Effects of high- and low-fiber diets on fecal fermentation and fecal microbial populations of captive chimpanzees.код для вставкиСкачать
American Journal of Primatology 71:548–557 (2009) RESEARCH ARTICLE Effects of High- and Low-Fiber Diets on Fecal Fermentation and Fecal Microbial Populations of Captive Chimpanzees SVETLANA KIŠIDAYOVÁ1, ZORA VÁRADYOVÁ1, PETER PRISTAŠ1, MÁRIA PIKNOVÁ1, KATARÍNA NIGUTOVÁ1, KLÁRA J. PETRŽELKOVÁ2,3, ILONA PROFOUSOVÁ2,4, KATEŘINA SCHOVANCOVÁ2,5, JIŘÍ KAMLER2,6, AND DAVID MODRÝ4,7 1 Institute of Animal Physiology, Slovak Academy of Sciences, Kosˇice, Slovak Republic 2 Department of Mammal Ecology Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Brno, Czech Republic 3 Liberec Zoo, Liberec, Czech Republic 4 Department of Parasitology, Faculty of Veterinary Medicine of University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic 5 Department of Botany and Zoology, Masaryk University, Brno, Czech Republic 6 Faculty of Forestry and Wood Technology MZLU, Brno, Czech Republic 7 Biology Center, Institute of Parasitology, Academy of Sciences of the Czech Republic, České Budeˇjovice, Czech Republic We examined fiber fermentation capacity of captive chimpanzee fecal microflora from animals (n 5 2) eating low-fiber diets (LFDs; 14% neutral detergent fiber (NDF) and 5% of cellulose) and high-fiber diets (HFDs; 26% NDF and 15% of cellulose), using barley grain, meadow hay, wheat straw, and amorphous cellulose as substrates for in vitro gas production of feces. We also examined the effects of LFD or HFD on populations of eubacteria and archaea in chimpanzee feces. Fecal inoculum fermentation from the LFD animals resulted in a higher in vitro dry matter digestibility (IVDMD) and gas production than from the HFD animals. However, there was an interaction between different inocula and substrates on IVDMD, gas and methane production, and hydrogen recovery (Po0.001). On the other hand, HFD inoculum increased the production of total short-chain fatty acids (SCFAs), acetate, and propionate with all tested substrates. The effect of the interaction between the inoculum and substrate on total SCFAs was not observed. Changes in fermentation activities were associated with changes in bacterial populations. DGGE of bacterial DNA revealed shift in population of both archaeal and eubacterial communities. However, a much more complex eubacterial population structure represented by many bands was observed compared with the less variable archaeal population in both diets. Some archaeal bands were related to the uncultured archaea from gastrointestinal tracts of homeothermic animals. Genomic DNA in the dominant eubacterial band in the HFD inoculum was confirmed to be closely related to DNA from Eubacterium biforme. Interestingly, the predominant band in the LFD inoculum represented DNA of probably new or yet-to-be-sequenced species belonging to mycoplasms. Collectively, our results indicated that fecal microbial populations of the captive chimpanzees are not capable of extensive fiber fermentation; however, there was a positive effect of fiber content on SCFA production. Am. J. Primatol. 71:548–557, 2009. r 2009 Wiley-Liss, Inc. Key words: chimpanzee; fiber; diet; in vitro fecal fermentation; DGGE; archaea; eubacteria INTRODUCTION Great apes are generally classified as herbivores [Milton, 1999; Milton, 2003b] and their intestine is a complex system composed of three main components containing important microbes that play various physiologic roles in the host animal. In great apes, microbial fermentation of digesta occurs in the lower intestine and results in the production of short-chain fatty acids (SCFAs) and gas. SCFAs and fermentative gas are produced predominantly in herbivorous animals, especially in the forestomach of ruminants [Bergman, 1999]. The SCFAs are also produced in the lower digestive tract of humans and all animal species, with the intestinal fermentation pattern r 2009 Wiley-Liss, Inc. Contract grant sponsor: Grant Agency of Czech Republic GAČR; Contract grant number: 524/06/0264; Contract grant sponsor: Grant Agency Ministry of Education of Slovak Republic and the Slovak Academy of Sciences VEGA; Contract grant number: 2/ 0009/08; Contract grant sponsor: MVTS project; Contract grant number: SK-CZ-0086-07. Correspondence to: Svetlana Kis̆idayová, Institute of Animal Physiology, Slovak Academy of Sciences, Soltesovej 4-6, 04001 Košice, Slovakia. E-mail: firstname.lastname@example.org Received 25 October 2008; revised 17 March 2009; revision accepted 17 March 2009 DOI 10.1002/ajp.20687 Published online 14 April 2009 in Wiley InterScience (www. interscience.wiley.com). Chimpanzee Fecal Microflora Fermentation Capacity / 549 similar to that in the rumen [Bergman, 1999]. Wild chimpanzees are estimated to take 87–98% of their annual diet from plant sources [Milton, 1999; Milton & Demment, 1988]. Chimpanzee diet is highly variable from one population to the next, but in all cases dominated by ripe fruit [Newton-Fisher, 1999]. Fiber fractions in the diets of free-ranging chimpanzees on a dry-weight basis were 33.6% of neutral detergent fiber (NDF) and 19.6% of acid detergent fiber (ADF) [Conklin-Brittain et al., 1998]. Chimpanzees are also known to eat termites, ants, and vertebrate prey, but such foods tend to make up only a small percentage (4–6%) of their annual diet [Goodall, 1986; Stanford, 2008]. The ecosystem of colon of great apes is considered to be similar to that of humans [Jenkins et al., 1998; Milton, 2003a], except for the presence and variable prevalence of ciliates of genus Troglodytella in the large intestine and cecum of all wild African great apes [File et al., 1976; Goussard et al., 1983; Imai et al., 1991; Muehlebein, 2005; Murray et al., 2000]. Several studies were conducted on in vitro fermentation with primate feces, in orangutans Pongo abelii [Schmidt et al., 2005], western chimpanzees Pan troglodytes verus [Ushida et al., 2006], three species of lemurs [Campbell et al., 2002], and vervet monkeys Chlorocebus aethiops [Costa et al., 1989]. The effects of high-fiber diets (HFDs) on fecal fermentation in wild and captive chimpanzees remain to be investigated. Understanding the role of intestinal microflora on nutrient utilization could prove useful for diet formulation. Great apes and other primates housed in zoological institutions are typically fed diets consisting of combinations of commercially manufactured primate biscuits and readily available fresh produce. These fruits and vegetables have been cultivated for human consumption and are high in soluble sugars and low in fiber compared with the foods selected by free-ranging primates [Schmidt, 2002]. Primate biscuits usually contain maximum 30% NDF and 20% ADF. The dietary fractions that have replaced the structural fiber in the diets of captive primates are readily available sugars and starch [Schmidt et al., 2000]. Some researchers have suggested that increasing the structural fiber and decreasing the levels of readily available carbohydrates in some herbivorous primate diets may decrease the incidence of obesity and other related health problems often seen in captive great apes and other primates [Schmidt et al., 2000]. However, no data on fiber fermentation capacity of chimpanzee gut microbiota are available to predict the effects of changes in fiber diet content of captive chimpanzees on fermentation parameters and microbial population. The objectives of the present in vitro study were as follows: (1) to examine the fermentation capacity of captive chimpanzees fecal microflora under conditions of low fiber or high fiber diets when meadow hay (MH), wheat straw (WS), barley grain (BG), and amorphous cellulose (AC) were used as substrates for in vitro gas production technique; (2) to determine the effects of low-fiber diet (LFD) and HFD on the populations of eubacteria and archaea in the feces of captive chimpanzees. METHODS Animals and Diets This study was conducted in the Liberec Zoo (Czech Republic) from July 25, 2007 to September 14, 2007. Two captive male chimpanzees (P. troglodytes; Kongo, 42 years of age; Tedy, 15 years of age; 6072.0 kg) were selected. Intestinal tract of both chimpanzees was naturally faunated with ciliate protozoa of Troglodytella abrassarti. Chimpanzees were fed two diets summarized in Table I, showing the mean values per animal and day. Changes in the diet were approved by the personnel in the Liberec Zoo as increasing fiber in diet of chimpanzees needed to be implemented. There were several caveats regarding the methodology and restrictions when working with zoo animals. For welfare and enrichment purposes, primate curators and keepers fed the animals a variable diet of different fruit and vegetable components. The components for the experimental HFD and basal LFD were carefully selected and fed according to the experimental nutritional parameters. Ssniff Pri biscuits (Ssniff Spezialdiäten GmbH, Soest, Germany), bakery products, and potatoes were not fed every day. In addition, the chimpanzees received browse of branches of Salix carpea, which comprised approximately 5% of their HFD diet. Tedy and Kongo were fed a basal LFD for 10 days for adaptation purposes, TABLE I. Chimpanzees Daily Intake of Experimental Diets (Means of Dry Matter, g/day and Chimpanzee) Intake Food item LFD HFD s Ssniff Pri biscuits 28 0 0 813 Nutrazus Leaf-Eater Primate biscuits Fruit—LFD (banana, plum, peach, 432 0 nectarine, watermelon, grapes, apple) Fruit—HFD (kiwi, pineapple, pears) 0 48 Fruit—both diets (apple, orange) 47 31 Vegetable—LFD (onion, carrot, kohlrabi) 39 0 Vegetables—HFD (root parsley, 0 63 Chinese leaves, spring onion, white charlock, lettuce, leek, corn cob, pepper, root celery, tomato) Bakery products 46 0 Potatoes (fresh and boiled) 48 0 Browse (Salix caprea) 0 ad libitum LFD, low-fiber diet; HFD, high-fiber diet. Ssniffs Pri biscuits, bakery products and potatoes were not fed every day. Am. J. Primatol. 550 / Kišidayová et al. which was followed by a 10 day experimentation LFD period with collection of feces. Subsequently, this period was followed by two consecutive 10-day transition periods to HFD. Finally, the experiment was concluded by a 10-day experimental period with HFD that included the collection of feces. Ethical considerations prohibited us from separating the animals throughout the study period. Therefore, the animals were separated only during the feeding trials and nights as fecal samples were collected every morning. Water was available ad libitum. Each diet item was weighed before the feedings and the food remainders were removed from their cages and their weight determined. Samples of each food item were frozen and analyzed for crude protein, lipid, starch, ash, ADF, acid detergent lignin (ADL), and NDF by standard methods [AOAC, 1996]. Starch was hydrolyzed with hydrochloric acid and analyzed as free sugars by polarimetry, whereas proteins were determined by using the Carres agents. The resulting values were corrected using optically active compounds dissolved in an ethanol–water mixture (Table II). Hemicellulose was calculated as NDF–ADF. Cellulose was calculated as ADF–ADL. Despite the fact that fecal microbes do not completely represent the microbial population of the cecum and colon, the fecal fermentation technique is a quite valuable non-invasive tool to approximate cecal and colonic fermentation. Collection of fecal samples was non-invasive and did not include any disturbance of the animals. The described research adhered to the legal requirements of the country, in which it was conducted. In vitro Feces Fermentation Hungate tubes (Belco Glass, Inc., Vineland, NJ) were used as fermentation vessels for the pressure transducer (PT) method of this in vitro gas production technique [Varadyova et al., 2005]. Fermentation TABLE II. Mean Nutritional Composition of LowFiber Diet (LFD) and High-Fiber Diet (HFD) of Chimpanzees and Fermentation Substrates of Wheat Straw (WS), Meadow Hay (MH) and Barley Grain (BG) Component LFD HFD WS MH BG % of dry matter NDF ADF ADL Cellulose Hemicellulose Crude protein Starch Lipids Ash 14.05 6.89 1.95 4.94 7.16 6.66 7.59 1.04 3.67 25.75 15.66 0.84 14.82 10.09 21.28 1.04 5.05 7.74 71.7 56.1 9.9 46.2 15.6 3.8 0.01 1.1 6.0 57.6 36.76 7.59 29.17 20.84 0.89 0.04 2.3 8.0 26.13 6.74 1.36 5.38 19.39 2.21 59.9 2.1 3.66 NDF, neutral detergent fiber; ADF, acid detergent fiber; ADL, acid detergent lignin; Cellulose 5 ADF–ADL; hemicellulose 5 NDF–ADF. Am. J. Primatol. experiments were carried out for each diet regime and lasted 24 hr. Inoculum Fresh fecal samples were collected after the morning feeding and transported to the laboratory in a pre-warmed thermos box continuously kept at 3871.01C. Feces (200 g/diet type) of both experimental animals were pooled and diluted in 300 ml of McDougall’s buffer [McDougall, 1948]. The feces were thoroughly mixed and filtered through four layers of gauze and kept at 3871.01C under CO2. After that, 10 ml of fecal inoculum from LFD or HFD animals were added anaerobically into 15 ml Hungate tubes containing 0.08 g of substrate. Substrates The fiber-digesting capacity of chimpanzee fecal microflora was tested with AC (100% of cellulose of initial dry matter), WS (46% of cellulose), MH(29% of cellulose), as well as capacity to starch digesting with BG (5% of cellulose), as shown in Table II. The WS, MH, and BG were ground and sieved through a 0.15–0.4 mm screen, bulked, and stored in sealed plastic containers until required. Six replicate fermentation tubes of each substrate were used for experimental groups of LFD and HFD inocula, each with inoculum, buffer, and substrate; and six were also used for the controls of LFD and HFD inocula, omitting the substrate. Gas measurements The inoculated fermentation tubes were filled up with CO2, closed with a butyl rubber stopper, plastic sealed and placed at 3971.01C in an incubator. The volume of accumulated gas was measured after 24 hr of incubation. The metering system of PT consisted of a three-way valve, a mechanical manometer fitted to a transducer (Premagas, Stara Tura, Slovakia), and a gas-tight syringe and needle. The three-way valve was connected to the transducer to measure pressure in the Hungate tubes and a gas-tight syringe was used to measure the volume of gas produced. The third port was connected by a hose to a needle, which was used to punch the rubber stopper on the Hungate tube. Gases from each fermentation Hungate tube were collected in a 2 ml glass syringe (for each Hungate tube separately) at the end of incubations and immediately analyzed for methane concentration using gas chromatography. Fermentation parameters analyzed SCFAs in the fermentation medium at the end of the 24 hr incubation period were determined by gas chromatography [Cottyn & Boucque, 1968] using crotonic acid as the internal standard with PerkinElmer Clarus 500 gas chromatograph (Perkin-Elmer, Inc. Shelton, CT). Chimpanzee Fecal Microflora Fermentation Capacity / 551 Methane from fermentation gas was analyzed using gas chromatograph (Perkin-Elmer Clarus 500). The percentage of methane was expressed per 1 ml of the gas volume. In vitro dry matter digestibility (IVDMD) was determined from the difference of the substrate weight before and after incubation and dry matter of control group. Contents of the fermentation vessels were centrifuged at 3500g for 10 min, residues were washed twice with distilled water, re-centrifuged, and dried at 1051C to constant weight [Mellenberger et al., 1970]. Hydrogen recovery was calculated as follows: 2H recovery (%) 5 2H accepted/2H released 100, where 2H accepted 5 (4M12P12B14V14C) and 2H released 5 (2A1P14B13V16C) with acetate (A), propionate (P), butyrate (B), valerate (V), caproate (C), and methane (M); expressed as net molar production rates. This stoichiometric recovery expresses the validity of metabolic hydrogen transfer based on the rumen fermentation model [Demeyer & Degraeve, 1991). Prokaryotic Fecal Population Analysis To examine bacterial population changes in the feces of chimpanzees fed LFD or HFD, a polymerase chain reaction/denaturizing gradient gel electrophoresis (PCR/DGGE) approach was used. Processed feces (transported for in vitro fermentation, pooled, diluted, filtered, and frozen at 601C) were used later for DNA isolation. Total DNA isolation The following methods of DNA isolation from chimpanzee feces were used for optimal DNA recovery: the E.Z.N.A. bacterial DNA kit (Omega Biotek, Norcross, GA), standard sodium dodecylsulphate lysis and subsequent chloroform extractions [Pospiech & Neumann, 1995], and the Chelex method [Regensbogenova et al., 2004]. Total DNA obtained by these methods was used as a template for PCR amplification of small subunit rRNA gene sequences from the chimpanzee feces microbial community. PCR amplification of 16S rRNA gene fragments for DGGE analysis Purified total DNA was used as a template for PCR amplification of eubacterial and archaeal 16S rRNA gene sequences. All PCR reactions were performed in a 50 ml PCR mix containing either 250 ng of purified genomic DNA or 1 ml of 10 diluted PCR product from first amplification, 200 mM each dNTP, 1 reaction buffer, 1.25 U Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), and 25 pmol each primer using Techne Progene thermal cycler (Techne, Cambridge, UK). Archaeal community The archaeal 16S rRNA genes from total community DNA was amplified using archaea-specific primers. The amplicons of approximately 180 bp spanned the hypervariable V3 region of 16S rDNA and were used for DGGE analysis of archaeal community structure. To obtain PCR samples for DGGE analysis of archaeal community, a nested PCR–DGGE approach was used. Nearly full length archaeal 16S rDNA was amplified with the primers ArcF7 and ArcR1326 [van Hoek et al., 2000]. The PCR parameters were 941C for 3 min followed by 35 cycles of 941C for 45 sec, 501C for 45 sec, and 721C for 1.5 min. In the second round of amplification, the obtained PCR products were used as a template to amplify the V3 region of the archaeal 16S rRNA gene by using the GC-clamp primer 344F(GC) and S-Univ-0518-a-A-17 [Muyzer et al., 1993]. Amplification was performed under the following conditions: the initial denaturation step at 941C for 3 min followed by 30 cycles of denaturation at 941C for 45 sec, annealing at 451C for 45 sec, extension at 721C for 30 sec, and final cycle step at 721C for 10 min. Eubacterial community For DGGE analysis of eubacterial microbial community, the V6–V8 regions of 16S rRNA genes were amplified with the pair of universal bacterial primers 968-GC-f and 1401-r [Nübel et al., 1996] using total DNA isolated from the chimpanzee feces. Thermal cycling conditions were as follows: denaturation at 941C for 3 min, 35 cycles of denaturation at 941C for 45 sec, annealing at 501C for 45 sec, extension at 721C for 45 sec, and the final extension at 721C for 5 min. Before the DGGE analysis, the presence of PCR products was confirmed by electrophoresis in an agarose gel stained with ethidium bromide and viewed under UV trans-illumination. DGGE of PCR products generated either with 968f-GC/1401r or 344F(GC)/S--Univ-0518-a-A-17 primer set was performed using the DCode Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA). PCR samples (40 ml) were applied directly onto 6% (for separation of eubacterial amplicons) or 9% (for separation of archaeal amplicons) polyacrylamide gels (40% Acrylamide-Bis 37.5:1) containing a linear denaturing gradient ranging from 35 to 55% denaturant (100% denaturant corresponded to 7 M urea and 40% (v/v) formamide). Electrophoresis was performed in 1 TAE (40 mM Tris, 20 mM acetate, 1 mM EDTA) at a constant voltage of 50 V at 601C for 17 hr. Afterward, the gels were incubated for 15 min in ethidium bromide (0.5 mg/ml), rinsed for 15 min in distilled water, and photographed using a MiniBis UV Vis Gel Am. J. Primatol. 552 / Kišidayová et al. Documentation system (Micro Photonics, Allentown, PA). Sequence analysis and comparisons Selected bands were excised from the DGGE gel using a clean scalpel and transferred to a microcentrifuge tube. DNA was recovered from polyacrylamide gel slices by the addition of 50 ml of water to each tube, which was then vortexed for 5 sec and centrifuged for 1 min. The supernatant was transferred to a clean tube and used as template in a final PCR reaction with the same primers as for the DGGE analysis. PCR products were cloned into the plasmid vector (pCR 2.1) using a TOPO TA cloning kit (Invitrogen). Recombinant clones were selected and plasmid DNA extracted using a plasmid miniprep kit (Qiagen, Hilden, Germany). Sequencing was performed on both strands at the Automated DNA Sequencing Service at Macrogen Inc., (Seoul, South Korea). The sequences of DGGE bands were submitted to the GenBank nucleotide sequence database. For the list of accession numbers see Table III. The sequences obtained were analyzed using the BLAST algorithms [Altschul et al., 1997] available at the National Centre for Biotechnology Information web page (http://www.ncbi.nlm.nih.gov/BLAST/). Statistics Statistical analysis was performed using analysis of variance (ANOVA) (Graphpad InStat, GraphPad Software, Inc., San Diego, CA) as a 4 2 factorial design, that represented four substrate groups (MH, WS, BG, and MC) and two inocula (LFD and HFD). Interaction between control and substrates were analyzed by a regular two-way ANOVA with Bonferroni post-test. The differences between the treatment and control means were considered to be significant when Po0.05. The means of acetate:propionate (A:P) ratios were compared by unpaired t-test. RESULTS Fermentation Parameters The interaction of the inocula and substrates (I S; Table IV) occurred in the IVDMD (F3,40 5 29.79; Po0.001), gas volume (F4,50 5 268.4; Po 0.001), methane production (F4,50 5 31.27; Po0.001), and hydrogen recovery (F3,40 5 8.6; Po0.001). No interaction of inoculum and substrate was detected in total SCFA production. The concentration of total SCFAs of the substrate treatment was increased for BG under both diet regimes (tLFD 5 7.36, Po0.001; tHFD 5 6.44, Po0.001), in contrast to the decreased total SCFAs at AC substrate (tHFD 5 2.48; Po0.05) compared with the fermentation control. Other experimental substrates had no effect on total SCFAs. Interaction of inoculum and substrate (I S; Table V) occurred in the concentrations of acetate (F4, 50 5 12.7, Po0.001), propionate (F4,50 5 3.1, Po0.05), n-butyrate (F4,50 5 11.4, Po0.001), iso-butyrate (F4,50 5 5.0, Po0.05), and n-caproate (F4,50 5 5.8, Po0.001). Concentrations of n-valerate (tHFD 5 2.5; Po0.05) and iso-valerate (tLFD 5 2.4; Po0.05) of the inoculum treatment were lower compared with the control for BG. The acetate: propionate (A:P) ratios produced in the fermentation process ranged from 2.6 to 3.2 and from 3.1 to 4.0 for TABLE III. Selected DGGE Bands Characterised During This Study DGGE band GB accession number A1 EU477159 A2 A3 EU477160 EU477161 A4 EU477162 A5 EU477163 E1 EU477164 E2 EU477165 Am. J. Primatol. Blastn best hit uncultured archaeon clone ConP1-14F Tetratrichomonas sp. uncultured methanogenic archaeon clone SRmetF14 methanogenic archaeon CH1270 methanogenic archaeon CH1270 uncultured bacterium clone RL186 uncultured bacterium clone RL184 % of sequence identity Blastn best hit from cultured organism where best hit is from uncultured source % of sequence identity 98 Picrophilus torridus 98 99 – Methanobrevibacter woesei 98 Picrophilus torridus 97 98 Picrophilus torridus 97 93 Bulleidia extructa 92 99 Eubacterium biforme 98 97 – 99 Chimpanzee Fecal Microflora Fermentation Capacity / 553 TABLE IV. Fermentation Parameters (Means) as Influenced by Substrate and Different Diet After 24 hr of Incubation With Chimpanzee Feces Substrate (S) Inoculum (I) Control LFD HFD LFD HFD LFD HFD LFD HFD LFD HFD AC WS MH BG S.E.M. Significance Control Control Control Control vs. vs. vs. vs. IVDMD (%) Gas volume (ml/g DM) Methane (10 2 ml/ml) Total SCFAs (mmol/l) 2H recovery (%) 28.0 5.1 23.6 5.3 43.8 25.0 73.2 83.8 2.0 272 100 235 100 266 96 364 178 624 375 1.8 9.0 1.6 2.1 0.4 6.3 0.4 7.0 1.4 6.0 3.1 0.3 40.9 61.9 44.3 58.4 46.7 60.6 47.6 62.9 56.3 71.0 0.8 56.0 41.3 45.0 39.4 51.0 38.8 53.0 40.9 52.0 47.6 1.0 I IS AC WS MH BG – – – – – – – – – – – – ns – – – – ns ns AC, amorphous cellulose; WS, wheat straw; MH, meadow hay; BG, barley grain; SCFAs, short-chain fatty acids; IVDMD, in vitro dry matter degradability; LFD, chimpanzee feces inoculum from low-fiber diet; HFD, chimpanzee feces inoculum from high-fiber diet; Po0.05; Po0.01; Po0.001. TABLE V. Short-Chain Fatty Acids (Means) as Influenced by Substrate and Different Diet After 24 hr of Incubation With Chimpanzee Feces Molar proportion of SCFAs (mol%) Substrate (S) Inoculum (I) Control LFD HFD LFD HFD LFD HFD LFD HFD LFD HFD AC WS MH BG S.E.M. Significance Control Control Control Control vs. vs. vs. vs. AC WS MH BG I IS Acetate Propionate n-Butyrate iso-Butyrate n-Valerate iso-Valerate n-Caproate 45.5 58.2 44.3 57.4 50.2 58.3 50.1 57.6 48.4 51.6 0.7 13.0 18.5 11.2 18.5 13.7 18.7 14.1 18.6 15.6 19.7 0.5 27.9 15.5 26.7 16.2 26.2 15.2 26.5 16.4 27.3 22.4 0.6 2.8 1.4 1.5 1.4 2.6 1.6 2.5 1.4 1.0 1.0 0.2 2.0 2.5 2.2 2.5 1.7 2.4 1.6 2.4 1.9 1.8 0.2 3.1 2.5 3.1 2.5 2.8 2.5 2.7 2.3 2.1 1.8 0.3 3.8 1.5 4.6 1.5 2.8 1.4 2.6 1.4 3.6 1.4 0.2 ns ns ns ns ns ns ns ns – – – – – – – – – – – – – – – – – – – – AC, amorphous cellulose; WS, wheat straw; MH, meadow hay; BG, barley grain; LFD, feces inoculum from low-fiber diet; HFD, feces inoculum from highfiber diet; SCFAs, short-chain fatty acids; Po0.05; Po0.01; Po0.001. substrates incubated with HFD (mean 3.070.11) and LFD (mean 3.670.15) inocula, respectively, with the means significant different (t8 5 3.092; P 5 0.0148). Identification of Dominant Bacteria DGGE fingerprint of archaeal community is shown in Figure 1, column A. Band profiles indicated a community shift induced by the diet. Although the number of bands was practically the same on both diets, the intensity of bands was strongly affected by the diet in all but A1 band. The identity of the archaea represented by selected bands (A1–A5, Fig. 1, column A) was determined by sequencing of the bands excised from the gel, reamplified, cloned, and sequenced (Table III). Most of these organisms are related to the uncultured archaea from gastro- Am. J. Primatol. 554 / Kišidayová et al. Fig. 1. DGGE analysis of archaeal (column A) and eubacterial (column E) faecal populations of chimpanzees fed with low- (lane S) or high-fiber (lane F) diets. Bands selected for sequence analysis are marked. intestinal tracts of homeothermic animals (pig feces—A1, rumen of Svalbard reindeer—A3, chicken cecum—A4, A5). Surprisingly, organisms represented by band A2 were found to be Tetratrichomonas-like protists of the order Trichomonadida. The eubacterial community was analyzed by DGGE analysis of approximately 450 bp V6–V8 region of 16S rRNA amplicons (Fig. 1, column E). Band profile comparison showed evident shift in the composition that was induced by the diet. However, much more complex eubacterial population structure represented by many weak bands was observed compared with a less variable archaeal population. The identity of two weakly dominant eubacterial species represented by E1 and E2 bands was determined by the sequence comparison. Although the E2 band (predominant on HFD) is closely related to Eubacterium biforme, the E1 band representing the dominant eubacterial species on LFD shows only limited similarity to the existing records in GenBank database and probably represents new, up to now unrecognized species belonging to mycoplasms. DISCUSSION Apes are considered to be hindgut fermenters and their large colon enables them to retain plant material for sufficient time to allow gut bacteria and possibly also ciliates to ferment ingested fiber [Collet et al., 1984; Milton & Demment, 1988; Remis & Dierenfeld, 2004]. Depending on the variability in fiber intake, geographic and seasonal diet variation may influence the extent of hindgut fermentation in colonic retrieval of dietary energy by the great apes Am. J. Primatol. [Conklin-Brittain et al., 1998; Popovich et al., 1997; Rothman et al., 2008]. Gorillas could hypothetically obtain considerable part of metabolizable energy (57%) through colonic fiber fermentation [Popovich et al., 1997] in comparison with 2–9% for humans [McNeil, 1984]. However, Rothman et al.  found out that the total diet digestibility of wild eastern gorillas on diets with approximately 40% NDF was about 60%, indicating that the Popovich’s  estimates of fiber digestibility are likely too high. The high extent of fiber fermentation was reported in captive orangutans, where fecal bacteria digested 86–88% of soybean hull and ground corncob substrates [Schmidt et al., 2005]. Milton & Demment  discovered that chimpanzees on an experimental HFD containing 34% NDF, which was similar to the amount of NDF in the diet of wild chimpanzees from Kibale NP [Conklin-Brittain et al., 1998], digested 54.3% of this diet. On the contrary, only 5–20% of high fiber substrates (AC, WS, and MH) was digested by fecal bacteria from the captive chimpanzees fed HFD in our experiment. However, the content of cellulose in HFD in our experiment was two-times higher than in the study of Milton and Demment  at the expense of lower contents of hemicellulose and lignin. Despite interactions between the type of inoculum and tested substrates, we observed that higher amounts of cellulose (>15%) affected the fermentation parameters. Significant effects of the source of dietary fiber on substrate fermentability by gut microflora were also previously observed in cats, dogs, horses, pigs, cattle, and humans [Sunvold et al., 1995]. The HFD in our experiment leads to a depression in IVDMD, which was most apparent for AC and WS fermentation substrates. This is in an agreement with the results of an in vivo digestibility study on chimpanzees, where the depression in digestibility with the HFDs was greatest on the slowest digesting fraction, cellulose [Milton & Demment, 1988]. These differences in IVDMD of MH, WS, and AC incubated with HFD inoculum observed in comparison with LFD inoculum were probably associated with rapidly (hemicellulose) and slowly (cellulose) digested fractions of the substrates. Our in vitro fermentation experiment showed that gas and methane production was lower for HFD inoculum vs. LFD inoculum. The significant differences in gas, methane, and SCFA production between the diets reflected the shift in bacterial population observed by PCR/DGGE analysis (Fig. 1). Methanogenesis represent loss of the part of feed energy. It would be advantageous in terms of animal nutrition to shift the intestine fermentation toward greater acetogenesis at the expense of methanogenesis. In HFD, the lower hydrogen recovery values were associated with lower methanogenesis. The lower values for methane and hydrogen recoveries in HFD inoculum than in LFD inoculum may indicate the dominance of reductive acetogen- Chimpanzee Fecal Microflora Fermentation Capacity / 555 esis rather than methanogenesis, which would be similar to the in vitro fermentation studies of the foods eaten by wild chimpanzees in Bossou [Ushida et al., 2006]. When Albizia zygia gum was used as a substrate, acetate was the most abundant produced acid, followed by lactate and propionate with the proportion of acetate:propionate:n-butyrate 5 62:20:6 [Ushida et al., 2006]. We did not measure lactate in our experiment, but the proportion of acetate:propionate:n-butyrate was 48:12:27 and 57:19:17 for LFD and HFD, respectively. Rumen ciliates have important contributions to the production of n-butyrate in the rumen [Michalowski, 1987]. We speculate that the wild chimpanzee population and/or activity of T. abrassarti (T. a.) might have been depressed. However, no data on T. a. population in Ushida study was provided and we could not estimate the contribution of T. a. to the fermentation in our experiments. This could be evaluated by experiments with defaunated animals and/or by experiments on axenic T. a. in vitro cultures. However, cultivation of T. a. has not yet been successful. Hydrogen recovery yields in our experiment were attributed to the formation of acetate and butyrate as well as the activity of hydrogen utilizing methanogens. In humans, the reductive acetogenesis demonstrates an important pathway for H2 disposal in the colon of subjects harboring low numbers of methanogens [Bernalier et al., 1996; Lajoie et al., 1988; Miller & Wolin, 1996]. In addition, some digestive ecosystems such as the guts of termites [Breznak & Switzer, 1986] or the ostrich hindgut [Fievez et al., 2001] can be dominated by reductive acetogens rather than methanogens. In the rumen, methanogens functioning as a hydrogen sink may be replaced by reductive acetogens [Demeyer et al., 1989; Demeyer & Degraeve, 1991], but reductive acetogenesis is not as efficient as methanogenesis [Fonty et al., 2007]. The acetate:propionate (A:P) ratios produced in our fermentation experiments ranged from 2.6 to 3.2 for substrates incubated with HFD. An A:P ratios ranging from 1.8 to 2.8 was observed when soybean hulls and ground corncobs were fed to the orangutan hindgut microflora in the continuous culture system [Schmidt et al., 2005]. These differences in A:P ratio may be related to greater acetogenesis in chimpanzees than in orangutans resulting from different activities of microbial populations and capability of extensive fiber digestion by orangutans at HFD. In monogastric animals, the importance of SCFA for nutrient energy supply is related to the dietary fiber content and is still a contentious issue as several aspects other than SCFA production should be taken into account when evaluating fiber for nutrition [Dierick et al., 1989]. From a nutritional point of view, SCFAs can contribute to energy supply but with lower efficiency compared with glucose. The principal SCFA, acetate, propionate, and butyrate, are metabolized by the colonic epithelium (butyrate), liver (propionate), and muscle (acetate) [Cummings & MacFarlane, 1997]. Butyrate as a major energy source in epithelial cells improves the health of the intestines. In our study, lower IVDMD, gas and methane production of MH, WS, and AC with HFD inoculum demonstrate the lower microbial activity of HFD inoculum vs. LFD inoculum. These differences indicate that experimental chimpanzees may have lower potential for fermentation of substrates with higher contents of cellulose. It is evident that as the cellulose content of the substrate increased, the amount of gas decreased. Differences in gas production observed between substrates were also caused by gas production associated with the rapidly digesting portion of the substrates, probably hemicellulose and starch. Future studies on the effects of type of fiber in diets of both captive and wild chimpanzees on fermentation parameters are necessary. Little information is available on the intestinal microflora of great apes in the captivity or wilds to enable comparison with our results [Frey et al., 2006; Ley et al., 2008; Uenishi et al., 2007]. PCR-based methods such as temperature gradient gel electrophoresis and amplified ribosomal DNA restriction analyses of the bacterial 16S rRNA gene were applied previously on feces of wild and captive chimpanzees [Uenishi et al., 2007]. This study revealed that Clostridium leptum subgroup bacteria, Lactobacillus gasseri-like bacterium, and Bifidobacterium pseudocatenulatum- or B. catenulatum-like bacterium are common intestinal bacteria for both captive and wild chimpanzees. In this study, a denaturating gradient gel electrophoresis method was used to determine the effect of low- and HFD on the diversity of eubacterial and archaeal populations in feces of captive chimpanzees. This method has already been used to investigate the variability of microorganisms in different environments, including the microbial populations in the gastrointestinal tract [Muyzer et al., 1993; Vanhoutte et al., 2004]. Our results demonstrated a clear diet induced shift in the composition of both archaeal and eubacterial communities. Although the archaeal population consisted of up to 10 species, a much more complex eubacterial population structure, represented by numerous DGGE bands, was observed in both diet regimes. Structural composition was analyzed by sequencing selected bands. No similarity with the results of Uenishi et al.  in microbial structural composition of feces in our experimental animals was observed. Predominant archaeal species in chimpanzee feces are related to archaea from other animals. Surprisingly, the microorganism represented by band A2 was found to be Tetratrichomonas-like, indicating limited specificity of archaeal PCR primers used. Tetratrichomonads are anaerobic protists found in the intestine of many animals, including humans. These microorganisms are routinely culti- Am. J. Primatol. 556 / Kišidayová et al. vated on media with starch addition [Clark & Diamond, 2002]. The DGGE analysis confirmed that Tetratrichomonads dominated in LFDs rich in starch. These results are consistent with the findings of Frey et al.  and Ley et al. . These authors analyzed bacterial diversity in fecal samples of great apes, showing that the predominant chimpanzee fecal eubacteria belonged to the Firmicutes division. The predominant eubacterial species of chimpanzee on HFDs E. biforme is related to the corresponding bacterium from human intestine [Downes et al., 2000; Wang et al., 1996]. This can either reflect a similarity between human and chimpanzee microbiota or transmission of bacteria between humans and great apes in Zoo conditions. However, we could retrieve no information on fibrolytic activities of these bacteria. Future studies involving cultivation and biochemistry are necessary to reveal the contributions of the microbial members of chimpanzee’s main gut to fibrolytic activities. Limited capacity of the adaptation to diets with increased contents of cellulose was observed in experimental captive chimpanzees. Although chimpanzees are fairly herbivorous, they are also the most omnivorous of the apes that may affect the ability or requirement for fermentation. It seems that chimpanzee’s intestine fermentation is more similar to that of humans than gorillas and orangutans. Variability of the great ape’s diets opens two main research questions: (i) differences in fermentation and microbial fauna between wild and captive great apes and (ii) geographic and seasonal variations in fermentation and microbial fauna in wild great apes. Hominid diet before fire was characteristic by several nutritional constrains including a possible energy shortage and high fiber content [Stahl, 1984; Wrangham & Conklin-Brittain, 2003]. In addition, hind-gut fermentation has probably played a more important role for our ancestors than in modern humans. Better understanding of the diet and digestive physiology of great apes may provide valuable insights into the evolution of human dietary practices. ACKNOWLEDGMENTS We would like to express our sincere thanks to Liberec Zoo for enabling us to conduct this study; namely to David Nejedlo, Son̆a Rohlová, Jir̆ina Kyzliková, Karel Kestler, Pavlı́na Passiánová, Nataša Petřiková, Luboš Melichar and Jindriška Skřivánková. We also thank to Dr. Peter Siroka for SCFAs and methane analysis. The authors acknowledge with thanks the provided valuable comments and language revision of the manuscript by Dr. J. M. Rothman from the Department of Anthropology, Hunter College at the City University of New York (USA) and Dr. E. Collakova, Department of Plant Pathology, Physiology and Weed Science, Virginia Tech, Blacksburg, Virginia. This research complied Am. J. Primatol. with host country and institutional policies of ethical research and treatment of non-human primates. REFERENCES Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. AOAC. 1996. Official methods of analysis of AOAC International. Gaithersburg, MD: Association of Official Analytical Chemists International. Bergman EN. 1999. Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Physiol Rev 70:567–590. Bernalier A, Lelait M, Rochet V, Grivet JP, Gibson GR, Durand M. 1996. Acetogenesis from H2 and CO2 by methane- and non-methane-producing human colonic bacterial communities. FEMS Microbiol Ecol 19:193–202. Breznak JA, Switzer JM. 1986. Acetate Synthesis from H2 plus CO2 by Termite Gut Microbes. Appl Environ Microbiol 52:623–630. Campbell JL, Williams CV, Eisemann JH. 2002. Fecal inoculum can be used to determine the rate and extent of in vitro fermentation of dietary fiber sources across three lemur species that differ in dietary profile: Varecia variegata, Eulemur fulvus and Hapalemur griseus. J Nutr 132:3073–3080. Clark CG, Diamond LS. 2002. Methods for cultivation of luminal parasitic protists of clinical importance. Clin Microbiol Rev 15:329–341. Collet JY, Bourreau E, Cooper RW, Tutin CEG, Fernandez M. 1984. Experimental demonstration of cellulose digestion by Trogodytella gorillae, an intestinal ciliate of lowland gorillas (Gorilla gorilla gorilla). Int J Primatol 5:328. Conklin-Brittain NL, Wrangham RW, Hunt KD. 1998. Dietary response of chimpanzees and cercopithecines to seasonal variation in fruit abundance. II. Macronutrients. Int J Primatol 19:971–998. Costa MA, Mehta T, Males JR. 1989. Effects of dietary cellulose and psyllium husk on monkey colonic microbialmetabolism in continuous culture. J Nutr 119:979–985. Cottyn BG, Boucque CV. 1968. Rapid method for the gas chromatographic determination of volatile fatty acids in rumen fluid. J Agric Food Chem 16:105–107. Cummings JH, MacFarlane GT. 1997. Role of intestinal bacteria in nutrient metabolism. J Parenter Enteral Nutr 21:357–365. Demeyer DI, Degraeve K. 1991. Differences in stoichiometry between rumen and hindgut fermentation. J Anim Physiol Anim Nutr Suppl 22:50–61. Demeyer DI, Degraeve KG, Durand M, Stevani J. 1989. Acetate: a hydrogen sink in hindgut fermentation as opposed to rumen fermentation. Acta Vet Scand 86:68–75. Dierick NA, Vervaeke IJ, Demeyer DI, Decuypere JA. 1989. Approach to the energetic importance of fiber digestion in pigs.1. Importance of Fermentation in the Overall Energy Supply. Anim Feed Sci Technol 23:141–167. Downes J, Olsvik B, Hiom SJ, Spratt DA, Cheeseman SL, Olsen I, Weightman AJ, Wade WG. 2000. Bulleidia extructa gen. nov., sp. nov., isolated from the oral cavity. Int J Syst Evol Microbiol 50:979–983. Fievez V, Mbanzamihigo L, Piattoni F, Demeyer D. 2001. Evidence for reductive acetogenesis and its nutritional significance in ostrich hindgut as estimated from in vitro incubations. J Anim Physiol Anim Nutr 85:271–280. File SK, McGrew WC, Tutin CE. 1976. The intestinal parasites of a community of feral chimpanzees, Pan troglodytes schweinfurthii. J Parasitol 62:259–261. Fonty G, Joblin K, Chavarot M, Roux R, Naylor G, Michallon F. 2007. Establishment and development of Chimpanzee Fecal Microflora Fermentation Capacity / 557 ruminal hydrogenotrophs in methanogen-free lambs. Appl Environ Microbiol 73:6391–6403. Frey JC, Rothman JM, Pell AN, Nizeyi JB, Cranfield MR, Angert ER. 2006. Fecal bacterial diversity in a wild gorilla. Appl Environ Microbiol 72:3788–3792. Goodall J. 1986. The chimpanzees of Gombe: patterns of behavior. Cambridge, MA: The Belknap Press of Harvard University Press. 673p. Goussard B, Collet JY, Garin Y, Tutin CEG, Fernandez M. 1983. The intestinal entodiniomorph ciliates of wild lowland gorillas (Gorilla gorilla gorilla) in Gabon, West Africa. J Med Primatol 12:239–249. Imai S, Ikeda SI, Collet JY, Bonhomme A. 1991. Entodiniomorphid ciliates from the wild lowland gorilla with the description of a new genus and 3 new species. Eur J Protistol 26:270–278. Jenkins DJA, Kendall CWC, Ransom TPP. 1998. Dietary fiber, the evolution of the human diet and coronary heart disease. Nutr Res 18:633–652. Lajoie SF, Bank S, Miller TL, Wolin MJ. 1988. Acetate production from hydrogen and [13C]carbon dioxide by the microflora of human feces. Appl Environ Microbiol 54:2723–2727. Ley RE, Hamady M, Lozupone C, Turnbaugh PJ, Ramey RR, Bircher JS, Schlegel ML, Tucker TA, Schrenzel MD, Knight R, Gordon JI. 2008. Evolution of mammals and their gut microbes. Science 320:1647–1651. McDougall EI. 1948. Studies on ruminant saliva. 1. The composition and output of sheep’s saliva. Biochem J 43:99–109. McNeil NI. 1984. The contribution of the large-intestine to energy supplies in man. Am J Clin Nutr 39:338–342. Mellenberger RW, Satter LD, Millet MA, Baker AJ. 1970. An in vitro technique for estimating digestibility of treated and untreated wood. J Anim Sci 30:1005–1011. Michalowski T. 1987. The volatile fatty acid production by ciliate protozoa in the rumen of sheep. Acta Protozool 26:335–345. Miller TL, Wolin MJ. 1996. Pathways of acetate, propionate, and butyrate formation by the human fecal microbial flora. Appl Environ Microbiol 62:1589–1592. Milton K. 1999. A hypothesis to explain the role of meat-eating in human evolution. Evol Anthropol 8:11–21. Milton K. 2003a. Micronutrient intakes of wild primates: are humans different? Comp Biochem Physiol A Mol Integr Physiol 136:47–59. Milton K. 2003b. The critical role played by animal source foods in human (Homo) evolution. J Nutr 133:3886S–3892S. Milton K, Demment MW. 1988. Digestion and passage kinetics of chimpanzees fed high and low fiber diets and comparison with human data. J Nutr 118:1082–1088. Muehlenbein MP. 2005. Parasitological analyses of the male chimpanzees (Pan troglodytes schweinfurthii) at Ngogo, Kibale National Park, Uganda. Am J Primatol 65:167–179. Murray S, Stem C, Boudreau B, Goodall J. 2000. Intestinal parasites of baboons (Papio cynocephalus anubis) and chimpanzees (Pan troglodytes) in Gombe National Park. J Zoo Wildl Med 31:176–178. Muyzer G, de Waal EC, Uitterlinden AG. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700. Newton-Fisher NE. 1999. The diet of chimpanzees in the Budongo Forest Reserve, Uganda. Afr J Ecol 37:344–354. Nübel U, Engelen B, Felske A, Snaidr J, Wieshuber A, Amann RI. 1996. Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacteriol 178:5636–5643. Popovich DG, Jenkins DJA, Kendall CWC, Dierenfeld ES, Carroll RW, Tariq N, Vidgen E. 1997. The western lowland gorilla diet has implications for the health of humans and other hominoids. J Nutr 10:2000–2005. Pospiech A, Neumann B. 1995. A versatile quick-prep of genomic DNA from Gram-positive bacteria. Trends Genet 11:217–218. Regensbogenova M, Kisidayova S, Michalowski T, Javorsky P, Moon-Van Der Staay SY, Moon-Van Der Staay GWM, Hackstein JHP, McEwan NR, Jouany JP, Newbold JC, Pristas P. 2004. Rapid identification of rumen protozoa by restriction analysis of amplified 18S rRNA gene. Acta Protozool 43:219–224. Remis MJ, Dierenfeld ES. 2004. Digesta passage, digestibility and behavior in captive gorillas under two dietary regimens. Int J Primatol 25:825–845. Rothman J, Dierenfeld E, Hintz H, Pell A. 2008. Nutritional quality of gorilla diets: consequences of age, sex, and season. Oecologia 155:111–122. Schmidt DA. 2002. Fiber enrichment of captive primate diets. Dissertation. Columbia: University of Missouri. 101p. Schmidt DA, Dempsey JL, Kerley MS, Porton IJ. 2000. Fiber in ape diet: a review. Proceedings of The Apes: Challenges for the 21st Century. p 177–179. Schmidt DA, Kerley MS, Dempsey JL, Porton IJ, Porter JH, Griffin ME, Ellersieck MR, Sadler WC. 2005. Fiber digestibility by the orangutan (Pongo abelii): in vitro and in vivo. J Zoo Wildl Med 36:571–580. Stahl AB. 1984. Hominid dietary selection before fire. Curr Anthropol 25:151–168. Stanford CB. 2008. Chimpanzee and red colobus: the ecology of predator and prey. Cambridge, MA: Harvard University Press. 296p. Sunvold GD, Hussein HS, Fahey GC, Merchen NR, Reinhart GA. 1995. In vitro fermentation of cellulose, beet pulp, citrus pulp, and citrus pectin using fecal inoculum from cats, dogs, horses, humans, and pigs and ruminal fluid from cattle. J Anim Sci 73:3639–3648. Uenishi G, Fujita S, Ohashi G, Kato A, Yamauchi S, Matsuzawa T, Ushida K. 2007. Molecular analyses of the intestinal microbiota of chimpanzees in the wild and in captivity. Am J Primatol 69:367–376. Ushida K, Fujita S, Ohashi G. 2006. Nutritional significance of the selective ingestion of Albizia zygia gum exudate by wild chimpanzees in Bossou, Guinea. Am J Primatol 68:143–151. van Hoek AHAM, van Alen TA, Sprakel VSI, Leunissen JAM, Brigge T, Vogels GD, Hackstein JHP. 2000. Multiple acquisition of methanogenic archaeal symbionts by anaerobic ciliates. Mol Biol Evol 17:251–258. Vanhoutte T, Huys G, de Brandt E, Swings J. 2004. Temporal stability analysis of the microbiota in human feces by denaturing gradient gel electrophoresis using universal and group-specific 16S rRNA gene primers. FEMS Microbiol Ecol 48:437–446. Varadyova Z, Baran M, Zelenak I. 2005. Comparison of two in vitro fermentation gas production methods using both rumen fluid and faecal inoculum from sheep. Anim Feed Sci Technol 123–124:81–94. Wang RF, Cao WW, Cerniglia CE. 1996. PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples. Appl Environ Microbiol 62:1242–1247. Wrangham R, Conklin-Brittain NL. 2003. Cooking as a biological trait. Comp Biochem Physiol A Mol Integr Physiol 136:35–46. Am. J. Primatol.