Changes in Growth and Function of Chick Small Intestine Epithelium
Due to Early Thermal Conditioning
Z. Uni,*,1 O. Gal-Garber,* A. Geyra,* D. Sklan,* and S. Yahav†
*The Faculty of Agricultural, Food and Environmental Quality Sciences, Department of Animal Sciences, Rehovot, Israel; and
†Institute of Animal Sciences, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel
pression and activity of BBM enzymes. The association
between ambient temperature, feed intake, growth rate,
and plasma T3 levels was reflected in the structure and
function of the intestinal tract. The results suggest that
thermal conditioning at an early age influences T3 concentrations, which in turn alter the intestinal capacity to proliferate, grow, and digest nutrients. However, these experiments were not able to discriminate between effects
due to feed intake and those due to thermal conditioning.
The treatments modulated changes in the intestinal tract
following thermal treatment.
ABSTRACT The effect of exposure to heat at 3 d of age
on small intestine functionality and development was
assayed by measuring villus size, proliferating enterocytes, and brush-border membrane (BBM) enzyme expression and activity. Results showed that thermal conditioning caused an immediate effect characterized by lowered triiodothyronine (T3) level, reduced feed intake, and
depressed enterocyte proliferation and BBM enzyme activity. A second series of effects, observed 48 h posttreatment, was characterized by elevated T3, increased feed
intake, increased enterocyte proliferation, and higher ex-
(Key words: small intestine, thermal conditioning, growth)
2001 Poultry Science 80:438–445
during their life span (Arjona et al., 1988, 1990; Yahav and
Hurwitz, 1996; Yahav et al., 1997). Short-term exposure to
heat stress during the first week of life results in growth
retardation followed by immediate compensatory growth
with higher feed intake that completely counteracts the
decrease in weight gain (Yahav and Hurwitz, 1996; Yahav
et al., 1997; Yahav and Plavnik, 1999). Early-age thermal
conditioning thus resulted in a significant and sustained
reduction in plasma T3 levels (Yahav and Plavnik, 1999).
A positive linear correlation has been found in chickens
and turkeys between plasma triiodothyronine (T3) levels
and feed intake and also with growth rate (Klandorf and
Harvey, 1985; Yahav et al., 1995, 1996, 1998). Plasma concentration of T3 is also inversely related to environmental
temperature (Hillman et al., 1985; May et al., 1986; Iqbal et
al., 1990), probably as a result of modulation of peripheral
deiodination (Rudas and Pethes, 1984; Kuhn et al., 1987).
The association between ambient temperature, feed intake, growth rate, and plasma T3 levels may be associated
with changes in the structure and function of the intestinal
tract as suggested by Mitchell and Carlisle (1992).
The effect of environmental thermal changes during
the first 10 d posthatch on the small intestine has not been
described. It can be hypothesized that environmental heat
INTRODUCTION
The epithelium of the small intestine is composed of a
continuously renewable population of cells. The stem
cells, located in the crypt region, produce enterocytes that
migrate up the villi and are extruded at the villus tip into
the intestinal lumen. Previous reports have shown that
the continuous proliferation, migration, differentiation,
and maturation of the crypt stem cells is regulated by a
variety of factors, including luminal nutrients, trophic
gastrointestinal hormones, growth factors, and cytokines
(Potten and Loeffler, 1987; Henning et al., 1994; Johnson
and McCormack, 1994).
In chickens, the small intestine of the newly hatched
chick undergoes maturation and dramatic morphological,
biochemical, and molecular changes during the first 10 d
posthatch (Uni et al., 1996, 1998a,b). In a manner similar
to other vertebrates, such as lamprey, amphibian tadpole,
and turtle (Wurth and Mussachia, 1964; Marshall and
Dixon, 1977; Hansen and Youson, 1978), the proliferation
of intestinal epithelial cells in chickens is not restricted
to the crypt but also occurs along the villus during the
first week posthatch (Uni et al., 1998a).
It is well documented that thermal conditioning at an
early age modulates responses of chickens to heat stress
Abbreviation Key: ALP = alkaline phosphatase; AP = aminopeptidase; BBM = brush-border membrane; GAPDH = glyceraldehyde-3phosphate dehydrogenase; PCNA = proliferating cell nuclear antigen; SI
= sucrase-isomaltase; TC = thermally conditioned; T3 = triiodothyronine.
Received for publication May 11, 2000.
Accepted for publication October 31, 2000.
1
To whom correspondence should be addressed: [email protected].
438
CHANGES IN GROWTH AND FUNCTION
stress at an early age will alter intestinal mucosal morphology and influence the proliferation and differentiation of intestinal epithelial cells. The purpose of the present study was to examine the effect of thermal conditioning on small intestine growth and function by measuring
villus size, proliferating enterocytes, and brush-border
membrane (BBM) enzyme expression and activity. In the
present experiment, the association between T3 and small
intestinal function is examined.
MATERIALS AND METHODS
Animals and Experimental Design
Male broiler chickens (Cobb) were obtained from a
commercial hatchery. Chicks were raised in battery
brooders in a temperature-controlled room under standard brooding conditions (Yahav et al., 1996). The experiment included (a) a nontreated control group (n = 80)
and (b) a thermally conditioned (TC) group exposed for
24 h to 36 ± 1 C and 70% relative humidity at the age of
3 d (n = 80), after which birds were returned to the battery
brooder and raised under regular conditions. Water and
feed were provided ad libitum. Feed in mash form was
formulated according to National Research Council (1994)
requirements. At daily intervals, BW and blood samples
were taken on an individual basis. The BW was recorded
for all birds, whereas blood was taken from 10 chicks per
treatment. On Day 3 birds were bled prior to TC treatment, and birds bled on Day 4 were bled at the end of
TC treatment. A sample of whole blood was taken from
the brachial vein and centrifuged at 3,000 rpm for 10 min.
The plasma was stored at −20 C for further analysis.
Tissue Sampling
On Days 3, 4, 5, 6, and 7 posthatch, chicks were killed
with an intercardiac overdose of sodium pentobarbital
(0.2 g/kg). From each chick, the jejunum (from the end of
the duodenum to Meckel’s diverticulum) was removed.
Segments (1 cm long) were taken from the middle of the
section, flushed twice with cold PBS, and placed in three
separate tubes: 1) frozen in liquid nitrogen and then
stored at −80 C for RNA expression analysis, 2) fixed in
a 4% neutral-buffered formalin solution for histology and
staining, and 3) stored at −20 C for determination of BBM
enzyme activity.
T3 Analysis
Radioimmunoassay (RIA) of T3 was carried out on
plasma samples using a diagnostic kit2 with an intra-
2
Coat-A-Count-Canine T3, Diagnostic Products Corporation (DPC),
Los Angeles, CA 90045-5597.
3
Zymed Labs, San Francisco, CA 94080.
4
Photoshop 4.0, Adobe System Incorporation, CA 95110.
5
NIH Image, Bethesda, MD 20892.
6
Sigma Chemical Co., St. Louis, MO 63178-9916.
7
BioRad, Hercules, CA 94547.
439
assay variation of 5.0 to 5.9%. The DPC kit was previously
validated for domestic fowl (Yahav et al., 1998) as described by Bar and Hurwitz (1979).
Histology and Proliferating Cell
Nuclear Antigen Staining
Jejunal sections were fixed in a 4% formalin-buffered
saline solution and embedded in paraffin. All histological
studies were performed on 5-µm sections, using standard
procedures with uniform conditions of fixation and staining with hematoxylin and eosin. Staining for proliferating
cell nuclear antigens (PCNA)3 was as previously described (Greenwell et al., 1991; Uni et al., 1998a). Morphometric indices were determined by computer analysis.4,5
The morphometric variables included villus height
(from the tip of the villus to the villus-crypt junction),
villus width (the width at half height), and number of
enterocytes in the G1-S-G2 phase, expressed as the number of PCNA-positive cells per 100 cells. The percentage
of proliferating cells in the crypt was estimated in 10
crypts from each chick as the mean number of PCNApositive nuclei of total crypt epithelial cells. The percentage of proliferating cells in the villus was estimated from
10 villi from each chick as the number of PCNA-positive
cells in a villus column per total number of cells in that
column.
Villus volume was calculated as a cylinder from villus
height and width at half height. The results of the morphometric determinations were from at least 10 well-oriented crypt villus structures from each chick. These data
were then pooled to calculate the mean value of each
variable, and expressed as mean ± SE.
Enzyme Activity Assays
Enzyme activities were assayed in homogenized jejunal
tissue (250 mg tissue/5 mL of 50 mM sodium phosphate
buffer, pH 7.2). Sucrase (EC 3.2.1.48) activity was assayed
colorimetrically by using sucrose as a substrate (Dahlquist, 1964; Palo et al., 1995b) and was expressed as mmole
glucose released per 1 min/g of jejunal protein. Aminopeptidase (AP) activity (EC 3.4.11.2) was determined by
hydrolysis of L-leucine-p-nitroanilide6 to p-nitroanilide
and L-leucine for 15 min at 37 C. The p-nitroanilide was
determined spectrophotometrically at 405 nm according
to Maroux et al. (1973), and AP activity was defined as
the production of 1 µmol p-nitroanilide/min per g jejunal protein.
Alkaline phosphatase (ALP) activity was determined
by measuring the hydrolysis of p-nitrophenol spectrophotometrically (Sigma kit 104;6 Palo et al. 1995b). One
unit of ALP activity was defined as the production of 1
µmol nitrophenol/min per g jejunal protein. Total protein
was determined using the BioRad7 protein assay for protein concentration following detergent solubilization.
440
UNI ET AL.
TABLE 1. Effect of thermal conditioning (TC) at the age of 3 d on BW, feed intake and plasma
Triiodothyronine (T3) concentration of male broiler chicks
BW (g)1
Age
Control
(d)
3
4
5
6
7
80.7
96.7
115
137
162
±
±
±
±
±
0.69
0.58a
1.53a
1.52
2.28
Feed intake (g/d)2
TC
80.3
93.6
113
135
157
±
±
±
±
±
0.44
0.53b
0.75b
1.78
1.88
Control
TC
NT3
25.5
31.9
36.6
37.7
NT3
23.1
24.7
41.0
43.0
±
±
±
±
0.58a
1.44a
0.24b
0.96b
Plasma T3 (pg/ml)
Control
±
±
±
±
0.58b
0.26b
1.33a
1.36a
2,652
2,403
2,463
2,237
2,335
±
±
±
±
±
TC
98
139a
88a
212b
119b
2,671
1,944
2,080
2,818
2,678
±
±
±
±
±
73
160b
89b
57a
94a
Values followed by different letters differ significantly (P < 0.05).
The measurement of BW was determined in the morning between 0600 and 0800 h.
2
Feed intake was measured at 0600 to 0800 h for the previous 24 h.
3
Not tested.
a,b
1
RNA Isolation, Probe Preparation,
and Dot-Blot Hybridization
Briefly, total RNA was isolated from chicken jejunal
tissue using TRI-REAGENT-RNA/DNA/protein isolation reagent8 according to the manufacturer’s protocol.
The integrity of the RNA was verified by ethidium bromide staining, and the RNA concentration was determined spectrophotometrically. About 10 µg mRNA from
each sample was spotted on Hybond-N nylon membrane
for dot-blot analysis, according to Amersham’s protocol.9
Prehybridization (42 C), hybridization (42 C), and washing (57 C) steps were according to the procedures recommended by Amersham for Hybond-N membranes. To
examine the RNA expression of AP, a 522-bp cDNA fragment from the chicken intestine AP gene (Gal-Garber and
Uni, 2000) was used as a probe. For examining the RNA
expression of sucrase-isomaltase (SI), a 786-bp cDNA
fragment from the chicken intestine sucrase-isomaltase
gene (Uni, 1998) was used as a probe. The amount of
total RNA per spot was determined through rehybridization with a probe for the constitutively expressed transcript for glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). The three probes were labeled with 32P-dCTP
by the random prime labeling method.10 After a highstringency wash (0.1X SSC/0.1% SDS at 57 C), blots were
exposed for 24 h at −70 C to film11 in the presence of an
intensifying screen. The abundance of SI and AP transcripts was normalized to the density of GAPDH transcripts by laser densitometer scanning.
the main effects were reduced to chicken, treatment, and
residual error term. Treatment means were compared by
t-test, and values were considered statistically different
at P < 0.05. Results are reported as means with their
standard error.
Statistical Analysis
All results were analyzed using the general linear models procedure of SAS (1986). The model included the main
effects of pen, animals within pens, heat treatment, and
a residual error term. Because there was no effect of pens,
8
TRI-REAGENT Molecular Research Center, Inc., Cincinnati, OH
45212.
9
Amersham, Arlington Heights, IL 60005.
10
Promega Corp., Madison, WI 53711-5399.
11
XAR-5 film, Eastman-Kodak, Rochester, NY 14650.
FIGURE 1. In vivo effect of thermal conditioning at 3 d posthatch
on the percentage of proliferation cell nuclear antigen (PCNA)-positive
cells in the crypts (A) and villi (B). Results are means, and bars are SE
from six chicks. Bars are shown when they do not fall within the symbols.
CHANGES IN GROWTH AND FUNCTION
RESULTS
BW and Feed Intake
Prethermal conditioning BW (Day 3) was similar in
both treatments (Table 1). After the thermal treatment,
BW was significantly reduced in the TC chickens as compared to control chickens. This pattern continued until 5
d of age. Feed intake was also reduced in TC chicks during
the thermal treatment until 5 d of age. Thereafter, feed
intake of TC chicks increased significantly relative to that
of control chicks.
Effect of Thermal Conditioning
on Plasma T3 Levels
Plasma T3 concentrations also were similar in both
groups before initiation of thermal conditioning (Table 1).
After thermal treatment, plasma T3 concentrations were
significantly reduced in treated chicks. After 5 d this pattern changed, and plasma T3 concentration became significantly higher in the TC chicks on Days 6 and 7.
Effect of Thermal Conditioning on
Epithelial Cell Proliferation
The proliferative status of crypts and villi was evaluated by determining the percentage of PCNA-positive
441
cells. At 4 d of age, immediately after heat exposure, a
rapid reduction in the percentage of PCNA-positive crypt
cells was observed in the TC treatment as compared to
the control group (40 vs. 63%) (Figure 1A). This reduction
continued, so that at 24 h postthermal treatment (at 5 d
of age), only 26% of the crypt cells were PCNA-positive
compared to 58% in the control group. Similarly, in the
villi, the percentage of PCNA-positive cells in the TC was
half that of the control chicks (13 vs. 25%) at 5 d of age
(Figure 1B). A rapid elevation in the percentage of PCNAstained cells then occurred between 24 and 48 h after
thermal conditioning in the crypts and villi (Figure 1)
before decreasing to control levels on Day 7.
Effect of Thermal Conditioning
on Villi Dimensions
To examine the effects of the thermal treatment on small
intestinal mucosal morphology, jejunal sections stained
with hematoxylin-eosin were examined by light microscopy. Jejunal sections obtained from control chicks
showed the normal pattern of mucosa with relatively
wide villi, narrow spaces between them, and elongated
columnar epithelial cells. In contrast, the jejunal mucosa
from thermally treated chicks 24 h posttreatment exhibited primarily narrow villi with wide spaces between
them (Figure 2). Villus volume in the TC chicks was significantly lower 24 h posttreatment (Day 5) than that in
FIGURE 2. Representative light microscopy (400×) of intestinal villi from the jejunum of chicks at 6 d of age. (A) Thermal conditioning group;
(B) control group. Sections were immunostained for proliferating cell nuclear antigen (PCNA) and counter-stained with hematoxylin. The dark
nuclei are PCNA-positive cells in mid-G1, S-phase, and G2 of the cell cycle. CPT = crypt region, G = goblet cell, E = enterocyte cell, PE = PCNApositive enterocyte, IEL = intestinal epithelial lymphocyte. Nearly 50% of the cells in A are proliferating (dark staining), whereas in B only a few
of the cells are designated as PCNA-positive. Bar = 10 µm.
442
UNI ET AL.
levels, reduced feed intake, and depressed enterocyte proliferation and BBM enzyme activities. In addition, there
was a second series of effects observed 48 h posttreatment,
which were characterized by elevated plasma T3 levels,
increased feed intake, increased enterocyte proliferation,
and higher expression and activity of BBM enzymes.
FIGURE 3. In vivo effect of thermal conditioning (TC) at 3 d posthatch
on the jejunal villus volume. Results are means and bars are SE from
six chicks. Bars are shown when they do not fall within the symbols.
C = control.
control chicks. However, a rapid elevation in villus volume size was observed thereafter. Significantly higher
villus volume was measured on Days 6 and 7 (48 to 72
h postthermal treatment) in the TC chicks (Figure 3).
Effect of Thermal Conditioning
on Sucrase, AP, and ALP Activities
The effect of thermal conditioning on jejunal BBM enzyme activities is shown in Figure 4. Sucrase specific
activity (Figure 4A) had not changed significantly at 5 d of
age but was markedly elevated 48 and 72 h posttreatment
(Days 6 and 7, respectively) by 60 and 45%, respectively.
On Day 5 (24 h postthermal treatment) AP- and ALPspecific activity in the jejunum of the TC group decreased
by approximately 30 and 50%, accordingly (Figure 4 B,C),
as compared to control chicks. This trend changed 48 and
72 h posttreatment (Days 6 and 7) when AP increased by
15 to 20% (Figure 4B) and ALP increased by ∼30% (Figure
4C) in the TC group as compared to the control group.
Effect of Thermal Conditioning on SI
and AP mRNA Expression
Figure 5 shows representative dot blots from the TC
and C groups and the change in the RNA expression
pattern of two BBM enzymes. On Day 5 (24 h postthermal
treatment) the AP mRNA levels decreased by approximately 70% (Figure 5B,E), whereas SI mRNA expression
levels increased 2.3-fold (Figure 5A,D). At 72 h postthermal treatment (Day 7) both BBM enzymes, AP and SI,
showed higher RNA expression in the TC birds than in
the controls.
DISCUSSION
In this study, thermal conditioning had immediate effects, which were characterized by lowered plasma T3
FIGURE 4. Effect of thermal conditioning treatment on jejunal activity
of sucrase (A), aminopeptidase (B), and alkaline phosphatase (C). Columns with different letters differ significantly (P < 0.05) between thermally conditions (TC) and control (C) groups. Results are means and
bars are SE from six chicks.
CHANGES IN GROWTH AND FUNCTION
443
FIGURE 5. Representative dot-blot analysis of two brush-border membrane enzymes in the jejunum of chicks from groups control (C) and
thermally conditioned (TC) at Days 4, 5, 6, and 7 posthatch. Panel A: Expression of sucrase-isomaltase (SI) in dot-blot analysis by using the 786bp jejunum reverse-transcriptase polymerase chain reaction (RT-PCR) product as a probe in a representative run. Panel B: Expression of aminopeptidase in dot-blot analysis using the 522-bp jejunum RT-PCR product as probe in a representative run. The same blots were rehybridized with
radioactive glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference probe (Panel C). Exposure time was 24 h at −70 C. (Panel D) The
ratio of sucrase-isomaltase (SI) to GAPDH. (Panel E) The ratio of aminopeptidase (AP) to GAPDH. Results are means ± SE of six replicates per
treatment. *Columns with significant differences (P < 0.05) between TC and C groups.
The chicks’ immediate response to thermal conditioning consisted of a 25% reduction in proliferating cells in
the crypts and a decrease in villus volume. These reductions were completely reversed during the recovery stage
(48 h postthermal conditioning), in which almost 40% of
the villus cells in the TC group were PCNA-positive. This
change might have been due to the accelerated rate of
mitotic activity in the crypts, and, as the rate of proliferation accelerated, cells reached the villus before completely
maturing. At 72 h postthermal treatment, the increased
number of cells in the villus underwent hypertrophy,
which led to elongation of the villus and resulted in differences in the villus volume between TC and control
groups.
The BBM enzymes were also affected by the thermal
treatment. Two possible mechanisms may affect BBM en-
zymes expression in the small intestine: a) primary regulation at the mRNA level and b) an increased rate of
turnover of the enzyme. In this study, the higher SI and
AP mRNA expressions during the recovery stage contribute to the synthesis of new SI and AP proteins and an
elevation in the digestive and absorptive capacity accordingly. However, different patterns of RNA expression
were found for AP and SI. Previous reports have shown
differential regulation of BBM enzymes; for example Torp
et al. (1993) reported that in pig small intestine, BBM AP
and lactase-phlorizin are differentially regulated.
In our study, changes in plasma T3 concentrations occurred with the thermal treatment, which were associated
with alteration in the activities of intestinal ALP and AP.
These results are in agreement with previous studies that
demonstrated effects of T3 on expression, activity, and
444
UNI ET AL.
regulation of BBM enzymes and transporters (Hodin et
al., 1992; Giannella et al., 1993; Jumarie et al., 1996). In
the rat, T3 induced AP expression at the transcriptional
level mediated by DNA cis-elements located within the
2.4-kb segment of the reporter gene (Hodin et al., 1996).
Moreover, T3 increased ATPase mRNA expression in rat
jejunal tissue via its response element located at the beta1-subunit of the Na-K-ATPase sequence (Matsumura et
al., 1992; Giannella et al., 1993).
A significant linear correlation has been demonstrated
between plasma T3 concentration and feed intake (Klandorf and Harvey, 1985; Yahav et al., 1996, 1998). This relationship leads to the conclusion that changes in small
intestinal morphology and enterocyte dynamics could result directly from changes in T3 levels or, alternatively,
via changes in feed intake that then influence T3 concentration.
It has been shown that T3 administration, by injection
or orally, increases crypt cell proliferation, BBM enzyme
activity, and small intestinal mucosal thickness (Tutton,
1976; Wall et al., 1970; Watson and Tuckerman, 1971;
Hodin et al., 1992; 1996). From our findings it can be
speculated that environmental heat or lower feed intake
influenced T3 levels that, in turn, might have altered the
intestinal capacity to proliferate, grow, and probably
absorb.
One result of thermal conditioning at an early age in
chicks is compensatory villus volume growth. Indeed,
we observed a greater surface area and higher mRNA
expression and activity of BBM enzymes in the small
intestine of the TC group in the second (compensatory
growth) phase, indicating higher digestive capacity of the
small intestinal tissue. These changes may explain the
changes in growth retardation followed by accelerated
growth, which were observed in previous experiments
by Yahav and Hurwitz (1996) and Yahav et al. (1997).
In other studies, adjustment of the small intestine to
different nutritional and environmental conditions has
been demonstrated. In migrating birds, the digestive tract
is enlarged to allow high rates of food processing (Piersma
and Lindstrum, 1997). Changes in the digestive organs
size may be induced by offering feeds with different textures (Piersma et al., 1996), by feed restriction (Palo et al.,
1995a), or by exposure to chronically elevated ambient
temperature (Mitchell and Carlisle, 1992). Feed restriction
has been found to change hormonal concentrations
(McMurtry et al., 1988), fat metabolism (Rosebrough et
al., 1986; Zhong et al., 1995), and digestive enzyme activity
(Palo et al., 1995b).
The current research demonstrates the effect of thermal
conditioning on gastrointestinal growth and function and
suggests a possible mechanism for this effect. However,
it should be mentioned that the effects due to thermal
treatment or due to feed intake were not separated in the
present experiment.
The thermal treatment was performed at a critical period of gastrointestinal tract development, when the main
process in the developing tissue is hyperplasia and before
the tissue reaches an equilibrium state of continuous cell
proliferation, migration, and differentiation (Uni et al.,
1996; Uni., 1999). It is suggested that heat conditioning
at 3 d posthatch alters T3 levels, which affects the small
intestine by changing cell proliferation and BBM enzyme
expression and activity. These changes modulate the intestinal tract for compensatory growth 48 h posttreatment.
ACKNOWLEDGMENT
This research was supported by a grant from the Binational Agricultural Research and Development Fund
(BARD).
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