Comparative investigations on digestion in grazing (Ceratotherium simum) and browsing (Diceros bicornis) rhinoceroses

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DOI: 10.1016/j.cbpa.2010.03.006 · Source: PubMed
Abstract
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
P. Steuer
a
, M. Clauss
b
, K.-H. Südekum
a
, J.-M. Hatt
b
, S. Silinski
c
, S. Klomburg
d
, W. Zimmermann
e
, J. Fickel
f
,
W.J. Streich
f
, J. Hummel
a,
a
Institute of Animal Science, University of Bonn, 53115 Bonn, Germany
b
Clinic for Zoo Animals, Exotic Pets and Wildlife, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland
c
Westfälischer Zoologischer Garten Münster, 48161 Munster, Germany
d
Zoo Osnabrück, 49082 Osnabruck, Germany
e
Zoologischer Garten Köln, 50735 Cologne, Germany
f
Leibniz-Institute of Zoo and Wildlife Research (IZW), 10315 Berlin, Germany
abstractarticle info
Article history:
Received 18 December 2009
Received in revised form 8 March 2010
Accepted 9 March 2010
Available online 12 March 2010
Keywords:
Black rhino
White rhino
Fiber digestion
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 reected in adaptations of digestive physiology of the
respective feeding types. In this study, feeding trials with four black and ve white rhinos were conducted in
four zoos. The animals had ad libitum access to the same batch of grass hay (second cut; neutral detergent
ber (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 ber;
12 mm) and uid (Co-EDTA) were quantied. 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 24 h, indicating that white rhinos
have a superior capacity to digest NDF. Average MRT for uids 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
particle
/MRT
uid
) 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 ber digestibility. The more
selective retention of particles by the white rhino corresponds with the higher digestion of ber 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.
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
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 classied 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
reected 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
ber 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) 380388
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: jhum@itw.uni-bonn.de (J. Hummel).
1095-6433/$ see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpa.2010.03.006
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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
feeding types.
1.2. Grazing and browsing rhinos
The white rhinoceros (Ceratotherium simum) is classied 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 at 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 130133) 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
conrmed (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
(MRT
particle
), a higher selectivity factor (SF = MRT
particle
/MRT
uid
),
higher ber digestibility and smaller average fecal particle size (better
chewing efciency).
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 34 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 ber (NDF): 22% DM) per day and
animal for management purposes.
During the collection period, food intake was quantied, 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 ber (ADF) and acid detergent lignin (ADL) according
to Van Soest et al. (1991). All ber 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 uid 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
24h
[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
24h
[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 quantied 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 quantied 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 quantied
in triplicates using a wet sieving machine (Vibrotronic Type VE 1,
Retsch Technology, Haan, Germany). Samples were sieved for 10 min
(water ow 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 neness according to Poppi et al. (1980), but by using
sieve aperture size instead of consecutive numbers for sieves.
Cobalt EDTA and chromium-mordanted ber (12 mm) were used
to quantify retention times for the uid 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
Table 1
Study animals.
Zoo Animal Sex Age at trial [years] Body weight estimated [kg]
Black rhinoceros
Köln B1 F 11.7 1300
B2 M 11.0 1300
Zürich B3 F 9.7 1200
B4 F 5.0 1200
White rhinoceros
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) 380388
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
particle
/MRT
uid
(Lechner-
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 bre 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-tting 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 (MannWhitney) to test for differences between
the two species were performed using SPSS 16 software (SPSS,
Chicago, IL, USA). The signicance level was set to α =0.05.
3. Results
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
0.75
for black rhinos. For white rhinos
DMI ranged from 56 to 90 g/kg BW
0.75
. In the in vitro fermentation
test, GP of NDF residues of rhino feces was signicantly higher for
black compared to white rhinos at the time intervals of 1218 and 18
24 h (p = 0.016), while no difference was apparent for the earlier or
later time intervals (Fig. 1).
The uid marker was excreted faster than the particle marker in both
species (see Figs. 2 and 3 for excretion curves). Mean retention time for
uid (MRT
uid
) 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
particle
ranged
from 34 to 43 h for black (39±4 h) and from 38 to 49 h for white
rhinoceroses (43 ± 5 h). While MRT
uid
and MRT
particle
did not differ
between the two species (p=0.111 MRT
uid
, p=0.286 MRT
particle
), SF
for black rhinos (1.2±0.1) was signicantly 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 signicant
relationship between these traits (R=0.75; R
2
=0.57; F
1.13
=16.995;
p=0.001) (Fig. 4).
Average fecal particle size quantied 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 signicant (p = 0.016).
4. Discussion
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 interspecic 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,
Table 2
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
0.75
] [g/kg BW] [g/kg OM]
Black rhinoceros
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
White rhinoceros
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) 380388
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 xed for feeding types to an extent that makes it detectable even
when the diet is identical for both. While the intraspecic variability
in intake was considerable in our study, a comparison between the
two rhino taxa does not support the view of a strict interspecic
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
1.0
according to Parra (1978) and Demment and Van Soest
(1985)), or intake related to metabolic body size (BW
0.75
), 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 ll 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 ll (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
study.
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 rst 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
reect 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 signicant 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 ber 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) 380388
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 ber fraction of white rhino feces
would be lower than that of black rhinos (indicating a more
comprehensive ber digestion already having taken place in the
animal gut) was indeed met for fermentation times of 1224 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 1224 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 (012 h),
intermediate (1224 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 ber. It
should be emphasized here that while the ranking of the samples will
not be inuenced by the in vitro conditions, these conditions will have
some inuence on the degradation kinetics of the NDF samples. For
example, a factor accelerating fermentation in the in vitro system
signicantly 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 ber 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 ber fraction can be regarded as a useful tool
for investigations on differences in the digestive physiology of
herbivores.
4.3. Ingesta retention
Due to the slow fermentatio n rate of ber, 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
particle
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
particle
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
particle
of
3234 h for ponies and 2932 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 rst 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
Table 3
Means standard deviation) of defecation rate, mean retention time of uid and
particles (MRT
uid
and MRT
particle
) and selectivity factor (SF = MRT
particle
/MRT
uid
)in
the whole gastrointestinal tract, and average fecal particle size (WAPS = weighted
average of particle size).
Defecations
[d
1
]
MRT
uid
[h]
MRT
particle
[h]
SF WAPS
[mm]
Black rhinoceros
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
a
B4 3.0± 0.9 31 38 1.2 6.8 ± 0.98
a
Mean± SD 3.3± 0.4 34±4 39 ± 4 1.2 ± 0.1 6.1±0.79
White rhinoceros
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
W3
b
2.1± 0.6 22 40 1.8 8.4 ± 1.12
W4
b
2.9± 0.4 28 38 1.4 7.4 ± 0.73
W5
b
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
a
On a diet based on browse leaves, WAPS was 5.9 ±0.65 for B3 and 8.1 ± 2.29 for B4.
b
Retention times in white rhinoceroses 35 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
0.75
, and W5 of 18.9 kg DM or 55 g DM/kg BW
0.75
.
Fig. 4. Relation of selectivity factor and percentage of grass in diet (R= 0.75; R
2
=0.57; F
1,13
=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) 380388
intestine plus the distal large intestine are interpreted as a plug ow
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
particle
of the
study rhinos and subtracting the transit times by trend results in a
difference in the MRT
particle
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-
stulated 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 ber 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
2
developing from the buffer and that one mol of
CO
2
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) rst introduced the selectivity factor (SF,
the quotient between MRT
particle
and MRT
uid
), 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 uids and particles will be
inuenced 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 inuenced).
In this study, a signicant 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: 1.11.3) or Indian rhinos (Polster, 2004:
1.41.6) t into this pattern, and the clear distinction between the
rhinos can be considered to be a major result of this study.
The signicant 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
particle
allows more extensive
use of the slowly digestible dietary ber 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 benet for the animal (Foose, 1982;
Hummel et al., 2006) and therefore has to be cleared from the
digestive tract relatively fast.
A longer MRT
uid
in browsing species may be more difcult to
explain. Clauss et al. (2006b) interpreted the shorter MRT
uid
in
grazing ruminants as a consequence of a higher uid throughput,
necessary to achieve the physical mechanisms for the otation 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 ber fraction (e.g.
pectins) is generally regarded to be more important in browse than in
grass (see Robbins, 1993; page 248). However, soluble ber fractions
like pectins are generally regarded to have a high fermentation rate
(Van Soest et al., 1991; Hall et al., 1998), which diminishes the
benecial effect of longer retention times.
Table 4
Comparison of data of feeding studies on rhinos on grass hay based diets (N 75% grass); NDF = neutral detergent ber; CP = crude protein; DM = dry matter; aD = apparent
digestibility; MRT
particle/uid
= mean retention time of particles/uid in the gastrointestinal tract.
N Grass in diet Diet composition Daily DM intake aD NDF MRT
particle
MRT
uid
[%] NDF, [% DM] CP, [% DM] [kg] [g/kg BW
0.75
] [%] [h] [h]
White rhinoceros
5 100 72 4.8 19.7±3.44 70 ± 3 48± 1 63/65
a,b
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
a,c
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
Black rhinoceros
3 100 75 4.5 15.7±3.72 69 ± 12 41 ±3 60
d
Foose (1982)
2 100 62 7 –– 33 ––Ullrey et al. (1979)
2 76 46.1 8.9 19.1 94 45± 2 2841 2534 Clauss et al. (2005a),
Fröschle and Clauss unpubl.
e
4 95 63.6 10.2 15.4±2.53 73±10 39 ± 4 34±4 This study
a
n=2.
b
Marker: fuchsin-stained particles.
c
Marker: Cr
2
O
3
; MRT calculated in Clauss et al. (2005a).
d
n=1.
e
As cited in Clauss and Hatt (2006) and Castell (2005).
385P. Steuer et al. / Comparative Biochemistry and Physiology, Part A 156 (2010) 380388
The longer MRT
uid
could also be due to the fact that the high
fraction of soluble ber (e.g. pectins), which have a water-binding
effect, increase the viscosity of the uid phase and hence ultimately
slow down its passage; a physiological adaptation to a higher uid
throughput (e.g. in the form of increased saliva production) might
therefore not have an advantageous effect in browsers. A considerable
soluble ber fraction will also occur in grazing hindgut fermenting
species, since a signicant 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 ber composition of the diet
might have facilitated an adaptation to a higher uid ow through the
GIT, which improves washing of soluble, absorbable nutrients out of
the digesta plug towards the absorptive gut surface (Lentle et al.,
1996).
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 uid 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
signicant when applying phylogenetic control, the level of the
correlation and its signicance 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 tting 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 t 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 at 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 reecting 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 inuence.
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 inuence on different
parameters of digestive physiology. While a recent data collection
could not nd an inuence 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
BW
0.19
, while Fritz et al. (2009) found a scaling to BW
0.22
. 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 (7.411.5 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.
5. Conclusions
The higher selectivity factors (MRT
particle
/MRT
uid
) of white rhinos
are consistent with data available for ruminants, and indicate a
more selective retention of particles compared to uid 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 ber.
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.
Acknowledgements
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 efciency in the mammalian dentition
phylogenetic and ontogenetic impact on the masticatory apparatus.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.cbpa.2010.03.006.
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    • "The relative abundance of eudicots in the bison diet raises questions about the degree to which bison should be considered obligate grazers [4,83,84]. Their broad mouth, massive shoulders, and low-slung head allow them to crop vegetation close to the ground858687, and their large reticulorumen facilitates digesting large amounts of low-nutrient graminoids [88]. However, our work shows that North American bison, like wisent and cattle, supplement their diet with more nutritious forbs and woody species throughout the growing season. "
    [Show abstract] [Hide abstract] ABSTRACT: North American bison (Bison bison) are becoming increasingly important to both grassland management and commercial ranching. However, a lack of quantitative data on their diet constrains conservation efforts and the ability to predict bison effects on grasslands. In particular , we know little about the seasonality of the bison diet, the degree to which bison supplement their diet with eudicots, and how changes in diet influence gut microbial communities, all of which play important roles in ungulate performance. To address these knowledge gaps, we quantified seasonal patterns in bison diet and gut microbial community composition for a bison herd in Kansas using DNA sequencing-based analyses of both chloroplast and microbial DNA contained in fecal matter. Across the 11 sampling dates that spanned 166 days, we found that diet shifted continuously over the growing season, allowing bison to take advantage of the seasonal availability of high-protein plant species. Bison consumed more woody shrubs in spring and fall than in summer, when forb and grass intake predominated. In examining gut microbiota, the bacterial phylum Tenericutes shifted significantly in relative abundance over the growing season. This work suggests that North American bison can continuously adjust their diet with a high reliance on non-grasses throughout the year. In addition, we find evidence for seasonal patterns in gut community composition that are likely driven by the observed dietary changes.
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    • "The results obtained from the dikdik and gazelle species support the concept that the difference between solute and particle retention is a species-specific, and hence a heritable, characteristic. This concept is supported by comparative analyses of this difference among ruminants (Clauss and Lechner-Doll, 2001; Clauss et al., 2006b), perissodactyls (Clauss et al., 2010c; Steuer et al., 2010), or mammals in general (Müller et al., 2011 ). In domestic ruminants, it has also been demonstrated that retention time characteristics are heritable traits: In cattle, 'frothy bloat' (a disease where rumen contents become extremely frothy) is linked to low saliva production (Mendel and Boda, 1961; Gurnsey et al., 1980) and long fluid retention in the rumen (Majak et al., 1986; Okine et al., 1989 ). Selective breeding against bloat susceptibility can be successful (Morris et al., 1997), a finding that indicates the potential for selective breeding for increased saliva production and hence increased rumen fluid throughput. "
    [Show abstract] [Hide abstract] ABSTRACT: Digesta flow plays an important role in ruminant digestive physiology. We measured the mean retention time (MRT) of a solute and a particle marker in the gastrointestinal tract (GIT) and the reticulorumen (RR) of five gazelles and one dikdik species. Species-specific differences were independent from body mass (BM) or food intake. Comparative evaluations (including up to 31 other ruminant species) indicate that MRT GIT relate positively to BM, and are less related to feeding type (the percentage of grass in the natural diet, %grass) than MRT RR. The MRTparticleRR is related to BM and (as a trend) %grass, matching a higher RR capacity with increasing BM in grazers compared to browsers. MRTsoluteRR is neither linked to BM nor to %grass but shows a consistent phylogenetic signal. Selectivity factors (SF; MRTparticle/MRTsolute, proxies for the degree of digesta washing) are positively related to %grass, with a threshold effect, where species with >20% grass have higher SF. These findings suggest that in different ruminant taxa, morphophysiological adaptations controlling MRTsoluteRR evolved to achieve a similar SF RR in relation to a %grass threshold. A high SF could facilitate an increased microbial yield from the forestomach. Reasons for variation in SF above the %grass threshold might represent important drivers of ruminant diversification and await closer investigation. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Jan 2015
    • "It has been suggested that a higher degree of digesta washing removes microbes faster from the fermentation chamber and hence selects for a microbial population that grows faster and is metabolically more active and efficient (reviewed in Clauss et al., 2010a; Müller et al., 2011). This matches the observation that high SF are linked to grass intake (Clauss et al., 2006Clauss et al., , 2010b Steuer et al., 2010), because grass contains comparatively high levels of digestible, yet slowly-digestible, fibre (Hummel et al., 2006 ). Yet, more experimental work on the relationships between various aspects of digestive physiology and digestibility proxies is required before such hypotheses can be accepted. "
    [Show abstract] [Hide abstract] ABSTRACT: In herbivore ecophysiology, comparative chewing efficiency has only recently received increased attention. This measure is best assessed on un-processed forage-only diets; corresponding comparative datasets are missing. We measured a faecal mean particle size (MPS [mm]) in 14 large herbivore species (body mass (M) range 60-4000kg; 8 ruminants and 6 hindgut fermenters) fed a consistent grass hay diet, in which intake, digesta mean retention times (MRT [h]) and digestive efficiency (as digestibility of faecal fibre measured by 96h cumulative in vitro gas production GP96h [ml per 200mg faecal fibre], and metabolic faecal nitrogen MFN [% organic faecal matter]) had been quantified simultaneously. MPS was generally lower in ruminants than in hindgut fermenters and increased with M in the total dataset, but was nearly constant among closely related taxa (e.g. within ruminants, within equids) irrespective of M. MPS (but not MRT) was significantly correlated to GP96h, whereas MRT (but not MPS) was significantly correlated to MFN, suggesting different effects of these factors on different aspects of digestibility. Combinations of measures including MPS mostly explained digestibility better than other combinations. The phylogenetic signal λ, which was mostly 1 when linking any single measure to digestibility, was estimated 0 in models that linked digestive efficiency to combinations of measures. These results support the intuitive concept that species diversification in large herbivores is tightly related to digestive physiology, and that chewing efficiency as measured by faecal particle size is an integral aspect of this scenario. Copyright © 2014 Elsevier Inc. All rights reserved.
    Full-text · Article · Oct 2014
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