Rumen content stratification in the giraffe (Giraffa camelopardalis)

Comparative Biochemistry and Physiology, Part A 203 (2017) 69–76

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Rumen content stratification in the giraffe (Giraffa camelopardalis) Cathrine Sauer a,b,1, Marcus Clauss c,⁎, Mads F. Bertelsen b, Martin R. Weisbjerg a, Peter Lund a a b c

Department of Animal Science, AU Foulum, Aarhus University, Blichers Allé 20, PO Box 50, DK-8830 Tjele, Denmark Center for Zoo and Wild Animal Health, Copenhagen Zoo, Roskildevej 32, DK-2000 Frederiksberg, Denmark Clinic for Zoo Animals, Exotic Pets and Wildlife, Vetsuisse Faculty, University of Zurich, Winterthurerstr. 260, CH-8057 Zurich, Switzerland

a r t i c l e

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Article history: Received 5 April 2016 Received in revised form 30 August 2016 Accepted 30 August 2016 Available online 01 September 2016 Keywords: Ruminant Browser Digestion Fermentation Feeding type Anatomy Physiology

a b s t r a c t Ruminants differ in the degree of rumen content stratification, with ‘cattle-types’ (i.e., the grazing and intermediate feeding ruminants) having stratified content, whereas ‘moose-types’ (i.e., the browsing ruminants) have unstratified content. The feeding ecology, as well as the digestive morphophysiology of the giraffe (Giraffa camelopardalis), suggest that it is a ‘moose-type’ ruminant. Correspondingly, the giraffe should have an unstratified rumen content and an even rumen papillation pattern. Digesta samples were collected from along the digestive tract of 27 wild-caught giraffes kept in bomas for up to 2 months, and 10 giraffes kept in zoological gardens throughout their lives. Samples were analysed for concentration of dry matter, fibre fractions, volatile fatty acids and NH3, as well as mean particle size and pH. There was no difference between the dorsal and ventral rumen region in any of these parameters, indicating homogenous rumen content in the giraffes. In addition to the digesta samples, samples of dorsal rumen, ventral rumen and atrium ruminis mucosa were collected and the papillary surface enlargement factor was determined, as a proxy for content stratification. The even rumen papillation pattern observed also supported the concept of an unstratified rumen content in giraffes. Zoo giraffes had a slightly more uneven papillation pattern than boma giraffes. This finding could not be matched by differences in physical characteristics of the rumen content, probably due to an influence of fasting time ante mortem on these parameters. © 2016 Elsevier Inc. All rights reserved.

1. Introduction The stratification of rumen content is a hallmark of domestic ruminant digestive physiology (Hummel et al., 2009). In the original research on anatomical differences between browsing and grazing ruminants (Hofmann, 1973), the absence of a distinct rumen content stratification in browsing ruminants was a side finding (Hofmann, 1989; Renecker and Hudson, 1990). The dichotomy between stratified rumen content in grazers and unstratified content in browsers was subsequently emphasized as a major physiological difference (Clauss et al., 2003), and anatomical differences between the feeding types were interpreted based on this characteristic. The concept that adaptations served to reinforce the presence or absence of stratification (Clauss et al., 2008) was modified after feeding experiments showed that the one mechanism most intuitively linked to stratification - the particle sorting mechanism - works in a very similar way in ruminants with and without rumen content stratification (Lechner et al., 2010). A newer concept considers the presence or absence of rumen content stratification as a consequence of either a low or high fluid throughput through the ⁎ Corresponding author. E-mail address: [email protected] (M. Clauss). 1 Present address: Chester Zoo, Caughall Road, Upton-by-Chester, Chester CH2 1LH, United Kingdom.

http://dx.doi.org/10.1016/j.cbpa.2016.08.033 1095-6433/© 2016 Elsevier Inc. All rights reserved.

reticulorumen and classifies ruminant digestive tracts as either ‘moose-type’ or ‘cattle-type’ (Clauss et al., 2010). ‘Moose-type’ ruminants have unstratified rumen contents and a low fluid throughput, possibly because they are rate-limited in the production of viscous saliva with tannin-binding proteins (Hofmann et al., 2008), and are strict browsers. ‘Cattle-type’ ruminants have a fluid throughput through the forestomach that is faster than that of ingesta particles, stratified rumen contents, and typically include grass in their natural diet, i.e., are intermediate feeders or grazers (Codron and Clauss, 2010). The current hypothesis is that the evolutionary driver for a ‘cattletype’ physiology is the increased microbial yield and increased microbial efficiency facilitated by an increased harvest of microbes by the continuous ‘washing’ of forestomach contents (Dittmann et al., 2015; Hummel et al., 2015), making the stratification of rumen contents a mere side effect. Rumen content stratification can be determined by various methods. In live animals, ultrasound examination can distinguish between stratified content with a dorsal gas dome and unstratified content without such a dome (Tschuor and Clauss, 2008). In fistulated animals, sampling of digesta from different regions of the rumen can demonstrate differences in dry matter (DM) content, particle size, and other measurements of fermentation processes indicative of content stratification (Hummel et al., 2009; Lechner et al., 2010). Other quantitative methods have been applied as well, such as a device that measured the resistance

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met by a weight pulled through the different stratification layers to determine “ingesta consistency” in fistulated animals (Welch, 1982). Differences in rumen content characteristics indicating stratification are difficult to demonstrate in dead animals, as there is a risk of mixing the rumen content during carcass handling. The stratification is maintained if carcasses are frozen in the natural resting position (Clauss et al., 2016). Careful handling and dissection of the fresh carcass, i.e., maintaining the natural position as much as possible, does allow sampling of the forestomachs in a way that differences between sampling locations can be assessed in terms of DM content and particle size (Clauss et al., 2009a) or pH (Ritz et al., 2013). Even if careful sampling of forestomach content to identify the degree of stratification is not an option, content stratification is still reflected in the rumen papillation pattern (Clauss et al., 2009b), as papillae growth is stimulated by the presence of volatile fatty acids and thus represents an integrated measure of content stratification over the last months. In contrast, rumen content characteristics will by nature only reflect the last few meals. Characteristics of the digestive tract anatomy suggest that the giraffe (Giraffa camelopardalis) is a ‘moose-type’ ruminant (Sauer et al., 2016). This matches observations in free-ranging animals that are strict browsers (Leuthold and Leuthold, 1972; Pellew, 1984) that only very rarely consume grass (Seeber et al., 2012). Browsing animals, such as the giraffe, are typically considered more difficult to maintain in captivity as compared to grazing animals (Müller et al., 2011). In captivity, browsing ruminants generally excrete larger faecal particles than grazing ruminants (Clauss et al., 2002). In contrast, no such difference is evident when comparing free-ranging grazers and browsers (Hummel et al., 2008; Lechner et al., 2010). One possible interpretation of these findings is that whereas the teeth of both feeding types are adapted to their respective natural diets (Heywood, 2010; Kaiser et al., 2010), those of browsers are less suited to comminute the food they receive in captivity. This is potentially exacerbated by the excessive tooth wear experienced by giraffes in captivity (Clauss et al., 2007). In giraffes, inadequate chewing efficiency might explain the apparently high prevalence of digestive tract phytobezoars (conglomerates of plant particles) in captive animals (Hummel and Clauss, 2006). Another indicator of sub-optimal diets in captivity was the drastic reduction in both number and size of rumen papillae observed in two captive giraffes by Hofmann and Matern (1988). In this study, we investigated the rumen contents stratification in giraffes kept in zoos or in short-term boma confinement. Boma giraffes were expected to have relatively homogenous rumen content with little difference between the dorsal and ventral rumen in terms of DM concentration, particle size, pH and measurements of fermentation. This absence of stratification was assumed to be matched by a homogenous rumen papillation pattern. Given reports of differences between free-ranging and captive animals (Hofmann and Matern, 1988), we hypothesized that giraffes kept and raised in zoos would have more stratified rumen contents and a less even rumen papillation pattern, as well as larger digesta particle sizes, than giraffes recently caught from the wild. 2. Materials and methods Samples were collected from 27 giraffes (25 males and 2 females) caught from the wild and kept in bomas, ranging in body mass from 280 to 660 kg (mean ± SD: 468 ± 95 kg), and 10 zoo giraffes (6 males and 4 females) ranging in body mass from 182 to 1225 kg (mean: 703 ± 272 kg), though not all samples were collected from every individual due to practical and time limitations. Wild giraffes were caught in South Africa or Namibia and housed in bomas by Wildlife Assignments International Ltd., Hammanskraal, South Africa, for approximately 2 months prior to euthanasia and dissection. During this period, the giraffes were group housed and received a diet of fresh locally cut savannah browse, leafy lucerne hay

(Medicago sativa), and Boskos pellets (based on ground native trees and various protein, energy and mineral sources, Wes Enterprises Ltd., South Africa). Water was freely available. All giraffe consumed food during the stay in the boma, but it was not possible to quantify individual intake. With permission from the Gauteng Province of South Africa, the boma giraffes were euthanized following various physiological experiments conducted by the Danish Cardiovascular Giraffe Research project (e.g. Smerup et al., 2016). Due to the experimental procedures, many boma giraffes were fasted overnight prior to anaesthesia and subsequent euthanasia. The duration of the fasting time was estimated based on when the giraffes last had access to feed and the time of euthanasia. The estimated fasting time for boma giraffes ranged from 0 to 48 h with a mean duration of 18 ± 11 h. Captive giraffes from six Danish and one Swedish zoo were culled for management reasons or because of chronic osteoarthritis. These animals were all group fed on diets consisting of hay (in 6 cases lucerne hay only, in 3 cases grass hay only, and one animal had access to both hay types), various concentrate pellets and as much browse as possible. Limited amounts of other feeds including Boskos pellets, pelleted dried sugar beet pulp, linseeds, oats, maize and various fruits and vegetables were used by individual institutions. All giraffes, except for one, had been housed separately overnight prior to culling. In an attempt to estimate digesta retention post mortem, the animals had been offered roughly 1 kg labelled silage and 0.5 kg labelled pellets at 18, 12 and 6 h before euthanasia. However, only 2 of the giraffes ate the labelled feeds – the rest essentially fasted; therefore, no results on marker distribution in the gastrointestinal tract were produced. Most giraffes were fed their regular ration of concentrate pellets on the morning of euthanasia. Therefore, two fasting times were estimated for each zoo giraffe, one representing time since last access to roughage and one representing time since last access to concentrate pellets. The average durations for zoo giraffes were 14 ± 7 (range: 0.5–20 h) fasting time for roughage and 2 ± 1 h for pellets, respectively. 2.1. Protocol for sampling and analyses Dissections generally started within 30 min after euthanasia and the sampling procedure in most cases took b1.5 h for wild-caught giraffes and b2.5 h for captive giraffes. Giraffes were kept in an upright position during transport to the dissection facility and during weighing. After weighing, the animal was placed on its right side and the intestines were ligated and removed. The forestomach was gently rolled over its ventral edge onto the left side, as illustrated in Fig. 1. Great care was taken to avoid any ‘kneading’ or pressure on any forestomach region (in particular, the blindsacs and the reticulum), to minimize mixing of rumen contents. The gastrointestinal tract sections were separated as depicted in Fig. 2, after ligating each section. Before collecting digesta samples, each section of the gastrointestinal tract, i.e., the reticulorumen, omasum, abomasum, small intestine, cecum and large intestine, was weighed full. Digesta samples were collected for determining DM and fibre concentrations in the dorsal rumen, ventral rumen, reticulum, omasum, abomasum and rectum (Figs. 1 and 2), as described by Clauss et al. (2009a). DM was determined by drying samples at 60 °C for 48 h until constant weight; however, samples collected from 13 of the boma giraffes were dried at 100 °C for 24 h. To quantify the effect of the drying temperature on the results of the subsequent fibre analysis, samples (n = 5) from one boma giraffe were manually divided into two representative subsamples that were dried at 60 °C for 48 h or at 100 °C degrees for 24 h. Prior to fibre analysis, the dried samples were ground to pass a 1 mm screen (Retsch Ultra Centrifugal Mill ZM 200, Haan, Germany). The content of neutral detergent fibre (NDF) was determined using heat-stable α-amylase and sodium sulphite in an ANKOM200 Fibre Analyzer (ANKOM Technology, Macedon, NY, United States). After the NDF procedure, ANKOM sample bags containing the fibre content of each

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Fig. 1. Schematic transects of a cranially viewed rumen changing position from upright to lying on its left side. The content depicted is highly stratified with a gas dome (white), a fibre mat (straw-like), slurry beneath the mat (light grey) and a pool of dense particles at the bottom (dark grey). Dorsal (D) and ventral (V) samples of digesta were collected at the positions indicated on the right most figure. This figure is for the illustration of the sampling method and is not representative for the rumen contents observed in giraffes.

sample were placed in a Dacron bag (38 μm pore size) and incubated in rumen fistulated cows for 288 h to determine the fraction of indigestible NDF (iNDF) as NDF residue after incubation and repeated NDF boiling (Lund et al., 2007). Ash content in the iNDF fraction was determined as the residue after dry-ashing the samples at 525 °C for 6 h, and both NDF and iNDF concentrations were corrected for ash content. NDF and iNDF results from samples dried at 100 °C were corrected using linear regression equations derived from the identical subsamples dried at 60 °C or 100 °C, viz. NDF60 °C (in % of DM) = 0.61 × NDF100 °C (in % of DM) + 9.17, R2 = 0.86 and iNDF60 °C (in % of DM) = 0.76 × iNDF100 °C (in % of DM) − 1.84, R2 = 0.98. At each sampling location (dorsal and ventral rumen, reticulum, omasum, abomasum, rectum), a sample of approximately 30 g wet weight was collected for determination of particle size distribution, and stored frozen. These samples were analysed using a wet sieving technique (Retsch AS Digit 200, Haan, Germany). Samples were soaked in 1 L of water for a minimum of 12 h prior to analysis. Each sample was then poured over a stack of 9 sieves with a linear reduction in mesh size (16, 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.063 mm). The sample was mechanically sieved for 10 min at a vibration amplitude of 2 mm and a water throughput of 0.3 L/min. Particles not retained on the smallest screen were not quantified. After sieving, the fraction of particles retained on each sieve was carefully transferred to pre-weighed containers and

dried at 100 °C for 24 h. Mean particle size (in mm) of each sample was calculated using the weighted average equation for the discrete mean by Fritz et al. (2012), using an assumed maximum particle size of 32 mm. Rumen fluid samples for VFA and NH3 analysis were collected at each reticuloruminal location, i.e., dorsal rumen, ventral rumen and reticulum. The fluid was filtered from the solid content of the sample by the use of a filter bag with 0.5 mm pore size (Grade Blender Bags, VWR, Denmark). Immediately after sampling, the filter bag was stored at 5 °C for approximately 2 h before the fluid and the solid part were fully separated. The concentration of NH3 in the rumen fluid was analysed by making the sample alkaline with KOH, after which NH3 was determined by titration after distillation using the Kjeltec2400 system (Foss Analytical, Hillerød, Denmark). VFA concentrations were determined by gas chromatography, as described by Jensen et al. (1995) with some modifications (Canibe et al. 2007). Samples for pH measurements were collected from the dorsal rumen, ventral rumen, reticulum, omasum, abomasum, and rectum. The pH value was measured using a portable pH meter (model IQ150, IQ Scientific Instruments, Inc.). The time from euthanasia until measurement of pH was noted for each sample. Reticulorumen and omasum pH was usually measured within 20–25 min of each other. For the relatively dry samples (omasum content and faeces), a small amount of demineralized water was mixed into the sample to make it just fluid enough for the pH meter to be able to measure pH. After sampling, the content of each gastrointestinal section was removed. The stomach sections were rinsed with water, squeezed and allowed to drip-dry for at least 15 min before determining empty weights, while the intestines were weighed without rinsing. The wet weight of digesta in each gastrointestinal section was determined by difference. Mucosa samples from the dorsal rumen wall, the ventral rumen wall and the wall of the Atrium ruminis were collected at the positions marked with black circles on Fig. 2 and stored in 10% formalin until dissection. A1.5 × 1.5 cm piece was cut from each mucosa sample using a metal template. All papillae were subsequently cut from the base of the square piece of mucosa and sorted according to size (small, medium or large) based on visual judgement by the dissector (CS). Number of papillae of each size was counted, and 10 papillae of each size were randomly chosen and measured using a digital calliper. For each papilla, length and width, determined at the midpoint (i.e., at length/2), was measured. All measurements were recorded to the nearest 0.01 mm. The surface enlargement factor (SEF) of the rumen mucosa papillae was calculated as:

SEF ¼ Fig. 2. Locations for sample collection in the stomachs. Locations for digesta sampling are marked with a white square (□), while locations for mucosa sampling are marked with a black circle (●). DR = dorsal rumen, AR = atrium ruminis, RE = reticulum, OM = omasum, A = abomasum, VR = ventral rumen.

ðno:of papillae  MPSAÞ þ base surface ; base surface

where MPSA = mean papillae surface area measured in cm2 (i.e., 2 × mean papillae height (cm) × mean papillae width (cm)), and base surface = area of the piece of mucosa dissected (i.e., 2.25 cm2).

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2.2. Statistical analyses

3.2. Digesta particle size

For each measurement, repeated measures ANOVA were used to determine differences between boma and zoo giraffes by models that included fasting time or log-transformed body mass as a covariate and origin (boma/zoo) as a between-subjects factor (Table S1; with stepwise exclusion of non-significant factors), with Sidak post hoc tests to adjust for multiple comparisons. Similarly, simple repeated measures ANOVA with Sidak post hoc tests were used to determine differences between forestomach locations. Linear regressions of logtransformed data served to identify scaling relationships with body mass. Additional pair-wise comparisons were made with t-tests. All statistical analyses were performed using the statistical software SPSS 22.0 (IBM, Armonk, NY). The significance level α was set to 0.05.

There was no difference in mean particle size between boma and zoo giraffes at the reticuloruminal sampling locations (Fig. 4A, P = 0.391–0.993), whereas zoo giraffes had larger particles than boma giraffes in the omasum (1.28 ± 0.35 mm vs. 0.81 ± 0.30 mm, P = 0.016) and in the faeces (0.85 ± 0.12 mm vs. 0.57 ± 0.10 mm, P = 0.001). Larger faecal particles in captive versus free-ranging giraffes have previously been reported (0.75 versus 0.35 mm, respectively) (Hummel et al., 2008). This difference between captive and free-ranging animals have also been found in another browser, the moose, whereas no similarly extreme difference is evident in grazing ruminants (Hummel et al., 2008; Lechner et al., 2010). This has been linked to the fact that diets fed to captive browsers likely differ more from natural diets than diets fed to captive grazers, and to reports of excessive tooth wear in captive giraffes (Clauss et al., 2007). Worn down teeth reducing chewing efficiency in captive individuals should result in larger particles in the reticulorumen as well. Although boma giraffes generally had longer fasting times, many of them had been under anaesthesia and thus unable to ruminate for up to 8 h prior to euthanasia, whereas the zoo giraffes were able to ruminate up until the time of euthanasia. The extended rumination in zoo giraffes may have counterbalanced any original differences in reticulorumen particle size when compared to boma giraffes. There was no effect of fasting time on mean particle size (P = 0.655). This finding may be a result of the combination of shorter rumination time and smaller initial particle size in boma individuals versus longer rumination time and larger initial particle size in captive individuals, cancelling the effect of fasting time. Within individual giraffes, mean particle size did not differ between dorsal and ventral rumen (boma: P = 1.000, zoo: P = 0.724), again supporting the hypothesis of unstratified rumen content in giraffe. In boma giraffe, there were no differences between rumen and reticulum mean particle size (dorsal rumen-reticulum: P = 0.082, ventral rumen-reticulum: P = 0.164), and the omasum contained smaller particles than any preceding location (P ≤ 0.002). In zoo giraffes, although a numerical reduction of mean particle size was observed from the reticulorumen to the omasum (Fig. 4A), it was not significant (P ≥ 0.132). When compared to other species of ruminants (Clauss et al., 2009a), giraffes had remarkably smaller particles in all reticulorumen locations (Fig. 4B). In addition, the magnitude of particle size reduction from the reticulorumen to the omasum, indicating selective retention of large particles in the reticulorumen, was much lower than in the other three species, yet the particle size in the omasum was similarly small. This is likely due to two reasons. Firstly, the animals had an unknown

3. Results and discussion Measurements not directly related to rumen contents stratification (such as contents weights or measures in the abomasum and lower digestive tract) are given in Appendix A (incl. Figs. S1–S7).

3.1. Digesta dry matter content The difference in DM concentration between the dorsal and ventral rumen regions was not significant (boma: P = 0.290, zoo: P = 0.637), while the reticular DM concentration was lower than in the rumen in boma (P ≤ 0.020) but not zoo giraffes (P ≥ 0.063). DM concentration in the omasum was higher than in the RR (boma: P b 0.001, zoo: P = 0.001, Fig. 3A). The absence of a difference in DM concentration between the dorsal and ventral part of the rumen indicates homogenous rumen content in the giraffe, as expected. When compared to other ruminants, i.e., addax (Addax nasomaculatus), bison (Bison bison), and moose (Alces alces) (Clauss et al., 2009a), the lack of difference in DM concentration between the dorsal and ventral rumen in giraffes is similar to findings in moose, another browsing ruminant (Fig. 3B), whereas the grazing, ‘cattle-type’ ruminants showed a clear difference in dorsal and ventral DM concentration, as evidence of a stratified rumen content. Overall, DM concentrations were lower in this study than previously reported for giraffes (reticulorumen: 13.5–13.8%, n = 9) (Maloiy et al., 1982; Clemens and Maloiy, 1983), which is probably due to the long fasting times in this study and to some extent to dietary differences between studies, both of which have been documented to affect digesta DM (Hummel et al., 2009).

Fig. 3. Dry matter concentration of digesta samples collected along the digestive tract of the giraffe and other ruminant species. DR = dorsal rumen, VR = ventral rumen, RE = reticulum, OM = omasum. Bars represent means and standard deviations. A) Dry matter concentration in giraffe digesta. Samples from 16 boma and 9 zoo giraffes. No common superscripts denote significant differences between sampling locations (small letters for boma, capital letters for zoo). B) Dry matter concentration in forestomach digesta of boma giraffes (this study), compared to addax (Addax nasomaculatus), bison (Bison bison), and moose (Alces alces) (from Clauss et al., 2009a). No common superscripts denote significant differences between sampling locations within a species.

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Fig. 4. Mean particle size of digesta samples collected along the digestive tract of the giraffe and other ruminant species. DR = dorsal rumen, VR = ventral rumen, RE = reticulum, OM = omasum. Bars represent means and standard deviations. a) Mean particle size of giraffe digesta in samples from 10 boma and 5 zoo giraffes. No common superscripts denote significant differences between sampling locations (small letters: boma, capital letters: zoo); *denotes difference between boma and zoo. b) Mean particle size in forestomach digesta of giraffes (this study), compared to addax (Addax nasomaculatus), bison (Bison bison), and moose (Alces alces) (from Clauss et al., 2009a). No common superscripts denote significant differences between sampling locations within a species.

proportion of pelleted (and hence, ground) feed in their diet. Secondly, the majority of both the boma and zoo giraffes in this study had been fasted for a substantial period of time, while continuing to ruminate and thereby reducing particle size (McLeod and Minson, 1988). Nevertheless, differences in particle size between dorsal and ventral rumen content have been documented in cattle for up to 24 h after feeding (Hummel et al., 2009) and in goats 12 h after feeding (Clauss et al., 2016), suggesting that the absence of particle size stratification in the giraffes of the present study may indeed indicate an absence of reticulorumen contents stratification. Although 75% of particle size reduction has been attributed to masticatory activity (initial chewing and rumination; McLeod and Minson, 1988), microbial fermentation of the feed particles will reduce the size as well (McLeod and Minson, 1988; Krämer et al., 2013). Thus, the reduction in particle size from the omasum to the faeces observed in this study (Fig. S3) indicates considerable microbial activity in the hindgut of the giraffe; however, the extent of this fermentation remains to be investigated. 3.3. Digesta fibre content The average iNDF:NDF ratio in the whole digestive tract was significantly higher in boma than in zoo giraffes (0.65 ± 0.08 vs. 0.46 ± 0.12, P ≤ 0.008 in all location-wise comparisons, Fig. S4). However, origin was not significant in the overall model, but there was a significant effect of fasting time (P = 0.048). Fasting time differed significantly in this comparison (887 ± 632 min in boma vs. 114 ± 74 min in zoo, P b 0.001), and was positively related to the iNDF:NDF ratio, indicating that more time for digestion led, expectedly, to a higher proportion of indigestible material. The higher iNDF:NDF ratio observed in boma giraffes is thus a consequence of their longer fasting time. Another contributing factor could have been differences in diets, as the boma diet was likely less digestible compared to the zoo diet, i.e., the boma diet probably had a higher iNDF concentration to start with. Within individual giraffes, there was no difference in iNDF:NDF ratio between the forestomach locations (boma: P = 0.069–1.000; zoo: P = 0.968–1.000). The lack of difference between the dorsal and ventral rumen indicates homogenous rumen content. 3.4. Digesta concentrations of VFA and NH3 Zoo giraffes generally had numerically higher concentrations of total VFAs (P = 0.064–0.090), of acetic acid (P = 0.064–0.097), propionic acid (P = 0.062–0.088), butyric acid (P = 0.026–0.045) and NH3 (P = 0.025–0.039) in the rumen and reticulum compared to boma giraffes (Fig. S5), again indicating more digestible diets and shorter fasting times. There was no difference between sampling locations for

concentrations of total VFA, any individual VFAs or NH3 (P ≥ 0.125, Fig. S5). Boma giraffes had a higher mean proportion of acetic acid (0.73 ± 0.02 vs. 0.69 ± 0.05, P = 0.023) and lower proportion of butyric acid (0.10 ± 0.01 vs. 0.14 ± 0.02, P = 0.001) than zoo giraffes, while the proportion of propionic acid was the same (0.17 ± 0.02 vs. 0.18 ± 0.03, P = 0.487). To summarise, the molar proportions of acetic:propionic:butyric acid found is this study were 73:17:10 for boma giraffes and 69:18:14 for zoo giraffes. Ratios of 76:14:9 and 73:14:13 have previously been documented in wild giraffes (Maloiy et al., 1982, n = 6; Clemens and Maloiy, 1983, n = 3). The difference in the proportion of propionic acid could be due to the inclusion of pelleted feeds in the present study. Commercial dairy cows have an average ratio of 63:22:11 (101 different diets, Morvay et al., 2011). The average reticuloruminal concentration of NH3 in the present study (boma giraffes: 18.5 ± 6.9 mg/100 mL and zoo giraffes: 33.7 ± 14.3 mg/100 mL) was in the range of values previously reported for 6 free-ranging giraffes of 24.6 ± 2.1 mg/100 mL (Maloiy et al., 1982), while the total VFA concentration (boma giraffes: 47.8 ± 18.8 mmol/L and zoo giraffes: 82.7 ± 42.7 mmol/L) in this study was lower than previously reported for giraffes (Maloiy et al., 1982, 158.3 ± 3.5 mmol/L; Clemens and Maloiy, 1983, 106.4 ± 10.6 mmol/L). Factors like differences in diet composition (Hummel et al., 2006), amounts fed and time passed since last feeding (Brask et al., 2015), greatly influence the concentration of fermentation products in the rumen of domestic ruminants, and are likely the cause of the large variation observed among giraffes in this study and also when comparing between studies. Differences in sampling protocols presumably contribute as well, with variation in lag time from death to sampling, the presence/absence of a fixation agent (e.g., H2SO4 used by Maloiy et al., 1982; Clemens and Maloiy, 1983) and potential differences in analytical procedures between studies. 3.5. Digesta pH The pH in the dorsal rumen, ventral rumen and omasum decreased (P = 0.022, 0.030 and 0.033, respectively) with time since euthanasia, while abomasum pH increased (P b 0.001, Fig. S6A). Fasting time had a (positive) effect only on the pH of the dorsal and ventral rumen (P = 0.022 and 0.049, respectively). When accounting for the longer time since euthanasia in zoo giraffes, there was no difference between boma and zoo giraffes in pH of the ventral rumen (P = 0.062) and abomasum (P = 0.277), but a difference was found in the dorsal rumen (P = 0.036) and the omasum (P = 0.021). The pH of the reticulum was unaffected by both time since euthanasia (P = 0.248) and giraffe origin (P = 0.170). Similar to this study, Ritz et al. (2013) observed a decrease of rumen pH with longer time since euthanasia in

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roe deer (Capreolus capreolus), suggesting a continuation of microbial activity after death while absorption of VFA terminates at the time of euthanasia. Within individual giraffes, there was no difference in pH between any forestomach locations (Fig. 5, boma: P = 0.084–0.998, zoo: P = 0.628–1.000), and thus no indication of a higher fermentation activity at either site. The pH measured in this study was comparable to values previously reported for reticuloruminal and abomasal content (pH of 6.5 and 3.6, respectively) of six free-ranging giraffes (Maloiy et al., 1982). The numerically lower pH in the omasum as compared to the rumen (Fig. 5) resembles findings reported in both red deer (Cervus elaphus) and fallow deer (Dama dama), with rumen – omasum differences of 0.7 and 0.5, respectively (Prins et al., 1972). The pH in the reticulum was unaffected by time since euthanasia in this study – an observation made in roe deer as well (Ritz et al., 2013). Perhaps the greater amount of buffering saliva in the reticulum makes this location less sensitive to time effects compared to other forestomach locations.

3.6. Rumen papillation There was a negative relationship between body mass and papillae density (number of papillae per cm2) (P = 0.003), with a scaling exponent that included − 1.0 in the 95% confidence interval for the dorsal and the ventral rumen, and came close to that value (− 0.95) for the Atrium ruminis (Fig. S7). Accounting for body mass, papillae density was lower in zoo giraffes than in boma giraffes (P = 0.022, Table 1). In boma giraffes, papillae density in the ventral rumen was lower than both in the dorsal rumen (P b 0.001) and the Atrium ruminis (P = 0.040; Table 1). In zoo giraffes, papillae density in the Atrium ruminis was higher than in the ventral rumen (P = 0.005) but not in the dorsal rumen (P = 0.081; Table 1). The average area of individual papilla of each location was positively correlated to body mass (all P b 0.001), with a scaling exponent that always included 1.0 in the 95% confidence interval (Fig. S7). Accounting for body mass, papilla area was much greater in zoo than in boma giraffes (P b 0.001, Table 1). In zoo giraffes, there was no difference in average papilla area between sampling locations (P = 0.688–0.850), whereas papillae in the ventral rumen were larger on average than papillae in the dorsal rumen in boma giraffes (P = 0.007, Table 1). There was no significant relationship between body mass and SEF (P = 0.261), with a scaling exponent that always included 0 in the 95% confidence interval (Fig. S7). SEF was greater in zoo than in boma giraffes (P = 0.014, Table 1). In spite of numerical differences (Table 1), there were no significant differences between forestomach

Fig. 5. pH in digesta samples collected in the forestomach of 6 boma and 7 zoo giraffes. DR = dorsal rumen, VR = ventral rumen, RE = reticulum, OM = omasum. Bars represent mean values with standard deviation; *denotes differences between boma and zoo (accounting for fasting time and time between euthanasia and measurement). There were no significant differences between forestomach locations in neither boma nor zoo giraffes.

Fig. 6. Relation between dorsal to ventral difference in rumen digesta dry matter concentration and relative SEF Dorsal rumen (in % SEF Atrium ) in giraffes (this study) and other ruminants (from Codron and Clauss, 2010). Each point represents the mean of a species.

locations for the SEF within boma (P = 0.085–0.996) or zoo giraffes (P = 0.147–0.992). Characteristics of the rumen papillae are typically influenced by both body size and diet (e.g. Josefsen et al., 1996; Wang et al., 2009). In this study, both effects were apparent. To our knowledge, the relationship between the intraruminal papillation pattern and body mass has hardly been investigated quantitatively, and the fact that the scaling exponents of papillae density and papilla area cancel out each other, resulting in no scaling of SEF, is a very interesting side finding. The observation that older, and hence larger animals have lower papillae densities and larger papillae was reported repeatedly (Berg et al., 1986; Josefsen et al., 1996; Mathiesen et al., 2000). A parsimonious explanation could be that rumen papillae are already developed in the embryo (e.g. Franco et al., 2011), and that their number is comparatively fixed. Notably, the rumen of neonate ruminants is homogenously covered with papillae, even at locations that have no papillae in the adult forms of some species such as the dorsal rumen or the ruminal pillars (Hofmann, 1973). Growth of both the rumen wall and the papillae necessarily leads to a reduction of the number of papillae per defined unit of area (such as cm2), while their total number may stay comparatively stable (Berg et al., 1986). Variation in papillae density between animals of similar age or size might then mainly reflect conditions that prevent development of all preformed papillae. This explanation matches observations that dietary manipulations that increase papilla size or papillae surface often do not increase papillae density (Shen et al., 2004; Wang et al., 2009; Xu et al., 2009). In our study, zoo giraffes were larger than boma giraffes (mean body mass of 687 ± 318 vs. 475 ± 88 kg) and had a lower density of papillae, but with each individual papilla having a larger surface area, which resulted in overall higher SEF. The fact that the difference between zoo and boma giraffes mostly persisted even when correcting for body mass indicates an additional dietary effect, with a more digestible diet in zoo giraffes as mentioned repeatedly above. When Clauss et al. (2009b) evaluated SEF data from 59 ruminant species, it was noted that the dorsal rumen-SEF value of 24 found in wild giraffes by Hofmann (1973) was unusually high (Table 1; possibly because the smaller papillae were not included in the measurement), as 11.0 was the second highest dorsal rumen-SEF found in the entire dataset (for grey duiker, Sylvicapra grimmia). The dorsal rumen-SEF found in this study (average of 13.5) appear more moderate; though still the highest of all ruminant species. When dorsal rumen-SEF and ventral rumen-SEF were expressed in % of the SEF of the Atrium ruminis (termed relative SEF), both were numerically higher in boma than zoo giraffes (Table 1), though not statistically different (P = 0.108 and P = 0.438, respectively). The relative SEF differed between the dorsal and the ventral rumen in boma giraffes

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Table 1 Characteristics of rumen mucosa papillae in boma and zoo giraffes. Boma giraffes

Zoo giraffes

Measure

Location

This study1 n = 12

H. (1973)2 n=6

This study1 n=7

H.M. (1988)1 n=2

Papillae density (papillae/cm2)

DR AR VR

80.0 ± 21.6a 74.9 ± 17.7a 52.8 ± 20.2b

24 26 19

40.7 ± 26.9ab 54.9 ± 24.0a 34.9 ± 16.1b

17/15 9/15 14/12

Papillae area (mm2)

DR AR VR

16.1 ± 4.1b 18.1 ± 5.9ab 19.5 ± 3.8a

– – –

41.3 ± 14.9 44.6 ± 16.8 51.1 ± 32.2

– – –

SEF

DR AR VR

13.5 ± 3.7 14.1 ± 3.1 11.4 ± 5.1

24 30 18

15.1 ± 4.1 22.5 ± 6.3 15.8 ± 5.5

2.7/1.9 4.95/4.59 2.47/2.74

Relative SEF (% of SEFAR)

DR VR

100.1 ± 33.2 87.2 ± 44.0

80.0 60.0

73.5 ± 32.5 72.7 ± 24.2

53.9/41.4 49.9/59.7

H. (1973) = Hofmann (1973), H.M. (1988) = Hofmann and Matern (1988), DR = dorsal rumen, AR = atrium ruminis, VR = ventral rumen, SEF = surface enlargement factor. No common superscripts denote significant differences between sampling locations within a group. 1 Three sizes of papillae were recognized and all were included in the papillae count. 2 Three sizes of papillae were recognized, but only the two largest were included in the papillae count.

(P = 0.033), but not in zoo giraffes (P = 0.957). No effect of body mass could be demonstrated for the relative SEF (P = 0.603). The relative SEF was negatively related to the difference in dry matter concentration between the contents of the dorsal and the ventral rumen (Fig. 6), suggesting that these two measures of rumen contents stratification are linked as expected. The relative SEF of boma giraffes were numerically much closer to 100% than in zoo giraffes, indicating slightly more stratified rumen content in the zoo giraffes. This difference matches previous observations in wild and captive giraffes (Table 1), and supports the assumption that feeding regimes in captivity might lead to more stratified rumen content in giraffe (Hummel and Clauss, 2006). This finding might be related to the difficulty of providing captive giraffe with adequate amounts and ‘quality’ of roughage. Lucerne hay is considered an indispensable diet component for captive giraffes, due to the fact that these animals require a source of structurally effective fibre, yet usually do not ingest grass hay in relevant amounts but typically accept lucerne hay more readily. However, maintaining giraffe on a diet of lucerne hay alone may still be problematic due to low ad libitum intakes (Hatt et al., 2005). As a dicot, lucerne shares some characteristics with browse, such as a higher degree of lignification than grass, as well as similar fermentation and physical fractionation patterns (Troelsen and Campbell, 1968; Gussek et al., 2016). Nevertheless, cattle fed only lucerne hay still display a pronounced rumen content stratification (Hummel et al., 2009). Possibly, only a dietary regime consisting predominantly of browse, which is very difficult to achieve in captivity, would result in the particularly homogenous papillation pattern observed in free-ranging animals.

4. Conclusion The physical characteristics of the rumen content of boma and zoo giraffes were investigated in this study. No difference between the dorsal and ventral rumen content was evident in any measurement (DM, particle size, iNDF:NDF, VFA, NH3 or pH), thus indicating an unstratified rumen content in the giraffe. This observation was further supported by the finding of an even rumen papillation pattern, as evidenced by high relative SEF of both the dorsal and ventral rumen, in boma giraffes. A numerical difference in relative SEF indicated a slightly less homogenous rumen content in zoo giraffes, but this finding could not be substantiated by physical characteristics of the rumen content. Nevertheless, it indicates that the diet these captive animals received in the last weeks before sampling, and hence most likely during their whole life in captivity, induces some degree of rumen content stratification not observed in animals from the wild.

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