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Published December 8, 2014 Mucosal surface area and fermentation activity in the hind gut of hydrated and chronically dehydrated working donkeys J. C. Sneddon,*1 E. Boomker,† and C. V. Howard‡ *Liverpool John Moores University, Liverpool L3 3AF, United Kingdom; †University of Pretoria, Pretoria 0110, South Africa; and ‡University of Liverpool, Liverpool L69 3BX, United Kingdom difference was not significant. Total mucosal and crypt surface area per unit volume of gut (Sv, m2/m3) was greater in dehydrated donkeys for the cecum (253 ± 23.0 vs. 161 ± 13.5, P < 0.01), the ventral colon (286 ± 6.2 vs. 171 ± 9.8, P < 0.01), the dorsal colon (276 ± 18.2 vs. 256 ± 11.0, P < 0.05), and the descending colon (260 ± 20.3 vs. 191 ± 15.2, P < 0.05). Enhanced fermentation activity and enhanced mucosal absorptive or secretory capacity within the hindgut during chronic dehydration was associated with an observed maintenance of appetite. These adaptations in the hindgut are valuable physiological attributes for working donkeys in semiarid regions where they are frequently exposed to chronic dehydration. ABSTRACT: The effects of mild chronic dehydration on fermentation rate and mucosal surface area in the cecum, dorsa and ventral colon, and descending colon of the hindgut were investigated in South African donkeys (n = 11) in agricultural work. Dehydration representing a 6% drop in BW (n = 6) was associated with increased fermentation activity in the cecum (252 ± 22.9 vs. 161 ± 13.5 mol/g of DMⴢ h−1, P < 0.01) and enhanced fluid retention in the ventral colon (0.81 ± 0.026 vs. 0.73 ± 0.034 mL/g gut, P < 0.05). Fermentation activity in the next segment of the hindgut, the ventral colon, of dehydrated donkeys was also greater numerically (92.5 ± 22.60 vs. 77.9 ± 10.40 mol/g of DMⴢ h−1), but this Key words: dehydration, donkey, fermentation, hind gut, morphology 2006 American Society of Animal Science. All rights reserved. INTRODUCTION J. Anim. Sci. 2006. 84:119–124 amounts to 20% of the total body water pool in fully hydrated animals (Argenzio, 1991; Kasirer-Izraely et al., 1994; Meyer, 1996a,b). Experimental observations on donkeys have established that the gut fluid pool depletes by up to 50% during a severe (25%) dehydration (Maloiy and Clemmens, 1980; Kasirer-Izraely et al., 1994); however, sufficient fluid is retained to maintain appetite (Izraely et al., 1989; Rutagwenda et al., 1990; Yousef, 1991). It is unknown whether there are any associated morphological changes in the equine mucosal wall; however, dehydration has been reported to have a degenerative morphological effect on the rumen mucosa in ruminants (Norgaard and Skadhauge, 1989). This study hypothesized that absorptive capacity indexed by gross morphological change in the mucosal surface and fermentation activity indexed by gas production of incubated digesta would be appreciably altered in the hindgut by a dehydration level comparable with that of agricultural work in working donkeys in southern Africa (Mueller and Houpt, 1991; Nengomasha et al., 1999). The donkey, Equus asinus, is an arid-adapted equine of economic importance to the subsistence agricultural sector in semiarid regions of Southern Africa (Kneale, 1996; Nengomasha et al., 1999; Starkey and Fielding, 2000). A major benefit of donkeys in this capacity is the ability to work while withstanding severe dehydration (Yousef, 1991). This is achieved by reduced water and energy turnover rates, reduced sweating rate, reduced water excretion, and maintained feed intake (Maloiy and Boarer, 1971; Izraely et al., 1989; Yousef, 1991). In addition, plasma volume is maintained by drawing on the substantial fluid reservoir in the hindgut (Yousef et al., 1970; Kasirer-Izraely et al., 1994). In ruminants, water constitutes up to 85 to 90% of total gastrointestinal fill (Silanikove, 1994), and similar data are recorded for equine hindgut (Kasirer-Izraely et al., 1994; Meyer, 1996a,b). Studies on the equine hindgut postmortem revealed that the water content MATERIALS AND METHODS Experimental Animals and Husbandry 1 Corresponding author: [email protected] Received January 17, 2005. Accepted September 6, 2005. Eleven donkeys (Equus asinus) were purchased from local users in North West Province, Gauteng, 119 120 Sneddon et al. Table 1. Weight and age data of experimental animals Identity number 2 6 7 8 9 Mean (SEM) 1 3 4 5 12 13 Mean (SEM) Age, yr Weight, kg 201 5 5.5 2.5 4.5 7.5 (3.16) 5 2 3.5 2.5 201 10 7.2 (2.82) 126 180 182 145 208 156 (13.2) 162 128 128 156 170 140 148 (7.3) Treatment category Hydrated Hydrated Hydrated Hydrated Hydrated Dehydrated Dehydrated Dehydrated Dehydrated Dehydrated Dehydrated 1 Estimated age. South Africa. The animals ranged in age between 2 to 20 yr and between 120 to 208 kg in weight (Table 1). The ages of the donkeys were reported by the owners and corroborated by dental patterns when possible. The animals were situated at the Onderstepoort Veterinary Research Unit, Faculty of Veterinary Science, University of Pretoria, from June to August, when there was no thermal stress on the animal (ambient temperature range 15 to 20°C). The donkeys were maintained in an earthen-floored outside paddock with ad libitum access to Teff hay (Eragrostis teff) and allowed access to water when individually stabled at night. This regimen emulated the husbandry conditions provided by the owners. Six donkeys were 50% water-restricted for a week before the experiment to attain a constant 6% dehydration, and the remaining 5 donkeys remained normally hydrated (Table 1). Donkeys were under continual veterinary supervision and were clinically assessed and weighed daily to quantify their level of dehydration. Clinical measures used in the assessment of dehydration included weighing, skin elasticity, capillary refill time, heart rate, and the general demeanor of the animal. Ethical Considerations The experimental protocol was approved by the Ethics Committee of the Faculty of Veterinary Science, University of Pretoria, before experiments commenced. The animals were used for a variety of physiological, anatomical (dental), and parasitological observations that had no impact on this study. The donkeys were humanely killed under veterinary supervision with a captive bolt at the Department of Pathology, Faculty of Veterinary Science, University of Pretoria. Measurement of Hindgut Fluid Reservoir Regions of the hindgut investigated were split into gross anatomical areas: the cecum, the ventral colon, Figure 1. Schematic representation of equine gut anatomy indicating positions of ligatures (dark bars indicated with the three arrows) at junctions between the cecum (C) and ventral colon (VC), between the ventral and dorsal colon (DC), and between the dorsal and descending colon (DSC; adapted from Argenzio, 1975). the dorsal colon, and the descending colon. The digestive tract was removed by ventral incision within 5 min of death, and individual regions of hindgut were ligated at anatomical points shown in Figure 1. Percentage water content of digesta was determined by drying 1- to 5-g samples of digesta to constant weight (Faichney and White, 1983). Percentage of water content was then multiplied by total volume of digesta in the hindgut region to obtain total fluid volume (Faichney and White, 1983). Volume of digesta within each gut region was measured to the nearest milliliter, carefully removing adhered digesta with a known volume of distilled water. Differences in individual gut size were accounted for by dividing total fluid content by mass of hindgut region (g) to give fluid content per unit weight of hindgut tissue (mL/g) within each hindgut region. Determination of Fermentation Indices Samples (n = 6) of digesta (approximately 400 g) were taken from each region of the hindgut immediately, and these samples were placed in a water bath at 39°C. The digesta were allowed to ferment anaerobi- 121 Hind gut morphology in dehydrated donkeys cally, and the production of gas was monitored by water displacement in a manometer over a period of 30 min. The digesta samples were then dried to a constant mass. Fermentation indices were calculated as mol/ g of DMⴢh−1. Tissue Sampling for Stereological Measurements on Hindgut Wall Each region of the hindgut wall was subjected to a brief (10 min) examination for gastrointestinal parasites using an isotonic saline wash; the washed tissue was then weighed. Gut tissue from each anatomical region was weighed to an accuracy of 0.01 kg. The washed tissue was then placed, with the serosa down, on a stainless steel table, and the mucosa was covered with a nylon net grid. The net had 2.0- × 2.0-cm holes so that 0.5- × 1.5-cm samples of tissue could be taken randomly from points within the area of the net. Random number values generated by a calculator were kept within number of net squares covering the length and breadth of each hindgut region. Tissue samples were adhered, with the serosa down, on a piece of Whatman filter paper and were dropped into 10% buffered formalin. The samples were heat-sealed in plastic in isotonically buffered formalin reservoirs until they were histologically processed, microtomed, and stained as transvers sections using an automatic processor and haematoxylin and eosin stain. This sampling was completed within 30 min of death. Sufficient samples were taken to provide 30 individual vertical sections per hindgut region, which in turn provided an average of 30 measurements per hindgut region for estimation of mucosal surface area, and mucosal and serosal volumes. Thirty samples per hindgut region were deemed sufficient as the average CV for the surface area of mucosal surface and crypt per unit volume was less than 1% across all hindgut regions in all donkeys. This was calculated using the method for ratio estimators (Howard and Reed, 1998). Gut density was estimated from mass and volume using fluid displacement (Scherle, 1970). The volume of tissue in each hindgut section was equivalent to its mass; average gut tissue density was not found to be significantly different from 1.0. Stereological Calculations The stereological procedures and calculations used to estimate the combined mucosal and crypt surface area, mucosal and serosal volumes, and mucosal height of each hindgut region on isotonically preserved tissue were based on those described by Baddeley et al. (1986). The transverse sections were placed under a microfiche reader (17.4× magnification) for estimation of mucosal and serosal volumes (mL), together with mucosal height (m). For the latter estimation, an appropriate graticule (one division = 0.5 mm at 17.4× magni- fication) was used, and an average of 6 readings were taken per slide using 30 individual slides. To estimate gross surface area of the combined mucosa and crypt per unit volume of gut tissue (m2/m3) a light microscope magnification (198×), and the cycloid grid (length of cycloid arc = 3.5 cm) were used (Baddeley et al., 1986). Surface amplification or rugosity of the mucosa was calculated by multiplying combined mucosa and crypt per unit volume of gut tissue by mucosal height. For a complete mathematical explanation of how transverse sections can be used to calculate surface area, volumes, and mucosal height within a 3-dimensional space, refer to Baddeley et al. (1986). Statistical Methods Data were normally distributed as defined by a Kruskall Wallis test (Sokal and Rohlf, 1969). The effects of dehydration were statistically tested using SPSS 12.0.1 (Apache Software Foundation, Inc., Chicago, IL) on all measured variables using Student’s Ttest. A probability value of 0.05 was taken as significant. RESULTS Donkeys lost an average of 6.3 ± 1.13% (n = 6) of their BW under the dehydration regimen. This weight loss was assumed to be water loss because feed intake in dehydrated donkeys appeared to remain normal throughout the dehydration period. In addition, the water loss was assumed to occur equally from all body water pools because the total mass (g) of the hindgut tissue remained constant when expressed as a percentage of total BW: 20.6 ± 0.27% and 21.4 ± 1.32% of BW in hydrated and dehydrated donkeys, respectively. Values for percentage fluid content of digesta were within the range of 79 to 92% in both hydrated and dehydrated donkeys for all gut regions. Dehydration produced greater values for the combined mucosal and crypt surface area per unit volume of gut (m2/m3) in all hindgut regions (P < 0.05 to P < 0.01, Table 2), and there were increases in combined mucosal and crypt surface area when BW was accounted for in the cecum (P < 0.05) and in the ventral colon (P < 0.01). Gut fermentation activity was greater in dehydrated donkeys in the cecum (1.56-fold greater). Gut fluid content was also enhanced in the ventral colon in dehydrated donkeys (P < 0.05). Surface amplification or rugosity of the mucosal surface was only greater in the descending colon of dehydrated donkeys (P < 0.05). Mucosal absorptive capacity thus seemed to be enhanced by an increase in surface area even though there was a decrease in mucosal depth in dehydrated donkeys, particularly in the cecum and ventral colon (Table 2). −1 Numbers in parenthesis indicate SE. *P < 0.05.; **P < 0.01. 1 Fermentation activity, mol/g of DMⴢ h Hydrated Dehydrated Gut fluid content, mL/g of gut Hydrated Dehydrated Combined mucosal & crypt surface area per unit volume of gut tissue, m2/m3 Hydrated Dehydrated Combined mucosal & crypt surface area/BW, cm2/kg Hydrated Dehydrated Surface amplification, mucosal rugosity index Hydrated Dehydrated Mucosal height, m Hydrated Dehydrated Gut tissue volume/mass, mL/g Hydrated Dehydrated Volume mucosal tissue, mL Hydrated Dehydrated Volume serosal tissue, mL Hydrated Dehydrated Response measures 1.9 × 104 (1.69 × 103) 1.6 × 104 (3.33 × 103) 4.8 × 103 (4.36 × 102) 3.8 × 103 (1.72 × 102)* 1.4 × 104 (1.41 × 103) 1.2 × 104 (8.55 × 103) 1.2 × 103 (60.0) 1.1 × 103 (1.10 × 102) 3.6 × 103 (4.39 × 102) 2.6 × 103 (1.71 × 102)* 614 (16.8 473 (36.3)** 10.5 (0.72) 13.6 (1.24)* 1.9 × 104 (9.70 × 103) 3.3 × 104(1.70 × 103)** 171 (9.8) 286 (6.2)** 0.73 (0.034) 0.81 (0.026)* 78 (10.4) 93 (22.6) Ventral colon 4.8 × 103 (4.30 × 102) 3.6 × 103 (2.45 × 102)* 663 (63.3) 548 (24.6) 10.6 (1.17) 13.4 (1.50) 4.6 × 103 (3.70 × 102) 6.8 × 103 (7.91 × 102)* 161 (13.5) 253 (23.0)** 0.73 (0.262) 0.73 (0.307) 161.4 (13.5) 252.7 (22.9)** Cecum 6.2 × 103 (6.97 × 102) 5.8 × 103 (5.21 × 102) 2.3 × 103 (1.84 × 102) 1.9 × 103 (1.75 × 102) 9.1 × 103 (7.85 × 102) 7.9 × 103 (5.64 × 102) 580 (56.5) 526 (39.1) 13.1 (1.03) 14.6 (1.61) 1.2 × 104 (1.08 × 103) 1.6 × 104 (1.60 × 103) 256 (11.0) 276 (18.2)* 0.77 (0.049) 0.78 (0.015) 148 (25.5) 113 (16.9) Dorsal colon 1.9 × 103 (4.32 × 102) 1.5 × 103 (2.42 × 102) 711 (172.5) 586 (106.4) 2.8 × 103 (5.55 × 102) 2.0 × 103 (3.37 × 102) 812 (20.5) 592 (17.4)** 15.4 (0.91) 15.3 (1.13) 3.2 × 103 (7.69 × 102) 2.7 × 103 (6.80 × 102) 191 (15.2) 260 (20.3)* 1.07 (0.090) 1.07 (0.139) 56 (5.9) 42 (15.4) Descending colon Table 2. Response measures (mean ± SE) for fermentation and absorptive capacity in the hindgut of hydrated (n = 5) and dehydrated (n = 6) donkeys1 122 Sneddon et al. Hind gut morphology in dehydrated donkeys DISCUSSION The objectives of this study were to investigate the effects of a chronic (6%) dehydration, equivalent to that experienced during agricultural field work, on the mucosal morphology, and on fermentation activity in the hindgut of working donkeys in Gauteng, South Africa. The donkeys lost an average of 6.3 ± 1.13% of their BW under the dehydration regimen. This level of dehydration has been observed in donkeys conducting typical field work in southern Africa (Nengomasha et al., 1999). Total gut mass as a percentage of BW remained about 20% in the current study, as in Kasirer-Izraely et al. (1994), in hydrated and dehydrated donkeys. Interestingly, this proportion was greater than the value of 11% for equines in general (Adolph, 1949). The relatively large proportion of gut to BW in donkeys compared with nonarid equines can be explained by their digestive strategy. In terms of adaptation to semiarid and arid environments, donkeys seem to be superior in that both DMI per unit of BW and digestive efficiency on high-fiber diets are greater than those of other equines under dry grazing conditions (Izraely et al., 1989). This enhanced digestive efficiency has been attributed to greater DMI, slower gastrointestinal transit time, longer retention times of feed residues on high-fiber diets, and enhanced recycling of urea (Pearson and Merritt, 1991; Mueller et al., 1994). Under conditions where the gastrointestinal tract is filled with poor quality fodder, the proportion of total BW occupied by the gut can double (Kasirer-Izraely et al., 1994). The morphological changes in the hindgut mucosa and enhanced fluid retention, particularly in the cecum and ventral colon, collectively reflect enhanced fermentation activity in these gut regions in dehydrated donkeys (Table 2) and agree with similar observations in a previous study on donkeys (Izraely et al., 1989). Contrary to observations reported for ruminants (Norgaard and Skadhauge, 1989), the morphological changes in the form of increased mucosal and crypt surface area (and decreased tissue depth in mucosa and serosa) in all hindgut regions seem to correspond with the enhanced fermentative capacity, particularly in the cecum and ventral colon (Table 2). Similar morphological adaptations have been described in the gastrointestinal tracts of other aridadapted mammals adapted to poor-quality, high-fiber diets (Buret et al., 1993). Physiological challenge in the form of extensive large colon resection has been reported to increase crypt surface area in the equine hindgut (Bertone et al., 1989). Rapid morphological changes of the gut mucosa in response to sudden changes in nutrient status have long been established in other mammals and reptiles (Diamond and Ferraris, 1993; Secor et al., 1994). The donkey is also an unusual ungulate in that feed intake is not severely reduced during dehydration 123 (Maloiy, 1970; Houpt, 1993). Given access to water after withdrawal of food and water, donkeys have been reported to eat first and then drink (Houpt, 1993). There was evidence from the current study to suggest that the digestive efficiency in the hindgut was maintained via fluid retention, which enhanced fermentation rates during dehydration. Fermentation activity might have been augmented via retention of fluid in the cecum and ventral colon (Table 2). When the amount of digesta in a hindgut region was taken into account by multiplying fermentation index by gut fluid content (mL/g gut) and gut tissue mass (Table 2), total fermentation activity per gut section was greatest in the ventral colon (the most capacious hindgut section) followed by the dorsal colon, the cecum, and the descending colon. The pattern of VFA production and absorption observed in the equine hindgut was first established by Argenzio (1975). The prime sites of VFA production are the cecum and ventral colon, and the prime site of absorption is the dorsal colon (Argenzio, 1975; 1991). The morphological changes in the hindgut mucosa and enhanced fluid retention, particularly in the cecum and ventral colon, collectively reflect enhanced fermentation activity in these gut regions in dehydrated donkeys (Table 2) and agree with similar observations in a previous study with donkeys (Izraely et al., 1989). Contrary to observations reported for ruminants (Norgaard and Skadhauge, 1989), the morphological changes in the form of increased mucosal and crypt surface area (and decreased tissue depth in mucosa and serosa) in all hindgut regions seem to correspond with the enhanced fermentative capacity, particularly in the cecum and ventral colon (Table 2). Similar morphological adaptations have been described in the gastrointestinal tracts of other aridadapted mammals adapted to poor-quality, high-fiber diets (Buret et al., 1993). Physiological challenge in the form of extensive large colon resection has been reported to increase crypt surface area in the equine hindgut (Bertone et al., 1989). Rapid morphological changes of the gut mucosa in response to sudden changes in nutrient status have long been established in other mammals and reptiles (Diamond and Ferraris, 1993; Secor et al., 1994). The experimental animals were all healthy and in draft work, and the parasite burdens in the gastrointestinal tract were typical of such animals. Parasites were largely confined to the stomach and small intestine (Wells et al., 1998) and thus had no influence on the randomized tissue sampling techniques used for stereological measurements. Three of the selected animals were considerably older than the remainder (Table 1), although the precise age of these animals was doubtful. Working donkeys in Southern Africa are hardy animals and frequently are found working into their late teens (Kneale, 1996; Wells et al., 1998). There is no evidence in the literature to indicate that the effects of mild dehydration on the hindgut mucosa 124 Sneddon et al. would have been appreciably influenced by age in healthy donkeys. 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