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Effects of high- and low-fiber diets on fecal fermentation and fecal microbial populations of captive chimpanzees.

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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: kisiday@saske.sk
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. [2008]
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
[1997] 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
[1988] 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 [1988] 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. [2007] 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. [2006] and Ley et al. [2008]. 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.
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