The Intraruminal Papillation Gradient in Wild Ruminants of Different Feeding Types: Implications for Rumen Physiology

JOURNAL OF MORPHOLOGY 270:929–942 (2009)

Marcus Clauss,1* Reinhold R. Hofmann,2 Jo¨ rns Fickel,2 W. Ju¨ rgen Streich,2 and Ju¨ rgen Hummel3

1Clinic for Zoo Animals, Exotic Pets and Wildlife, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland 2Research Group for Evolutionary Genetics, Leibniz-Institute for Zoo and Wildlife Research (IZW), Berlin, Germany 3Department of Animal Nutrition, Institute of Animal Science, University of Bonn, Bonn, Germany

ABSTRACT Browsing and grazing ruminants are species between seasons (Hofmann, 1973; Langer, thought to differ in the degree their rumen contents are 1974; Ko¨nig et al., 1976; Hofmann, 1982; Hofmann stratified—which may be due to different characteristics and Schnorr, 1982; Hofmann, 1984, 1985; Smolle- of their respective forages, to particular adaptations of Wieszniewski, 1987; Hofmann et al., 1988a; Hofmann the animals, or both. However, this stratification is diffi- and Nygren, 1992; Josefsen et al., 1996; Forsyth and cult to measure in live animals. The papillation of the rumen has been suggested as an anatomical proxy for Fraser, 1999; Mathiesen et al., 2000; Kamler, 2001), stratification—with even papillation indicating homoge- or between free-ranging and captive individuals (Hof- nous contents, and uneven papillation (with few and mann and Matern, 1988; Marholdt, 1991; Hofmann small dorsal and ventral papillae, and prominent papil- and Nygren, 1992; Lentle et al., 1996). lae in the atrium ruminis) stratified contents. Using the Differences in the degree of papillation among surface enlargement factor (SEF, indicating how basal different rumen regions in the same animal have mucosa surface is increased by papillae) of over 55 rumi- been recognized for a long time in cattle, where nant species, we demonstrate that differences between especially the dorsal rumen wall completely lacks the SEFdorsal or SEFventral and the SEFatrium are signifi- papillae. In contrast, Martin and Schauder (1938) cantly related to the percentage of grass in the natural noted that the rumen of some deer species is diet. The more a species is adapted to grass, the more distinct this difference, with extreme grazers having evenly papillated. Differences in the papillation unpapillated dorsal and ventral mucosa. The relative among different rumen regions in different rumi- SEFdorsal as anatomical proxy for stratification, and the nant species were noted repeatedly (Garrod, 1877; difference in particle and fluid retention in the rumen as Langer, 1973), and put into a systematic perspec- physiological proxy for stratification, are highly corre- tive by Hofmann (1973), with extensive photo- lated in species (n 5 9) for which both kind of data are graphic documentation. Examples of papillation available. The results support the concept that the strat- extremes are given in Figure 1, where the rumina ification of rumen contents varies among ruminants, of roe deer (Capreolus capreolus), steenbok (Raphi- with more homogenous contents in the more browsing cerus campestris) and gerenuk (Litocranius wal- and more stratified contents in the more grazing species. leri) display a comparatively even papillation in all J. Morphol. 270:929–942, 2009. Ó 2009 Wiley-Liss, Inc. regions, whereas the rumina of hartebeest (Alcela- KEY WORDS: grazer; browser; rumen; rumen papillae; phus buselaphus), gemsbok (Oryx gazella) and surface enlargement factor reedbuck (Redunca redunca) show a papillation

INTRODUCTION R. R. Hofmann is currently at: Trompeterhaus, 15837 Baruth/ Mark, Germany. In ruminants (Hofmann, 1969; Langer, 1973), hippopotamids (Langer, 1975), and several rodent spe- Contract grant sponsor: German Wildlife Foundation (Deutsche cies (Vorontsov, 2003), the portion of the forestomach Wildtierstiftung e.V.). where bacterial fermentation occurs, and hence vola- *Correspondence to: Marcus Clauss, Clinic for Zoo Animals, tile fatty acids (VFA) are produced, is characterized Exotic Pets and Wildlife, Vetsuisse Faculty, University of Zurich, by an absorptive mucosa whose surface is consider- Winterthurerstr. 260, 8057 Zurich, Switzerland. ably enlarged by papillae. In ruminants, it was dem- E-mail: [email protected] onstrated that the development of these papillae is stimulated by the presence of VFA (Warner et al., Received 14 September 2008; Revised 17 December 2008; 1956; Sander et al., 1959; Sakata and Tamate, 1978, Accepted 19 December 2008 1979). Because VFA production is also a function of Published online 26 February 2009 in diet quality, the number and size of forestomach pap- Wiley InterScience (www.interscience.wiley.com) illae reﬂect variation in diet quality, e.g., within a DOI: 10.1002/jmor.10729

Ó 2009 WILEY-LISS, INC. 930 M. CLAUSS ET AL.

Fig. 1. Papillation of the rumen (A: atrium ruminis, D: dorsal rumen, V: ventral rumen) of six ruminant species fixed in situ: on the left side, from top to bottom, the browsers roe deer (Capreolus capreolus), steenbok (Raphicerus campestris; rod indicating the cardia) and gerenuk (Litocranius walleri) show a comparatively even papillation in all regions of the rumen; on the right side, from top to bottom, hartebeest (Alcelaphus buselaphus), gemsbock (Oryx gazella) and reedbock (Redunca redunca) show an uneven ruminal papillation, with large papillae in the atrium and smaller papillae and even unpapillated areas in the dorsal or ventral rumen. Magnification adapted for qualitative comparison; historical photographs, no scale bars available (from Hofmann, 1969; Hofmann et al., 1976; Hofmann and Schnorr, 1982). anterior 5 left side of photographs. that is distinct in the Atrium ruminis and around cific ruminal areas, such as the dorsal rumen wall, the Ostium intraruminale (i.e., in the middle the atrium, or the ventral rumen wall, reflect dif- rumen layer), less distinct towards the more dorsal ferences in the degree of ingesta stratification in and ventral layers, and even completely absent in the rumen (Hofmann, 1973; Langer, 1974; Hof- the most dorsal and ventral regions. It has been mann, 1989; Josefsen et al., 1996; Mathiesen proposed that differences in the papillation of spe- et al., 2000). Actually, differences the stratification

Journal of Morphology PAPILLATION PATTERNS IN RUMINANTS 931 of rumen contents have been found in parallel to 2008a; Schwarm et al., 2008), and has been pro- differences in papillation in several wild ruminant posed to be a major driver of particular anatomical species (Clauss et al., 2009, in press). adaptations in grazers, such as strong rumen pil- In cattle and other grazing ruminants, the lars (Clauss et al., 2003) and large omasa (Clauss rumen contents are stratified, with a distinct gas et al., 2006a). dome of CO2 and methane above the ‘‘fiber mat,’’ However, the presence or absence of a rumen which itself floats on the liquid layer, at the very contents stratification is actually difficult to inves- bottom of which very dense, fine sedimented par- tigate in live animals. Recently, the use of ultraso- ticles form a ‘‘sludge’’ layer (Grau, 1955; Capote nography has been shown to facilitate the differen- and Hentges, 1967; Hofmann, 1973; Tschuor and tiation between a gas dome and a fiber mat in the Clauss, 2008; Hummel et al., in press). In con- rumen of cattle, and allowed to demonstrate the trast, the rumen contents of several browsing spe- absence of a gas dome in a browse-fed captive cies anecdotally appeared rather homogenous, moose (Tschuor and Clauss, 2008), but this tech- ‘‘frothy,’’ without a distinct separation of gas, par- nique has obvious limitations in terms of applic- ticles, fluids, and sludge (Hofmann, 1969, 1973; ability over a wide range of species; the same is Nygren and Hofmann, 1990; Renecker and Hud- true for comparative measurements of fluid and son, 1990; Clauss et al., 2001). As papillation particle passage through the gastrointestinal tract. growth is induced by VFA, the presence of a dor- In contrast, using the rumen mucosa papillation sal gas dome and a ventral sludge layer could pre- as a surrogate measure for the stratification of the vent papillae formation in these locations, rumen contents is an attractive alternative. The because gas and sludge will displace any VFA papillation of a certain rumen region can be easily that could accumulate in these regions. Actually, quantified by the use of the ‘‘surface enlargement differences in VFA content between different factor’’ (SEF; Schnorr and Vollmerhaus, 1967), rumen ingesta layers have been measured in which represents the factor by which the absorp- domestic cattle (Smith et al., 1956; Tafaj et al., tive surface of the rumen mucosa is increased due 2004). In contrast, in homogenous rumen con- to the papillae in comparison to the basal rumen tents, VFA can be assumed to be relatively evenly area; an unpapillated region of the rumen, thus, distributed throughout the ingesta, leading to an has a SEF of 1.0. For rumen contents stratifica- even ruminal papillation. tion, three rumen regions are of particular inter- Hofmann (1969, 1973, 1989) first proposed that est: the atrium ruminis which usually shows the the difference in rumen contents stratification was most distinct papillae development and hence the one of many differences between grazing and highest SEF (Hofmann, 1973; Langer, 1973), and browsing ruminants. When comparing different both the dorsal and the ventral rumen wall. The ruminant species according to the macroscopic difference between either the dorsal or the ventral appearance of their mucosa of ruminal regions rumen wall SEF and the SEF of the atrium rumi- such as the dorsal rumen, the atrium and the ven- nis thus becomes, in theory, a measure of the tral rumen (Fig. 2), it is evident that in species degree of rumen contents stratification, with a increasingly specialized in grass forage, the differ- small difference indicating homogenous, and a ences among the rumen regions become more large difference comparatively stratified rumen pronounced. The stratification of rumen contents contents. according to updrift and sedimentation into a ‘fiber For this contribution, published SEF data for mat’ with the larger, lighter particles above a fluid these three rumen regions were collated from the phase containing the smaller, denser particles, is literature, and supplemented with hitherto unpub- regarded as the main mechanism responsible for lished data of the second author (RRH). The fol- the sorting of particles in the reticulorumen in lowing predictions were made domestic ruminants (Sutherland, 1988; Beaumont and Deswysen, 1991; Lechner-Doll et al., 1991b; 1. There is no correlation between body mass Dardillat and Baumont, 1992; Kaske et al., 1992). (BM) and the SEF of the atrium ruminis, the Additionally, the continuous mixing of the rumen dorsal or the ventral rumen wall, because the contents, which forces small particles through the SEF is a measure of diet quality, which we did fiber mat, where they may also be retained (the not assume to be correlated to BM in such a ‘filter bed effect’) (Poppi et al., 2001; Faichney, broad-scale data collection. 2006) is also considered an important mechanism 2. There is a negative correlation of both the abso- responsible for the selective particle retention in lute SEF of the dorsal and the ventral rumen this organ. A well-developed stratification (with an wall, as well as of the relative SEF of these according filter-bed effect) was suggested to cause regions (in % of the Atrium ruminis SEF), with the differential outflow of fluids and particles in the percentage of grass (%grass) in the natural grazing wild ruminant species (Clauss and Lech- diet of the respective species, indicating an ner-Doll, 2001; Behrend et al., 2004; Hummel increase in rumen contents stratification with et al., 2005; Clauss et al., 2006b; Hummel et al., increasing adaptation to a grass diet.

Journal of Morphology 932 M. CLAUSS ET AL.

Fig. 2. Samples of mucosa from the dorsal rumen (top set), the atrium ruminis (middle set), and the ventral rumen (bottom set) of nine ruminant species (Giraffidae: Giraffe Giraffa camelopardalis; Cervidae: White-tailed deer Odocoileus virginianus, Fallow deer Dama dama,Pe`re David’s deer Elaphurus davidianus; Bovidae: Bushbuck Tragelaphus scriptus, Thomson gazelle Gazella thomsoni, Goat Capra hircus, Blackbuck Antilope cervicapra, African buffalo Syncerus caffer). Note that although the atrium ruminis is always heavily papillated, papillation of the dorsal and ventral wall appears to decrease from the browsing species (left) to the intermediate feeders (center) and the grazers (right). Magnification adapted for qualitative comparison: magnification was kept constant within but not between species; apparent differences in papilla size between species do not necessarily reflect real size differences; historical photographs, no scale bars available (from Hofmann, 1973; Hofmann et al., 2005; and previously unpubl. photographs).

3. There is a positive correlation between the rela- important changes in the ruminal mucosa papillation (Hofmann tive SEF (in % of the Atrium SEF) of the dorsal and Matern, 1988; Marholdt, 1991). The only exception were animals kept in a semi-natural setting on large pastures from and the ventral rumen wall, indicating that the Whipsnade Wild Animal Park, such as Chinese water deer (Hof- mechanism responsible for rumen contents mann et al., 1988a), Pe`re David’s deer, axis deer, hog deer, stratification is effective throughout the whole blackbuck, and nilgai. The SEF measurements were performed rumen. on formalin-fixed samples by counting the number of papillae 4. There is a negative correlation between the rel- for a given basal area, and measuring their height and width, as described in the literature (Hofmann, 1973; Langer, 1973). ative SEF (in % of the Atrium SEF) of the dor- When data on BM were available, it was directly taken from sal rumen wall, as an anatomical measure of the sources; if only SEF but no BM data was available, BM rumen contents stratification, and the selective data was taken from Clauss et al. (2002). All data represent particle retention (as compared to the fluid means calculated on the basis of individual measurements, or, in the case of many publications, on the basis of average values retention) in the reticulorumen (Clauss et al., per season. The number of individuals investigated per species 2006b) as a physiological measure; in other is given in Table 1. words, the more the rumen mucosa reflects con- As in more recent evaluations of the influence of adaptation tents stratification, the larger the difference to the natural diet in ruminants (Clauss et al., 2008a), the per- between fluid and particle passage from the centage of grass in the natural diet (%grass) was used to characterize species on a continuous scale. The bulk of the respec- rumen in the same species. tive data was taken from Van Wieren (1996) and from the data collection that formed the basis of Owen-Smith [(1997), data kindly provided by the author], which were supplemented by METHODS several other publications (Table 1). Whenever seasonal data was available, the %grass used to characterize a species repre- The SEF is calculated as the sum of surface of the papillae sents the mean of the values from different seasons. It should and the basal area divided by the basal area. Data on the SEF be noted that this literature data was collated using a variety of the dorsal rumen wall, the ventral rumen wall, and the of sources and methods, and does not represent the actual diet atrium ruminis were collated from both the literature and hith- ingested by the individuals measured in this study. erto unpublished observations. When data was available from Data on the ratio between the mean retention time of par- both the literature and the second author, data from the second ticles: fluids in the reticulorumen were taken from Clauss et al. author was preferred for a maximum of data consistency. For (2006b). Data for both papillation and this ratio were available this data collection, only measurements from free-ranging ani- for cattle, sheep, goat, giraffe, moose, roe deer, wapiti, ibex, and mals were used, because the diets fed in captivity may lead to mouflon.

Journal of Morphology TABLE 1. Mean body mass (BM) and surface enlargement factors of ruminant species of different feeding type collated from different sources, and an estimation of the percentage of grass (% grass) in the natural diet of the species according to literature sources

SEF in % SEF atrium BM % Species (kg) Dorsal Ventral Atrium Dorsal Ventral Source Grass Source

Impala Aepyceros melampus 55 3.9 5.0 15.3 25 32 A (25) 60.0 1 Hartebeest Alcelaphus buselaphus 174 1.0 1.8 19.0 5 9 A (9) 96.7 2 Moose Alces alces 258 9.6 9.5 15.3 63 62 J (25) 2.0 1 Springbok Antidorcas marsupialis 41 2.6 3.0 14.8 17 20 K (7) 30.0 4 Pronghorn Antilocapra americana 40 4.5 5.2 9.0 50 58 B (2) 15.0 1 Blackbuck Antilope cervicapra 33 1.7 2.3 6.3 27 37 B (1) 75.0 1 Axis deer Axis axis 85 1.5 1.9 7.0 22 27 B (2) 70.0 1 Hog deer Axis porcinus 45 1.9 3.1 6.0 32 52 B (1) 50.0 5 Bison Bison bison 335 1.0 2.1 15.9 6 13 B (1) 84.0 1 Cattle Bos taurus 600 1.0 1.5 14.2 7 11 L (1) 79.0 2 Nilgai Boselaphus tragocamelus 220 2.4 2.6 8.8 28 30 B (3) 59.5 2 Domestic goat Capra hircus 31 2.5 2.6 6.7 37 38 H (53) 28.0 1 Alpine ibex Capra ibex 60 3.1 3.7 14.7 21 25 B (1) 60.0 1 Roe deer Capreolus capreolus 25 6.2 6.5 9.0 69 72 C, D, E, 9.0 1 F (58) Serau Capricornis crispus 37 3.6 3.6 12.5 29 29 O (10) 70.0 1 Red duiker Cephalophus harveyi 16 4.5 7.5 11.0 41 68 A (2) 1.0 3 Red deer Cervus elaphus 170 3.7 3.7 19.0 19 19 C (12) 47.0 1 Wapiti Cervus elaphus canadensis 300 2.9 3.4 8.8 33 39 B (1) 64.0 1 Sika deer Cervus nippon 70 1.9 3.3 12.6 15 26 B (14) 50.0 1 Sambar Cervus unicolor 200 4.0 4.7 14.8 27 32 M (12) 45.0 1 Wildebeest Connochaetes taurinus 182 1.0 7.5 36.5 3 21 A (5) 90.0 1 Fallow deer Dama dama 60 2.4 4.3 16.2 15 27 G (11) 46.0 1 Tsessebe Damaliscus lunatus 119 1.0 1.0 16.0 6 6 A (5) 99.3 2 Pere Davids Deer Elaphurus davidianus 120 1.2 2.3 13.3 9 17 B (3) 75.0 7 Grant’s gazelle Gazella granti 55 3.7 5.5 20.0 19 28 A (13) 50.0 1 Thomson’s gazelle Gazella thomsoni 21 2.5 2.5 16.5 15 15 A (15) 85.5 2 Giraffe Giraffa camelopardalis 750 24.0 18.0 30.0 80 60 A (4) 0.2 2 Himalayan tahr Hemitragus jemlahicus 55 2.5 3.0 9.9 25 30 P (39) 75.0 8 Chinese water deer Hydropotes inermis 11 3.3 3.7 6.7 50 55 I (58) 50.0 7 Waterbuck Kobus ellipsiprymnus 201 1.0 1.0 32.0 3 3 A (5) 80.0 2 Uganda kob Kobus kob 79 1.0 1.0 19.0 5 5 A (5) 95.0 9 Gerenuk Litocranius walleri 43 7.5 12.0 19.0 39 63 A (8) 0.0 2 Gu¨ nther’s dikdik Madoqua guentheri 4 7.5 16.5 19.0 39 87 A (6) 5.0 10 Kirk’s dikdik Madoqua kirki 5 6.5 12.5 17.5 37 71 A (6) 17.0 3 Muntjac Muntiacus reveesi 15 4.5 4.8 6.9 65 69 N (33) 10.0 1 Suni Neotragus moschatus 6 7.3 9.0 11.5 63 78 A (4) 0.0 6 Mule deer Odocoileus hemionus 80 5.1 6.9 10.7 48 65 B (4) 11.0 1 White-tailed deer Odocoileus virginianus 70 4.0 4.6 7.0 57 65 B (3) 9.0 1 Klipspringer Oreotragus oreotragus 11 10.5 10.0 17.5 60 57 A (3) 5.0 1 Beisa oryx Oryx beisa 145 1.7 2.5 11.0 16 23 B (2) 83.0 12 Gemsbok Oryx gazella 182 1.0 1.5 12.5 8 12 A (3) 82.0 2 Oribi Ourebia ourebi 16 1.0 — 15.0 7 — A (5) 48.5 2 Sheep Ovis ammon domesticus 31 2.1 2.2 6.5 33 34 H (14) 50.0 1 Mouﬂon Ovis ammon musimon 40 2.6 3.5 8.6 30 41 G (10) 69.0 11 Dall’s sheep Ovis dalli dalli 56 2.2 6.1 19.4 11 32 B (2) 56.0 1 Mongolian gazelle Procapra gutturosa 29 4.2 4.5 15.5 27 29 R (38) 28.0 13 Reindeer Rangifer tarandus 62 9.5 11.6 9.5 99 122 Q (36) 36.0 1 Steenbok Raphicerus campestris 11 6.0 6.0 18.0 33 33 A (18) 10.0 1 Mountain reedbuck Redunca fulvolufula 24 1.0 1.0 16.0 6 6 A (6) 99.0 2 Bohor Reedbuck Redunca redunca 45 1.0 1.0 20.0 5 5 A (3) 80.0 1 Barasingha Rucervus duvauceli 200 1.7 2.4 9.0 19 26 B (2) 80.0 1 Chamois Rupicapra rupicapra 50 3.5 4.3 15.5 22 28 B (16) 74.0 1 Grey duiker Sylvicapra grimmia 14 11.0 11.0 23.5 47 47 A (6) 5.0 2 African buffalo Syncerus caffer 599 1.0 2.0 35.0 3 6 A (4) 90.0 1 Eland Taurotragus oryx 465 3.8 8.0 35.0 11 23 A (4) 50.0 3 Bongo Tragelaphus euryceros 250 4.1 — 11.1 37 — B (3) 20.0 14 Lesser Kudu Tragelaphus imberbis 91 8.0 9.5 31.0 26 31 A (4) 10.0 2 Bushbuck Tragelaphus scriptus 50 7.0 9.0 24.5 29 37 A (4) 10.0 1 Greater Kudu Tragelaphus strepsiceros 214 9.0 13.5 40.0 23 34 A (3) 5.0 1

Sources for SEF data (number of individuals investigated in parentheses): A, (Hofmann, 1973); B, (Hofmann, unpubl.); C, (Hof- mann et al., 1976); D, (Ko¨nig et al., 1976); E, (Hofmann et al., 1988b); F, (Wen-Jun and Hofmann, 1991); G, (Geiger et al., 1977); H, (Hofmann et al., 1987); I, (Hofmann et al., 1988a); J, (Hofmann and Nygren, 1992); K, (Hofmann et al., 1995); L, (Schnorr and Vollmerhaus, 1967); M, (Stafford, 1995); N, (Pfeiffer, 1993); O, (Jiang, 1998); P, (Forsyth and Fraser, 1999); Q, (Mathiesen et al., 2000); R, (Jiang et al., 2003). Sources for %grass data: 1, (Van Wieren, 1996); 2, (Owen-Smith, 1997); 3, (Gagnon and Chew, 2000); 4, (Bigalke, 1972); 5, (Dhungel and O’Gara, 1991); 6, (Heinichen, 1972); 7, (Geist, 1999); 8, (Schaller, 1973); 9, (Field, 1972); 10, (Hofmann and Stewart, 1972); 11, (Stubbe, 1971); 12, (Skinner and Chimimba, 2005); 13, (Campos-Arceiz et al., 2004); 14 (Kingdon, 1982). 934 M. CLAUSS ET AL. Relationships among species were inferred from a phyloge- the natural diet are usually not found (Van Wie- netic tree based on the complete mitochondrial cytochrome b ren, 1996; Clauss et al., 2003; Sponheimer et al., gene. Respective DNA sequences were available from GenBank (http://www.ncbi.nlm.nih.gov) for all ruminant species investi- 2003) or very weak (Gagnon and Chew, 2000), gated (except for Gazella thomsoni, which was therefore omitted because browsers are found across the body size from the analyses). Sequences were aligned using CLUSTALX range (Sponheimer et al., 2003). In our case, the (Thompson et al., 1997), visually controlled and trimmed to significance was retained even if phylogeny was identical lengths (1,143 bp). To select the best-fitting nucleotide taken into account (Table 2). substitution model for the data, a combination of the software packages PAUP* (v.4.b10; Swofford, 2002) and MODELTEST There were significant correlations between BM (v.3.7; Posada and Crandall, 1998) was used. Analysis was and all parameters investigated (Table 2). Signifi- based on a hierarchical likelihood ratio test approach imple- cant correlations were also evident for %grass with mented in MODELTEST. The model selected was the general all parameters except SEF . Similarly, Van time-reversible (GTR) model (Lanave et al., 1984; Tavare´, 1986) atrium with an allowance both for invariant sites (I) and a gamma (G) Wieren (1996) already had not found a significant distribution shape parameter (a) for among-site rate variation correlation between %grass and the SEFatrium. (GTR1I1G) (Rodriguez et al., 1990). The nucleotide substitu- Phylogenetic control generally did not change the tion rate matrix for the GTR1I1G model was similarly calcu- results (Table 2). Correlations of %grass with SEF lated using MODELTEST. Parameter values for the model parameters generally had markedly higher corre- selected were: -lnL 5 15642.5273, I 5 0.4613, and a50.8093 (eight gamma rate categories). The phylogenetic reconstruction lation coefficients than the rather weak correla- based on these parameters was then performed using the maxi- tions with BM (Table 2; compare Fig. 4a–d). The mum likelihood (ML) method implemented in TREEPUZZLE reindeer (Rangifer tarandus) was an evident out- (v.5.2; Schmidt et al., 2002). Support for nodes was assessed by lier to the general pattern, showing an unusually a reliability percentage after 100,000 quartet puzzling steps; only nodes with more than 50% support were retained. The high degree of papillae uniformity in its rumen for resulting tree is displayed in Figure 3. The basal polytomy for its feeding type as intermediate feeder. familial relationships (Bovidae, Cervidae, Giraffidae and Antilo- Partial correlations (controlling for BM) between capridae) was resolved assuming it to be a soft polytomy (Purvis %grass and SEF parameters (except SEFatrium) and Garland, 1993). To meet the input requirements for the were highly significant (Table 2), indicating that phylogenetic analysis implemented in the COMPARE 4.6 program (Martins, 2004), we resolved the remaining polytomies to the correlations between %grass and the SEF pa- full tree dichotomy by introducing extreme short branch lengths rameters were not caused by a mediating effect of (l 5 0.00001) at multifurcating nodes. Taxa grouping in the BM. The relative SEFdorsal and the SEFventral (in % bifurcating process followed the phylogenies proposed by Pitra of the SEF of the atrium) were positively corre- et al. (2004) for Cervidae and by Fernandez and Vrba (2005) for all other taxa. lated to each other (Fig. 4e), with a generally The subjects of the comparative analyses were individual spe- higher value (by 6% of the SEFatrium) in the rela- cies, each characterized by its respective SEF as described tive SEFventral (raw data: R 5 0.909, P < 0.001; above. To achieve normal data distribution, data for BM, phylogenetically controlled: R 5 0.857, P < 0.001; SEFdorsal, SEFatrium and SEFventral were ln-transformed. Statis- n 5 56). The three species that appeared to devi- tical analyses were performed with and without accounting for phylogeny, to test for the validity of a general, functional ate the most from this pattern were the giraffe, hypothesis, and to then discriminate between convergent adap- with a higher relative SEFdorsal, and the two dik- tation and adaptation by descent. Data were analyzed by corre- dik species (Madoqua guentheri and M. kirki), in lation and partial correlation analysis (controlling for the which the relative SEF was disproportionately influence of body mass). To additionally include phylogenetic dorsal information, we used the Phylogenetic Generalized Least- lower than the relative SEFventral. Squares approach (Martins and Hansen, 1997; Rohlf, 2001) in Among the few species (n 5 9) for which com- which a well-developed standard statistical method was parative data on both the relative SEFdorsal and extended to enable the inclusion of interdependencies among the passage characteristics (SF 5 selectivity factor, species due to the evolutionary process. To test the robustness mean retention time of particles/mean retention of the results, the comparative analysis was performed for both a set of phylogenetic trees involving branch lengths (tree 1) and time of fluids in the reticulorumen) were available, another set with equal branch lengths (tree 2). As there were there was a significant, negative correlation no relevant differences in the results, only the tests using tree between these parameters both for the raw data 1 are given here. The COMPARE 4.6 program (Martins, 2004) (R 520.917, P < 0.001) and the phylogenetically served for the phylogenetically controlled calculations. The other statistical calculations were performed with the SPSS controlled test (R 520.922, P < 0.001) (Fig. 4f). 12.0 software (SPSS, Chicago, IL). The significance level was set to a50.05. DISCUSSION In general, the hypotheses concerning the corre- RESULTS lation of the patterns of the ruminal papillation In this dataset, there was a significant, positive with the %grass in the natural diet were con- correlation between body mass and %grass in the firmed. If the ruminal papillation pattern is natural diet (Table 2). On average, grazing rumi- accepted as a proxy for the stratification of rumen nants are larger than browsing ruminants (Bell, contents, the results therefore indicate that the 1971; Case, 1979; Bodmer, 1990; Van Wieren, contents of more grazing wild ruminants tend to 1996; Pe´rez-Barberıà and Gordon, 2001), but corre- be more stratified than those of more browsing lations between BM and the proportion of grass in ruminants.

Journal of Morphology PAPILLATION PATTERNS IN RUMINANTS 935

Fig. 3. Fifty percent majority rule maximum likelihood tree (100,000 puzzling steps), depicting the phylogenetic relationships among complete mitochondrial cytochrome b sequences from 58-ruminant taxa as used in the phylogenetically controlled statistics in this study (accession codes from GenBank available at: http://www. ncbi.nlm.nih.gov). The families of the Girafﬁdae, Antilocapridae, Cervidae, and Bovidae (from Hippotraginae to Bovinae) are represented.

Large interspecific comparisons such as this one curacy in the results of our analysis. Finally, dif- are, by necessity, limited by certain factors that ferences in the handling of rumen samples (mea- need to be stated: The data on the natural diet surements on fresh samples or after storage in for- composition was not generated from the same ani- malin of varying concentrations) will influence the mals, or not even the same animal populations, results (Lentle et al., 1997). Some of these varia- that were used for the papillation measurements; tions may have been attenuated by the use of the same limitation applies to the use of body ratios (SEF dorsal in % of SEF atrium). However, weight data derived from other individuals. our study was not concerned with the detailed Whether the habitat from which the individuals positioning of individual species in the comparison, used for this study were taken was representative but with a broad general pattern that is clearly for the ‘‘typical’’ habitat of the species could not be evident across the wide range of species from assessed. The fact that papillation characteristics different taxonomic groups. as well as the natural diet are subject to seasonal The results confirm that the rumen contents of variation could have introduced a source of inac- free-ranging wild ruminants induce a more pro-

Journal of Morphology TABLE 2. Results of statistical analyses for correlations and partial correlations (controlling for body mass) between the surface enlargement factors (SEF) of different areas of the rumen mucosa, the proportion of grass (%grass) in the natural diet, and body mass, using either raw data from Table 1 or phylogenetically controlled statistics

%Grass SEFdorsal SEFventral SEFatrium SEFdorsal (% SEFatrium) SEFventral (% SEFatrium) n 58 58 56 58 58 56 Raw data Body mass R 0.401 20.281 20.281 0.289 20.323 20.487 P 0.002 0.032 0.036 0.028 0.013 <0.001 % Grass R – 20.872 20.818 20.006 20.726 20.737 P <0.001 <0.001 0.966 <0.001 0.001 % Grass (Partial correlation) R – 20.864 20.802 20.138 20.688 20.677 P <0.001 <0.001 0.305 <0.001 <0.001 Phylogeny-controlled Body mass R 0.469 20.277 20.210 20.294 20.328 20.471 P <0.001 0.030 0.115 0.023 0.012 <0.001 % Grass R – 20.871 20.649 0.010 20.689 20.626 P <0.001 <0.001 0.929 <0.001 0.001 % Grass (Partial correlation) R – 20.861 20.643 20.158 20.639 20.576 P <0.001 <0.001 0.245 <0.001 <0.001

R 5 correlation coefﬁcient.

Fig. 4. Correlations between the surface enlagement factor (SEF) of the dorsal and ventral rumen mucosa and a) body mass (BM, kg) or b) the proportion of grass in the natural diet (%grass), between the relative SEF of the dorsal and ventral rumen mucosa (in % of the SEF of the atrium ruminis) and c)BMord) %grass, e) between the SEFdorsal (in % of the SEFatrium) and the SEFventral (in % of the SEFatrium), f) between the SEFdorsal (in % of the SEFatrium) and the ‘‘selectivity factor,’’ the quotient of the mean retention time (MRT) of particles in the reticulorumen (RR) over the MRT of ﬂuid in the RR (Clauss et al., 2006b). PAPILLATION PATTERNS IN RUMINANTS 937

TABLE 3. Surface enlargement factor in the rumen of several wild intermediate feeding ruminants in relation to the predominant composition of their actual diet

SEF in % SEF SEF atrium

Species Diet Dorsal Ventral Atrium Dorsal Ventral

Impala Aepyceros melampus Browse 6.0 6.5 18.0 33 36 Grass 1.8 3.4 12.5 14 27 Thomson’s gazelle Gazella thomsoni Browse 3.5 3.5 16.5 21 21 Grass 1.5 1.5 16.5 9 9 Eland Taurotragus oryx Browse 6.0 12.0 35.0 17 34 Grass 1.7 4.0 35.0 5 11 Grant’s gazelle Gazella granti Browse 5.5 7.0 20.0 28 35 Grass 1.9 4.0 20.0 10 20

Data from Hofmann (1973). nounced difference in the intraruminal papillation nants (Schwarm et al., 2008); correspondingly, no pattern in those species that are characterized by a elaborate stratification of contents would be higher proportion of grass in their natural diet. In expected in this group, and reports on the papilla- addition to the quantitative findings collated in this tion of the hippopotamus do not indicate a stratifica- study, there are qualitative observations on addition as seen in grazing ruminants (Langer, 1988). tional species that are in accord with the pattern We propose that it is a higher fluid content in the observed here. For example, Agungpriyono et al. grazing ruminant forestomach as compared to the (1992) observed that the rumen of the lesser mouse- hippopotamids (Thurston et al., 1968) that facili- deer (Tragulus javanicus) was completely evenly tates the stratification of the contents by allowing papillated, and Clauss et al. (2006c) observed the particle movements according to density. same in captive okapi (Okapia johnstoni). Both spe- One obvious explanation for differences in papil- cies are known to avoid grass consumption in the lation between ruminant species is the sheer effect wild (Nordin, 1978; Hart and Hart, 1988). In con- of body size and hence rumen size—the larger a trast, the rumen of three more recently domesti- rumen, the more distinct the characteristics of its cated bovini, the gaur (Bos frontalis), the yak (Bos contents’ different layers could be. The significant grunniens) and the zebu (Bos indicus) were reported correlations between BM and SEF parameters, to resemble that of domestic cattle (Sarma et al., which had not been expected, would support this 1996), presumably indicating an absence of papillae hypothesis. However, these findings can probably in the dorsal area. Similar observations were made be explained by the fact that BM was correlated to in other bovini such as water buffalo (Bubalus buba- %grass in this dataset. Low proportions of grass in lis) (Hemmoda and Berg, 1980), lowland anoa the natural diet, and SEF parameters indicating (Bubalus depressicornis) (Clauss et al., 2008b), ban- unstratified rumen contents, do occur across the teng (Bos javanicus) and European bison (Bison whole body size range (up to the largest ruminant, bonasus) (Clauss, pers. obs.), the puku (Kobus var- the giraffe), indicating that the consistency of the doni) (Stafford and Stafford, 1990) and the lechwe rumen contents itself, irrespective of its volume, (Kobus leche) (Stafford and Stafford, 1991). All these must be considered responsible for the demon- animals are assumed to ingest high proportions of strated effects. Similarly, the physiological measure grass in their natural diet. Similar to our study, of rumen contents stratification, which is the differ- Enzinger and Hartfiel (1998) demonstrated quanti- ence between particle and fluid retention in the tatively that the rumen mucosa of captive roe deer rumen, also does not follow a strict pattern with was evenly papillated, in contrast to that of fallow body size, either (Clauss and Lechner-Doll, 2001). deer, sheep and goats. The most prominent outlier But is this consistency of the rumen contents an in our data collection, the reindeer, has been effect of the ingested forage, or of physiological reported to have homogenous rumen contents adaptations of the different animal species? Intra- (Westerling, 1970; Hobson et al., 1976) and a very specific comparisons of SEF data, from individuals even rumen papillation (Soveri and Nieminen, of different intermediate feeder species ingesting a 1995; Josefsen et al., 1996; Mathiesen et al., 2000; browse- or a grass-dominated diet (Table 3) suggest Soveri and Nieminen, 2007). Whether this is caused a diet component in the formation of a stratified by the peculiar diet of reindeer, or by an exceptional rumen. In addition, Hofmann (1973) observed that rumen physiology, remains to be elucidated. In the a group of zebus—a species with an usually unpa- hippopotamids, which also have a voluminous for- pillated dorsal area—had a completely papillated estomach lined with a papillated mucosa, the ab- rumen when kept on lucerne pasture; similarly, he sence of differential particle retention contrasts observed that an oribi kept on a diet of concentrate with the selective large particle retention in rumi- and occasional browse had a completely papillated

Journal of Morphology 938 M. CLAUSS ET AL. rumen, in contrast to free-ranging specimens. rumen (Clauss et al., 2006b; Fig. 3f), sheep rumen Therefore, it can be suspected that certain charac- contents are naturally less distinctively stratified teristics of dicotyledonous forage, which need to be than those of cattle. Additionally, in direct compari- further elucidated, induce less stratification in the sons of animals feeding on the same pastures, rumen than monocotyledonous forage. Actually, in sheep ingest a higher proportion of browse than domestic ruminants, certain secondary plant com- cattle (Sanon et al., 2007). Given these observa- pounds in some dicotyledonous forages (lucerne, clo- tions, we hypothesize that sheep—and the zebu cat- ver) have been identified as the cause of ‘‘frothy tle mentioned above—are better adapted to less bloat’’ or ‘‘legume bloat,’’ a disease of domestic cattle stratified rumen contents, and that cattle, as which is characterized by an absence of rumen con- extreme grazers with a very distinct rumen content tents stratification (Clarke and Reid, 1970). On the stratification and a distinct difference in particle other hand, passage trials in a moose did not show and fluid passage from the rumen, have lost the a difference in the pattern of particle and fluid ability to cope with frothy rumen contents, most retention between diets including browse, legume, likely due to less flexible rumen motility patterns or grass hay (Renecker and Hudson, 1990), and (Colvin and Backus, 1988). thus suggest independence of rumen contents strat- The question arising from these observations is ification from dietary factors at least in this species. about the adaptive value of the described differen- Maybe this is an indication that at least moose have ces. Evidently, given the proportion of areas that evolved physiological adaptations that, directly or are un-papillated or only lined with small papillae, indirectly, prevent the formation of rumen contents the total absorptive surface of the whole rumen is stratification. less in grazers than in browsers of similar rumen The fact that both, the dorsal as well as the ven- size. This trend is most likely not countered by an tral rumen region show a difference in SEF as increase in SEF in the papillated rumen areas of compared with the atrium (Fig. 4e) supports our grazers, as there was no significant increase in hypothesis that a mechanism leading to this strati- SEFatrium with increasing %grass in the natural fied papillation must be operative throughout the diet (Table 2). This difference in absorptive area in whole rumen. The most probable mechanism the rumen puts measurements on the concentra- appears to be rumen content and rumen fluid vis- tion of volatile fatty acids (VFA) (Hoppe, 1977; cosity (Clauss et al., 2006b). Actually, rumen fluid Clemens and Maloiy, 1983; Hoppe, 1984; Prins of roe deer or moose was found to be of a higher et al., 1984) and on VFA production rates (Gordon viscosity than that of mouflon, bison (Clauss et al., and Illius, 1994) in the rumen contents of wild 2009, in press) or that of cattle (Hummel et al., in ruminants into a new perspective. Most of these press). However, this hypothesis has to be tested studies had not found a statistical difference in in a larger number of species. Reasons for their measurements between grazers and brows- the deviation of giraffe (with an unusually high ers. The concentration of VFA depends on produc- SEFdorsal) and dikdik (with unusually low SEFdorsal tion and absorption rate across the epithelium of as compared to the SEFventral) cannot be given here. the fermentation chamber (Lechner-Doll et al., One of the consequences of a higher fluid viscos- 1991a). Because a rise in VFA concentration trig- ity would be the entrapment of gas bubbles in the gers an increase in the epithelial growth in the rumen contents, rather than the formation of a dis- rumen (Nocek and Kesler, 1980; Goodlad, 1981; tinct dorsal gas dome. This would explain the Dirksen et al., 1984), the absorption capacity for ‘‘frothy’’ appearance of browsers’ rumen contents VFA will increase and hence actual VFA concentra- (see Introduction), and the absence of a distinct dor- tions measured after this adaptation period will be sal gas dome observed in a moose (Tschuor and leveled. Thus, large differences in VFA production Clauss, 2008). McCauley and Dziuk (1965) found rates may be masked and reflected by only very that in resting goats only little gas accumulated in small differences in VFA concentrations (Lechner- the dorsal rumen, and suggested that the gas was Doll et al., 1991a). Higher VFA production rates, trapped as ‘‘numerous bubbles’’ in the rumen con- which may be demonstrated in natural forages of tents instead. The frothy appearance of the rumen browsing ruminants (Hummel et al., 2006), there- contents of browsers suggests that the pathologic fore need not necessarily translate into higher process of ‘‘frothy bloat,’’ seen in domestic rumi- VFA concentrations in the rumen of these animals nants fed fresh legume forages (Clarke and Reid, because of their larger absorptive surface. 1970), may offer some comparative potential for the Ruminal VFA concentrations also depend on understanding of the differences in rumen physiol- rumen fluid volume and fluid outflow rate ogy between browsing and grazing ruminants (Lechner-Doll et al., 1991a). Some large grazing (Clauss et al., 2006b). Interestingly, domestic sheep ruminants, such as cattle (Clauss et al., 2006b; are less susceptible to legume-caused frothy bloat Schwarm et al., 2008), are characterized by a par- than cattle (Olson, 1940; Colvin and Backus, 1988). ticularly fast fluid passage through the rumen, Given the papillation pattern (Table 1) and the dif- which might reduce VFA concentrations. These ferential passage of fluid and particles from the animals might compensate for their comparatively

Journal of Morphology PAPILLATION PATTERNS IN RUMINANTS 939 lower absorptive surface in the rumen by an aptation in itself (in the sense of a convergent evo- increased absorption of VFA in their comparatively lution) would have to be studied in in vitro diges- larger omasum (Clauss et al., 2006a). This fast tion models in which important characteristics of throughput might additionally have the advantage the modeled ingesta—such as its viscosity—are of maximal use of microbial protein produced in manipulated. Note that our study does not claim the rumen (as suggested by Hummel et al., 2008a). that the stratification of rumen contents itself is an The distinct stratification induced by this high adaptation; it merely describes a notable difference fluid throughput could also render the rumination in the degree of stratification between the ruminant process more efficient, so that only such material feeding types that happens, that is reflected in the from the fiber mat is regurgitated that needs fur- rumen papillation pattern, and to which the ani- ther comminution. In two white-tailed deer (Odo- mals adapt by various anatomical and physiological coileus virginianus) (a browser, with a presumed means. To which degree these adaptations are ge- lack of a distinct rumen contents stratification), netically fixed or subject to modification due to the Dziuk et al. (1963) observed regurgitation and re- ingested forage remains to be demonstrated. Cur- swallowing of ingesta without rumination and sus- rent evidence suggests that both extremes—the pected that further mastication had been unneces- extreme grazers as well as the extreme browsers— sary for the respective regurgitated bolus. The are derived from more rudimentary intermediate authors noted that such observations (of ‘vain’ feeding types (Codron et al., 2008; DeMiguel et al., regurgitations) had not been made in cattle or 2008). The extremes in rumen contents stratifica- sheep. However, detailed studies on differences in tion and papillation therefore describe opposing regurgitation frequency and rumination in differ- rumen physiologies and emphasize the general flex- ent ruminant species are mostly lacking. ibility of the ruminant digestive system. At least two different extremes of rumen physiology are thus proposed by the observed differen- ACKNOWLEDGMENTS ces in rumen mucosa papillation and hence rumen contents stratification: The authors thank the numerous hunters and zoological institutions that helped collecting mate- 1. Browsers, ingesting a faster-fermenting forage, rial. Norman Owen-Smith kindly provided the have more viscous rumen contents and fluid, data collection from his 1997 publication. The either due to forage or saliva characteristics, authors also thank A. S. Hug for assistance in the and a comparatively slow fluid throughput; the preparation of photographic material and B. whole rumen acts as an absorptive organ, and Schneider for support in literature acquisition. shows no particular adaptations to a contents They thank anonymous reviewers for their useful stratification with a fiber mat. 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