strined through three lyers of compress guze (1000 m pore size, type 17, MedPro Novmed AG, Flwil, Switzerlnd). Fermenters were filled with 890 ml of rumen fluid nd 110 ml of McDougll buffer (Czerkwski nd Breckenridge, 1977). Flow rte of rtificil sliv ws set to 500 ml per dy, equivlent to dilution rte of 0.50 per dy. Two nylon bgs (70 mm 130 mm, 100 m pore size; Crro et l., 1995) were put into ech vessel. Initilly, only one of the bgs contined g DM of experimentl feed nd, for esier estblishment of fvourble fermenttion conditions, the other ws filled with 60 g of fresh squeezed solid rumen content (Czerkwski nd Breckenridge, 1977). After 24 h, bgs contining solid rumen content were replced with feed bgs. Dily therefter, those feed bgs which hd been incubted for 48 h were replced. Directly following this procedure, the fermenters were flushed with N 2 to immeditely re-estblish nerobic conditions (Czerkwski nd Breckenridge, 1977). After removl from the fermenters, feed bgs were gently squeezed nd wshed with cold tp wter until the outflow ws cler. Subsequently, fermenttion residues were stored t 20 C for subsequent processing Experimentl design nd diet preprtion Four diets were evluted (Tble 1). The control diet, without sponin rich fruits, supplied (DM bsis) 8.77 g per dy of low qulity medow grss hy (63 mg/g crude protein (CP), 630 mg/g neutrl detergent fibre (NDF)), 3.51 g per dy dried Archis pintoi ( tropicl psture legume; 163 mg/g CP, 430 mg/g NDF), 1.70 g per dy brley strw (32 mg/g CP, 830 mg/g NDF) nd 0.17 g dy ure. In order to consider in vivo limittions of feeding the tropicl fruits used in this experiments complete fruits of Spindus sponri were used t proportion of 100 mg/g diet DM, wheres 200 mg/g diet DM were pplied with the fruits of Enterolobium cyclocrpum nd Pithecellobium smn (synonymous to Albizi smn). These proportions correspond to threshold levels pplied in frm prctice (Díz et l., 1993; Zmor et l., 2001), which llows sufficiently pltble diet nd tht detrimentl effects of the sponins on metbolism of the rumen (Nvs-Cmcho et l., 1994) re voided. The fruits replced liquots of the bsl diet. Totl dily DM supply (14.15 g) nd dietry CP content (130 mg/g) were equl mong diets, the ltter by the use of different proportions of ure. Prior to use, ll ir dried feed ingredients, except ure, were ground in lbortory mill using 3 mm screen. Afterwrds,

4 82 H.D. Hess et l. / Animl Feed Science nd Technology 109 (2003) Tble 1 Composition (mg/g DM) of fruits nd complete diets Tretments Control S. sponri E. cyclocrpum P. smn Diet ingredients Medow grss hy Archis pintoi hy Brley strw Ure Spindus sponri fruits 100 Enterolobium cyclocrpum fruits 200 Pithecellobium smn fruits 200 Anlysed composition Fruits Crude sponins Totl condensed tnnins Extrctble condensed tnnins n.d. c Orgnic mtter Neutrl detergent fibre Crude protein Ether extrct Non-structurl crbohydrtes Totl sugrs Complete diets Crude sponins b Totl condensed tnnins b Orgnic mtter Neutrl detergent fibre Crude protein Ether extrct Non-structurl crbohydrtes Assuming sponins nd condensed tnnins originte from the fruits only. b Not determined. c Not detected. fine forge prticles were removed by 0.25 mm screen in order to minimise losses through wshing out of the nylon bgs. This procedure ws omitted with the sponin rich fruits becuse it might hve creted frctions with different sponin contents. The four diets were evluted simultneously with funted nd defunted rumen fluid during four 10 dys periods. For tht purpose, on dy 1 of ech experimentl period the content of every other fermenter ws defunted with Synperonic NP9 (ICI, Middlesbrough, UK) dministered t concentrtion of 2.5 ml/l rumen fluid (Dohme et l., 1999). This ensured complete defuntion within 24 h. The first 4 dys of ech period served s n dpttion phse nd the lst 6 dys s the mesurement phse. This ws the minimum time for dpttion suggested by Czerkwski nd Breckenridge (1977), nd ws previously found to be sufficient for significnt response of methnogenesis to dietry mnipultions nd defuntion (Dohme et l., 1999; Mchmüller et l., 2002).

5 2.3. Anlyticl procedures H.D. Hess et l. / Animl Feed Science nd Technology 109 (2003) For lter nlysis of fermenttion vribles, dily smples of fermenter fluid were drwn directly from ech vessel with syringe 4 h before feed introduction. Fluid ph nd mmoni concentrtion were determined with ph meter (model 632, Metrohm, Herisu, Switzerlnd) equipped with the respective electrodes. Concentrtions of voltile ftty cids (VFA) were mesured using gs chromtogrphy (GC Str 3400 CX, Vrin, Sugrlnd, TX, USA) ccording to Tngermn nd Ngengst (1996). Fermenttion gses were collected in gs proof bgs (TECOBAG, 5 l, PETP/AL/PE-12/12/75 qulity, Tesserux Continer GmbH, Bürstdt, Germny), nd concentrtions of methne, hydrogen, oxygen nd nitrogen were nlysed with gs chromtogrph (model 5890 Series II, Hewlett Pckrd, Avondle, PA, USA) equipped with TCD nd FID detector. The column (Crboxen-1000; Fluk, Buchs, Switzerlnd) ws 4.5m 2.1 mm in size, nd gs smples of 150 l were injected by gs-tight syringe (1725 RN, Hmilton, Bonduz, Switzerlnd). Argon ws used s crrier gs. Crbon dioxide ws estimted s the difference between totl gs volume nd the remining gses. The gs volume in the collection bgs ws quntified by the corresponding replcement of wter. The hydrogen blnce ws clculted by the eqution proposed by Demeyer (1991) considering VFA nd methne. Products such s lctte, formte nd succinte re not considered in this eqution. Counts of cilite protozo (dily) nd totl bcteri (on dys 7 nd 10) were determined in the fermenter fluid smples using 0.1 mm nd 0.02 mm depth Bürker counting chmbers (Blu Brnd, Wertheim, Germny), respectively. Prior to counting, smples were fixed by ddition of 0.1 ml/ml (protozo) nd 0.99 ml/ml (bcteri) of Hyem solution (HgCl 2, 2.5 mg/ml; N 2 SO 4, 25.0 mg/ml; NCl, 5.0 mg/ml). For determintion of methnogen counts, smples of fermenter fluid were collected t the end of ech experimentl period, frozen in liquid nitrogen nd stored t 70 C. Methnogen counts were determined by the fluorescence in situ hybridiztion technique (FISH) outlined by Sthl et l. (1995) nd modified by Soliv et l. (2003). The oligonucleotide probe S-D-Arch A-20 lbelled with rective fluorescent dye Cy3 t the 5 end (Microsynth GmbH, Blgch, Switzerlnd) ws employed, nd Fluoro Gurd TM (Bio Rd, Hercules, CA, USA) ws used s the ntifde regent. Fluorescence signls were exmined with microscope (BX-60, Olympus Opticl AG, Volketswil, Switzerlnd) equipped for epifluorescence mesurements nd processed with softwre for utomtic counting (nlysis, version 3.1, Soft Imgine System GmbH, Uster, Switzerlnd). Fermenttion residues were lyophilised for 72 h prior to lbortory nlysis. The diets, tropicl fruits nd residues were ground in lbortory mill using 1 mm screen. Contents of DM, totl sh nd ether extrct were determined ccording to stndrd methods (Numnn nd Bssler, 1997), with the nlysis of sh free NDF being crried out with the use of -mylse (Termmyl 120L, type S, Novo Nodirsk A/S, Bgsværd, Denmrk) but without sodium sulphite. A C/N nlyser (type FP-2000, Leco Instrumente GmbH, Kirchheim, Germny) ws used to determine N. Non-structurl crbohydrtes were defined s the orgnic mtter (OM) not incorported in NDF, CP nd ether extrct. Dt on pprent in vitro OM, CP nd NDF degrdtion were clculted from the contents of OM, CP nd NDF in the nylon bgs before nd fter incubtion.

6 84 H.D. Hess et l. / Animl Feed Science nd Technology 109 (2003) Fruits of S. sponri, E. cyclocrpum nd P. smn were further nlysed for their contents of crude sponins, tnnins nd sugrs. Sponin contents were nlysed in smples ground through 2 mm screen fter extrction by procedure bsed on methods described by Wll et l. (1952) nd Hostettmnn nd Mrston (1995). Briefly, in the first step the lipid frction ws removed from the ground mteril by extrction with petroleum ether. Afterwrds, the deftted mteril ws soked in ethnol (96%). The ethnol phse ws concentrted in rotry evportor to 1/3 of the originl volume. The residue ws suspended in distilled wter nd shken with butnol to extrct sponins from the queous phse. The butnol soluble extrct ws dried nd extrcted solids were dissolved in methnol followed by precipittion of the sponins by ddition of diethyl ether nd recovery of sponins by filtrtion. The residue ws gin suspended in methnol, precipitted with cetone nd recovered by filtrtion. Finlly, the precipitte ws re-suspended in methnol nd evported to obtin dry powder, which ws considered to correspond to the crude sponin frction of the fruits. Fruits were nlysed for their contents of extrctble nd totl condensed tnnins ccording to the butnol/hcl procedure suggested by Terrill et l. (1992), with the extrction completed with n queous methnol (700 ml/l), formic cid (5 ml/l) nd scorbic cid (0.5 g/l) solution (Telek, 1989). Purified tnnins of ech fruit, obtined s recommended by Hgermn nd Butler (1980), were used s stndrds. Contents of totl sugrs mesured in the fruits were determined fter hot extrction with 80% ethnol, subsequent filtrtion nd ddition of n orcinol/sulphuric cid regent by colorimetric mesurement using continuous-flow utonlyser (AutoAnlyser 2, Brn nd Luebbe GmbH, Norderstedt, Germny) Sttisticl nlysis Dt were nlysed using the generl liner model (GLM) procedure of SAS (version 6.12, SAS Institute, Cry, NC, USA). A two-fctoril model (four diets two protozol tretments) using verge vlues from dys 5 to 10 ws pplied. The four experimentl periods were considered s replictes (n = 4). For multiple comprison mong subgroup mens (diet protozol tretment), dt were dditionlly nlysed with mono-fctoril model (eight tretments). Since the interction mostly remined insignificnt in vribles describing rumen fluid chrcteristics nd in hydrogen blnce, these dt re presented s overll diet mens nd protozol tretment mens, respectively, together with the probbility of error (P) of the interction. All multiple comprisons mong mens were performed with Tukey s method. The MIXED procedure of SAS with the rndom nd repeted sttements s recommended by Littell et l. (1998) ws used to compre tretments in the evolution of methne relese with time. 3. Results 3.1. Composition of diets The crude sponin contents differed between S. sponri nd the other two fruits (Tble 1). The clculted differences in sponin content mong the complete diets were smller, but still comprised four-fold level of sponins supplied by S. sponri compred

7 H.D. Hess et l. / Animl Feed Science nd Technology 109 (2003) to tht provided by E. cyclocrpum nd P. smn. While the fruits of E. cyclocrpum nd P. smn contined 24 nd 45 mg/g of extrctble condensed tnnins, no extrctble tnnins were found in the fruits of S. sponri. Correspondingly, the content of totl condensed tnnins (extrctble nd bound) ws highest in P. smn, intermedite in E. cyclocrpum nd lowest in S. sponri. All three fruits contined reltively high proportions of totl sugrs of mg/g. Diets vried to some extent in content of fibre nd non-structurl crbohydrtes, depending on proportion nd composition of the fruits. Similr dietry contents of CP were ttined s intended Rumen fluid chrcteristics The men rumen fluid ph cross ll diets ws 6.84, nd effects of fruits nd defuntion were minor (Tble 2). Rumen fluid mmoni declined (P <0.05) by 17% with P. smn, compred to the other tretments. Totl VFA concentrtions nd molr proportions of cette were similr mong diets. When compred to the control diet, inclusion of the fruits showed no effect on the molr proportion of propionte. However, propionte proportion ws higher (P <0.05) with S. sponri versus E. cyclocrpum nd P. smn. Molr proportions of n-butyrte nd n-vlerte were not influenced by S. sponri, but incresed (P < 0.05) with E. cyclocrpum nd P. smn. iso-butyrte nd iso-vlerte proportions declined (P <0.05) by bout 25% with P. smn, but not with the other fruits. Ammoni nd totl VFA concentrtions were reduced (P <0.001) by defuntion. In defunted rumen fluid, molr proportions of propionte nd n-vlerte were incresed t the pprent expense of cette nd n-butyrte. Defuntion hd no effect on molr proportions of iso-butyrte nd iso-vlerte. No interctions between diet nd protozol sttus occurred with the exception of the molr proportion of iso-vlerte. This vrible ws reduced (P <0.05) by defuntion in the S. sponri diet from to of totl VFA but remined unchnged by defuntion in ll other diets (dt not shown in Tble). All fruits ffected totl protozol counts, but in contrsting wys. S. sponri reduced (P <0.05) the number of protozo by 54% wheres E. cyclocrpum nd P. smn incresed (P <0.05) protozol counts by 55% when compred to the control. Totl bcteril counts verged ml 1, nd were similr mong diets. Defuntion incresed (P <0.01) totl bcteril counts by 18%. Methnogen counts, determined t the end of ech 10-dy period, were not ffected by diet or defuntion Nutrient degrdtion nd methne emissions The three sponin rich fruits differed in their effects on nutrient degrdtion nd methne emission (Tble 3). The use of E. cyclocrpum nd P. smn incresed (P <0.05) pprent OM degrdtion by 7 10%. Degrdtion of NDF ws reduced (P <0.05) by 12 15% when fruits of P. smn nd S. sponri were included. Effects on NDF degrdtion were smller nd not significnt with fruits of E. cyclocrpum. Apprent CP degrdtion ws higher (P <0.05) with E. cyclocrpum thn with the control diet nd the other fruit diets. The pprent degrdtion of OM nd NDF during 48 h of incubtion ws not ffected by defuntion, but the pprent CP degrdtion incresed (P <0.001) by bout 10%. There were some trends for interctions of diet with protozol sttus, prticulrly in NDF

8 Tble 2 Effect of sponin rich fruits nd defuntion on in vitro rumen fluid chrcteristics nd hydrogen blnce (verges of dys 5 to 10) Diets Protozol sttus S.E.M. Interction (P-vlue) Control S. sponri E. cyclocrpum P. smn Funted Defunted Rumen fluid vribles ph 6.85 b c 6.83 bc 6.83 z 6.84 z Ammoni (mmol/l) b 8.5 z 5.9 y Voltile ftty cids Totl (mmol/l) z 78.5 y Molr proportions Acette z y Propionte b b b y z n-butyrte b b z y iso-butyrte b z z n-vlerte b b y z iso-vlerte b z z Microbil counts Protozo (10 3 ml 1 ) 6.3 b 2.9 c Bcteri (10 9 ml 1 ) b y 3.7 z Methnogens (10 8 ml 1 ) c z 2.3 z Hydrogen blnce Produced (mmol per dy) 78.3 bc 74.2 c b 80.8 z 76.9 y Utilised (mmol per dy) 60.2 b 55.4 c b 66.0 z 56.8 y Recovered (%) 79.6 b 75.3 b b 83.8 z 74.7 y S.E.M.: stndrd error of the men. Mens by diet group, n = 8. Mens by protozol sttus groups, n = 16. Diet group men vlues nd protozol sttus group mens within the sme line shring no common letters re different t P<0.05. b Averges of dys 7 nd 10 only. c Averge of dy 10 only. 86 H.D. Hess et l. / Animl Feed Science nd Technology 109 (2003) 79 94

9 Tble 3 Effect of sponin rich fruits nd defuntion on pprent in vitro nutrient degrdtion nd methne emissions (verges of dys 5 to 10) Protozol sttus Diets Men S.E.M. Interction (P-vlue) Control S. sponri E. cyclocrpum P. smn Apprent nutrient degrdtion rtes Orgnic mtter Funted 0.41 c 0.41 bc b 0.43 Z Defunted 0.42 bc 0.40 c Z Men 0.42 B 0.40 B 0.46 A 0.45 A Neutrl detergent fibre Funted b 0.30 b 0.29 b 0.31 Z Defunted b 0.31 b 0.30 b 0.30 Z Men 0.33 A 0.28 B 0.30 AB 0.29 B Crude protein Funted 0.49 d 0.49 cd 0.53 bcd 0.49 cd 0.50 Y Defunted 0.54 bc 0.56 b cd 0.55 Z Men 0.51 B 0.53 B 0.55 A 0.50 B Methne emissions mmol per dy Funted 7.64 b 6.58 bc Z Defunted 4.71 de 3.35 e 5.29 cd 4.64 de 4.50 Y Men 6.18 B 4.96 C 7.05 A 6.64 AB mmol/g orgnic mtter degrded Funted 1.44 b 1.23 b Z Defunted 0.86 c 0.64 c 0.86 c 0.78 c 0.79 Y Men 1.15 A 0.93 B 1.17 A 1.14 A mmol/g neutrl detergent fibre degrded Funted 2.53 b 2.53 b Z Defunted 1.58 cd 1.55 d 2.16 bc 1.96 bcd 1.81 Y Men 2.05 B 2.04 B 2.93 A 2.87 A mmol/mmol crbon dioxide Funted b Z Defunted 0.10 c 0.08 c 0.09 c 0.09 c 0.09 Y Men 0.12 A 0.10 B 0.12 A 0.12 A S.E.M.: stndrd error of the men. Mens by sub-group, n = 4. Sub-group mens within the sme vrible shring no common lower cse letters re different t P<0.05. Diet group men vlues nd protozol sttus group mens within the sme vrible shring no common cpitl letters re different t P<0.05. H.D. Hess et l. / Animl Feed Science nd Technology 109 (2003)

10 88 H.D. Hess et l. / Animl Feed Science nd Technology 109 (2003) degrdtion (P = 0.07). Accordingly, decline in NDF degrdtion ws only observed with S. sponri in combintion with defuntion. Dily methne relese ws reduced by bout 20% with S. sponri (14 nd 29% with funted nd defunted rumen fluid, respectively) nd ws incresed by 14% with E. cyclocrpum (P <0.05). With P. smn, methne relese did not differ (P >0.05) from the control diet. When relted to pprently degrded OM, use of E. cyclocrpum nd P. smn hd no effect on methne relese, but the difference (P <0.05) from the control diet persisted with S. sponri. In contrst, methne relese per grm of pprently degrded NDF ws similr with S. sponri nd the control diet, but ws incresed with E. cyclocrpum nd P. smn. This ws mostly due to vritions in funted rumen fluid, wheres effects were smller with defunted rumen fluid (interction P<0.01). The rtio of methne to crbon dioxide ws lower (P <0.05) with the S. sponri diet thn with ll other diets, indicting tht vrition of gs relese pttern only occured with this diet. Among diets, dily methne relese (verge of dys 5 to 10) ws reduced (P <0.001) through defuntion by more thn 40% nd ws ssocited with corresponding shift towrds lower methne to crbon dioxide rtio. This effect ws even lrger during the first dys of the mesurement periods (Fig. 1). According to the results of the repeted mesurement nlysis, methne relese from the control diet ws lower (P <0.05) from dys 2 to 5 thn from dys 8 to 10. With S. sponri supplementtion, vlues were lower from dys 3 to 6 thn on dy 9. Fig. 1, only presenting dily methne relese of the control nd the S. sponri diet, lso illustrtes tht trends to lower methne relese with S. sponri did not strt before dy 4 but persisted from tht point. It is evident tht the S. sponri effect occurred in both funted nd defunted rumen fluid. The hydrogen blnce (Tble 2) reflected vritions in dily methne relese. Hydrogen production, utilistion nd recovery were highest with E. cyclocrpum nd lowest with S. sponri, while vlues were intermedite in the control diet nd with P. smn. Defuntion reduced ll hydrogen blnce trits (P <0.05). Methne (mmol/d) b b b b b b b b b b b b b b b b b b b Dy of Experiment Fig. 1. Evolution of methne relese: ( ) control diet/funted; ( ) control diet/defunted; ( ) Spindus sponri/funted; ( ) S. sponri/defunted. Group mens obtined within the sme experimentl dy shring no common letter re different t P<0.05. (Verticl brs show stndrd errors of mens.)

11 H.D. Hess et l. / Animl Feed Science nd Technology 109 (2003) Discussion In contrst to other sponin rich diet ingredients of tropicl plnt origin such s Yucc schidiger (e.g., Sen et l., 1998; Hristov et l., 1999) nd Sesbni sesbn (Newbold et l., 1997; Teferedegne et l., 1999), the three tropicl sponin rich fruits used in the present study hve not been intensively investigted. For S. sponri pericrp (i.e. the outer prt of the fruit), sponin contents s high s 385 mg/g were reported by Nvs-Cmcho et l. (1994). Sponin contents of E. cyclocrpum leves were 130 mg/g (Nvs-Cmcho et l., 1994), nd fruits of P. smn were found to contin 105 g sponins/kg (Restrepo nd Jiménez, 1999). Sponin contents therefore were expected to be higher for S. sponri compred with the other two fruits, which ws ccounted for by dietry inclusion of twice the mounts of E. cyclocrpum nd P. smn compred to S. sponri. The crude sponin contents nlysed in the btches used in the present study were considerbly lower thn these published results. Additionlly, the clculted proportion of sponins in the diet with S. sponri ws four times higher thn in the diets with E. cyclocrpum nd P. smn which were similr in sponin content. All three fruits contined some condensed tnnins. However, contents were lower thn those reported from other tropicl forge trees such s Cllindr clothyrsus nd Acci mngiu (Jckson et l., 1996). Assuming tht condensed tnnins originted from the fruits only, the concentrtions in the complete diets were below 15 mg/g. It is therefore highly unlikely tht effects of the sponins, which re effective t much lower concentrtions (Śliwiński et l., 2002), would hve been msked by effects of tnnins. It lso seems very unlikely tht tnnins inctivted the sponins by forming complexes with them since tnnins hve higher ffinity for protein, which ws present in high proportions in the diets. The reltively high content of CP nd non-structurl crbohydrtes, prticulrly sugrs, indictes tht the fruits represent vluble feed ingredients supplying not only sponins but lso high mounts of digestible nutrients Effects of the sponin rich fruits Most of the rumen fermenttion vribles recorded were influenced by the dietry inclusion of the sponin rich fruits. Extent nd nture, however, vried with individul fruits. The OM of the diets contining fruits of E. cyclocrpum nd P. smn ws degrded to higher extent thn tht of the control diet nd the diet contining S. sponri. This is probbly relted to the different NDF content of the fruits nd, consequently, of the diets. The E. cyclocrpum nd P. smn diets contined less NDF nd more non-structurl crbohydrtes which re more redily fermented. In the cse of E. cyclocrpum, the higher CP degrdtion lso might hve contributed to the reltively higher OM degrdtion. The low NDF degrdtion in the diet contining S. sponri t levels of OM nd CP degrdtion similr to the control diet could be relted to specific inhibition of fibre degrding microbes due to high concentrtions of sponins in the diet. From in vitro results, Wng et l. (2000) suggested tht sponins my ffect different bcteri species in different wys nd tht cellulolytic bcteri seem to be more susceptible to sponins thn others. Since pprent CP degrdtion is influenced by microbes ttched to plnt cell wlls, this could msk effects on CP degrdtion, lthough rumen fluid mmoni concentrtions with the S. sponri diet did not differ from the control diet.

12 90 H.D. Hess et l. / Animl Feed Science nd Technology 109 (2003) Even though the fruits hd effects on OM nd NDF degrdtion, totl VFA concentrtion nd the molr proportion of cette were similr mong diets. However, S. sponri incresed propionte nd reduced n-butyrte when compred with E. cyclocrpum nd P. smn. This difference between the fruits could hve been either direct effect of the different sponin concentrtions (Wng et l., 2000) or result from their different contents of structurl nd non-structurl crbohydrtes (Frnce nd Siddons, 1993). Decresed rumen protozol counts with supplementtion of sponin rich extrcts (Hristov et l., 1999) or sponin rich forges (Díz et l., 1993; Nvs-Cmcho et l., 1994, 2001; Newbold et l., 1997; Teferedegne et l., 1999; Hess et l., 2003) hve been reported. In the present study, the nti-protozol effect of fruits of S. sponri ws confirmed. One possible mechnism to explin the effect of sponins on protozo is chnge in cell membrne permebility (Klit et l., 1996). Of ll rumen microbes, protozo re prticulrly susceptible to sponin-induced chnges in cell membrne properties (Moss et l., 2000) Supplementtion with E. cyclocrpum nd P. smn did increse protozol counts in the present study. Also, Nvs-Cmcho et l. (1999, s cited by Nvs-Cmcho et l., 2001b) reported incresed protozo popultions in vivo when the level of P. smn fruits in the diet ws incresed from 0 to 200 mg/g, but found reduced popultions with higher levels. This, however, impired niml performnce due to low rumen ph nd reduced forge degrdbility. Sen et l. (1998) suggested tht sponins could enhnce nutrient trnsport into microbil cells when given t low doses while the mssive shift in cell membrne permebility occurring with high doses is detrimentl to the microbes, potentilly explining the differing response to the fruits investigted here. A possible smll dverse sponin effect on protozo might lso hve been msked in the E. cyclocrpum nd P. smn diets by higher nutrient supply, in the form of sugrs, in these diets compred to control. Prcticl implictions of results found with higher doses of E. cyclocrpum nd P. smn would be of limited vlue, since they exceed common threshold levels pplied by frmers in Centrl Americ in the dry seson (Zmor et l., 2001). The use of S. sponri reduced methne relese wheres E. cyclocrpum nd P. smn incresed methnogenesis reltive to the control diet. This could be the result of dose dependence, s low proportions of the sponin rich Yucc schidiger extrct lso remined without cler effect ginst methne (Śliwiński et l., 2002), but lso my be specific for the type of sponin. S. sponri, for instnce, contins sponins with high molluscicidl ctivity which re probbly genus specific nd hve not been reported for the two other two fruits (Ribeiro et l., 1995). Although effects of other secondry ingredients of the S. sponri fruits cnnot be totlly excluded, this seems unlikely becuse the biologiclly very ctive sponins mde up mjor proportion in these fruits. The present results illustrte tht the development of the methne-suppressing effect of S. sponri requires severl dys, but then seems to remin constnt Effects of defuntion Effects of defuntion on rumen fermenttion hve been intensively investigted nd reviewed, including its effect on methnogenesis (e.g. Jouny et l., 1988; Jouny, 1994; Mthison et l., 1998; Moss et l., 2000). Most defuntion induced chnges found in the present study were expected from those previous studies, except for pprent OM nd

13 H.D. Hess et l. / Animl Feed Science nd Technology 109 (2003) NDF degrdtion which ws not ffected by defuntion. In spite of this observtion, the concentrtion nd dily mount of totl VFA ws reduced by elimintion of protozo. This reduction ws ccompnied by lower molr proportion of cette nd n-butyrte nd n incresed proportion of propionte. Acette nd n-butyrte re the mjor fermenttion end products of protozo (Jouny, 1994), nd therefore defuntion shifted VFA ptterns towrds higher propionte production (10.1 mmol per dy versus 12.4 mmol per dy in funted nd defunted rumen fluid, respectively). Even though the pprent CP degrdtion ws higher in defunted rumen fluid, the mmoni concentrtion ws reduced by bout 30%. This ws probbly the consequence of the termintion of the bcteri-degrding ctivity of the protozo (Jouny, 1994) nd suggests tht the CP degrded ws more efficiently used in defunted rumen fluid for microbil protein synthesis. This is further supported by the compenstory increse of totl bcteril counts cused by defuntion. Dily methne relese ws reduced by defuntion, s hd been found in some other studies (e.g. Whitelw et l., 1984; Dohme et l., 1999), lthough the extent of this effect seems to depend on the type of diet used (Kreuzer et l., 1986; Ushid et l., 1997). Since fibre degrdtion ws similr in funted nd defunted rumen fluid in the present study, the lower methne relese cnnot be ttributed to reduced fibre degrdtion in the defunted stte. Similr to results of Dohme et l. (1999), the methne suppression by defuntion ws not ccompnied by reduction in methnogen count. However, comprison of the dt is limited by the different methnogen counts suggesting tht methnogen cultivtion, s pplied in Dohme et l. (1999), ws probbly incomplete in contrst to the FISH technique used in the present study. The missing response in methnogen count to defuntion suggests tht defuntion reduced methne production becuse of lower hydrogen supply, thus reducing ctivity per methnogen. Prt of the high initil methne suppressing effect of defuntion ws trnsient, presumbly due to the estblishment of new blnce between hydrogen producers other thn protozo nd methnogens Interction between supplementtion with sponin rich fruits nd defuntion Interctions between diet nd protozol sttus were mostly wek. This is stonishing in the cse of methne relese since non-dditive effect of the fruits nd of defuntion ws expected from the direct effects of the fruits on protozo. Furthermore, the fruits obviously influenced dily methne relese in similr wy s they ffected protozol counts, suggesting tht inhibition of methnogenesis by S. sponri ws primrily the result of its nti-protozol ctivity. However, the inhibitory effect of S. sponri ws not dependent on the presence of protozo in rumen fluid. The effect ws numericlly higher in defunted versus funted rumen fluid. Also the dely in effect of S. sponri supplementtion on methnogenesis by 4 5 dys effectively negtes ny connection between sponin induced depression of protozol popultion nd reduced methnogenesis. Lck of interctions with defuntion ws described in the context of methne nd protozo suppression cused by dietry coconut oil (Dohme et l., 1999). Therefore, it seems likely tht sponins or other constituents of S. sponri directly influence ctivity of methnogens. As this ws not reflected in reduced methnogen counts, decrese in ctivity per methnogen hs to be ssumed. Effects of dietry mnipultions on the methne-producing cell ctivity of individul methnogens hve been proposed (Soliv et l., 2003). Finlly, differentil effects

14 92 H.D. Hess et l. / Animl Feed Science nd Technology 109 (2003) of sponins on individul bcteril species, s demonstrted by Wng et l. (2000) with Yucc sponins, could help to explin decresed methnogenesis unffected by protozol popultions. 5. Conclusions Results confirm tht fruits of the tropicl multi-purpose tree Spindus sponri hve nti-protozol effects nd indicte tht they hve the potentil to reduce methne relese from ruminl fermenttion. Since the depression of methne relese due to S. sponri ws found in both funted nd defunted rumen fluid, it cn be ssumed tht this effect is not medited through ssocited effects on protozo. The fruits of Enterolobium cyclocrpum nd Pithecellobium smn, in the proportions used in this study, seem to hve low vlue in reducing totl rumen protozol counts nd methne relese. Results lso illustrte tht combintions of S. sponri supplementtion nd defuntion would be prticulrly promising strtegies to suppress methne emissions. However, n importnt constrint is suggested by the fct tht there is still no simple nd effective on frm defuntion method (Moss et l., 2000). Certin limittions in generlistion of the results could be given by the fct tht the rumen fluid used in this study ws from single donor cow only. Further reserch is necessry to confirm effects of S. sponri under vrying dietry conditions s well s in vivo with reference to niml performnce. Acknowledgements This study ws supported by the Swiss Agency for Development nd Coopertion (SDC) through their Reserch Fellow Prtnership Progrmme. The uthors re grteful to Prof. Wnner nd Prof. Brun, Fculty of Veterinry Medicine, University of Zurich, nd their lbortory stff for nlysing the VFA nd llowing ccess to the rumen-fistulted donor cow. References Crro, M.D., Lebzien, P., Rohr, K., Effect of pore size of nylon bgs nd dilution rte on fermenttion prmeters in semi-continuous rtificil rumen. Smll Rumin. Res. 15, Czerkwski, J.W., Breckenridge, G., Design nd development of long-term rumen simultion technique (Rusitec). Br. J. Nutr. 38, Demeyer, D.I., Quntittive spects of microbil metbolism in the rumen nd hindgut. In: Jouny, J.P. (Ed.), Rumen Microbil Metbolism nd Ruminnt Digestion. INRA, Pris, Frnce, pp Díz, A., Avendño, M., Escobr, A., Evlution of Spindus sponri s defunting gent nd its effects on different ruminl digestion prmeters. Livest. Res. Rurl Dev. 5, 1 6. Dohme, F., Mchmüller, A., Estermnn, B.L., Pfister, P., Wsserfllen, A., Kreuzer, M., The role of the rumen cilite protozo for methne suppression cused by coconut oil. Lett. Appl. Microbiol. 29, Dominguez Bello, M.G., Escobr, A., Rumen mnipultion for the improved utiliztion of tropicl forges. Anim. Feed Sci. Technol. 69, Finly, B.J., Estebn, G., Clrke, K.J., Willims, A.G., Embley, T.M., Hirt, R.P., Some rumen cilites hve endosymbiotic methnogens. FEMS Microbiol. Lett. 117,

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