Schwarm, A; Ortmann, S; Wolf, C; Streich, W J; Clauss, M (2009). More efficient mastication allows increasing intake without compromising digestibility or necessitating a larger gut: Comparative feeding trials in banteng (Bos javanicus) and pygmy ( liberiensis). Comparative Biochemistry and Physiology - Part A, Molecular and Integrative Physiology, 152(4):504-512. Postprint available at: University of Zurich http://www.zora.uzh.ch Zurich Open Repository and Archive Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch

Originally published at: Winterthurerstr. 190 Comparative Biochemistry and Physiology - Part A, Molecular and Integrative Physiology 2009, 152(4):504-512. CH-8057 Zurich http://www.zora.uzh.ch

Year: 2009

More efficient mastication allows increasing intake without compromising digestibility or necessitating a larger gut: Comparative feeding trials in banteng (Bos javanicus) and (Hexaprotodon liberiensis)

Schwarm, A; Ortmann, S; Wolf, C; Streich, W J; Clauss, M

Schwarm, A; Ortmann, S; Wolf, C; Streich, W J; Clauss, M (2009). More efficient mastication allows increasing intake without compromising digestibility or necessitating a larger gut: Comparative feeding trials in banteng (Bos javanicus) and pygmy hippopotamus (Hexaprotodon liberiensis). Comparative Biochemistry and Physiology - Part A, Molecular and Integrative Physiology, 152(4):504-512. Postprint available at: http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch

Originally published at: Comparative Biochemistry and Physiology - Part A, Molecular and Integrative Physiology 2009, 152(4):504-512. More efficient mastication allows increasing intake without compromising digestibility or necessitating a larger gut: Comparative feeding trials in banteng (Bos javanicus) and pygmy hippopotamus (Hexaprotodon liberiensis)

Abstract

The digestion of plant material in mammalian herbivores basically depends on the chemical and structural composition of the diet, the mean particle size to which the forage is processed, and the ingesta retention time. These different factors can be influenced by the , and they can presumably compensate for each other. The pygmy hippopotamus, a non-ruminating foregut fermenter, has longer mean retention times than ruminants; however hippos do not achieve higher (fibre) digestibilities on comparable diets, which could be due to ineffective mastication. We performed feeding trials with six pygmy hippos (Hexaprotodon liberiensis) and six banteng cattle (Bos javanicus) on a grass diet. As predicted, both achieved similar dry matter, organic matter, crude protein and gross energy digestibilities. However, neutral and acid detergent fibre digestibility was lower in pygmy hippos. Apparently, in these species, fibre digestibility was more influenced by particle size, which was larger in pygmy hippos compared to banteng, than by retention time. In spite of their higher relative food intake, the banteng in this study did not have greater relative gut fills than the hippos. Ruminants traditionally appear intake-limited when compared to equids, because feed particles above a certain size cannot leave the rumen. But when compared to nonruminating foregut fermenters, rumination seems to free foregut fermenters from an intrinsic food intake limitation. The higher energy intakes and metabolic rates in wild cattle compared to hippos could have life-history consequences, such as a higher relative reproductive rate. 1

1 More efficient mastication allows increasing intake without compromising digestibility or

2 necessitating a larger gut: comparative feeding trials in banteng (Bos javanicus) and pygmy

3 hippopotamus (Hexaprotodon liberiensis)

4

5 Angela Schwarma,b*, Sylvia Ortmanna, Christian Wolfc, W. Jürgen Streicha, Marcus Claussd

6

7

8 aLeibniz Institute for Zoo and Wildlife Research (IZW) Berlin, Alfred-Kowalke-Str. 17, 10315

9 Berlin, Germany, [email protected]

10 bFreie Universität Berlin, 14195 Berlin, Germany

11 cHelmholtz Centre Berlin for Materials and Energy, 14109 Berlin, Germany

12 dClinic for Zoo , Exotic Pets and Wildlife, Vetsuisse Faculty, University of Zurich,

13 Winterthurerstr. 260, 8057 Zurich, Switzerland, [email protected]

14

15 *corresponding author

16

17 Running head: Feeding trials with two foregut fermenters

18 2

19 Abstract

20 The digestion of plant material in mammalian herbivores basically depends on the

21 chemical and structural composition of the diet, the mean particle size to which the forage is

22 processed, and the ingesta retention time. These different factors can be influenced by the

23 animal, and they can presumably compensate for each other. The pygmy hippopotamus, a non-

24 ruminating foregut fermenter, has longer mean retention times than ruminants; however hippos

25 do not achieve higher (fibre) digestibilities on comparable diets, which could be due to

26 ineffective mastication. We performed feeding trials with six pygmy hippos (Hexaprotodon

27 liberiensis) and six banteng cattle (Bos javanicus) on a grass diet. As predicted, both species

28 achieved similar dry matter, organic matter, crude protein and gross energy digestibilities.

29 However, neutral and acid detergent fibre digestibility was lower in pygmy hippos. Apparently,

30 in these species, fibre digestibility was more influenced by particle size, which was larger in

31 pygmy hippos compared to banteng, than by retention time. In spite of their higher relative food

32 intake, the banteng in this study did not have greater relative gut fills than the hippos. Ruminants

33 traditionally appear intake-limited when compared to equids, because feed particles above a

34 certain size cannot leave the rumen. But when compared to nonruminating foregut fermenters,

35 rumination seems to free foregut fermenters from an intrinsic food intake limitation. The higher

36 energy intakes and metabolic rates in wild cattle compared to hippos could have life-history

37 consequences, such as a higher relative reproductive rate.

38

39 Key words: Mean retention time; Particle size; Foregut fermentation; Ruminant; Herbivore

40

41

42 3

43 Introduction

44 Herbivores rely on symbiotic microorganisms for the digestion of plant material. These

45 microorganisms are located in voluminous anatomical structures where the fermentative

46 digestion takes place; hence, these structures are mostly referred to as fermentation chambers

47 (Stevens and Hume 1998). Gut bacteria are considered to be very old organisms in an

48 evolutionary sense, and they do not differ fundamentally in their biology and ecology between

49 host species and thus in the ability to digest similar plant material (Van Soest 1994). Therefore,

50 the extent and rate of the fermentation process for a given forage type will basically depend on

51 three factors: the chemical composition of the diet, the ingesta particle size, and the ingesta

52 retention time.

53 Forage digestibility is considered to be inversely related to the amount of cell wall and

54 lignification (Karasov and Martínez del Rio 2007; in vitro e.g. Hummel et al., 2006; in vivo e.g.

55 Barboza 1993), because lignin is indigestible, and because the digestible portions of cell wall are

56 fermented at a slower rate than other nutrients. The advantage of small over large ingesta

57 particles is the larger relative surface area exposed to microbial attack resulting in higher (fibre)

58 digestion rates for small particles (Bjorndal et al., 1990, cf. Clauss and Hummel 2005; in vitro

59 e.g. Gerson et al., 1988, in vivo e.g. Bowman and Firkins 1993). Furthermore, if ingesta

60 retention time, and thus the time for microbial fermentation, is short, digestive efficiency

61 decreases (Clauss et al., 2007b; in vitro e.g. Hummel et al., 2006, in vivo e.g. Udén et al., 1982).

62 Animals have evolved different adaptations to optimize digestive efficiency, i.e. altering

63 diet composition, particle size and retention time. The chemical composition of the ingested

64 diet can be influenced by diet selection, i.e. selecting food items with a lower fibre and lignin

65 content (Illius and Gordon 1993; Sprent and McArthur 2002). Food particle size is primarily

66 reduced by mastication – during ingestion and, in ruminants, during rumination (Wilson et al.,

67 1989). The degree of comminution, and thus chewing efficiency, is a multifactorial function of 4

68 tooth morphology, efficiency of masticatory movement, diet properties, the time spent chewing

69 and the number of chews per quantity of food (Pérez-Barbería and Gordon 1998a). In this

70 regard, ruminants are particularly interesting: they possess molars with enamel ridges that form

71 a complex tridimensial structure of characteristic crescent-shaped cusps (Pérez-Barbería and

72 Gordon 1998b), and they submit the ingesta to repeated mastication, achieving equal or finer

73 ingesta particles than other “similar-sized” herbivores (Van Soest 1994). The period of ingesta

74 retention finally is a physiological characteristic of a species and will vary, both within and

75 between species, with food intake level (Clauss et al., 2007a; Clauss et al., 2008) and with the

76 volume of the gastrointestinal tract (Langer and Snipes 1991; Karasov and McWilliams 2005).

77 It is reasonable to assume that these different factors (diet composition, particle size and

78 retention time) can compensate for each other. For example, Karasov et al. (1986) explained

79 that herbivorous reptiles (lizards) achieve almost the same digestibilities on the same diet as

80 nonruminant (woodrat, mouse). The lack of a masticatory apparatus was presumably

81 outbalanced by lower food intakes and longer ingesta retention times in reptiles. Among

82 mammals, Clauss et al. (2005) outlined that, compared to horses, Indian rhinoceros (Rhinoceros

83 unicornis) have longer, and elephants have similar ingesta retention times; yet, this does not

84 result in higher digestibility coefficients in the rhino, and elephants nevertheless achieve even

85 lower digestibilities. The authors speculated that this was due to a reduced ingesta particle size

86 reduction in these two herbivore groups as compared to horses. However, studies in which all

87 the relevant variables were measured simultaneously are rare.

88 We investigated the digestive efficiency in two different foregut fermenters, the non-

89 ruminating pygmy hippopotamus (Hexaprotodon liberiensis) and the ruminating banteng cattle

90 (Bos javanicus). Hippos have longer mean retention times than ruminants (common and

91 pygmy hippo: Foose 1982; Clauss et al., 2004; pygmy hippo: Schwarm et al., 2008a); however

92 they do not achieve higher (fibre) digestibilities on comparable diets (common hippo: Arman 5

93 and Field 1973; Abaturov et al., 1995; common and pygmy hippo: Foose 1982; Schwarm et

94 al., 2006), which could be due to ineffective mastication (Arman and Field 1973; Clauss et al.,

95 2004). So far, the knowledge on digestion coefficients in hippos is based on low sample sizes,

96 without direct comparisons to ruminants. Thus, to facilitate an interspecific comparison we

97 investigated six pygmy hippo and six banteng on a constant diet by measuring intake, nutrient

98 composition of the offered and ingested food, particle size, nutrient digestibility and retention

99 time.

100 We predicted that pygmy hippos would display similar nutrient digestibilities as banteng

101 on the same diet. Longer ingesta retention times in hippos would outbalance their less effective

102 ingesta mastication. These longer ingesta retention times could be a consequence of instrinsic

103 characteristics as a lower food intake, or a greater gut volume, or both. Based on previous

104 reports on hippo food ingestion (reviewed in Clauss et al., 2007b) and gut fill (reviewed in

105 Clauss et al., 2003), we predicted that pygmy hippos would display both, a lower food intake

106 and a greater gut fill than the ruminant.

107

108 Material and Methods

109 The trials were performed with six pygmy hippos and six banteng at the Zoological

110 Gardens of Berlin (ZGB) and Halle (ZGH) in summer 2005 and 2006. Body mass (BM) of the

111 pygmy hippos was measured at the beginning and the end of each trial period, whereas BM of

112 the banteng were estimated by the keepers by visual judgement (height and width) and age and

113 sex as reference. Details of the animals are summarized in Table 1.

114 The animals were fed fresh grass only, the staple diet at both zoos during summer. Grass

115 (C3-species) was harvested from mixed swards of the surrounding countryside. Since the

116 grass diet was usually supplemented with fruits and vegetables in hippos and with sugar beet

117 pulp in banteng, an adaptation period of 14 days was allowed to pass before the trial started. It 6

118 was planned to study each animal in two trials on different intake levels (with a second

119 adaptation period of 5 days in between) – ad libitum (high intake, HI), and, subsequently, at

120 approximately 75 % of the individual ad libitum intake (low intake, LI). Each trial lasted 7 days.

121 Due to a shortage of grass (the weather being too hot for sufficient regrowth or too wet for the

122 mechanical harvest, respectively), one pygmy hippo (animal 6) and three banteng (animals 10-

123 12) could only be assessed at one intake level (HI). For the same reason, some animals had to be

124 fed grass hay (soaked in water) at one day during one trial (animal 2, HI: at day one after marker

125 feeding; animal 4, LI: day six; animal 7, LI: day one; animal 8, LI: day two; animal 9, HI: day

126 two).

127 All animals were fed separately. Due to the husbandry techniques at the respective zoos,

128 not all animals could be kept separately at all times, and feeding regimens differed. Three

129 pygmy hippos (animals 1-3) received food once daily, approximately at 17:00 hours and had

130 access to the food until the next morning. The other pygmy hippos (animals 4-6) as well as three

131 banteng (animals 7-9) received food twice daily, at approximately 08:30 and 17:00 hours. These

132 animals had access to the food until the next meal was offered. Three banteng (animals 10-12)

133 were kept together between the feeding times; in these animals, the individual access to food

134 was limited from 08:30 to 11:00 and from 15:30 to 18:00 hours. Food items offered and

135 leftovers were quantified on a daily basis by weighing, and representative samples (for each

136 individual) of food offered and leftovers were stored frozen (-20°C) until analysis. During the

137 day (approximately 08:00-18:00), the pygmy hippos were kept on land with no access to a water

138 pool. During the night (approximately 18:00-08:00), the pygmy hippos had free access to a

139 water pool, with the exception of animal 1 on the high intake level. Drinking water was always

140 accessible for ad libitum consumption by all animals.

141 Two of three banteng that were kept together received a coloring agent in their ration

142 (animal 10: titanium dioxide 40 g/d; animal 11: brilliant blue food colour, Sensient Food Colors, 7

143 Geesthacht, Germany, 2 g/d; both fed twice daily; the blue color was mixed in approx. 200 g

144 sugar beet pulp for better acceptance), so that faeces could be ascribed to the individual animals.

145 Defaecations were collected completely in regular intervals, cleaned from sand (when

146 contaminated), weighed, thoroughly mixed, and an aliquot (200-400 g) was taken and stored

147 frozen. Faeces voided by hippos into the water pool at night were not sampled.

148 For nutrient analyses food and faecal samples were dried at 40°C and 60°C,

149 respectively. Dry matter (DM) content of food and faecal subsamples was determined by

150 drying at 103°C to constant weight. Samples were ground with a ‘Nossener mill’ (Gebrüder

151 Jehmlich, Nossen, Germany, 1 mm round perforated screen). Food samples were additionally

152 ground afterwards with a laboratory mill (A11basic, IKA, Staufen, Germany) to minimize the

153 presence of long glumes. Subsamples of all ground samples of food provided, food left over and

154 faeces, respectively, were pooled, resulting in three samples per animal and trial. Pooled

155 samples were analysed (in duplicate) according to Naumann and Bassler (1976) for their content

156 of crude ash (CA) in a muffle furnace and of nitrogen (N) by the Dumas method in a Elementar

157 rapid N III Analyser (Elementar Analysensysteme, Hanau, Germany). Crude protein (CP) was

158 calculated as CP = 6.25*N. Neutral detergent fibre (aNDFom), acid detergent fibre (ADFom) and

159 acid detergent lignin (ADLsa) were analysed according to Van Soest (1967) and Van Soest et al.

160 (1991) using the Ankom200 Fibre Analyser. Concerning the fibre terminology, the subscript

161 “a” stands for an assay with heat stable amylase, “om” for an expression exclusive of

162 residual ash, and “sa” for solubilisation of cellulose with sulphuric acid (Udén et al. 2005).

163 Organic matter was calculated as 100-CA, hemicelluloses (HC) as aNDFom-ADFom and

164 celluloses (C) as ADFom-ADLsa. In order to measure metabolic faecal nitrogen (MFN), the

165 nitrogen content in the faecal aNDFom residue was determined according to Mason and

166 Frederiksen (1979):

167 MFN (% total faecal nitrogen, TFN) = MFN (% faecal dry matter, DM) * 100/ N (% faecal DM) 8

168 With MFN (% faecal DM) = N (% faecal DM) – NDF-N (% faecal DM)

169 With NDF-N (% faecal DM) = aNDFom (% faecal DM) * NDF-N (% faecal aNDFom residue)/

170 100

171 MFN is defined as the nitrogen fraction in the faeces of bacterial and animal origin. Faecal

172 aNDFom N represents undigested plant N.

173 Gross energy (GE) was determined by bomb calorimetry (IKA-Labortechnik,

174 C5003control, Staufen, Germany). The nutrient content of the ingested diet was calculated by

175 substracting the content in the leftovers from the content of the offered diet.

176 In a previous paper (Schwarm et al. 2008a), we reported data from the same trials on

177 fluid, small (2 mm) and large (10 mm) grass hay particle passage time (= mean retention time:

178 MRT) in the gastrointestinal tract. Here, the data on small particle MRT (chromium-mordanted

179 fibre) is used as one of twenty data columns in Table 2 to test relationships between digestive

180 variables.

181 One faecal subsample per animal and diet treatment was used for the determination of

182 the mean faecal particle size (MPS) using wet-sieving technique (Retsch VS 1000, Haan,

183 Germany) with sieve mesh sizes of 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.063 mm. After thawing,

184 5-15 g of the samples were soaked in tap water overnight (stored in the fridge) and stirred with a

185 spoon before sieving for 10 minutes at an amplitude of 45 (approx. 1.5 mm). The particles of

186 each fraction were dried at 103°C to constant weight together with a subsample for DM content

187 determination. The MPS of each faecal sample was calculated numerically after fitting a suitable

188 function to the respective sample data (x-axis: sieve mesh size, y-axis: cumulative percentage of

189 retained particles) using the software TableCurve 2D (Systat Software UK Ltd., London, UK).

190 We did not use the modulus of fineness (MOF) as in Clauss et al. (2004) because the MOF

191 variable comprises a linear expression (number of sieve) of an exponentially increasing particle 9

192 size due to sieve mesh sizes of 0.5,1,2,4 etc. mm. Consequently, with actual increasing particle

193 size the MOF does not increase accordingly.

194 The apparent nutrient digestibility (aD), the digestible energy intake (DEI) and the true

195 protein digestibility (TPD) were calculated as in Clauss et al. (2005) and Mason and Frederiksen

196 (1979). For these calculations, the total amount of excreted faeces is required, which could only

197 be estimated in pygmy hippos (due to the water pool access in the night) using ADLsa as an

198 internal (indigestible) marker. On the basis of the known ADLsa content of the individually

199 ingested grass, the amount of excreted faeces was extrapolated. In banteng total faeces

200 production could be measured by total faecal collection. In one pygmy hippo (animal 1, HI) total

201 faeces collection was possible because for this animal, no water pool was available at the time.

202 Total faeces production in banteng and the one pygmy hippo was also controlled by estimation

203 of faeces excretion with ADLsa.

204 The indigestible gut content (VN, kg DM) and the total gut content (V, kg DM) were

205 calculated according to Holleman and White (1989):

206 VN = F * MRT

207 With F (faeces output, kg DM/h) = total daily faeces output/24

208 and with MRT = mean (2 mm) particle retention time through the whole digestive tract (h).

209 V = VN + ((VN * (aD DM/100))/(2(1 – (aD DM/100))) assuming linear absorption of ingested

210 food with time spent in the tract.

211 With VN = indigestible gut content (kg DM) and aD DM = apparent dry matter digestibility

212 (Table 2).

213 Wherever possible, nonparametric methods were used, because the normality

214 presumption for parametric tests cannot reliably be tested with very small sample sizes. Thus,

215 the Mann-Whitney-U-Test was used for comparisons between species; the high and low intake

216 levels were compared separately. Monotonous associations between pairs of variables were 10

217 measured by calculating Spearman´s correlation coefficient (SCC). The latter analysis should be

218 considered as exploratory since all trials, and thus repeated measurements per individual, were

219 combined. To avoid misunderstandings, we have added quotation marks when designating these

220 results as “significant”. Linear and multiple regression analysis were used for the calculation of

221 intraspecies relationships and ANCOVA for interspecies comparisons of interrelations between

222 OM or aNDFom digestibility (dependents) and MRT, MPS and relative DMI (rDMI)

223 (independents). The significance level was set to α=0.05. All statistical calculations were

224 performed with the SPSS 12 software (SPSS, Chicago, IL).

225

226 Results

227 The animals appeared to be healthy throughout the study and did not lose weight

228 (measured in pygmy hippos, and judged by external appearance in banteng, see Table 1).

229 The grass at Berlin and Halle zoo contained on average 27 ± 13 % and 34 ± 2 % dry

230 matter, respectively. The dry matter fraction of the grass offered to the animals (≠ ingested

231 diet) at Berlin and Halle zoo had on average (mean ± SD) a similar chemical composition with

232 93 ± 3 and 92 ± 1% organic matter (OM), 12 ± 3 % and 12 ± 3 % crude protein (CP), 63 ± 2 and

233 60 ± 1% neutral detergent fibre (aNDFom), 34 ± 2 and 33 ± 1% acid detergent fibre (ADFom), 3 ±

234 1 and 5 ± 1% Lignin and 18.5 ± 0.4 and 18.0 ± 0.3 MJ/kg DM gross energy, respectively. The

235 trials at Berlin zoo were performed in two different years, however the dry matter content of

236 the grass was comparable between years (mean ± SD: 24 ± 4 vs. 31 ± 6) and the differences

237 in nutrient content were small.

238 At the high intake level, pygmy hippos ingested a diet (≠ offered) of significantly lower

239 aNDFom, ADFom and GE content than banteng, and at the low intake level, the CP and GE

240 content in the diet ingested by pygmy hippos was lower (Table 2). At the low intake level, the

241 average rDMI in pygmy hippos of 19 ± 12 g/(kg0.75*d) (n=5) was significantly lower than that of 11

242 banteng (47 ± 9 g/(kg0.75*d), n=3; Table 2). At the high intake level the same pattern was

243 obvious, the average rDMI in pygmy hippos was 35 ± 13 g/(kg0.75*d) (n=6) and in banteng 51 ±

244 23 g/(kg0.75*d) (n=6). An influence of fibre content on intake will be more evident when

245 different feeds of different fibre concentrations are compared. Nevertheless, in pygmy hippos,

246 aNDFom-content in the ingested diet and rDMI were positively correlated, in contrast to banteng

247 (Table 3); the correlation in pygmy hippos could result from the larger range of the aNDFom-

248 content in the ingested diet (51-63% DM) as compared to banteng (61-66% DM).

249 In pygmy hippos, mean retention time (MRT) and rDMI were negatively correlated, in

250 contrast to banteng (Fig. 1a including statistics in the figure legend); again, the range of MRT

251 was larger in pygmy hippos (59-124 h) as compared to banteng (50-61 h). As expected, there

252 was a “significant” correlation between rDMI and relative digestible energy intake (rDEI) in

253 both species (Fig. 1b).

254 The mean faecal particle size was significantly higher in hippos compared to banteng at

255 both intake levels (Table 2), accounting for the proportion of larger faecal particles in pygmy

256 hippos that was absent in banteng (Fig. 2). The mean particle size (MPS) was not related to the

257 rDMI in either species, but in banteng, a negative correlation between MPS and aNDFom-content

258 in the ingested diet was found (Table 3).

259 There was no significant difference in the nutrient digestibility of the banteng calculated

260 with ADLsa as internal marker or determined by total faecal collection (Table 2, last rows). In

261 the pygmy hippo in which total faecal collection was possible, nutrient digestibility was within

262 the range of the other hippos. When comparing digestibility coefficients between pygmy hippos

263 and banteng (HI: n=6 vs 6, LI: n=5 vs 3), there was no difference in DM, OM, CP and GE

264 digestibility; however banteng achieved significantly higher aNDFom and ADFom digestibilities

265 than hippos at the high intake level (66 and 62% in banteng compared to 52 and 47% in hippo,

266 respectively); the same difference was evident for the low intake level. Metabolic faecal 12

267 nitrogen (MFN, non-dietary nitrogen) was significantly higher in pygmy hippos compared to

268 banteng at HI (72 vs. 66% of total faecal nitrogen) and showed the same trend at LI. True

269 protein digestibility (TPD) did not differ significantly between species. The digestibility of DM,

270 OM, aNDFom and ADFom was not associated with the rDMI in either species, and OM

271 digestibility was not associated with the aNDFom or ADFom content of the ingested diet (Table

272 3). In pygmy hippo, apparent digestibility of aNDFom and the proportion of lignified fibre in the

273 ingested diet (ADLsa in aNDFom) were negatively correlated, in contrast to banteng (Table 3).

274 Again, the range of lignified fibre in the ingested diet was larger in pygmy hippos (4.1-9.9%

275 ADLsa in aNDFom) as compared to banteng (3.5-7.0).

276 OM or aNDFom digestibility was not associated with MRT in either species (Table 3).

277 However, OM and aNDFom digestibility and MPS were negatively correlated in banteng, in

278 contrast to hippos (Table 3), although the MPS range was much smaller in the ruminant.

279 Regression analysis within each of the species did not yield significant relationships between

280 OM or aNDFom digestibility (dependents) with MRT, MPS and rDMI (independents), neither

281 uni- nor multivariat. The additional inclusion of species as categorical variable in an ANCOVA

282 analysis also led to insignificant results.

283 The species did not differ significantly in their relative dry matter gut fill, which

284 constituted on average 1.8% of body mass (Table 2).

285

286 Discussion

287 Validity of results

288 The limitations of digestion studies have to be considered when interpreting the results.

289 The grass was harvested over the whole experimental period from different swards and

290 therefore differed in plant community and maturity. However, the nutrient composition of 13

291 both the grass offered and ingested, was mostly similar among species, and therefore, an

292 interspecies comparison in nutrient digestibility is feasible.

293 One problem was that the food access in three banteng (animal 10, 11, 12) was time

294 limited (due to husbandry techniques), and it turned out that the supposed ad libitum DMI (HI)

295 was actually a comparatively low intake level for these three animals (omitting them from the

296 mean gives a rDMI of 71 instead of 51 g/(kg0.75*d). Accordingly, differences between the

297 species in relative food intake did not reach significance for the HI level. The significant

298 difference in rDMI at the low intake level between banteng and hippo is not attributed to the

299 very low rDMI of one hippo (animal 5; 7 g/(kg0.75*d)). Omitting that hippo from the mean gives

300 a rDMI of 22 instead of 19 g/(kg0.75*d). The sample size of each species at the LI level is

301 below 6 (5 hippos, 3 banteng), and thus conclusions drawn from the LI level are limited.

302 Hippos 1-3 were fed less frequently (once vs. twice a day) and had less time to feed (14

303 vs. 24 hours) than hippos 4-6, however they had similar or even higher rDMIs than hippos 4-6.

304 Thus, it can be assumed that trial limitations as feeding frequency and time of food access were

305 not substantial contributors to the response of the animals.

306 In one pygmy hippo (animal 5, HI, LI) and one banteng cattle (animal 8, LI) the

307 DM digestibility was much lower than the OM digestibility (≥ 20 percentage units, Table

308 2). This can be attributed to the contamination of the faeces by sand. Contaminations

309 caused by sand ingestion could not be removed. Omitting that hippo and banteng does not

310 affect the conclusions.

311 In banteng, which could not be weighed, the results on rDMI, rDEI and total gut fill

312 depend on accuracy of weight estimation. In fact, the results are within the range of feral and

313 domesticated cattle (on comparable diets) given in the literature (Poppi et al., 1980a; Foose

314 1982; Solaiman et al., 1990; Prigge et al., 1990).

315 14

316 Digestive performance

317 As predicted, both species achieved similar dry matter, organic matter, crude protein and

318 gross energy digestibilities. However, fibre digestibility was lower in pygmy hippos, although

319 they had longer retention times than banteng. Apparently, in these species, fibre digestibility

320 was more influenced by particle size, which was larger in pygmy hippos compared to banteng,

321 than by retention time. As predicted, pygmy hippos displayed a lower rDMI compared to

322 banteng.

323 In contrast to common hippos that achieve greater relative gut capacities than ruminants

324 (Langer 1988; Clauss et al., 2003), and in contradiction to our prediction, the pygmy hippos did

325 not have a greater relative gut fill than the ruminating banteng. The longer retention times

326 measured in hippos must therefore be considered as a consequence of their lower rDMIs

327 (causing a lower rate of gut emptying), not of a potentially greater gut fill. Whether changes in

328 gut fill could represent an adaptive strategy of pygmy hippos or banteng was not investigated in

329 this study.

330 We had expected that multivariate analyses, using the digestibility of OM or aNDFom as

331 the respective dependent variables, and MRT, MPS and rDMI as influence factors, would

332 demonstrate both significant relationships within each species and a compensatory effect of

333 MRT and MPS on digestive efficiency, since both species have similar OM digestibilities but

334 differ in MRT, MPS, rDMI and aNDFom digestibility. However, we could not find such

335 relationships for the individuals in either species. To understand compensatory effects, further

336 investigations and analyses on the species rather than on the individual level (n ≥ 6 species) with

337 a larger variety in both MRT and MPS are required.

338 The fibre digestibilities in banteng were similar to those in feral and domesticated cattle

339 on comparable (forage only) diets (grass hay: Foose 1982; Solaiman et al., 1990; fescue hay:

340 Goetsch et al., 1987). In correspondence to results from Wilson et al. (1989) in domestic cattle, 15

341 MPS was lower on diets with higher fibre (aNDFom) content in banteng, presumably because of

342 an increase in rumination activity. It must be assumed that an influence of particle size on OM

343 and aNDFom digestibility could only be found in banteng because a critical MPS for fast

344 digestion is exceeded severalfold in pygmy hippos. Pygmy hippos can only crush their food due

345 to the interlocking canines that allow vertical movements of the jaws only. In contrast, banteng

346 are ruminants, thus they can not only move their jaws laterally which permits grinding of the

347 food, but submit the plant material to repeated chewing. Additionally, Bovinae possess

348 specialised selenodont cheek teeth (crescent-shaped cusps) with high crowns (hypsodont),

349 whereas hippos have bunodont molars (cheek teeth with low, rounded cusps) with low crowns

350 (brachyodont) (Thenius 1989; Archer and Sanson 2002; Boisserie and Lihoreau 2006). The

351 result of this difference is obvious in Fig. 2. The figure also demonstrates that the critical size

352 for passage from the rumen of the banteng is defined by a sieve mesh size between 1 and 0.5

353 mm for the majority of the particles, similar to findings in domestic cattle (Poppi et al.,

354 1980b), whereas there is no such effect in the stomach of the hippo.

355 The effect of different particle sizes of the same feed on the efficiency of the in vitro

356 fermentation has been intensively studied (e.g. Robles et al., 1980; Cherney et al., 1988;

357 Bjorndal et al., 1990). Bjorndal et al. (1990) showed that, after 48 hours, small, 2 mm long,

358 grass blades had been digested twice as much as large, 10 mm, particles (in vitro OM

359 digestibility). In the study of McLeod and Minson (1969) the in vitro DM digestibility of 2 mm

360 long grass still increased during the fermentation period of 48 to 96 h. It can be expected that the

361 conditions in vivo are less defined, because a mixture of different sized particles (see Fig. 2) is

362 fermented simultaneously, leading to an average correlation between time and digestibility. It

363 would be interesting to digest the used forage as a mixture of different sized particles for a time

364 period longer than 48h in vitro. 16

365 The metabolic faecal nitrogen loss did not differ between the investigated species at the

366 low intake level and was even higher in pygmy hippos at the high intake level compared to

367 banteng. This result was in contrast to our expectations, which were based on earlier findings

368 indicating comparatively lower metabolic faecal losses in hippos as compared to ruminants

369 (Schwarm et al., 2006). Because hippos lack a caecum and a differentiated colon (Stevens and

370 Hume 1995) and have presumably little additional bacterial fermentation in the lower digestive

371 tract as opposed to ruminants (Clemens and Maloiy 1982; Clemens and Maloiy 1983), less

372 bacterial nitrogen should be produced in the hindgut and hence be excreted in these animals.

373 However, these comparisons had not been based on experiments in which identical diets were

374 used. Additionally, when the dietary crude protein content (ingested) was plotted against the

375 dietary content of apparently digestible crude protein (“Lucas test”, cf. Van Soest 1967; Robbins

376 1993; Van Soest 1994), no difference in the pattern between the species was evident (Fig. 3, see

377 legend for statistics). This indicates a similarity in the endogenous (faecal) losses of protein

378 (reflected by the negative intercept, hippo: y = 0.92x-3.9, banteng: y = 0.99x-3.6) – the majority

379 of which will be derived from bacteria (Mason 1969; Van Soest 1994). Apparently, foregut

380 fermenting herbivores that rely on bacterial fermentation have a high degree of similarity

381 regarding metabolic faecal losses, regardless of the differentiation of their lower digestive

382 tract. This assumption is fortified by the result of a study were no differences in metabolic

383 faecal losses were found within different foregut fermenters and even between foregut and

384 hindgut fermenters (Schwarm et al. 2008b).

385 rDMI and MRT were negatively correlated in pygmy hippos (Fig. 1a), in contrast to

386 banteng. Negative DMI-MRT relationships can usually be found in cattle (e.g. Colucci et al.,

387 1982; Shaver et al., 1986). However, the range in rDMI in the ruminant of this study was

388 smaller compared to other studies.

389 17

390 Energy intake

391 The negative relationship of MRT and rDMI in hippos indicates that they cannot

392 increase food intake without compromising digestive efficiency by accelerating ingesta passage.

393 Therefore, hippos are limited in the extent by which they can increase energy uptake by

394 increasing food intake (Clauss et al., 2007b). Correspondingly, although there was some overlap

395 in the digestible energy intake in hippos and banteng of this study, the hippos did not attain

396 similarly high energy intakes as the ruminants (Fig. 1b). This matches reports on lower

397 metabolic requirements of hippos as compared to other mammalian species (Eltringham 1999;

398 Schwarm et al., 2006) and the statement of Reilly et al. (2001) that an increased chewing

399 efficiency is related to the development of higher metabolic rates in birds and mammals.

400 What are the consequences of a supposed difference in metabolic rate between ruminants

401 and hippos? It has been suggested that the reproductive potential of a species is linked to its

402 metabolic rate (McNab 2006). When comparing longevity and reproduction variables such as

403 calving interval and age of first conception in hippos and banteng/wild cattle (Table 4), and

404 taking into account that empirical evidence for (female) reproductive senescence in feral

405 animals is equivocal (e.g. Gaillard et al., 1994; Loison et al., 1999), then the difference in the

406 number of calves produced during the lifetime of a pygmy hippo (18 calves), common hippo (18

407 calves) and banteng/wild cattle (19 calves) is only marginal. But on a relative scale, pygmy and

408 common hippos deliver fewer calves compared with banteng/wild cattle per lifetime year (0.5

409 and 0.4 vs. 0.8 calves/year, respectively), and produce less biomass (relative calf weight: 47 and

410 53 vs. 201 g/kg body weight0.75 of the mother/lifetime year, respectively; Table 4). Thus, it

411 could be speculated that the lower energy intake and metabolic rate in hippos compared to

412 ruminants result in a lower reproductive rate. Moreover, banteng give birth to a relatively

413 heavier calf compared to hippos (Table 4), which is probably important for predator avoidance

414 and the migratory behaviour of many ruminant species. Whether differences in metabolic rate 18

415 actually correlate with differences in life history patterns needs to be elucidated in comparative

416 studies.

417

418 Putting ruminants into a comparative perspective

419 Traditionally, our view of ruminants has been dominated by the comparison with the

420 hindgut (colon) fermenting equids (Janis 1976). Compared to equids, ruminants appear intake-

421 limited due to their elaborate forestomach physiology, because feed particles above a certain

422 size cannot leave the rumen. For other (nonruminant) foregut fermenters, like sloths (Clauss

423 2004) or hippos (Clauss et al., 2004), it has been suggested that a different particle retention

424 mechanism in the forestomach might lead to a differential excretion of larger particles as a

425 means of clearing less digestible bulk from the forestomach, similar to the excretion of larger

426 particles from the hindgut of caecum fermenters or horses. However, although a selective

427 excretion of larger particles was demonstrated in hippos (Schwarm et al., 2008a), data on the

428 daily food intake of nonruminant foregut fermenters (collated e.g. in Clauss et al., 2007a) do not

429 suggest that these animals have a comparatively high food intake; on the contrary, both sloths

430 and hippos are known for their low food intake compared to ruminants (Foley et al., 1995;

431 Clauss et al., 2007b). Using the example of primates, Clauss et al. (2008) outlined that for

432 nonruminant foregut fermenters, a low intake-slow passage strategy is the only physiological

433 option, whereas hindgut fermenters can, on a species level, either pursue the same strategy or a

434 high intake-short passage strategy. Due to the differential digestion kinetics of those substrates

435 that could also be digested auto-enzymatically (simple sugars, starches, which ferment

436 relatively fast) and those that cannot (plant material, which ferments more slowly), foregut

437 fermenters will always lose most of the auto-enzymatically digestible substrate (e.g. cell content

438 in grazers) to the microbial symbionts in their forestomach, and therefore an efficient fibre

439 utilisation via long retention times is the only logical option open to them. By contrast, hindgut 19

440 fermenting species, which always use the auto-enzymatically digestible substrate prior to

441 bacterial fermentation, can either have a low or a high throughput strategy. With this

442 background, our comparison of banteng and hippos put the ruminants into a new perspective.

443 Both banteng and hippos retain large particles (measured as 10 mm particles applied orally) for

444 a similar length of time when fed ad libitum (Schwarm et al., 2008a). However, due to the

445 design of the hippopotamus forestomach, smaller particles are retained for a similar length of

446 time or even longer. By contrast, the morphophysiological design of the ruminant forestomach

447 allows a concomitant faster excretion of the smaller particle fraction (Schwarm et al., 2008a),

448 thereby clearing the forestomach faster and, ultimately, allowing the higher intake in banteng

449 compared to hippos documented in this study and in ruminants compared to nonruminant

450 foregut fermenters in general (Clauss et al., 2007a). Note that in spite of the higher relative food

451 intake, the banteng in this study did not have a greater relative gut fill than the hippos. In other

452 words, although ruminants are often thought to be intake-limited as compared to hindgut

453 fermenters (Janis 1976), ruminating foregut fermenters are not intake limited when compared to

454 nonruminating foregut fermenters. Thus, rumination could be described as a strategy that frees

455 foregut fermenters from an intrinsic food intake limitation. The selective particle passage in

456 ruminants is the result of a ‘sorting mechanism’ that allows a comparatively fast clearing of the

457 forestomach. We conclude that the efficiency of this sorting mechanism therefore permits

458 ruminanting foregut fermenters to achieve comparatively high energy intakes and hence also

459 metabolic rates, whereas nonruminant foregut fermenters likely share a low metabolic rate as a

460 common feature (Schwarm et al., 2006; Clauss et al., 2007a; Munn et al., 2008).

461

462 Acknowledgments

463 This study was supported by grants from the Freie Universität Berlin (NaFöG) to AS and

464 the German Science Foundation (DFG) to SO (OR 86/1-1). We thank Ragnar Kühne, Timm 20

465 Spretke, Uwe Fritzmann, Kurt Goedicke, Conny Hofmann and colleagues from the Zoological

466 Gardens of Berlin and Halle, Heidrun Barleben, Doreen Pick and Erin Bambury from the IZW

467 Berlin, Olaf Dibowski, Stephan Kohlmüller and their colleagues from the Landeslabor

468 Brandenburg, Germany, and Julia Fritz from the Ludwig-Maximilian Universität München for

469 their engaged support. Sincere thanks to the anonymous reviewers for their valuable comments.

470 21

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668 Tables 669 Table 1. Details of the pygmy hippos (Hexaprotodon liberiensis) and banteng (Bos javanicus) 670 studied at the Zoological Gardens of Berlin (ZGB) and Halle (ZGH). In pygmy hippos body 671 mass was measured four times (before and after each trial) and given values are means (± 672 SD). Year of Species Animal Born Sex BM (kg) Facility trial 1 1985 male 248 ± 4 ZGB 2005 2 1983 female 225 ± 3 ZGB 2005 3 1997 female 238 ± 2 ZGB 2006 Pygmy hippo 4 1998 female 203 ± 3 ZGH 2006 5 1976 female 202 ± 4 ZGH 2006 6 2000 male 196 ZGH 2006 7 2002 male 550a ZGB 2005 8 2004 female 220 a ZGB 2005 9 2004 male 200 a ZGB 2005 Banteng 10 1996 female 700 a ZGB 2006 11 2001 female 600 a ZGB 2006 12 1997 female 650 a ZGB 2006 673 BM = body mass 674 a Estimated 26

675 Table 2. Relative dry matter intake (rDMI) and nutrient composition and apparent nutrient digestibility of the ingested diets (OM: organic matter, 676 CP: crude protein, EE: ether extract, NDF = aNDFom: neutral detergent fibre, ADF = ADFom: acid detergent fibre, GE: gross energy) ingested by 677 pygmy hippo (Hexaprotodon liberiensis) and banteng (Bos javanicus) and digestible energy intake (DEI), metabolic faecal nitrogen (MFN), true 678 protein digestibility (TPD), mean retention time (MRT) of 2 mm particles, mean particle size (MPS), total dry matter gut fill (TGF) and the P- 679 values of the interspecies comparison (Mann-Whitney-U analysis). The comparison of the digestibility in banteng calculated with total faecal 680 collection or ADLsa as internal marker is displayed as well. * = total faecal collection, † MRT data from the publication Schwarm et al. (2008a) Nutrient composition of the ingested diets Nutrient digestibility of the ingested diets MFN TPD MRT† MPS TGF

Species Animal Diet rDMI OM CP EE NDF ADF GE DM OM CP EE NDF ADF GE DEI 2 mm (MJ/ (MJ/kg (g/kg0.75) (%DM) (%) (kg0.75 (%TFN) (%) (h) (mm) (%BM) DM) *d)) 1* HI 18 95.6 12.7 1.9 58.0 30.9 18.7 60 62 69 57 53 47 61 0.21 73 92 79 10.65 1.1 1 LI 14 95.3 10.2 1.6 59.2 30.4 18.1 73 75 73 65 71 66 73 0.18 73 93 114 6.89 1.1 2 HI 43 90.2 16.2 1.7 59.3 30.3 18.0 69 73 78 52 71 65 71 0.55 72 94 75 9.77 2.2 2 LI 14 94.5 13.0 1.9 58.4 30.5 18.1 45 50 57 47 38 33 46 0.13 69 88 110 5.32 1.3 3 HI 50 96.0 12.9 2.2 63.0 34.4 18.9 50 53 63 26 44 39 49 0.46 75 91 66 7.71 2.6 Pygmy 3 LI 95.1 7.8 1.7 60.8 34.9 18.3 53 55 57 43 40 37 52 0.38 77 9.40 hippo 40 65 85 2.4 4 HI 37 92.7 15.4 2.5 58.8 32.4 18.4 41 46 62 27 34 29 42 0.29 71 89 71 9.97 2.4 4 LI 19 91.6 10.8 2.7 58.1 31.8 17.6 49 51 44 42 43 39 46 0.15 75 86 124 8.84 1.8 5 HI 21 92.3 17.8 2.8 52.0 27.6 18.4 25 66 68 43 58 53 62 0.23 70 90 104 8.88 2.1 5 LI 7 92.1 11.7 4.5 50.8 27.7 18.2 9 43 21 52 30 23 37 0.05 70 77 120 5.06 1.0 6 HI 36 88.7 14.3 2.7 54.0 28.1 17.9 64 63 73 59 53 47 62 0.39 71 92 59 4.50 1.6 7* HI 60 94.8 11.6 1.6 64.9 33.0 18.8 57 62 69 64 57 50 61 0.69 70 90 58 0.42 2.1 7* LI 43 90.3 15.0 1.7 60.5 32.4 18.5 54 62 77 70 60 51 63 0.50 68 93 50 0.39 1.3 8* HI 79 90.1 15.2 2.3 63.2 32.1 19.0 68 77 79 49 79 76 76 1.13 70 94 54 0.37 3.0 8* LI 40 91.9 13.8 2.1 64.6 33.0 19.0 49 69 70 36 70 65 65 0.51 64 89 54 0.32 1.8 Banteng 9* HI 73 93.0 15.7 2.1 66.0 32.0 19.5 61 73 72 33 78 74 71 1.02 65 90 53 0.34 3.0 9* LI 56 91.6 13.9 2.1 63.4 33.3 19.1 64 73 73 33 75 73 71 0.75 64 90 50 0.31 2.1 10* HI 37 94.5 7.2 1.7 61.3 36.4 19.0 59 67 52 56 62 59 66 0.46 64 83 53 0.38 1.1 11* HI 28 94.6 7.1 1.7 61.8 36.6 19.0 58 65 46 47 61 58 61 0.32 65 81 61 0.52 1.0 12* HI 26 94.6 7.1 1.7 61.8 36.5 19.0 56 63 47 43 58 53 60 0.29 66 82 55 0.40 0.8

27

Table 2 continued Nutrient composition of the ingested diets Nutrient digestibility of the ingested diets MFN TPD MRT† MPS TGF

Species Animal Diet rDMI OM CP EE NDF ADF GE DM OM CP EE NDF ADF GE DEI 2 mm (MJ/ (MJ/kg (g/kg0.75) (%DM) (%) (kg0.75 (%TFN) (%) (h) (mm) (%BM) DM) *d)) mean ± SD Pygmy 92.6 14.9 2.3 57.5 30.6 18.4 0.36 76 8.58 2.0 n=6 HI 34±12 52±16 61±10 69±6 44±15 52±13 47±12 58±10 72±2 91±2 hippo ±2.9 ±2.0 ±0.4 ±4.0 ±2.6 ±0.4 ±0.14 ±16 ±2.24 ±0.6 93.6 10.7 1.9 63.2 34.4 19.1 0.65 0.41 1.9 Banteng n=6* HI 51±23 60±4 68±6 61±14 49±11 66±10 62±11 66±6 66±2 87±5 56±3 ±1.8 ±4.1 ±0.3 ±1.9 ±2.3 ±0.2 ±0.36 ±0.06 ±1.0 p (U-test) 0.240 0.699 0.093 0.065 0.015 0.041 0.004 0.699 0.180 0.485 0.699 0.041 0.041 0.394 0.132 0.004 0.132 0.004 0.002 0.699

Pygmy 93.7 10.7 2.5 57.5 31.1 18.1 0.18 109 7.10 1.5 n=5 LI 19±13 46±23 55±12 50±19 50±9 44±16 40±16 51±14 71±4 86±6 hippo ±1.7 ±1.9 ±1.2 ±3.9 ±2.6 ±0.3 ±0.12 ±19 ±1.98 ±0.6 91.3 14.2 2.0 62.8 32.9 18.9 0.59 0.34 1.8 Banteng n=3* LI 46±9 56±8 68±6 73±4 46±21 68±8 63±11 66±4 65±2 91±2 51±2 ±0.9 ±0.7 ±0.2 ±2.1 ±0.5 ±0.3 ±0.14 ±0.04 ±0.4 p (U-test) 0.036 0.071 0.036 1.000 0.071 0.250 0.036 0.393 0.250 0.071 0.571 0.143 0.143 0.250 0.036 0.071 0.143 0.036 0.036 0.571

Comparison of the digestibility in banteng calculated with total faecal collection or ADLsa as internal marker Banteng n=6* HI 60±4 68±6 61±14 49±11 66±10 62±11 66±6 Banteng n=6 HI 64±7 72±3 67±11 47±19 72±5 68±5 70±4 p (U-test) 0.310 0.240 0.485 0.937 0.394 0.394 0.240 681 682 28

683 Table 3. Spearman correlation coefficient (SCC) between pairs of variables measured in 684 pygmy hippo (Hexaprotodon liberiensis) and banteng (Bos javanicus). Variables were neutral 685 and acid detergent fibre in the ingested diet (aNDFom/ADFom ingested, %), relative dry matter 686 intake (rDMI, g/(kg0.75*d)), mean particle size (MPS), apparent digestibility (%) of dry matter 687 (DM), organic matter (OM), neutral detergent fibre (aNDFom), acid detergent fibre (ADFom), 688 acid detergent lignin (ADLsa) in aNDFom (%) and mean retention time (MRT, h). Both intake 689 levels (HI, LI) per species were combined (hippo: N=11, banteng: N=9); P-values were 690 calculated for exploration purposes. Hippo Banteng Variable 1 Variable 2 SCC p SCC p aNDFom ingested rDMI 0.65 0.031 0.56 0.116 MPS rDMI 0.40 0.228 -0.42 0.265 MPS aNDFom ingested 0.15 0.658 -0.75 0.020 aD DM rDMI 0.25 0.457 0.55 0.125 aD OM rDMI 0.21 0.536 0.49 0.183 aD aNDFom rDMI 0.21 0.530 0.55 0.125 aD ADFom rDMI 0.15 0.658 0.48 0.187 aD OM aNDFom ingested 0.18 0.593 0.38 0.313 aD OM ADFom ingested -0.30 0.370 -0.36 0.343 aD aNDFom ADLsa in aNDFom -0.74 0.009 -0.10 0.795 aD OM MRT -0.18 0.593 -0.28 0.471 aD aNDFom MRT -0.16 0.649 -0.41 0.279 aD OM MPS 0.13 0.709 -0.74 0.023 aD aNDFom MPS 0.15 0.658 -0.75 0.020 691 29

692 Table 4. Body mass (BM, kg), longevity (a) and reproduction variables as number of calves 693 at a time, birth weight (kg), calving interval (month) and age of first conception (years) of 694 banteng (Bos javanicus), wild cattle (Bovini), pygmy and common hippos (Hexaprotodon 695 liberiensis, Hippopotamus amphibius). Median values in squared brackets were used for 696 calculations of the absolute and relative reproductive output as calves/lifetime (= 697 reproductive lifetime), calves/lifetime year, and the produced relative calf weight (% BM and 698 g/kg metabolic body weight, MBW0.75, of the mother)/lifetime year. Banteng Wild cattle Pygmy hippo Common hippo Body mass (BM, kg) 600-9002, 400- - 180-2601 1400-32005 9003, 600-8004 [220] [2300] [700] [700] Birth weight (kg) 304 45 (35-50)7 5.7 (4.5-6.2)6, 25-553, 428 3.4-6.43 [41] [5.3] Number of calves at a - 17 19 19 time [1] Calving interval 13.511 157 19.4-21.710 228 (month) [21] Age of first 23, 2-34 4-513 3-512, 4-53 9 (7-15)14, 9-108 conception (years) [2.3] [4.5] [4.3] [9] Longevity (years) 263, 204 302 355 409, 455 [23] [42.5] Calves/lifetime 18.4 20.4 17.5 18.3 Calves/liftetime year 0.80 0.68 0.50 0.43 Produced relative calf 3.43 4.37 1.21 0.77 weight (% BM of the mother)/lifetime year Produced relative calf 176 225 47 53 weight (g/kg MBW0.75 of the mother)/lifetime 699 700 1Hlavacek et al., 2003, 2Sinclair et al., 2001, 3Nowak 1999, 4Buchholtz 1988, 5Eltringham 701 2001, 6Lang et al., 1988, 7Sinclair 1977, 8Smuts and Whyte 1981, 9(cf.)Eltringham 1999, 702 10Zschokke 2002, 11Mohamad et al., 2005, 12Lang 1975, 13Pienaar 1969,14Laws and Clough 703 1966 704

705 30

706 Figure Legends

707 Figure 1. Relationship between

708 (a) relative dry matter intake (rDMI, g/(kg0.75*d)) and particle (2 mm) average passage time in

709 the gastrointestinal tract (MRT GIT, h) in pygmy hippos (Hexaprotodon liberiensis) and

710 banteng (Bos javanicus). Spearman correlation coefficients (SCC): hippos: N=11, SCC=-

711 0.77, p=0.006. banteng: N=9, SCC=-0.30, p=0.440, and

712 (b) relative dry matter intake (rDMI, g/(kg0.75*d)) and relative digestible energy intake (rDEI,

713 MJ/(kg0.75*d)). Spearman correlation coefficients (SCC): hippo: N=11, SCC=0.93, p<0.001;

714 banteng: N=9, SCC=0.97, p<0.001.

715

716 Figure 2. Faecal particles of banteng (Bos javanicus, upper row) and pygmy hippo

717 (Hexaprotodon liberiensis, lower row), retained on each of the nine sieves

718 (16,8,4,2,1,0.5,0.25,0.125 and 0.063 mm). Fresh faecal samples (of which the initial dry

719 matter weight was calculated to be the same for both species) were separated by wet-sieving

720 technique (Retsch). The black bar scales 50 mm.

721

722 Figure 3. Relationship between dietary crude protein (CP, % dry matter) content and the

723 dietary content of apparently digestible crude protein (dCP, % dry matter) in pygmy hippos

724 (Hexaprotodon liberiensis) and banteng (Bos javanicus). Spearman correlation coefficients

725 (SCC): hippo: N=11, SCC=0.84, p=0.001; banteng: N=9, SCC=0.95, p<0.001.

726 31

140 Hippo 120 Banteng 100 GIT (h) 80

60 2mm particles 40 MRT 20

0 0 1020304050607080 rDMI (g/(kg0.75*d)) 727

728 Figure 1 a

1.2

1.0 Hippo Banteng

*d)) 0.8 0.75

0.6

0.4 rDEI (MJ/(kg 0.2

0.0 0 1020304050607080 rDMI (g/(kg0.75*d)) 729

730 Figure 1 b 731 732 733 734 735 736 737 738 739 740 741 742 32

743 744 mm 16 8 4 2 1 0.5 0.25 0.125 0.063 745 746 747 748 749 750 751 752 753 Figure 2 754 755 14

12 Hippo Banteng 10

8

6 dCP (%DM) 4

2

0 0 5 10 15 20 CP (%DM) 756 757 Figure 3 758