Phenotypic flexibility of structure and function of the digestive system of Japanese quail
1 Institute of Zoology and Evolutionary Biology, University of Jena,
Erbertstraße 1, D-07743 Jena, Germany
2 Institute of Zoology, Assiut University, Assiut, Egypt
* Author for correspondence at present address: Department of Biology II, University of Munich, Luisenstrasse 1416, D-80333 Munich, Germany (e-mail: starck{at}zi.biologie.uni-muenchen.de)
Accepted 13 March 2003
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Summary |
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Key words: Diet switching, dietary fibre content, cell proliferation, resting metabolic rate, hypertrophy, hyperplasia, Japanese quail, Coturnix japonica, gizzard, small intestine, liver
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Introduction |
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The phenotypic flexibility of the avian digestive system has been studied
earlier and it has been shown that intestine size and function respond to a
variety of nutritional factors as well as to changes in internal demands
(Savory and Gentle,
1976a,b
;
Karasov, 1996
;
Piersma and Lindström,
1997
; for reviews, see Starck,
1999a
; McWilliams and Karasov,
2001
). For the small intestine, a response time of just a few days
has been suggested based on measurements of the tissue turnover time, i.e.
100% replacement of all cells of a tissue. Depending on the age and segment of
the intestine, tissue turnover times range from 3 to 17 days
(Imondi and Bird, 1966
;
Uni et al., 2000
;
Lilja, 1987
;
Lilja and Amneus, 1987
;
Starck,
1996a
,b
).
It has also been shown that the gizzard of birds adjusts to changes in diet
composition (Spitzer, 1972
;
Starck, 1999b
;
Dekinga et al., 2001
). After
switching the diet from soft to hard food, the gizzard doubled in size within
6 days in Japanese quail (Starck,
1999b
) and within 68.5 days in red knots Calidris
canutus (Dekinga et al.,
2001
).
Here, we investigate the processes at the cell and tissue levels that underlie changes in organ size response to changes in dietary fibre composition, and also whether there are changes in resting metabolic rate associated with up- and downregulation of the gastrointestinal capacity. Although flexible responses to feeding a high-fibre diet have been reported previously, nothing is known about the underlying cellular processes, i.e. cell proliferation or increase in cell size. Flexible responses would vary considerably in complexity and timing depending on whether they were based on proliferation and differentiation of new cells, or on increase in cell size without the need for mitosis and differentiation.
The energy content of the food was reduced by adding non-digestible fibre
to standard food without changing its nutrient composition. The resulting
high-fibre diet was also coarser than standard diet and required more gizzard
grinding activity. As a result of switching from a standard diet to a
high-fibre diet, we expected the sizes of gizzard muscle and small intestine
to increase. In the small intestine, decreasing quality of the food may be
compensated by increasing intestinal length, circumference and surface
magnification. With increasing digestive load to the intestine we also
expected the muscle layer to thicken (Karasov,
1990,
1996
;
Martinez del Rio et al., 1994
;
McWilliams and Karasov, 2001
;
Hume, 2002
). Upregulation of
the digestive system incurs energetic costs through protein synthesis, so to
maintain a balanced daily energy budget, quail would also need to increase
food intake or to fuel flexible responses from adipose tissue.
Possible cellular mechanisms of upregulating the gizzard capacity are
hyperplasia, i.e. production of more cells, or hypertrophy, i.e. increased
cell size. There is no direct information in birds about the cellular
processes underlying an increase in mucosal surface, although it is well known
that maintenance of the gut is based on permanent cellular turnover, i.e. a
balance between cell proliferation in the intestinal crypts and continued cell
loss at the tip of the villi (Altmann,
1972; Johnson and McCormack,
1994
; Starck,
1996a
,b
).
In mammals, it has been shown that enlargement of the mucosal absorptive
surface is based on increased cell proliferation rates
(Williamson and Chir, 1978
;
Sakata and Engelhardt, 1983
;
Jacobs and Lupton, 1984
;
Goodlad et al., 1989; Engelhardt et al.,
1989
), while elevated levels of apoptosis result in decreased
mucosal epithelium (Fleming et al.,
1992
; Boza et al.,
1999
; Dunel-Erb et al.,
2001
). Recent studies (Raab et
al., 1998
; Mentschel et al.,
2001
) suggest that in pigs an increase in mucosal absorptive
surface may also be based on decreased apical apoptosis. Decreased apoptosis
would be energetically cheaper than increased cell proliferation. No
information is available for birds, but the similarity of the maintenance
system (i.e. cellular turnover) suggests that changes in the mucosal
epithelium are caused by a mechanism similar to that in mammals. Liver size
has been reported to decline when quail feed on a high-fibre diet
(Starck, 1999b
). Analysis of
liver composition permits an assessment of whether these changes are based on
changes in lipid content, i.e. declining energy stores, or on changes in cell
numbers, which would imply a changing metabolic capacity of the liver.
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Materials and methods |
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Food
Standard food (dry matter: 22.6% crude protein, 4.7% lipids, 12.2% ash,
3.8% sugar, 3.3% fibre, 45.2% starch, 4% calcium, 0.9% phosphate) was obtained
from the Department of Animal Production of the University
Stuttgart-Hohenheim, Eningen, Germany. The energy content of the fresh food
was 77 kJ g1. Non-digestible fibre (oat beards) was
purchased from a commercial mill in Lahr, Germany. The experimental diet
contained 40% or 45% non-digestible fibre by mass; consequently, the energy
content of the experimental food was reduced by the same amount. During all
phases of the study, food and water were offered ad libitum.
Feeding experiments
All quail were allowed to acclimatize to the standard food diet for 4 weeks
prior to feeding trials, before conducting the following feeding experiments.
(1) Control quail were fed the standard diet (control group, N=11)
and two experimental groups were fed a high-fibre diet containing 40%
non-digestible fibre for periods of 2 (N=6) and 4 weeks
(N=6). Quail from this experiment were used to study gross
morphological, histological and morphometric changes in response to diet
switching. (2) To study the cellular changes associated with changes in
gizzard size, 140 quail were acclimatized to the standard diet for 4 weeks.
Then, they were divided into a control group remaining on the standard diet
and an experimental group (70 birds in each group; beginning of experiment
defined as day 0). The experimental group was switched to a diet containing
45% non-digestible fibre. After 14 days, 10 birds from each group were killed,
and tissue samples were taken. The diet of the remaining experimental birds
was switched back to the standard diet until day 28, when a further 10 birds
from each group were killed. Diet-switching from standard diet to experimental
diet and back to standard diet was repeated three times, so that in total 7
pairs of groups were taken. (3) To study details of the time course of
structural reorganization of the gizzard, 50 quail were fed the standard diet
for 4 weeks. At day 0 of the experiment, all quail were switched to a diet
containing 45% non-digestible fibre. After 14 days, quail were switched back
to the standard diet for another 14 days. The entire feeding trial lasted 28
days, equivalent to one diet-switching period in the previous experiment. Five
quail were killed before diet-switching. During the course of the experiment,
groups of five quail were killed at intervals of 15 days, and tissue
samples were taken from the gizzard of these quail. Further details about the
experiment are given in Starck
(1999b), where data from the
same experiment were published. Here, we use tissue material that had not been
analyzed previously. An exposure time of 14 days was chosen in all experiments
because previous studies had shown that this period allows for full
acclimation of the digestive system to the changed diet
(Kloss, 1996
; Starck, 1999;
Dekinga et al., 2001
).
Dissections and histology
Animals were killed by cervical dislocation, then immediately dissected
macroscopically and organ (gizzard, liver, small intestine) fresh masses
determined using a laboratory scale (precision 0.01 g). The organs were
preserved in 5% paraformaldehyde in 0.1 mol l1 phosphate
buffer, pH 7.4, 4°C, for at least 48 h. Gut length was measured on the
preserved material using an electronic slide calliper (precision 0.05 mm),
which avoided stretching artefacts that can occur with fresh tissue. To
measure the cross-sectional area of gizzard muscles we cut the preserved
gizzards longitudinally from the pylorus to the isthmus. An image of the two
sides was recorded with a video camera and the image stored on computer. The
area of the muscle was then measured using an image analysis program. For
histology, tissue samples of the gizzard, small intestine and liver were taken
from the preserved material, washed in buffer, dehydrated through a graded
ethanol series to 96% ethanol and embedded in hydroxyethyl methacrylate
(Historesin). Embedded material was sectioned into short series of 50 sections
per sample (section thickness 2 µm), mounted on slides and stained with
Methylene-Blue Thionin. Histological sections were studied using a Jenaval
research microscope (Zeiss, Jena, Germany) equipped with a video camera and
connected to the image-analysis and morphometry system. Microphotographs were
taken with a digital camera (Nikon Coolpix 990, Japan). We used SigmaScanPro
(Vers. 4.0, Jandel Scientific, SPSS Inc., Chicago, USA) for imaging and
morphometry.
Morphometry
We measured 40 sections per tissue sample and took three measurements of
muscle layer (tunica muscularis) thickness per section, as a straight line
from the inner to the outer margin of the muscle layer. Epithelial surface
magnification was measured as the epithelial surface over a baseline defined
by the inner circular muscle layer. Measurements were made by tracing the
epithelial surface with a cursor on a digitizing tablet and calculation of its
total length divided by the length of the baseline, expressed as a
dimensionless ratio. There is no standardized quantitative means of assessing
the vascularization of a tissue, i.e. the number and density of blood
capillaries in a tissue. Injection of microspheres would be one option, but
the results depend on blood pressure, blood flow velocity, blood flow volume,
flow dynamics, peripheral shunting and branching patterns of larger vessels.
We therefore measured the area of tissue in histological sections between the
mucosal epithelium and the muscle layer and related it to the sum of the area
of the capillaries under that particular epithelium, also expressed as a
dimensionless ratio. This procedure does not give absolute values, but a
relative measurement of the number, size and density of vessels in a given
volume of tissue and, as such, allows us to compare vascularization in the
experimental and the control groups.
Fixation of tissue in isotonic and buffered paraformaldehyde, dehydration
to 96% ethanol and embedding in methacrylate minimizes embedding artefacts,
but the procedure may result in 10% shrinkage of tissue compared to the
original size (Böck 1989).
Since all tissue samples were treated identically in the present study,
however, direct comparisons could be made.
Cell proliferation and cellular hypertrophy
The mitotic index in histological sections is an indication of cell
division, the number of mitotic cells per cell population being a direct
measurement of cell proliferation. In birds, intestinal cell proliferation of
the mucosal epithelium is restricted to the intestinal crypts. We counted
mitotic cells in 300 randomly chosen intestinal crypts per gut segment and
animal to obtain an estimate of cell proliferation activity in the particular
region of the gut of the individual animal.
Cellular hypertrophy leads to increased cell size. In the avian gizzard, smooth muscle cells are arranged in a parallel orientation. We measured the average cross-sectional diameter of smooth muscle cells by counting the number of cells/nuclei crossed by a 1 mm long line perpendicular to the smooth muscle cells. Tissue samples were obtained from five quail per experimental day and from each quail we counted 10 sections. For each bird an individual mean value was calculated before subjecting the values to statistical evaluation to avoid inflation of degrees of freedom.
Lipid extraction
Gizzard and liver were dissected, cleaned from adhering tissue and dried at
55°C to constant mass. Organ fat was extracted in a Soxhlet apparatus
using petroleum ether as solvent. For each sample we ran at least 25 cycles to
ensure complete extraction of lipids. Lean dry mass (LDM) was calculated as
organ fresh mass (water mass+fat mass).
Respirometry
For the metabolic measurements we used five quail that were adjusted to the
standard food diet. Measurements were made daily (with few exceptions) between
10 h and 16 h with quail that were post-absorptive for 2 h. Respirometry
started on day 12 of the experiment. On day 17, the diet was switched to 40%
non-digestible fibre, and on day 31, back to the standard diet. The
measurements were terminated on day 36. We measured oxygen consumption in an
open flow system (FOX Sable Systems, Henderson, NV, USA) at 30°C, i.e. the
thermoneutral zone of quail (Feuerbacher,
1981; Kloss,
1996
), in the dark for 90 min. The air stream (250 ml
min1) was dried (silica gel blue, Roth GmbH, Germany) before
entering the metabolic chamber (chamber volume 250 ml). The air stream vented
from the metabolic chamber was dried again before entering the oxygen
analyzer. We estimated resting metabolic rates (RMR) by taking the lowest
value that did not change during a 10 min interval for more than 0.01%
O2-concentration. Metabolic data were analyzed with the Sable
Systems Inc. software using Withers
(1977
) equation 3a (assuming a
respiratory quotient of 0.83).
Statistics
None of the variables differed from normal distribution. Values are means
± standard deviation (S.D.). We used univariate analysis of covariance
(ANCOVA) with body mass as covariate to evaluate the effect of diet on
gizzard, intestine and liver morphometric parameters. For analysis of food
intake we used repeated-measures (R-M) ANCOVA with body mass as covariate,
food composition as inter-subject factor, and day as within-subject factor. We
used SPSS Version 11.0 for the analysis of variance (ANOVA). For the analysis
of metabolic measurements we first calculated a linear regression of oxygen
consumption against time over the whole course of the experiments to assess
long-term trends. Regressions were calculated for all birds together and then
grouped by individual quail to assess individual reactions. As there was no
indication of any consistent trend over time we analyzed the three
experimental periods (standard diet, experimental diet or reassumed standard
diet) separately by repeated-measures restricted maximum likelihood estimation
(REML) with body mass as covariate. These analyses were performed using
Genstat, 5th edition.
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Results |
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During the acclimation period, quail ingested on average 14.2±2.8 g (N=12) standard food per day. During experiment 1, control birds ate on average 14.8±1.0 g (N=6) food each day, although they ate less food on the first day compared to the last 5 days (Fig. 2). Diet composition had a highly significant effect on food intake (R-M ANCOVA with body mass as covariate; d.f.=1,9; F=53.851; P<0.001; body mass was not significant as a covariate F=3.758, P=0.85; Fig. 2). After switching to the high-fibre diet, experimental quail reduced their food intake to less than 50% of the control amount (6.3±1.2 g, N=6). After 3 days on the high-fibre diet, the quails resumed food intake rates similar to the rate before the diet switch. Resumption of the intake rate coincided with stabilization and regain of body mass on day 6 of experiment.
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Gizzard size changes
Gizzard fresh mass in quail fed the high-fibre diet for 14 days was 182% of
gizzard mass in the control group. Gizzard length and cross-sectional area of
gizzard muscles of experimental birds were 140% of those of controls. Lean dry
fraction (LDF) of the gizzard was 126% of the control value, indicating that a
major portion of the size change was based on increased protein fraction,
while water and lipids did not contribute much to the fresh mass increase
(Table 1). After 4 weeks
feeding on the high-fibre diet, gizzard fresh mass was 230% and gizzard length
was 173% of the size in control birds. The LDF reached 263% of control values.
ANCOVA with body mass as covariate showed that diet composition was a
significant factor for all measured parameters. In no case was body mass
significant as a covariate. Post-hoc comparisons among means showed
highly significant differences between control and experimental groups for all
measured parameters (Table 1).
Gizzard fresh mass and gizzard length increased with exposure time, i.e. were
significantly larger after 4 weeks than after 2 weeks exposure time. The other
measurements were not affected by the length of the feeding trial.
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Gizzard histology
The muscles of the avian gizzard consist of layers of smooth muscle cells
separated by thin sheets of connective tissue
(Fig. 3) resulting in an
`onion-structure' of the gizzard muscle. Within each muscle layer, the smooth
muscle cells show the typical elongated spindle shape with the peripheral
compartment of contractile fibres and the perinuclear cytoplasm. The nucleus
is cigar-shaped, about five times as long as wide.
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We analysed tissue material from experiment 2, in which quail were repeatedly switched at biweekly intervals between standard and high-fibre diet. The control group was kept in parallel without any changes of diet. Animals from both groups were measured at 2-week intervals. We compared the effects of the following factors on number of nuclei in the gizzard muscle: the factor `repeat' tested for differences between the repeated diet switches, the factor `group' tested for overall differences between experimental and control group, and the factor `food' tested for differences between high fibre and standard diet. The effect of diet change was assessed by the interaction term between `food' and `group'. No interaction would indicate that the groups did not differ, despite the differences in diet. Significant differences would indicate an effect of diet on the number of nuclei. We discarded body mass as a covariate from the analysis, because it was not significant in any of the previous analyses of gizzard size changes. Repeat was a significant factor (univariate ANOVA, d.f.=3,47; F=3.66; P=0.019), thus food composition and the number of repeated diet switches affected the structural response. It was not an increased response (upregulation) that was stronger with repeated diet switches but the decreased response (downregulation), which did not always return to control values (Fig. 4). This observation was consistent with gizzard mass not returning to its original value during downregulation. After switching from standard diet to high-fibre diet, the number of nuclei mm1 declined significantly, indicating an increase in smooth muscle cross section (Fig. 4A). The experimental and control groups differed in their overall number of nuclei mm1. Gizzards of control birds had significantly more nuclei mm1 than the experimental birds. During the entire experimental period, the control group was unchanged. Overall, the effect of food was significant, with higher numbers of nuclei in birds fed the standard diet (d.f.=1,47; F=15.08; P<0.001). However, the interaction between food and group was highly significant (d.f.=1,47, F=22.79; P<0.001), indicating a massive difference between control and experimental birds. In the experimental birds, switching back to standard diet resulted in an increase of nuclei mm1, i.e. a decrease of muscle cell cross section (Figs 3AC, 4). Changes in both directions could be elicited repeatedly. At a more detailed level, we observed a decline in number of nuclei mm1 within 1 day of switching to the high-fibre diet (Fig. 4B). 5 days after diet switching, the number of nuclei mm1 was at 50% of the level of the control group, and it remained this low as long as the high-fibre diet was offered. When the food was replaced by the standard diet, the number of nuclei mm1 increased, indicating a decrease in cell size. Subsequent values were not significantly different from each other, but differences were significant when longer intervals were compared (univariate ANOVA with food and day as factors, post-hoc REGWQ test at P=0.01), e.g. comparisons between days 1 and 4 after diet switching in either direction rendered significantly different values (Fig. 4B). Despite the changes in gizzard size, we did not observe any mitotic structures in satellite cells. Thus, cell proliferation can be excluded as a possible source of organ size increase.
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Gut morphometry
The duodenum of quail fed the high-fibre diet for 14 and 28 days was 115%
and 130% longer, respectively, than the length in control birds
(Table 1). The length of the
jejunoileum was 112% of the control length after 2 weeks and 123% after 4
week, and the length of the rectum had increased after 2 weeks (125%) and 4
weeks (138%) of experimental diet. All reported length differences were
statistically highly significant between control and experimental groups
(ANCOVA with body mass as covariate; see
Table 1 for statistics).
However, gut lengths of quail fed the high-fibre diet for 2 and 4 weeks were
not significantly different (ANOVA, post-hoc REGWQ test;
Table 1), so for histological
examinations we used only tissue material from quail fed for 2 weeks on the
high-fibre diet. The circumference of the gut and the thickness of the muscle
layer (Tunica muscularis) changed in response to diet composition. Both
measurements in quail fed the high-fibre diet were significantly higher,
except for the thickness of muscle layer in the rectum, than in control birds
(ANCOVA with body mass as covariate; see
Table 2 for statistics). The
surface area of the small intestine of quail fed the high-fibre diet was
enlarged in all three segments of the intestine
(Table 2). Enlargement of the
duodenal surface of quail fed the 40% non-digestible diet was 130% of the
control, and the jejunoileum and rectum were 151% and 149% of control levels,
respectively (Table 2).
|
When compared to control birds, the number of mitotic cells per intestinal crypt of quail fed the high-fibre diet was 140% in the duodenum, 155% in the jejunoileum and 117% in the rectum. Differences between controls and experimental group were significant for all parts of the gut (Table 2).
Vascularization
The vascularization of duodenal and jejunoileum tissue in quail fed the
high-fibre diet was 205% and 214% of control birds, respectively. However, the
effect of food composition was only marginally significant (ANCOVA with body
mass as covariate; Table 2). An
elevated level of vascularization in the rectum of experimental birds (137%)
was not significantly different from the control.
Liver mass, histology and organ composition
Liver fresh mass decreased to 78% and 79% of control values in quail fed
the high-fibre diet for 2 and 4 weeks, respectively. The differences between
experimental birds and control were significant (ANCOVA with body mass as
covariate; Table 3). The LDF of
the liver did not change in response to food composition; a slight decline in
LDF during a 2 week exposure to high fibre was not significant. Lipid content
was significantly lower in experimental animals than in control birds. Thus,
differences in liver fresh mass were based on changes in the lipid (and water)
components of liver but not on the protein component. Change in cell numbers
can be excluded as a cause of change in organ size.
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Metabolic rates
We measured oxygen consumption in five quail over a period of 24 days.
Measurements started 5 days prior to switching from standard food to the
high-fibre diet. Daily measurements continued during the 14 days of feeding
the high-fibre diet, and for another 5 days after switching back to standard
food (Fig. 5). We observed a
decline in metabolic rate just before diet switching that was significant when
analyzed by repeated measures REML of oxygen consumption over the 4 days of
pre-treatment with body mass as covariate: effect of day, Wald-statistics =
5.29 on 3 d.f., P=0.001; covariate body mass, Wald-statistics = 3.02
on 1 d.f., P=0.082). In addition, individual quail clearly differed
from each other (differences between quail, Wald-statistics = 13.15 on 4 d.f.,
P<0.001). This metabolic decline was associated with the birds
acclimating to the experimental conditions. There were no significant changes
in resting metabolic rate (RMR) after birds switched diets (oxygen consumption
over the 14 days of food treatment: effect of day, Wald-statistics = 1.19 on 9
d.f.; P=0.298; covariate body mass, Wald-statistics = 0.87 on d.f. 1;
P=0.351; oxygen consumption over the 6 days after shifting back to
the standard diet: effect of day, Wald-statistics = 1.54 on 5 d.f.;
P=0.173; covariate body mass, Wald-statistics = 0.69 on 1 d.f.;
P=0.406). In contrast, the differences between individual quail
remained stable (oxygen consumption over the 14 days of food treatment: effect
of quail, Wald-statistics = 15.51 on 4 d.f.; P<0.001; over the 6
days after shifting back to standard diet: effect of quail, Wald-statistics =
5.03 on 4 d.f.; P<0.001). The mean oxygen consumption of the five
quail among the three experimental intervals showed no effect of experimental
interval (pre-treatment, treatment, post-treatment) on oxygen consumption but,
again, clearcut differences between individual quail (effect of experimental
interval, Wald-statistics = 0.65 on 2 d.f.; P=0.521; effect of quail,
Wald-statistics = 2.58 on 4 d.f.; P=0.035; effect of covariate body
mass, Wald-statistics = 0.51 on 1 d.f.; P=0.476). This analysis
showed clearly that oxygen consumption differed between quail and was affected
by initial acclimation but not by the change of diet. Thus, once the animals
had successfully habituated to the experimental conditions, the resting
metabolic rates were constant throughout the experiment.
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Discussion |
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Normal or elevated intake rates were re-established after 6 days, i.e. when
organ size had been enlarged to accommodate the functional demands of the
changed diet composition. The amount and period of decline of food intake and
the later increase of intake rates were similar to those reported by Starck
(1999b) for a diet containing
45% non-digestible fibre. Because of the reduced intake, it is not the load to
the gastrointestinal tract that causes enlargement of the gizzard but the
quality of the food (reduced energy content, increased coarseness) that
triggers the observed phenotypic changes of the gastrointestinal tract.
Increased food intake only follows after the digestive tract has been
structurally reorganized and can accommodate increased loads of digesta.
Flexible changes of the gastrointestinal tract
Gizzard
The observed size changes of the muscular stomach support our earlier
findings (Starck, 1999b). Mean
gizzard mass was 4.8±0.5 g after 2 weeks experimental exposure, which
is within the prediction limits of 5.3±0.9 g
(Starck, 1999b
).
Interestingly, gizzard size continued to increase over a period of 4 weeks,
reaching a maximum size of 6.01±0.5 g. This adds a new perspective to
our earlier report that the maximum possible gizzard size in Japanese quail
was 6 g when fed a diet containing 45% non-digestible fibre for 2 weeks
(Starck, 1999b
). In the
present study, we found that an extended feeding period resulted in a further
increase in gizzard size, even when the original fibre-load was not strong
enough to elicit the maximum response. We conclude that the flexible response
of gizzard size is determined by a combination of diet quality and exposure
time. Extended exposure time causes continued organ growth until the upper
ceiling is reached.
Increase in gizzard mass was associated with an increase in lean dry fraction (LDF), while lipid proportions did not change. Changes in gizzard size were the result of changes in smooth muscle cell size, i.e. hypertrophy and cellular atrophy during enlargement and reduction, respectively. We did not detect any increase in mitotic index in several hundred histological slides examined. Therefore, we conclude that the increased gizzard size is solely due to hypertrophy of smooth muscle cells. Hypertrophy and cellular atrophy could be elicited repeatedly and were synchronized with changes in organ size.
Intestines
The high-fibre diet had a decreased energy content and increased coarseness
compared to standard food. The digestive modulation model
(Karasov, 1990;
Martinez del Rio et al., 1994
)
predicts an increase of intestine size (length, circumference, surface
magnification) in response to decreasing food quality. Taking the different
segments of the gastro-intestinal tract as cylinders, we calculated a volume
of 1821 mm3 for quail fed standard food, and 2795.6 mm3
for experimental animals, which is in agreement with the digestive modulation
model. The associated increase in vascularization indicates that `downstream'
transport mechanisms respond in concert with the mucosal epithelium. Increased
muscle layer thickness is certainly related to increased bulk.
The elevated levels of cell proliferation in the intestinal crypts after 2
weeks on the high-fibre diet show that enlargement of the mucosal epithelium
is based on proliferation of new cells. In birds, we do not observe apoptosis
at the tip of the villi. The extrusion rate of epithelial cells cannot be
measured directly, but we must expect that cell extrusion will also be
elevated because it balances cell proliferation as soon as the upregulated
state is reached. To our knowledge, this is the first study to examine changes
in mitotic index in bird intestine in response to diet switching, so our
conclusions are restricted to quail. For mammals, increased cell proliferation
(rats and sheep; Sakata and Engelhardt,
1983; Engelhardt et al.,
1989
) as well as a change in apoptosis rate (pigs;
Raab et al., 1998
;
Mentschel et al., 2001
) were
reported to be mechanisms that respond to diet switching and change the
mucosal surface area. In mice/rats, increasing the fibre content of food
induced mitosis in the small intestine
(Sakata and Engelhardt, 1983
;
Jacobs and Lupton, 1984
;
Engelhardt et al., 1989
;
Goodland et al., 1989
;
Fleming et al., 1992
). We
observed a similar pattern in quail but the underlying regulatory mechanisms
remain unresolved.
Liver
Liver fresh mass declined during the experiment, but this was exclusively
based on declining lipid content of the liver. LDF of the liver was constant
during the experiment, indicating that no cell proliferation had occurred.
This makes immediate sense because the liver is an intermediate store for
lipids, which were obviously metabolized to fuel the upregulation of the
gastrointestinal system. Interestingly, the lipid content of the liver of
quail fed the high-fibre diet for 4 weeks was intermediate between the levels
in controls and in quails fed the high-fibre diet for only 2 weeks. The 4-week
group could not be distinguished statistically from the others. A possible
interpretation is that a 4 week period allows time for better overall
acclimation of the digestive system to the high-fibre diet and for
reestablishment of a nutritional status equivalent to that on the standard
diet.
Metabolic rates
Diet switching did not significantly affect RMR and mean oxygen consumption
was 1.19±0.31 ml O2 h1
g1. Our measurements are the same as the mean values
reported by Kloss (1996), who
measured RMR of adult (male) quail in the thermoneutral zone as
1.18±0.22 ml O2 h1 g1.
She also compared quail fed different diets and showed that RMR was constant,
although digestive efficiency as well as morphological parameters of the
digestive tract changed. The constancy of RMR is an interesting contrast to
the flexible metabolic rates of growing quail
(Schew, 1995
;
Schew and Ricklefs, 1998
).
When young quail were temporarily malnourished their RMR decreased by 40%. The
young of songbirds also lowered their RMR when subjected to periods of fasting
or malnutrition (Konarzewski and Starck,
2000
; Brzek and Konarzewski,
2001
). There are two possible interpretations: (1) at some time
during ontogeny quail lose the flexibility of RMR, or (2) quail do not lose
their flexible RMR, but our experimental conditions did not meet the
requirements necessary to elicit a reduced RMR in adults. If flexible
responses to changes in diet are condition dependent, we may hypothesize that
well-fed quail simply use their adipose tissue stores to fuel the flexible
responses of their gastrointestinal tract. That way, they tolerate a
temporarily negative energy budget for a few days but they keep their RMR
constant. If, however, quail were in a poor condition, they might have no fuel
for the flexible responses and might have to reduce energy expenditure to
other organ systems, thus reducing their RMR. Both ideas need to be tested
before final conclusions can be drawn. Condition dependency of metabolic
response is indirectly supported by a study of emperor Aptenodytes
forsteri and king penguins A. patagonicus, which reduce RMR only
after extended fasting periods before entering starvation, characterized by
mobilization of body protein (Groscolas
and Cherel, 1992
). Also, Klaassen and Biebach
(1994
) showed that garden
warblers Sylvia borin reduce RMR during starvation.
Overall conclusions about organ size flexibility
The data presented in this study show that different processes account for
the organ size changes of gizzard, intestine and liver. In the gizzard, size
changes are based on up-(cellular hypertrophy) and downregulation (cellular
hypotrophy) of cell size. In contrast, size changes of the mucosal epithelium
of the intestine are based on changes in the balance of cell proliferation and
cell loss, and involve increased intestinal crypt cell proliferation. Finally,
liver size changes are based on changes in organ lipid contents. The costs of
flexibility of the digestive system are paid by metabolizing body fuel, while
the RMR stays constant. The condition dependency of the responses still needs
to be tested.
The limited amount of available data restricts a comparative perspective.
Studies on epithelial renewal of small intestine in birds and mammals showed
that a continuous proliferation of cells in the intestinal crypts not only
drives the renewal of the epithelium, but also allows for fast and reversible
changes of mucosal epithelial surface (Starck,
1996a,b
,
1999a
,b
).
Our data as well as published data on mammals indicate that elevated levels of
cell proliferation are important in increasing mucosal epithelium surface, but
rates of cell loss have not yet been examined.
Note that ectotherm sauropsids also show a considerable up- and
downregulation of their gastrointestinal system in response to feeding, but
that the underlying tissue mechanisms are different. In snakes (Starck and
Beese, 2001,
2002
) and crocodiles
(Starck et al., 2002
), the
pseudostratification of the mucosal epithelium allows for rapid, reversible
and repeated up- and downregulation of the small intestine capacity. The
tissue mechanism of upregulation in ectotherm sauropsids is energetically
cheap. The different mechanisms observed in ectotherm sauropsids, birds and
mammals suggest independent evolutionary origins of the flexibility of the
gastrointestinal system in each of the three taxa.
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