Comparative investigations on digestion in grazing (Ceratotherium simum) and browsing (Diceros bicornis) rhinoceroses
Rhinoceroses represent the largest extant herbivores with extensive dietary specialization for plant groups like browse (black rhino Diceros bicornis) or grass (white rhino Ceratotherium simum). However, it is not clear to what extent such diet selection patterns are reflected in adaptations of digestive physiology of the respective feeding types. In this study, feeding trials with four black and five white rhinos were conducted in four zoos. The animals had ad libitum access to the same batch of grass hay (second cut; neutral detergent fiber (NDF) 63% dry matter (DM), crude protein 10.2% DM). Total intake, fecal N content, in vitro digestibility of NDF residues of feces, fecal particle size and mean retention time (MRT) of particles (Cr-mordanted fiber; 1-2mm) and fluid (Co-EDTA) were quantified. The average daily DM intake was 70+/-12 g/kg BW(0.75) for white and 73+/-10 g/kg BW(0.75) for black rhinos. In the in vitro fermentation test fecal NDF residues of black rhinos resulted in higher gas productions at fermentation times of 12 to 24h, indicating that white rhinos have a superior capacity to digest NDF. Average MRT for fluids and particles was 28+/-4h and 43+/-5h in white and 34+/-4h and 39+/-4h in black rhinos. The selectivity factor (SF=MRT(particle)/MRT(fluid)) was higher for white (1.5+/-0.2) than for black rhinos (1.2+/-0.1) (p=0.016). In a comparison of 12 ruminant and 3 rhino species, SF was correlated to percentage of grass in diet (R=0.75). Mean fecal particle size was higher in white (9.1+/-1.94 mm) than in black rhinos (6.1+/-0.79 mm) (p=0.016). The results demonstrate differences between white and black rhinos in terms of retention times and fiber digestibility. The more selective retention of particles by the white rhino corresponds with the higher digestion of fiber measured indirectly. Furthermore there is indication for a general pattern of high SF in grazing ruminants and rhinos. The difference in fecal particle size between both rhino species might be due to the considerable difference in body weight.
Comparative investigations on digestion in grazing (Ceratotherium simum) and
browsing (Diceros bicornis) rhinoceroses
, M. Clauss
, K.-H. Südekum
, J.-M. Hatt
, S. Silinski
, S. Klomburg
, W. Zimmermann
, J. Fickel
, J. Hummel
Institute of Animal Science, University of Bonn, 53115 Bonn, Germany
Clinic for Zoo Animals, Exotic Pets and Wildlife, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland
Westfälischer Zoologischer Garten Münster, 48161 Munster, Germany
Zoo Osnabrück, 49082 Osnabruck, Germany
Zoologischer Garten Köln, 50735 Cologne, Germany
Leibniz-Institute of Zoo and Wildlife Research (IZW), 10315 Berlin, Germany
Received 18 December 2009
Received in revised form 8 March 2010
Accepted 9 March 2010
Available online 12 March 2010
Mean retention time
Fecal particle size
Rhinoceroses represent the largest extant herbivores with extensive dietary specialization for plant groups
like browse (black rhino Diceros bicornis) or grass (white rhino Ceratotherium simum). However, it is not
clear to what extent such diet selection patterns are reﬂected in adaptations of digestive physiology of the
respective feeding types. In this study, feeding trials with four black and ﬁve white rhinos were conducted in
four zoos. The animals had ad libitum access to the same batch of grass hay (second cut; neutral detergent
ﬁber (NDF) 63% dry matter (DM), crude protein 10.2% DM). Total intake, fecal N content, in vitro digestibility
of NDF residues of feces, fecal particle size and mean retention time (MRT) of particles (Cr-mordanted ﬁ ber;
1–2 mm) and ﬂuid (Co-EDTA) were quantiﬁed. The average daily DM intake was 70 ±12 g/kg BW
white and 73 ±10 g/kg BW
for black rhinos. In the in vitro fermentation test fecal NDF residues of black
rhinos resulted in higher gas productions at fermentation times of 12 to 24 h, indicating that white rhinos
have a superior capacity to digest NDF. Average MRT for ﬂuids and particles was 28 ± 4 h and 43 ± 5 h in
white and 34 ± 4 h and 39 ±4 h in black rhinos. The selectivity factor (SF =MRT
) was higher
for white (1.5 ±0.2) than for black rhinos (1.2 ±0.1) (p =0.016). In a comparison of 12 ruminant and 3
rhino species, SF was correlated to percentage of grass in diet (R =0.75). Mean fecal particle size was higher
in white (9.1±1.94 mm) than in black rhinos (6.1 ±0.79 mm) (p =0.016). The results demonstrate
differences between white and black rhinos in terms of retention times and ﬁber digestibility. The more
selective retention of particles by the white rhino corresponds with the higher digestion of ﬁber measured
indirectly. Furthermore there is indication for a general pattern of high SF in grazing ruminants and rhinos.
The difference in fecal particle size between both rhino species might be due to the considerable difference in
© 2010 Elsevier Inc. All rights reserved.
1.1. Digestive physiology of browsers and grazers
Among extant vertebrates, mammals have developed the largest
diversity of herbivores. In accordance with their selection of food
plants, they have been classiﬁed as grazing (focusing on leaves and
stems of grass), browsing (focusing on leaves and stems of trees,
shrubs or herbs) or intermediate feeding types (the latter switching
between the two extremes). The respective feeding niche can be
reﬂected in various aspects of biology (see Gordon and Prins, 2008 for
reviews). Morphological adaptations of feeding types have received
most attention in ruminants (Hofmann, 1973, 1989), and to some
extent in macropods (Sanson, 1989; Hume, 1999). On a physiological
level, an effective particle retention was postulated to be a particularly
adaptive evolutionary feature in grazers (Kay et al., 1980; Foose,
1982). This is explained by the higher proportion of slow fermenting
ﬁber in grass compared to browse (Short et al., 1974; Foose, 1982;
Hummel et al., 2006), and has been described for ruminants (Clauss
and Lechner-Doll, 2001; Hummel et al., 2005; Clauss et al., 2002b).
Furthermore, differences in tooth morphology can potentially lead to
a decrease in food comminution in browsing herbivores leading to
larger fecal particles in browsing ruminants (Clauss et al., 2002b) and
macropods (Lentle et al., 2003).
Comparable specialization has been reported for other herbivores
such as hyraxes (Deniro and Epstein, 1978) and rodents (Williams
Comparative Biochemistry and Physiology, Part A 156 (2010) 380–388
⁎ Corresponding author. University of Bonn, Institute of Animal Science, Endenicher
Allee 15, 53115 Bonn, Germany. Tel.: +49 228 732281; fax: +49 228 732295.
E-mail address: firstname.lastname@example.org (J. Hummel).
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journal homepage: www.elsevier.com/locate/cbpa
and Kay, 2001). Grazers and browsers are well documented in
perissodactyls in the fossil record with browsing taxa like Sinohippus
occurring well into the late Miocene (MacFadden, 2005). However,
rhinoceroses are the only group with extant representatives of both
1.2. Grazing and browsing rhinos
The white rhinoceros (Ceratotherium simum) is classiﬁed as a
typical grazing species, with a dietary proportion of herbs as low as 1%
and no intake of browse at all, while the black rhinoceros (Diceros
bicornis) has a proportion N 95% of dicot material in its diet (Owen-
Smith, 1988). Separation of the genera took place in the late Miocene
to early Pleistocene (6 to 2 million years ago) (Hooijer, 1969; Hooijer
and Pattersson, 1972; Hooijer, 1976; Groves, 1997). Both species can
be considered to re present the largest extant herbivores tr uly
specialized for a forage type (Owen-Smith, 1988; Shrader et al.,
2006), with only the common hippopotamus (Hippopotamus amphi-
bius) rivaling the white rhino as the largest specialized grazer. In
accordance with their feeding habit, adaptations of the chewing
apparatus have been described for rhinos. D. bicornis has a two-
phased chewing activity with a cutting ectoloph and more grinding
lophs on the lingual side, while C. simum has more hypsodont teeth
and shows a ﬂat grinding occlusal surface in the upper molars with
closely packed shearing blades and more cementum (Schaurte, 1966;
Fortelius, 1982; Thenius, 1989). Ceratotherium is also described to
have more pronounced lateral jaw movements, a longer relative
premolar row length, and a lower degree of blade sharpness (Thenius,
1989; Popowics and Fortelius, 1997; Palmqvist et al., 2003). Based on
his comprehensive comparative investigations on digestion in
ungulates, Foose (1982; page 130–133) postulated differing trophic
strategies for grazing and browsing rhinos: The latter are expected to
have a shorter retention time and a lower digestibility. Based on data
collected from various feeding trials, these assumptions seem to be
conﬁrmed (Clauss et al., 2005a, 2006a). Potentially related to that,
experience indicates that the black rhino can be considered a more
challenging herbivore to feed in captivity compared to its grazing
relative (Dierenfeld, 1995, 1999; Clauss and Hatt 2006).
In comparative physiological studies, the aim generally is to test
for adaptations to certain environmental factors, e.g. characteristics of
food plants. In this respect, a two-species approach inherently has
shortcomings: The most important is that differences between species
always are very likely, but need not be interpreted as adaptations but
simply as by-chance results of genetic separation, as outlined in detail
by Garland and Adolph (1994). Recommendations of the aforemen-
tioned latter paper on strategies to circumvent the shortcomings of a
two-species comparative study were followed as closely as possible
and are outlined in the discussion.
1.3. Aims of the study
In this study we intended to investigate whether the differences in
aspects of digestive physiology described for browsing and grazing
ruminants can also be found in rhinos. In detail, for the white rhino
(grazer) we expected a longer mean retention time of particles
), a higher selectivity factor (SF = MRT
higher ﬁber digestibility and smaller average fecal particle size (better
2. Material and methods
Five white and four black rhinos from four different zoological
institutions were available for the study (Table 1). Body weights were
estimated based on the known weight of one black rhino (not
included in this study), plus information from experienced zoo staff.
The animals were kept separately during the trials to allow individual
recording and sampling of food and feces, except for rhinos W3, W4
and W5, which were kept together for 3–4 h a day on the outside
enclosure. Col or markers (beetroo t a nd be tanin) were fed to
distinguish between individuals in this case.
For an adaptation period of 14 days and a collection period of a
minimum of 6 days, all animals had ad libitum access to a mixed hay
of temperate grasses (second cut). Hay from one identical batch was
used in all four facilities. Additionally black rhinos received 500 g and
white rhinos 600 g of a pelleted compound (crude protein (CP): 18%
dry matter (DM); neutral detergent ﬁber (NDF): 22% DM) per day and
animal for management purposes.
During the collection period, food intake was quantiﬁed, and
representative samples were taken from the diet (every second day)
and the feces (every day, representing app. 10% of daily fecal output,
the outer layer of each dung ball being removed to avoid contami-
nation of the sample). The fecal samples were frozen and freeze dried.
For chemical analysis, hay and dried feces were ground through a
1 mm sieve. Both the feed and fecal samples were analyzed for DM,
ash and CP (Dumas method). Feed samples were analyzed addition-
ally for ether extract (EE) according to Bassler (1976), and for NDF,
acid detergent ﬁber (ADF) and acid detergent lignin (ADL) according
to Van Soest et al. (1991). All ﬁber fractions are expressed as ash-
corrected values. In vitro fermentation of the hay was evaluated with
the Hohenheim Gas Test (HGT; Menke et al., 1979), using standard-
ized sheep rumen ﬂuid as the inoculum source. Metabolizable energy
(ME) and apparent organic matter digestibili ty (aD OM) for
ruminants were estimated from 24 h in vitro gas production (GP)
(plus nutrient composition) according to the following regression
equations: ME [MJ/kg DM]=0.72+0.1559 GP
[ml/200 mg DM] +
0.0068 CP [g/kg DM]+0.0249 EE [g/kg DM] (Menke and Steingass,
1988); aD OM [%] =0.889 GP
[ml/200 mg DM]+0.0448 CP [g/kg
DM]+0.0651 ash [g/kg DM]+14.88 (Menke and Huss, 1987).
Cell wall degradation was quantiﬁed using an approach compa-
rable to Prins et al. (1981) and Prins et al. (1983). NDF residues of hay
and feces were fermented in vitro in the HGT, with GP quantiﬁed at 4,
8, 12, 18, 24, 32, 48, 56, 72, 80 and 96 h (GP related to ash-corrected
NDF residue, expressed as ml/200 mg NDF).
From the undried fecal samples, fecal particle size was quantiﬁed
in triplicates using a wet sieving machine (Vibrotronic Type VE 1,
Retsch Technology, Haan, Germany). Samples were sieved for 10 min
(water ﬂow 2 L/min) over a cascade of sieves with apertures of 16, 8,
4, 2, 1, 0.5, 0.25, 0.125 and 0.063 mm. The mean fecal particle size was
expressed as weighted average of particle size (WAPS), calculated as
the modulus of ﬁneness according to Poppi et al. (1980), but by using
sieve aperture size instead of consecutive numbers for sieves.
Cobalt EDTA and chromium-mordanted ﬁber (1–2 mm) were used
to quantify retention times for the ﬂuid and the particle phase,
respectively (Udén et al., 1980). Markers were given in a pulse dose
mixed with two bananas or two small bread rolls, all ingested within
less than 10 min. Samples were taken from each defecation. One
Zoo Animal Sex Age at trial [years] Body weight estimated [kg]
Köln B1 F 11.7 1300
B2 M 11.0 1300
Zürich B3 F 9.7 1200
B4 F 5.0 1200
Osnabrück W1 F 35.2 2200
W2 M 29.4 2200
Münster W3 F 15.1 1900
W4 F 18.9 2200
W5 M 14.9 2400
F = female; M = male.
381P. Steuer et al. / Comparative Biochemistry and Physiology, Part A 156 (2010) 380–388
overnight sample was taken (the middle of this interval being used as
sampling time). The samples were dried at 103 °C and ground through
a 1 mm sieve. Marker concentration was measured after wet ashing
according to Behrend et al. (2004) with atomic absorption spectros-
copy (Perkin-Elmer 1100 B, Perkin Elmer, Wellesley, Massachusetts,
USA). MRT was calculated according to Thielemans et al. (1978). The
selectivity-factor (SF) was calculated as MRT
Doll et al., 1990).
To test for the generality of the relation of feeding type and SF, data
of 12 ruminant and 3 rhino species was compiled (for data see
Electronic appendix), all using Co-EDTA and Cr mordanted ﬁbre as
markers and allowing animals ad libitum diet access. Data were
analyzed by phylogenetically controlled regression analysis. The
subjects of the analysis were species. Relationships among them due
to the evolutionary process were inferred from a phylogenetic tree
based on the complete mitochondrial cytochrome b gene. Respective
DNA sequences were available from GenBank (http://www.ncbi.nlm.
nih.gov). Sequences were aligned using CLUSTALX (Thompson et al.,
1997), visually controlled and trimmed to identical length (1.143 bp).
To select the best-ﬁtting nucleotide substitution model for the data, a
combination of the software packages PAUP* (v.4.b10; Swofford,
2002) and MODELTEST (v.3.7; Posada and Crandall, 1998) was used.
Analysis was based on a hierarchical likelihood ratio test approach
implemented in MODELTEST. The model selected was the general
time-reversible (GTR) model (Lanave et al., 1984; Tavaré, 1986) with
an allowance both for invariant sites (I) and a gamma (G) distribution
shape parameter (α) for among-site rate variation (GTR + I + G)
(Rodriguez et al., 1990). The nucleotide substitution rate matrix for
the GTR + I + G model was similarly calculated using MODELTEST.
Parameter values for the model selected were: − lnL=x, I = xy, and
α = xyz. The phylogenetic reconstruction based on these parameters
was then performed using the maximum likelihood (ML) method
implemented in TREEPUZZLE (v.5.2; Schmidt et al., 2002). Support for
nodes was assessed by a reliability percentage after 100,000 quartet
puzzling steps; only nodes with more than 50% support were retained.
The basal polytomy for familial relationships was resolved assuming it
to be soft polytomy (Purvis and Garland, 1993). To meet the input
requirements for the phylogenetic analysis implemented in the
COMPARE 4.6 program (Martins, 2004), we resolved the remaining
polytomies to full tree dichotomy by introducing extreme short
branch length (l= 0.00001) at multifurcating nodes.
We used the Phylogenetic Generalized Least Squares approach
(Martins and Hansen, 1997; Rohlf, 2001) in which a well established
method was extended to enable the inclusion of interdependencies
among species due to the evolutionary process. To test the robustness
of the results, the comparative analysis was performed for both a set
of phylogenetic trees involving branch length and another set with
equal branch length. As there were no relevant differences in the
results, only the tests using the former tree are given here. The
COMPARE 4.6 program (
Martins, 2004) served for phylogenetically
controlled calculations. Other statistical calculations including a
nonparametric test (Mann–Whitney) to test for differences between
the two species were performed using SPSS 16 software (SPSS,
Chicago, IL, USA). The signiﬁcance level was set to α =0.05.
The DM content of the hay used in the study (one mixed sample per
institution) was 89.9 ± 0.9%, the nutrient composition (DM basis) was
63.4± 0.8% for NDF, 32.8±0.8% for ADF, 3.1 ± 0.7% for ADL, 10.2±
0.5% for CP, 2.0 ± 0.5% for EE and 8.2 ± 0.7% for ash. Standardized 24 h
in vitro GP was 44.6± 1.4 ml/200 mg DM. Metabolizable energy and
apparent organic matter digestibility of the hay were estimated to be
8.8± 0.3 MJ/kg DM and 65 ± 1.3% respectively.
Daily DM intake (DMI) was variable between rhinos (Table 2) and
ranged from 60 to 84 g/kg BW
for black rhinos. For white rhinos
DMI ranged from 56 to 90 g/kg BW
. In the in vitro fermentation
test, GP of NDF residues of rhino feces was signiﬁcantly higher for
black compared to white rhinos at the time intervals of 12–18 and 18–
24 h (p = 0.016), while no difference was apparent for the earlier or
later time intervals (Fig. 1).
The ﬂuid marker was excreted faster than the particle marker in both
species (see Figs. 2 and 3 for excretion curves). Mean retention time for
) ranged from 29 to 38 h for black rhinos (34±4 h) and
from 22 to 31 h for white rhinos (28±4 h) (Table 3). MRT
from 34 to 43 h for black (39±4 h) and from 38 to 49 h for white
rhinoceroses (43 ± 5 h). While MRT
did not differ
between the two species (p=0.111 MRT
, p=0.286 MRT
for black rhinos (1.2±0.1) was signiﬁcantly lower than for white rhinos
(1.5±0.2) (p= 0.016). In the phylogenetic regression analysis, litera-
ture data on SF and percentage of grass in diet revealed a signiﬁcant
relationship between these traits (R=0.75; R
p=0.001) (Fig. 4).
Average fecal particle size quantiﬁed via WAPS ranged from 5.1 to
6.8 mm for the black rhinoceroses (6.1 ± 0.79 mm) and from 7.4 to
11.5 mm for the white rhinoceroses (9.1±1.94 mm) (Table 3);
differences between the two species were signiﬁcant (p = 0.016).
4.1. Inferring on adaptation from comparative studies
A comparative study using a small sample size has to be careful in
its interpretation of differences as adaptations to environmental
factors (Garland and Adolph, 1994). The most important point of
criticism is that interspeciﬁc differences in any character are very
likely to be present, but need not necessarily be interpreted as
adaptations. A misinterpretation of random differences as adapta-
tions, or confounding reasons for characteristics (e.g. body weight vs.
feeding style) are possible in an approach using only a limited amount
of species. Establishing a correlation between the respective trait and
the environmental factor is a way to cope with this problem, but
obviously has a statistical requirement of at least 3 species.
Among the strategies to enhance the value of an approach using a
limited amount of species is a) to make explicit predictions on the
traits of interest which should be as independent as possible from
each other (see the aims section for a list of predictions for the
variables of our study); b) to choose species which evolved in
environments that differ as little as possible except for the
environmental factor of interest (a requirement satisfactorily met in
the rhino taxa investigated, since they can occur sympatrically); and
c) to give an indication of the quantity of the difference (see Hulbert,
Means (± standard deviation SD) of daily dry matter intake (DMI) and fecal nitrogen
(N) content (OM = organic matter).
Animal DMI Fecal N
[kg] [g/kg BW
] [g/kg BW] [g/kg OM]
B1 16.3± 3.14 75 ± 14 13±2.4 1.98
B2 18.1± 1.83 84 ± 8 14±1.4 2.03
B3 12.1± 1.06 60 ± 5 10±0.9 2.21
B4 14.9± 1.19 73 ± 6 12±1.0 2.77
Mean± SD 15.4 ± 2.53 73± 10 12 ± 1.6 2.25 ± 0.362
W1 22.6± 4.29 70 ± 13 10±2.0 2.98
W2 28.9± 3.00 90 ± 10 13±1.4 2.72
W3 19.8± 2.96 69 ± 11 10±1.6 2.74
W4 21.2± 1.96 66 ± 6 10±0.9 2.21
W5 19.2± 2.55 56 ± 7 8.0±1.1 2.14
Mean± SD 22.3 ± 3.90 70± 14 10 ± 1.8 2.56 ± 0.365
p (U-test) Not tested 0.556 0.191 0.286
382 P. Steuer et al. / Comparative Biochemistry and Physiology, Part A 156 (2010) 380–388
1984), and therefore its relevance for species performance (attempts
for quantifying relevance are made for each trait investigated).
4.2. Intake and digestibility
For browsing rhinos on a diet comparable to that in the wild, a
strategy of high intake/low digestibility can be expected (Foose, 1982;
Clauss et al., 2005a). Browse material contains considerably higher
amounts of lignin, rendering a larger part of this forage completely
indigestible, than in grass (Foose, 1982; Hummel et al., 2006). The
question would be if such a strategy of high intake can be considered
as ‘ﬁxed’ for feeding types to an extent that makes it detectable even
when the diet is identical for both. While the intraspeciﬁc variability
in intake was considerable in our study, a comparison between the
two rhino taxa does not support the view of a strict interspeciﬁc
difference in relation to feeding type. This is true for intake related to
body weight, a measure which relates intake to gut capacity (which
scales to BW
according to Parra (1978) and Demment and Van Soest
(1985)), or intake related to metabolic body size (BW
), which puts
intake more in relation to energy requirements. A lack of a difference
between the rhino taxa is in accordance with the results of Foose
(1982; Table 4).
In literature, different concepts of regulation of food intake are
reported. For ruminants, Conrad (1966) described diet intakes to be
regulated via energy dominantly in well digestible/high concentrate
diets, and by gut ﬁll dominantly in diets low in digestibility/high in
forage. For the giraffe, another large browsing herbivore, considerable
intake limitation has been described on a grass hay diet in comparison
to grazing bovids (Foose, 1982), probably due to intake limitation
related to gut ﬁll (Clauss et al., 2002a). No indication for a lower intake
in the browsing species was found for rhinos on a grass hay diet in this
It should be added here that our results are only valid for hay of the
quality used in this study (second cut, estimated OM digestibility for
ruminants 65%). A differing hay quality (e.g. a ﬁrst cut hay rich in
stems) would probably have challenged the intake capacity of the
species to a larger extent. If the quality of the study hay is put into
relation with the natural food resources, food quality in terms of NDF
and CP seems to be lower in the wild for white rhinos (n=6; NDF
74.6± 1.0% DM; CP 4.7 ± 1.1% DM; (Kiefer et al., 2003), while in black
rhinos NDF values of a level comparable to the study hay are generally
found in their natural forage (n = 24; 58 ±9% NDF; 12 ± 4% CP;
Dierenfeld et al., 1995).
Fecal N values are regarded to be an indicator of the production of
microbial biomass in the fermentation chambers, and therefore to
reﬂect the digestion of the diet (Mésochina et al., 1998 for horses;
Lukas et al., 2005 for ruminants). This method can be regarded as a
potential tool in the evaluation of diet quality in rhinos under free-
ranging conditions, especially in grazing taxa (see Leslie et al., 2008
for a recent review). Validity of the approach has also been shown for
browsing taxa such as the greater kudu (Tragelaphus strepsiceros)
(van der Waal et al., 2003). In this study, no signiﬁcant difference in
fecal N values was found between the rhino species, therefore giving
no indication for a difference in OM digestibility.
The studies of Ullrey et al. (1979) and Foose (1982) indicated a
higher ﬁber digestibility in white compared to black rhinos (Table 4).
Fig. 1. In vitro fermentation of NDF preparations (rhino feces and grass hay).
Fig. 2. Marker excretion pattern of a black rhinoceros (B3). Fig. 3. Marker excretion pattern of a white rhinoceros (W4).
383P. Steuer et al. / Comparative Biochemistry and Physiology, Part A 156 (2010) 380–388
The results of the in vitro fermentation of the NDF residues from rhino
feces and hay are therefore of particular interest. Our expectation that
the in vitro gas production from the ﬁber fraction of white rhino feces
would be lower than that of black rhinos (indicating a more
comprehensive ﬁber digestion already having taken place in the
animal gut) was indeed met for fermentation times of 12–24 h. If this
result is interpreted considering potential differences in retention
times between the species, it can be assumed that the better digestion
of the 12–24 h fraction of the in vitro test by white rhinos indicates a
superior retention capacity in this species. Both rhinos seem to digest
little of the slow fermenting NDF-fractions (in vitro fermentation
times N 24 h). The higher in vitro gas production in fecal compared to
grass hay NDF-residues for the slow-fermenting fractions can be
explained by the fact that the distribution of the faster (0–12 h),
intermediate (12–24 h) and slower (N 24 h) fermenting NDF fractions
is changed in the rhino feces in the direction of the slower fermenting
fraction, resulting in a higher proportion of slow fermenting ﬁber. It
should be emphasized here that while the ranking of the samples will
not be inﬂuenced by the in vitro conditions, these conditions will have
some inﬂuence on the degradation kinetics of the NDF samples. For
example, a factor accelerating fermentation in the in vitro system
signiﬁcantly is the necessary milling of the samples before the
analysis, while the use of dried material may delay the onset of
fermentation to some degree. Given our estimations for the retention
times in the part of the digestive tract where ﬁber fermentation takes
place (see below), fermentation seems to be rather faster under the in
vitro conditions compared to the GIT of the animal. The use of in vitro
fermentation of the fecal ﬁber fraction can be regarded as a useful tool
for investigations on differences in the digestive physiology of
4.3. Ingesta retention
Due to the slow fermentatio n rate of ﬁber, which is on a
comparable level with the passage rate from the fermentation
chamber of larger herbivores (Mertens, 1993), mean retention time
of food in the digestive tract can be considered a key parameter in
herbivores. Compared to other data on grass diets (≥ 75% grass in the
diet on a dry matter basis) (Table 4), the MRT
of the grazing
white rhino appears to be rather short. Data from studies with
comparable markers indicate longer retention times in Indian rhinos
(Clauss et al., 2005b: MRT
57 h for an animal on a 100% grass
forage diet). The study of Foose (1982) using Fuchsin stained particles
arrives at MRT of 61/71 h (Indian), 63/65 h (white) and 60 h (black)
for rhinos. Data on equids at ad libitum intake indicate MRT
32–34 h for ponies and 29–32 h for donkeys (Pearson et al., 2006).
In studies on differences in digestive/fermentative capacity of
herbivores, the major site of interest is generally the fermentative
chamber. Attempts have been made to give estimations for the
retention time in the fermentation chamber of perissodactyls (Moore-
Colyer et al., 2003). In this study, the approach of Udén et al. (1982a)
was followed (which in the latter study was applied to fecal marker
excretion curves after administering the markers into the caecum),
backed by additional considerations ( Grovum and Williams, 1973;
Martínez del Rio et al., 1994; Caton and Hume, 2000): In exponential
marker excretion models in ruminants, the time of ﬁrst marker
appearance in the feces has been interpreted as the retention time in
the tubular, non-mixing portions of the digestive tract, largely the
small intestine and portions of the large intestine. In an attempt to
translate this concept to the digestive tract of the rhino, the small
Means (± standard deviation) of defecation rate, mean retention time of ﬂuid and
) and selectivity factor (SF = MRT
the whole gastrointestinal tract, and average fecal particle size (WAPS = weighted
average of particle size).
B1 3.7± 1.2 29 34 1.2 5.1 ± 0.49
B2 3.6± 1.3 38 43 1.1 5.9 ± 0.38
B3 3.0± 1.1 36 40 1.1 6.6 ± 0.55
B4 3.0± 0.9 31 38 1.2 6.8 ± 0.98
Mean± SD 3.3± 0.4 34±4 39 ± 4 1.2 ± 0.1 6.1±0.79
W1 2.6± 0.5 30 49 1.6 11.5 ± 0.82
W2 3.4± 0.9 30 41 1.4 10.8 ± 0.64
2.1± 0.6 22 40 1.8 8.4 ± 1.12
2.9± 0.4 28 38 1.4 7.4 ± 0.73
2.7± 1.0 31 48 1.6 7.4 ± 0.54
Mean± SD 2.7± 0.5 28±4 43 ± 5 1.5 ± 0.2 9.1±1.94
p (U-test) 0.064 0.111 0.286 0.016 0.016
On a diet based on browse leaves, WAPS was 5.9 ±0.65 for B3 and 8.1 ± 2.29 for B4.
Retention times in white rhinoceroses 3–5 were measured on a different occasion
than the rest of the data for these animals; W3 and W4 had an average daily intake of
17.6 kg DM or 58 g/kg BW
, and W5 of 18.9 kg DM or 55 g DM/kg BW
Fig. 4. Relation of selectivity factor and percentage of grass in diet (R= 0.75; R
=16.995; p = 0.001); dotted line represents the linear regression for ruminants and
rhinos. Other ungulates like equids, the African elephant and particularly hippos do not seem to follow the pattern of the former two groups. (Ruminants: GC = Giraffa camelopardis,
OJ = Okapia johnstoni,CC=Capreolus capreolus,CH=Capra hircus, DS = Domestic sheep, BD = Bubalus depressicornis, CI = Capra ibex, OA = Ovis ammon musimon, AN = Addax
nasomaculatus, BB = Bubalus bubalis,BJ=Bos javanicus, DC = Domestic cattle; Camelids: BC = Bactrian camel; OC = one-humped camel; Hippos: PH = pygmy hippo;
CH = common hippo; Rhinos: BR = black rhino, IR = Indian rhino, WR = white rhino; Equids: DD = Domestic donkey, DH = Domestic horse; AE = African elephant).
384 P. Steuer et al. / Comparative Biochemistry and Physiology, Part A 156 (2010) 380–388
intestine plus the distal large intestine are interpreted as a plug ﬂow
reactor and the caecocolon as a mixing chamber. The subtraction of
the transit time from MRT can be regarded to result in a proxy for the
retention time in the mixing compartments (MC) of the GIT. In rhinos
this should dominantly correspond to the caecocolon, the major site of
fermentative activity. Applying this concept to the MRT
study rhinos and subtracting the transit times by trend results in a
difference in the MRT
MC (black rhino: 20± 2.6 h; white rhino:
28± 8.6 h; p =0.0635) and supports the idea of a higher fermentative
capacity in the white rhinoceroses of the study. These calculations also
suggest that MRT measurements for the whole GIT might mask
differences in MRT in the fermentation chambers. However, it must be
stated that one has to be careful when applying such concepts to
situations in which an in situ evaluation is not possible (i.e., in non-
ﬁstulated animals): The assumption of the concept that the
caecocolon actually works dominantly as a mixing chamber in rhinos
must be met.
As already outlined, a difference found between species should be
checked for possible omission of adaptation to different environmen-
tal factors. In our case, this means quantifying the consequence of the
measured longer retention time of food in the hindgut of the white
rhino in terms of ﬁber digestion and in terms of its energy budget.
Using the reciprocal values of the retention times in the hindgut as
passage rates (Hungate, 1966), and using the fermentation rate
calculated from our in vitro fermentations of the cell wall fraction of
the study hay, one may apply the approach of Ørskov and McDonald
(1979) and McDonald (1981), in estimating the proportion of feed
actually degraded in the hindgut. In our case, this means multiplica-
tion of the maximal gas production with the factor c/(c+ k)(c being
the fermentation rate, and k being the passage rate, both expressed as
%/h). Assuming 20 h as retention time for the black rhino and 28 h for
the white rhino results in actually realized gas productions of 16.1 ml
from the NDF fraction of 200 mg DM of the study hay in the black and
18.9 ml in the white rhino species. Assuming that around 50% of the
gas comes from CO
developing from the buffer and that one mol of
corresponds to one mol of short chain fatty acids produced
(Blümmel et al., 1999) and assuming proportions of 65% acetate, 20%
propionate and 15% butyrate (Wolin, 1960), this indicates a difference
of 0.38 MJ ME/kg DM of hay. In conclusion, the difference in retention
time results in a higher energy extraction on the size of 5% for a white
rhino per unit of ingested dry matter — without doubt a difference
relevant for the animal.
4.4. Selectivity factor
Lechner-Doll et al. (1990) ﬁrst introduced the selectivity factor (SF,
the quotient between MRT
), as a measure to
quantify differences in digestive strategies of ruminant feeding types.
Clauss and Lechner-Doll (2001) and Hummel et al. (2005) followed
this approach and arrived at the conclusion of generally lower SF in
browsing compared to grazing ruminants. The SF is considered a
useful tool to compare animals, since ﬂuids and particles will be
inﬂuenced in the same way by factors such as DMI or husbandry and
even social components (since feces play some role in marking
behavior of rhinos, daily defecation patterns can be inﬂuenced).
In this study, a signiﬁcant difference in SF between white (1.5 ±
0.2) and black rhinos (1.2 ± 0.1) was found. Data from other studies
on black (Clauss et al., 2005a: 1.1–1.3) or Indian rhinos (Polster, 2004:
1.4–1.6) ﬁt into this pattern, and the clear distinction between the
rhinos can be considered to be a major result of this study.
The signiﬁcant positive correlation between SF and the percentage
of dietary grass in a sample of 12 ruminant and 3 rhino species makes
an interpretation of SF as an adaptation to a diet high in grass
warranted. What could be the causes for, or the adaptive value of, the
observed differences in SF? A longer MRT
allows more extensive
use of the slowly digestible dietary ﬁber — a fraction that has been
stated to be far more prominent in grass compared to browse. The
black rhino represents a species with a very high intake of woody
twigs in its diet — a potentially almost completely indigestible food
item, which is of little energetic beneﬁt for the animal (Foose, 1982;
Hummel et al., 2006) and therefore has to be cleared from the
digestive tract relatively fast.
A longer MRT
in browsing species may be more difﬁcult to
explain. Clauss et al. (2006b) interpreted the shorter MRT
grazing ruminants as a consequence of a higher ﬂuid throughput,
necessary to achieve the physical mechanisms for the ﬂotation and
sedimentation described to be important for the functioning of the
fermentation chamber of grazing ruminants. In terms of energy
metabolism, it could be due to a higher relevance of the soluble
digesta fraction in browse; in fact, the soluble ﬁber fraction (e.g.
pectins) is generally regarded to be more important in browse than in
grass (see Robbins, 1993; page 248). However, soluble ﬁber fractions
like pectins are generally regarded to have a high fermentation rate
(Van Soest et al., 1991; Hall et al., 1998), which diminishes the
beneﬁcial effect of longer retention times.
Comparison of data of feeding studies on rhinos on grass hay based diets (N 75% grass); NDF = neutral detergent ﬁber; CP = crude protein; DM = dry matter; aD = apparent
= mean retention time of particles/ﬂuid in the gastrointestinal tract.
N Grass in diet Diet composition Daily DM intake aD NDF MRT
[%] NDF, [% DM] CP, [% DM] [kg] [g/kg BW
] [%] [h] [h]
5 100 72 4.8 19.7±3.44 70 ± 3 48± 1 63/65
– Foose (1982)
1 100 75.4 5.6 25.0 70 38 ––Foose (1982)
2 100 62 7 –– 67 ––Ullrey et al. (1979)
3 100 65.5 13.2 –– 57±2 49/53
– Kiefer (2002)
3 100 (fresh) 65.5 7.5 –– 43 ± 1 ––Kiefer (2002)
5 95 63.4 10.2 22.3±3.90 70±12 – 43 ± 5 28±4 This study
3 100 75 4.5 15.7±3.72 69 ± 12 41 ±3 60
– Foose (1982)
2 100 62 7 –– 33 ––Ullrey et al. (1979)
2 76 46.1 8.9 19.1 94 45± 2 28–41 25–34 Clauss et al. (2005a),
Fröschle and Clauss unpubl.
4 95 63.6 10.2 15.4±2.53 73±10 – 39 ± 4 34±4 This study
Marker: fuchsin-stained particles.
; MRT calculated in Clauss et al. (2005a).
As cited in Clauss and Hatt (2006) and Castell (2005).
385P. Steuer et al. / Comparative Biochemistry and Physiology, Part A 156 (2010) 380–388
The longer MRT
could also be due to the fact that the high
fraction of soluble ﬁber (e.g. pectins), which have a water-binding
effect, increase the viscosity of the ﬂuid phase and hence ultimately
slow down its passage; a physiological adaptation to a higher ﬂuid
throughput (e.g. in the form of increased saliva production) might
therefore not have an advantageous effect in browsers. A considerable
soluble ﬁber fraction will also occur in grazing hindgut fermenting
species, since a signiﬁcant fraction of dietary hemicelluloses — which
are generally found to be particularly prominent in grasses (Robbins,
1993; Hummel et al., 2006) — is probably turned soluble in the
proximal sections of the gut (Keys et al., 1969; Parra, 1978); in
addition, hemicellulose might have a lesser effect on the viscosity of
the digesta compared to pectins. Thus the ﬁber composition of the diet
might have facilitated an adaptation to a higher ﬂuid ﬂow through the
GIT, which improves washing of soluble, absorbable nutrients out of
the digesta plug towards the absorptive gut surface (Lentle et al.,
An alternative explanation attempt may be that water is absorbed
more completely in browsing compared to grazing species, therefore
slowing down the movement of a ﬂuid phase marker in the distal
parts of the GIT, the major site of water absorption. However, a lower
fecal dry matter content was not found for C. simum compared to D.
bicornis in this study (20.2 ±0.8 vs. 18±1.9%).
While for ruminants and rhinos, t he pattern of a positive
correlation of percentage of grass in the diet and SF can be regarded
as given (Fig. 4), this correlation is less evident when all further
ungulate data available (horse, donkey, African elephant, Bactrian
camel, one-humped camel, common hippo and pygmy hippo) are
added to the data set, resulting in 22 species altogether (Fig. 4 ).
Although the correlation of percentage of grass in diet and SF stays
signiﬁcant when applying phylogenetic control, the level of the
correlation and its signiﬁcance is considerably lower (R = 0.48 instead
of 0.75; p =0.022 instead of p = 0.001), and at visual inspection, the
relationship is far less evident than in the dataset of ruminants and
rhinos only, indicating that factors other than botanical dietary niche
(grazers and browsers) most likely play a role. Remarkably, the
ungulate groups not ﬁtting the pattern of ruminants and rhinos are
following either a strategy of considerably higher intake (equids,
elephants, with particularly low SF) or lower intake (camelids, hippos,
with particularly high SF; both latter groups additionally character-
ized by a relatively low metabolic rate). The hypothesis relating SF to
feeding type would ﬁt into this pattern insofar as browsing ruminants
(showing low SF) can be expected to realize a higher food intake/
lower digestibility than their grazing relatives, at least when feeding
on their natural diets. However, summing up this discussion, in
contrast to rhinos and ruminants, for ungulates as a whole no safe
conclusion on a potential relation of selective retention of particles in
the gut and feeding type can be drawn.
4.5. Fecal particle size
Studies like Lentle et al. (2003) on wallabies or
Clauss et al.
(2002b) on ruminants found larger fecal particle sizes in browsing
compared to grazing herbivores. This is coherent with characteristics
of teeth structure and the chewing apparatus in grazers and browsers,
like the tendency to have more enamel crests vertical to the direction
of mastication on the ﬂat occlusal surface in the former (Fortelius,
1982). In contrast to this, Fritz et al. (2007) found no difference in
fecal particle size between D. bicornis and C. simum, despite their
different feeding type (smaller fecal particle size was only found in R.
unicornis). These animals were fed their regular zoo diets, to some
extent reﬂecting the natural feeding habits of the rhinos. From the
background of these studies, the results of our study are unexpected,
since the black rhinos were found to have smaller fecal particle sizes
than white rhinos when being fed an identical diet of grass hay — in
contrast to the three studies mentioned above. While the particularly
high values in the two older white rhinos may indicate an impact of
age-related tooth wear in these animals, the difference between the
taxa holds true even after correction for this inﬂuence.
Fecal particle size can be regarded as a good measure to quantify
the degree of food comminution in the oral cavity. To allow the
comparison of the rhino feeding types under this latter perspective,
body size differences between the taxa need to be considered, since
body weight is discussed to be of relevant inﬂuence on different
parameters of digestive physiology. While a recent data collection
could not ﬁnd an inﬂuence on retention time in ungulates (Clauss
et al., 2007), fecal particle size has in fact been found to increase with
body weight (Udén and Van Soest, 1982; Clauss et al., 2002b; Fritz
et al., 2009). Based on the data collection of Udén (1978), Pérez-
Barbería and Gordon (1998) estimate a scaling of fecal particle size to
, while Fritz et al. (2009) found a scaling to BW
. The latter
data collection includes all guilds of mammalian herbivores, while the
former includes 3 ruminants, 2 equids and one lagomorph. Correcting
black rhino fecal particle size accordingly results in a fecal particle size
of app. 6.9 mm on average (range of the individual black rhinos 5.7–
7.7), the upper range overlapping with the values of white rhinos of
this study (7.4–11.5 mm). While the difference in fecal particle size
between the rhinos gets somewhat smaller when correcting for body
weights, it can be safely stated for the hay used in this study that there
was no indication at all for conspicuously larger fecal particle size in
the browsing rhino compared to its grazing relative.
• The higher selectivity factors (MRT
) of white rhinos
are consistent with data available for ruminants, and indicate a
more selective retention of particles compared to ﬂuid in the
digestive tract of the grazing rhino. While this relation seems to hold
true for ruminants and rhinos, the situation is more complicated
when all ungulate groups (e.g. hippos, camel ids, equids and
elephants) are included, potentially due to a much larger range of
food intake levels within the whole group than within ruminants
and rhinos only.
• Based on in vitro fermentation of the fecal NDF fraction, the white
rhino is a more comprehensive digester of ﬁber.
• The black rhino was found to have smaller fecal particle sizes in this
study; at least a part of this difference might be explained to be an
effect of body size.
We would like to sincerely thank the staff at the different rhino
facilities for their kind cooperation and help in this study. This
research was supported by the German Research Foundation (DFG,
HU 1308/4-1) and is publication no. 17 of the DFG Research Unit 771
“Function and enhanced efﬁciency in the mammalian dentition —
phylogenetic and ontogenetic impact on the masticatory apparatus”.
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