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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. Water intake values (measured using graded water managers) fell within 5.9 to 6.3% of
BW across all animals, suggesting that older animals
were not significantly different from younger ones in
terms of their hydration capacity.
In conclusion, it seems that the mild chronic dehydration experienced by the donkeys in the current
study produced morphological and physiological adaptations in the hindgut that enhanced the fermentation
capacity and absorptive capacity of the hindgut, particularly the cecum and ventral colon. These findings
support earlier studies reporting maintained food intake during dehydration in donkeys (Izraely et al.,
1989; Rutagwenda et al., 1990; Yousef, 1991). This
physiological attribute is an advantage in semiarid
regions when water supplies are scarce and interrupted.
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