Published December 8, 2014
Intestinal morphology and enzymatic activity in newly weaned pigs fed
contrasting fiber concentrations and fiber properties1
M. S. Hedemann,*2 M. Eskildsen,* H. N. Lærke,* C. Pedersen,†3 J. E. Lindberg,†
P. Laurinen,‡ and K. E. Bach Knudsen*
*Department of Animal Health, Welfare and Nutrition, Danish Institute of Agricultural Sciences,
Research Centre Foulum, 8830 Tjele, Denmark; †Swedish University of Agricultural Sciences,
Department of Animal Nutrition and Management, SE-750 07 Uppsala, Sweden;
‡MTT Agrifood Research Finland, Animal Production Research, FIN-05840 Finland
ABSTRACT: The main objective of this study was to
determine the effect of fiber source and concentration on
morphological characteristics, mucin staining pattern,
and mucosal enzyme activities in the gastrointestinal
tract of pigs. The experiment included 50 pigs from 10
litters weaned at 4 wk of age (BW 8.6 ± 1.4 kg) and
divided into 5 treatment groups. Diets containing fiber
of various physico-chemical properties and concentrations were formulated to contain 73, 104, or 145 g of
dietary fiber/kg of DM. The diets were based on raw
wheat and barley flours. Pectin and barley hulls, representing soluble and insoluble fiber sources, respectively, were used to increase the fiber concentration.
The pigs were fed the experimental diets for 9 d, and
then the pigs were euthanized and the entire gastrointestinal tract was removed. Tissue samples were taken
from the mid and distal small intestine and from the
mid colon. Inclusion of pectin in the diets significantly
decreased (P < 0.001) ADFI and ADG compared with
pigs fed no pectin. The villi and the crypts were shorter
in pigs fed pectin-containing diets, but the villous
height/crypt depth ratio was unaltered. Pectin significantly decreased the area of mucins in the crypts of the
small intestine, indicating that the pigs fed the pectin-
containing diet would probably be more susceptible to
pathogenic bacteria, although this cannot be separated
from the impact on ADFI. The lectin-binding pattern
of the intestinal mucosa was unaffected by diet. The
activity of lactase and maltase was increased in pigs
fed diets with high fiber content, whereas sucrase activity was increased in pigs fed the pectin-containing diets.
The activity of the peptidases, aminopeptidase N and
dipeptidylpeptidase IV, was increased when feeding
high fiber diets, whereas the activity of γ-glutamyl
transpeptidase remained unaffected by the experimental diets. In conclusion, the reduced feed intake observed with the pectin-containing diets could explain
the lower villous height and crypt depth observed in
this study. However, direct effects of pectin also are
possible, and thus further study is warranted. Feeding
pigs high insoluble fiber diets improved gut morphology
by increasing villi length and increased mucosal enzyme activity when compared with pigs fed pectin-containing diets. The mucin content as determined by
staining characteristics suggests that pigs fed high insoluble fiber diets might be better protected against
pathogenic bacteria than pigs fed diets high in soluble fiber.
Key words: digestive enzyme, fiber, gut morphology, mucin, pig
2006 American Society of Animal Science. All rights reserved.
INTRODUCTION
After the application of antibiotic growth promoters
for weaners ceased as of January 2000 in Denmark, a
1
Financial support provided by the Nordic Joint Committee for
Agricultural Research is gratefully acknowledged.
2
Corresponding author: [email protected]
3
Present address: South Dakota State University, Animal and
Range Science, Brookings 57007.
Received June 21, 2005.
Accepted January 16, 2006.
J. Anim. Sci. 2006. 84:1375–1386
dramatic increase in postweaning diarrhea has been
observed (Callesen, 2004). This has augmented the calls
for nutritional strategies that provide protection
against postweaning diarrhea.
The weaning process is a unique and difficult period
in a pig’s life when the animal is stressed by a change
in nutrition, removal from the dam, and a change in
physical environment. Associated with the weaning
process are changes in gut morphology and reduction
in enzyme activity of the small intestine, which causes
decreased digestive and absorption capacity (Pluske,
2001). Studies in older pigs have shown that the various
1375
1376
Hedemann et al.
types of plant carbohydrates behave differently in the
gastrointestinal tract of pigs. Inclusion of soluble nonstarch polysaccharides (NSP) in the diet can stimulate
the growth of a commensal gut flora, leading to increased production of short-chain fatty acids, and a
lower pH in the large intestine (Bach Knudsen et al.,
1991). Short chain fatty acids can inhibit the growth of
many pathogens, the majority of which prefer neutral
or slightly alkaline environment for growth (Gibson and
Wang, 1994), and low pH has been shown to have negative effect on the growth of bacterial pathogens like
Escherichia coli and Clostriduim perfringens (Wang
and Gibson, 1993). However, soluble NSP, in particular
when provided as purified sources, increases luminal
viscosity leading to slower absorption with a risk of
reduced ileal digestibility (Johansen et al., 1997). Insoluble NSP reduce the transit time and provide substrate
that is slowly degradable by the microflora in the distal
large intestine (Freire et al., 2000).
The study’s objectives are to investigate the interactions between dietary fiber (DF) concentration and components with varying physiochemical characteristics on
gut morphology and enzymatic activity in the intestinal
mucosa during the postweaning period.
MATERIALS AND METHODS
The experiment was approved by the ethical committee of the Uppsala Region, Sweden.
Animals and Housing
The study, carried out at the Swedish University of
Agricultural Sciences, utilized 50 castrated males
(Yorkshire × Landrace), 5 pigs/litter from 10 litters. The
pigs had access to a commercial cereal-based diet from
21 d of age and were weaned at 27 ± 4 d of age (BW
8.6 ± 1.4 kg) and assigned to a dietary treatment based
on BW and litter. The experiment was carried out in 5
replicates of 2 pigs per dietary treatment housed together in a pen. The pigs were weighed at the beginning
and end of the experiment (9 d).
Diets and Feeding
Five experimental diets were formulated to contain
carbohydrates with different physicochemical properties at different concentrations (Table 1). The carbohydrates of the basal low fiber diet (LF) were from raw
wheat and barley flours. Two diets with a medium DF
concentration (104 g of DM/kg) were made by including
either pectin (Genu pectin type B Rapid Set, CP Kelco,
Lille Skensved, Denmark) with approximately 75% degree of methylation at 71 gⴢkg−1 as-fed (MFP) or barley
hulls at 96 gⴢkg−1 as-fed (MFH) to the basal diet. Two
high fiber diets (145 g of DF/kg of DM) were formulated
by adding barley hulls at 191 gⴢkg−1 as-fed (HFH) or
pectin and barley hulls at 71 and 96 gⴢkg−1 as-fed, respectively (HFP), to the basal diet (Table 1). The pectin
source used in the MFP and HFP diets contained approximately 50% sucrose. To avoid differences in the
sucrose content of the diets, 2.1% sucrose was added to
the LF, MFH, and HFH diets. With the purpose of
balancing the reduced amount of protein from whey
protein concentrate and cereals, lysine, threonine, and
methionine were added to the diets to meet or exceed
the requirements for AA (NCPP, 2005). The pigs were
fed ad libitum, and water was supplied with a lowpressure water nipple. Daily feed consumption was recorded.
Collection of Samples
After sedation with a 1:1 mixture of of Stresnil (40
mg of azaperone/mL; Janssen-Cilag Pharma, Vienna,
Austria) and Zolitil (50 mg of tiletamine and 50 mg of
zolazepam/mL; Vibrac S.A., Carros, France) at 0.1 mL/
kg of BW, the pigs were euthanized 9 d after weaning
with an overdose of Pentobarbital Sodium (60 mg/mL;
Apoteket, Umeå, Sweden) at 100 mg/kg of BW. The
abdominal cavity was opened and the entire gastrointestinal tract was immediately removed. The small intestine was isolated, and the length was determined.
Tissue samples (5 cm) for microscopy were taken at 50
and 90% of the small intestinal length (SI50 and SI90,
respectively) and at 50% of the colon length (Co50).
The samples were taken in duplicate and transferred
to either 4% (wt/vol) neutral buffered formalin solution
(Bie & Berntsen, Rødovre, Denmark) or Clark’s fluid
(25% glacial acetic acid [Merck 63, Darmstadt, Germany] in absolute alcohol). All small intestinal samples
were taken from sections not lined with Peyer’s patches.
Additional samples for enzyme and protein determination were taken at 50 and 90% of the small intestinal
length, distal to the samples taken for microscopy. The
samples (10 cm) were opened lengthwise, rinsed carefully with ice-cold 0.9% NaCl, blotted dry, and stored
at −20°C until enzyme analysis.
Samples for Microscopy
After 24 h in the neutral buffered formalin, the tissue
samples were carefully cleaned of remaining digesta
using deionized water and then transferred to a fresh
solution of 4% neutral buffered formalin. Subsequently,
the samples were dehydrated, infiltrated with paraffin
wax, and stored. Six slides were prepared from each
sample, and each slide contained a minimum of 4 sections cut at 4-␮m thickness, at least 50 ␮m apart.
The sections were deparaffinized and hydrated, and
then were processed for carbohydrate histochemistry
using either the periodic acid-Schiff (PAS) reaction or
the alcian blue reaction at either pH 2.5 or pH 1.0
according to Kiernan (1990). For the PAS reaction, the
sections were placed in 1% periodic acid (Merck 524)
for 10 min. The sections were rinsed in water and placed
in Schiff’s reagent (Merck 1.09033) for 20 min. After a
rinse in water, the sections were counterstained with
1377
Dietary fiber and gut function in weaned pigs
Table 1. Composition of the 5 experimental diets
Diet1
Item
Ingredient, % as-fed
Barley flour
Wheat flour
Barley hulls
Pectin2
Sucrose
Whey protein concentrate
Fish meal
Vitamin/mineral premix3
Mono calcium phosphate
Limestone
L-Threonine
L-Lysine-HCl
D,L-Methionine
Calculated digestible AA, g/kg, as-fed
Lysine
Threonine
Methionine + cysteine
Tryptophan
Analyzed chemical composition, % DM
Dry matter
Crude protein (N × 6.25)
HCl fat
Ash
Sugars
Starch
Total NSP4
Total NCP5
Cellulose
Klason lignin
Dietary fiber
LF
MFH
21.6
54.0
20.1
50.1
9.6
2.1
14.7
4.5
1.3
0.6
1.1
2.1
10.7
4.5
1.3
0.6
1.0
MFP
21.3
53.2
HFH
18.4
46.1
19.1
7.1
0.04
10.7
4.5
1.3
0.7
1.1
0.11
2.1
6.7
4.5
1.3
0.5
1.0
0.26
0.04
HFP
19.6
49.1
9.6
7.1
6.7
4.5
1.3
0.6
1.0
0.03
0.33
0.07
14.4
11.3
8.8
3.0
12.4
9.4
7.7
2.5
12.6
9.2
7.5
2.5
11.8
7.5
7.0
1.9
12.0
7.6
7.1
2.0
90.2
22.3
2.8
5.5
3.5
58.5
6.5
5.7 (4.0)
0.8
0.8
7.3
90.0
21.7
3.4
4.6
3.9
52.7
8.4
7.6 (3.1)
0.8
2.1
10.4
89.9
21.6
2.8
4.3
3.9
52.2
8.6
8.5 (5.8)
0.1
1.8
10.4
90.2
22.1
3.3
5.1
3.7
49.0
11.9
8.5 (3.7)
3.3
2.8
14.7
90.2
21.4
3.0
4.6
3.8
49.4
12.4
12.1 (4.9)
0.3
1.9
14.3
1
LF = low fiber; MFH = medium fiber hulls; MFP = medium fiber pectin; HFH = high fiber hulls; HFP =
high fiber pectin.
2
GENU pectin type B Rapid Set (CP Kelco, Lille Skensved, Denmark). Contains approximately 50%
sucrose.
3
Provided the following quantities of vitamins and minerals per kilogram of complete diet: 5,174 IU of
vitamin A; 517 IU of vitamin D3; 50 mg of alpha-tocopherol; 1.9 mg of menadione; 1.9 mg of thiamine; 4.7
mg of riboflavin; 2.8 mg of pyridoxine; 0.02 mg of vitamin B12; 14 mg of D-pantothenic acid; 20 mg of niacin;
0.20 mg of biotin; 3.2 mg of folic acid; 103 mg of Fe (as FeSO4 7H2O); 91 mg of Zn (as ZnO); 23.4 mg of Mn
(as MnO); 22 mg of Cu (as CuSO4 5H2O); 0.2 mg of I (as KI); and 0.3 mg of Se (as Na2SeO3).
4
Nonstarch polysaccharides.
5
Noncellulosic polysaccharides; values in parentheses are soluble NCP.
Mayer’s hematoxylin, dehydrated, and covered with a
cover slip. For the alcian blue reactions, the sections
were placed in 1% alcian blue (Sigma A3157) in either
3% acetic acid (Merck 63), pH 2.5, or in 0.1 M HCl, pH
1.0, for 30 min. The sections were rinsed in 0.1 M HCl or
3% acetic acid (the solvent for the dye) and subsequently
washed in water. The sections were counterstained
with eosin, dehydrated, and covered with a cover slip.
The PAS reaction stains for neutral mucins, the alcian
blue reaction at pH 2.5 stains for carboxylated or sulfated types of acidic mucins, and the alcian blue reaction at pH 1.0 stains for sulfomucins (Kiernan, 1990).
Carbohydrate histochemistry was evaluated as described previously (Brunsgaard, 1997). Briefly, 15 welloriented villi and crypts were selected on each slide.
For each villous and crypt, the area of mucin granules
with a clear positive reaction for neutral mucins, acidic
mucins, or sulfomucins was determined using a com-
puter-integrated microscope and an image analysis system (Quantimet 500MC, Leica, Cambridge, UK). This
area included the mucous material present in the crypt
lumen. Because the histochemical procedure used in
our study stains the granules of all mucous cells (goblet
cells and crypt secretory cells) as well as the apical
secretion of these cells, these are all included in the
measurements.
The slides stained for neutral mucins were further
used to determine the area, height, and density of the
crypts and villi. Moreover, thickness of the muscularis
externa was measured. The area of crypts or villi was
determined as the area encircled by the basement membrane and the crypt mouth, the basement membrane
and the end of the villi, respectively, on 15 well-determined crypts or villi. The crypt depth was determined
as the distance between the basement membrane and
the crypt mouth, and the villous height was determined
1378
Hedemann et al.
as the distance between the crypt mouth and the end
of the villi. The density of crypts and villi was determined as the number of crypts or villi present over a
defined distance across the luminal part of the mucosa.
Mitotic Counts
After 24 h in Clarke’s fixative, the tissue samples for
mitotic counts were transferred to 70% ethanol and
stored at +5°C. The mitotic counts in the crypts were
performed as described by Goodlad (1994). The counts
were performed with a small piece (2 × 2 mm) of tissue
hydrated through a graded series of alcohols to 25%
ethanol. The samples were hydrolyzed for 14 min in 1
M HCl at 60°C, then placed in Schiff’s reagent (Goodlad,
1994). The crypts were displayed by microdissection
under a stereomicroscope (Leica MZ6, Leica Microsystems AG, Wetzlar, Germany), and the number of native
mitoses in 20 crypts was counted using a 40× objective
on the light microscope.
Lectin Histochemistry
Lectin histochemistry was performed as described
previously by Hedemann et al. (2005) using the following biotin labeled lectins: Galanthus nivalis lectin
(GNA) is specific for α-D-mannose; Maackia amurensis
lectin is specific for α-2,3 neuraminic acid; and Sambucus nigra lectin for α-2,6 neuraminic acid. The lectins
were supplied by EY Laboratories Inc., San Mateo, CA.
The slides processed for lectin histochemistry were
evaluated for staining frequency and intensity of the
goblet cells/mucous cells, and the cytoplasm and the
apical membrane of the epithelial cells. The evaluation
was done separately on the epithelial cells in the villi
and the crypts. The values used for scoring the frequency of cells with positive lectin reactivity were (1):
no cells; (2) between 0 and 25% of cells; (3) between 25
and 75% of cells; and (4) more than 75% of cells. The
values used for scoring intensity of lectin reactivity
were (1) none, (2) weak, (3) moderate, and (4) heavy
staining. The scorings were done using a 25× objective
on the light microscope by one individual who was
blinded to the treatments. The lectin score was calculated as the product of staining intensity and proportion
of stained cells of each segment in each pig. The lectin
scores were thus: 1 = no reactivity; 2 to 4 = low reactivity; 5 to 9 = moderate reactivity; >10 = high reactivity.
Enzyme Activity
After thawing, the mucosa of the intestinal segment
was scraped from the underlying muscular layers. The
mucosa was homogenized in aqueous Triton X-100 (6
mL/g of mucosa) using an Ultra Turrax T 25 (Janke &
Kunkel GMBH & Co. KG, Staufen, Germany) equipped
with a S25N-8G probe. A sample of the homogenate
was taken for the determination of alkaline phosphatase (AP) before the centrifugation. After centrifugation (20,000 × g, 60 min) the sediment was dissolved in
a similar volume of aqueous Triton X-100, and centrifugation was repeated. The activity of aminopeptidase N
(APN) was determined in both supernatants, whereas
the remaining enzymes were determined only in the
supernatant from the first centrifugation due to very
low activity in the second supernatant (M. S. Hedemann, unpublished data).
The procedure for determining the concentration of
protein in the homogenate (Lowry et al., 1951) was
modified and performed in a 96-microwell plate using
BSA (A 7638, Sigma Chemical, St. Louis) as the standard. The activity of APN, γ-glutamyl transpeptidase
and dipeptidylpeptidase IV was measured using L-alanine-4-nitroanilide (101014, Merck, Darmstadt, Germany), γ-L-glutamic acid 3-carboxy-4-nitroanilide (G
5008, Sigma), and glycyl-L-proline-4-nitroanilide
(L1880, Bachem Feinchemikalien AG, Bubendorf, Switzerland), respectively, as substrates (Hedemann et
al., 2003).
Lactase, maltase, and sucrase activities were determined according to Dahlqvist (1968) using a glucose kit
(166 391, Boehringer Mannheim, Mannheim, Germany) to determine the amount of liberated glucose.
The activity of AP was measured using the AP reagent
(245-20 ALP, Sigma).
The enzyme activities were expressed as units per
milligram of protein, except for AP that was expressed
as units per gram of mucosa. One unit was defined as
the amount of enzyme that hydrolyzed 1 ␮mol of the
substrate per min.
Chemical Analyses
All feed samples were milled through a 0.5-mm mesh
screen (Cyclotec 1093 Sample mill, Foss Tecator, Hoeganaes, Sweden) before analysis. The DM was determined by freeze drying followed by drying at 105°C for
20 h, and ash was determined by combustion at 525°C
for 6 h (AOAC, 1990). Crude protein (N × 6.25) was
determined by the Kjeldahl method (AOAC, 1990) using
a Kjell-Foss 16200 autoanalyser (Foss, Hillerød, Denmark). Total lipids (HCl fat) were extracted with diethyl
ether, after acid hydrolysis (Stoldt, 1952). Sugars (glucose, fructose, and sucrose) and fructans were determined by an enzymatic-colorimetric assay (Larsson and
Bengtsson, 1983) and starch by the enzymatic-colorimetric method as described by Bach Knudsen (1997).
Neutral NSP and constituent sugars were analyzed as
alditol acetates by gas chromatography and uronic
acids by colorimetry described in detail by Bach Knudsen (1997). Klason lignin was measured gravimetrically
as the residue resistant to 12 M H2SO4 (Theander and
Åman, 1979). Content of DF was calculated as
DF = total NSP + lignin.
Statistical Analyses
Performance data were analyzed using the GLM procedures of SAS (SAS Inst. Inc., Cary, NC). The pen was
1379
Dietary fiber and gut function in weaned pigs
Table 2. The ADFI, ADG, and G:F of pigs fed different fiber concentrations and sources postweaning1
Diet2
Item
ADG, g/d
ADFI, g/d
G:F
P-value4
P-value5
LF
MFH
MFP
HFH
HFP
SEM3
Diet
Beginning weight
Pectin
Fiber
P×F
186
302
0.58
166
283
0.56
100
218
0.29
204
322
0.58
58
180
0.08
26
25
0.10
0.003
0.003
0.009
0.001
0.001
0.001
0.002
0.001
0.005
0.60
0.57
0.28
0.15
0.12
0.31
1
Values are LSmeans of 5 observations per diet.
LF = low fiber; MFH = medium fiber hulls; MFP = medium fiber pectin; HFH = high fiber hulls; HFP = high fiber pectin.
3
Pooled SEM.
4
P-values obtained using diet as the main effect.
5
P-values obtained when excluding LF and separating the effect of fiber concentration (medium or high) and fiber source (pectin or no
pectin). P = pectin; F = fiber.
2
used as the experimental unit. The main effect of diet,
with beginning weight of the pigs as covariate, was
assessed. In addition, a supplementary model was applied to selectively separate the effects of fiber concentration and type of fiber. For this model, only diets
MFH, MFP, HFH, and HFP were included.
The morphological and histological responses and the
enzyme activities of the small intestine were analyzed
as repeated measurements using the mixed procedure
of SAS (Littell et al., 1996), with diet as the betweenanimal effect and segment as the within-animal effect
according to the following model:
Yijk = ␮ + αi + Uj + (αU)ij + ωk + (αω)ik + ε
RESULTS
Diets and Animal Performance
ijk,
[1]
where αi is the effect of diet (i =LF, MFH, MFP, HFH,
or HFP), Uj is the litter (j = 1,...,10), (αU)ij refers to the
individual pig, ωk is the segment (SI50, SI90), αωik is
the interaction between diet and segment, and the term
εijk ∼ N(0,σ2) represents the random error.
In addition to model 1, a supplementary model was
applied to selectively separate the effect of fiber concentration and type of fiber. For this model only diets MFH,
MFP, HFH, and HFP were included. The supplementary model was:
Yijkl = µ + αi + βj + (αβ)ij + Uk + ωl + (αω)il
+ (βω)jl +(αβω)ij l + ε
mucins in the crypts in the colon and the activity of
AP, the data were analyzed on a logarithmic scale to
obtain normality. The response estimates were subsequently transformed back to the original scale. Because
confidence intervals on the original scale are not symmetric around the response estimates, the confidence
limits rather than the SE are presented for those responses. Treatment differences are considered significant at α = 0.05.
[2]
ijkl,
where αi is the content of pectin (i = yes, no); βj is
the fiber concentration (j = medium, high); (αβ)ij is the
interaction between pectin and fiber concentration; Uk
is the litter (k = 1,...,10); ωl is the segment; αωil is the
interaction between pectin and segment; βωjl is the interaction between fiber concentration and segment;
αβωijl is the interaction between pectin, fiber concentration and segment; and ε ijkl ∼ N(0,σ2) represents the
random error. The morphological and histological responses of the colon were analyzed according to model
(1) and (2), but omitting the effect of segment and the
interactions between segment, pectin, and fiber concentration.
Results are expressed as least squares means and
SEM. For 3 responses, the area of neutral and acidic
As expected, there were differences in chemical composition of the diets relative to starch, polysaccharides
of NSP, and Klason lignin (Table 1). With increasing
DF concentrations, NSP increased whereas starch decreased. The pectin diets contained the most soluble
noncellulose polysaccharides and greatest amount of
cellulose was in the LF diet and the diets containing
barley hulls. The amount of sugars, where sucrose
makes up the major part (results not shown), did not
differ between the diets.
The diets affected ADG, ADFI, and G:F of the pigs
(Table 2). Pigs fed diets containing pectin had lower
ADG (P < 0.003), ADFI (P < 0.003), and G:F (P < 0.009)
than pigs fed the LF, MFH, or the HFH diets.
Gut Wall Architecture and Epithelial
Cell Proliferation
Villous height was affected by diet (Table 3). Feeding
pectin-containing diets reduced the villous height,
whereas no effect of the fiber concentration was observed. The number of villi per millimeter increased in
pectin-fed pigs compared with pigs fed MFH and HFH
(P < 0.008). There was no effect of fiber concentration
on villi density and no interaction between fiber and
pectin concentration. The villous height was significantly affected by the location in the small intestine
with height being shorter at SI90 than at SI50 in all
pigs. Villous height and ADG were positively correlated
(r = 0.55, P < 0.001) at SI50 and (r = 0.43, P < 0.003)
at SI90 (results not shown).
1380
Hedemann et al.
Table 3. Morphological characteristics and mitotic counts at 50 and 90% of the small intestinal length and at 50% of
the colon length of pigs fed different fiber concentrations and sources postweaning1
Diet2
Item
LF
MFH
MFP
P-value4
HFH
HFP
P-value5
SEM3
Diet
Segment
Pectin
Fiber
P×F
Segment
30
26
0.01
0.01
0.002
0.38
0.78
0.01
0.16
0.10
0.008
0.78
0.68
0.02
0.001
0.001
0.001
0.06
0.30
0.001
0.04
0.23
0.13
0.02
0.22
0.53
0.002
0.12
0.91
Villous height, ␮m
SI506
SI90
358
354
380
325
303
307
390
351
357
268
Villi density, No./mm
SI50
SI90
SI50
SI90
Co50
10.1
9.9
381
300
365
9.2
10.0
386
303
387
10.2
10.1
9.5
9.9
Crypt depth, ␮m
333
383
286
286
375
444
9.8
10.5
299
255
368
0.5
0.5
18
15
23
0.05
Crypt density, No./mm
SI50
SI90
Co50
19.2
19.0
15.4
19.4
18.9
14.9
19.3
20.2
16.2
19.5
19.3
14.1
20.3
20.6
15.7
0.7
0.05
0.92
0.79
1.1
0.01
1.19
1.06
0.10
0.10
0.57
0.04
0.79
0.09
0.86
0.21
7.4
6.9
2.8
0.7
0.12
0.19
0.09
0.04
0.76
0.16
0.3
0.52
0.15
0.62
0.99
Villous height/crypt depth ratio
SI50
SI90
0.96
1.20
SI50
SI90
Co50
8.7
8.4
2.6
1.00
1.09
0.94
1.10
1.05
1.24
Mitotic counts, No./crypt
9.1
8.5
2.7
8.2
8.2
2.4
8.8
7.4
2.9
1
Values are least square means and SEM (n = 10).
LF = low fiber; MFH = medium fiber hulls; MFP = medium fiber pectin; HFH = high fiber hulls; HFP = high fiber pectin.
3
Pooled SEM.
4
P-values obtained using diet as the main effect and analyzing data from SI50 and SI90 as repeated measurements.
5
P-values obtained when excluding LF and separating the effect of fiber concentration (medium or high) and fiber source (pectin or no
pectin) and analyzing data from SI50 and SI90 as repeated measurements. P = pectin; F = fiber.
6
SI50 and SI90 = samples taken at 50 and 90% of the small intestinal length, respectively; Co50 = sample taken at 50% of the colon length.
2
Pigs fed the pectin diets had shorter crypts (P < 0.001)
than pigs not fed pectin. In the colon, the crypt depth
was not affected by diet. However, using the supplementary model, a significant effect of pectin was observed
as pigs fed pectin-containing diets had shorter crypts.
Pigs fed pectin had greater crypt density at SI50, SI90,
and Co50 than pigs not fed pectin.
The crypt depth differed (P < 0.001) significantly between the locations in the small intestine, whereas the
number of crypts per millimeter did not differ between
SI50 and SI90.
The villous height/crypt depth ratio differed by location (P = 0.04) and ranged from 0.96 to 1.19 at SI50
and from 1.06 to 1.24 at SI90 but was unaffected by
dietary treatment.
The epithelial cell proliferation as determined by the
number of native mitoses was affected by the fiber content of the diets in the small intestine. Pigs fed HFH
and HFP had a lower number of native mitoses than
pigs fed LF, MFP, and MFH. Pectin tended to lower
the mitotic counts in the small intestine (P < 0.09),
whereas the mitotic counts in the colon were unaffected
by the experimental diets. The mitotic counts correlated
to the crypt depth in SI50 (r = 0.36, P = 0.01), but
there was not a correlation in the SI90 or Co50 sections
(results not shown).
The experimental diets did not affect the thickness
of the muscularis externa in the small intestine or the
colon (results not shown). The thickness was significantly affected (P < 0.001) by the position in the small
intestine with SI50 having 176 ± 6 ␮m and SI90 having
254 ± 13 ␮m. In the colon the average muscle thickness
was 188 ± 7 ␮m.
Mucin Staining Characteristics
and Lectin Histochemistry
In general, the staining area of sulfo, acidic, or neutral mucin on villi was not affected by diet, but there
was a tendency (P = 0.07) toward a decrease in the area
of neutral mucins on villi in pigs fed pectin diets (Table
4). Pectin significantly decreased the area of the mucins
in the crypts in the small intestine. Diet had no influence on the mucin-staining pattern in the colon.
There was a larger area of sulfomucins and acidic
mucins in the crypts at SI90 than at SI50 (P < 0.003)
and a tendency toward more neutral mucins in the
crypts at SI50 than at SI90 (P = 0.07). The area of
sulfo and neutral mucins on villi did not differ among
segments, but there were more acidic mucins on the
villi at SI90 than at SI50 (P < 0.03).
1381
Dietary fiber and gut function in weaned pigs
Table 4. Staining area of neutral, sulfo, and acidic mucins (␮m2 × 10−3) on the villi and in the crypts of pigs fed
different fiber concentrations and sources postweaning1
Diet2
Item
LF
MFH
MFP
P-value4
HFH
HFP
P-value5
SEM3 Diet Segment Pectin Fiber P × F
Segment
Area of neutral mucins on villi
SI506
SI90
0.72
0.84
1.04
0.79
SI50
1.53
1.13
0.72
0.76
0.94
0.87
0.80
0.68
0.14
0.12
0.41
0.49
0.07
0.96
0.96
0.28
0.99
0.16
0.02
0.07
0.05
0.61
0.36
0.09
0.98
0.74
0.56
Area of neutral mucins in crypts
SI90
Co50
1.20
1.29
1.27
1.06
0.87
1.22
0.90
3.457
3.55
3.64
4.22
3.26
(2.54 to 4.68) (2.85 to 4.44) (2.82 to 4.69) (3.26 to 5.48) (2.62 to 4.05)
0.13
0.48
Area of sulfomucins on villi
SI50
SI90
0.56
0.61
0.71
0.65
0.71
0.77
0.70
0.77
0.73
0.92
0.09
0.13
0.25
0.33
0.34
0.40
0.88
0.37
0.95
1.35
1.62
0.10
0.14
0.30
0.07
0.003
0.01
0.65
0.76
0.01
0.60
0.93
0.18
0.67
0.77
0.11
0.11
0.33
0.03
0.96
0.61
0.07
0.08
0.13
0.12
0.01
0.001
0.008
0.04
0.61
0.001
0.77
0.44
0.50
Area of sulfomucins in crypts
SI50
SI90
Co50
1.25
1.62
1.96
1.31
1.37
1.78
1.15
1.30
2.00
1.35
1.53
2.12
0.70
Area of acidic mucins on villi
SI50
SI90
0.45
0.65
0.62
0.73
0.45
0.63
0.51
0.66
Area of acidic mucins in crypts
SI50
SI90
Co50
1.45
1.45
1.10
1.36
1.07
1.86
1.74
1.50
1.58
1.46
2.42
2.38
2.91
2.51
3.127
(2.30 to 4.22) (1.86 to 3.13) (1.61 to 3.52) (2.42 to 3.49) (1.90 to 3.31)
0.33
1
Values are least square means and SEM (n = 10).
LF = low fiber; MFH = medium fiber hulls; MFP = medium fiber pectin; HFH = high fiber hulls; HFP = high fiber pectin.
Pooled SEM.
4
P-values obtained using diet as the main effect and analyzing data from SI50 and SI90 as repeated measurements.
5
P-values obtained when excluding LF and separating the effect of fiber concentration (medium or high) and fiber source (pectin or no
pectin) and analyzing data from SI50 and SI90 as repeated measurements. P = pectin; F = fiber.
6
SI50 and SI90 = samples taken at 50 and 90% of the small intestinal length, respectively; Co50 = sample taken at 50% of the colon length.
7
Values are back-transformed least square means (lower and upper limits of the 95% confidence interval; n = 10).
2
3
No effect of fiber concentration or interaction between
fiber concentration and pectin was observed on mucin
staining characteristics at either crypt or villi.
Diet did not affect the mucin staining area of the
total villi or crypt area except for the area of sulfomucins
on villi (results not shown). The mucin staining area
on the villi accounted for approximately 2% of the total
villous area (Table 5). The neutral mucins covered 6.7
and 7.9% of the crypt area in the SI50 and SI90 (P <
0.05), respectively. For sulfomucins and acidic mucins,
the mucin staining area of the total crypt area differed
significantly between the SI50 and SI90 being 75%
greater in the SI90 (P < 0.05). In the colon, neutral
mucins covered 19.4% of the crypt area and sulfomucins
and acidic mucins covered 9.7 and 14.5%, respectively.
The lectin histochemical scores at various sites along
the gastrointestinal tract are shown in Table 6. In general, a lectin score indicating no reactivity (1) or a low
lectin score (2 to 4) was observed in the small intestine
with GNA. The goblet cells on the villi in SI50 displayed
a low GNA score, whereas no GNA reactivity was observed in SI90. The cytoplasm on the villi in SI90 had
a low GNA score, which was significantly greater than
in SI50 where no GNA reactivity was observed. No
reactivity with Maackia amurensis lectin was observed
Table 5. Mucin staining area of total villous or crypt area
at 3 sites in the gastrointestinal tract of pigs1
Segment2
Neutral mucin
Sulfomucin
Acidic mucin
Mucin staining area of total villous area, %
SI50
SI90
2.4 ± 0.4
2.6 ± 0.2
SI50
SI90
Co50
6.7 ± 0.4a
7.9 ± 0.4b
19.4 ± 1.0
2.0 ± 0.1a
2.5 ± 0.2b
1.5 ± 0.1a
2.3 ± 0.2b
Mucin staining area of total crypt area, %
6.3 ± 0.3a
11.0 ± 0.5b
9.7 ± 0.6
6.8 ± 0.3a
12.2 ± 0.4b
14.5 ± 0.8
a,b
Least square means in a column with different superscript letters
differ (P < 0.05).
1
No dietary treatment differences were observed. Values are the
least square means ± SE of the least square mean for 50 animals
pooled across all dietary treatments.
2
SI50 and SI90 = samples taken at 50 and 90% of the small intestinal length, respectively; Co50 = sample taken at 50% of the colon
length.
1382
Hedemann et al.
Table 6. Lectin score of the mucous cells, and the apical membrane and the cytoplasm
of the epithelium in the small intestine and the colon of pigs1,2
Goblet cells/mucous cells
Segment3
Crypt
Apical membrane
Villi
Crypt
GNA
SI50
SI90
Co50
1.0 ± 0.0
1.0 ± 0.0
1.0 ± 0.0
2.4 ± 0.2a
1.0 ± 0.0b
SI50
SI90
Co50
1.8 ± 0.3
1.1 ± 0.1b
1.1 ± 0.1
Cytoplasm
Villi
Crypt
Villi
1.0 ± 0.0
1.0 ± 0.0
1.0 ± 0.0
1.0 ± 0.0
1.3 ± 0.2
1.1 ± 0.1b
2.3 ± 0.2a
1.8 ± 0.3a
1.2 ± 0.2b
1.2 ± 0.2
1.4 ± 0.2
2.8 ± 0.4
1.0 ± 0.0
1.1 ± 0.1
6.1 ± 1.2b
9.6 ± 1.2a
5.4 ± 0.5b
6.9 ± 0.6a
3.8 ± 0.4
1.6 ± 0.2
1.6 ± 0.2
4
1.1 ± 0.1
1.1 ± 0.1
—5
MAA4
a
1.0 ± 0.0
1.0 ± 0.0
1.2 ± 0.1
1.1 ± 0.1
5.9 ± 0.8
SNA4
SI50
SI90
Co50
8.5 ± 1.1
10.0 ± 0.8
5.4 ± 0.5
7.4 ± 1.0
10.8 ± 0.8a
b
3.2 ± 0.7b
5.4 ± 0.8a
2.9 ± 0.4
Means in a column with different superscript letters differ significantly (P < 0.05).
No dietary treatment differences were observed. Values are the means ± SE of the mean for 50 animals
pooled across all dietary treatments and illustrate intestinal morphological differences in relation to the
different lectins used.
2
The lectin score was calculated as the product of staining intensity and proportion of stained cells of
each segment in each pig. The lectin scores indicate: 1 = no reactivity; 2 to 4 = low reactivity; 5 to 9 =
moderate reactivity; and > 10 = high reactivity.
3
SI50 and SI90 = samples taken at 50 and 90% of the small intestinal length, respectively; Co50 = sample
taken at 50% of the colon length.
4
GNA = Galanthus nivalis lectin; MAA = Maackia amurensis lectin; and SNA = Sambucus nigra lectin.
5
An effect of diet was observed (P = 0.02): low fiber = 1.3; medium fiber hulls = 6.2; medium fiber pectin =
2.2; high fiber hulls = 1.6; and high fiber pectin = 3.3 (SEM = 1.1).
a,b
1
at most positions, but the goblet cells in SI50 the crypts
and the apical membrane of the villi showed significantly greater lectin score than the same positions in
SI90. The Sambucus nigra lectin score was greater in
the SI90 than in the SI50 at all sites, except for the
cytoplasm in the villi. Pigs fed MFH had a greater GNA
score of the apical membrane than pigs fed LF, MFP,
or HFH (P < 0.02), whereas the score of pigs fed HFP
did not differ from the others.
Mucosal Enzyme Activity
The specific activity of disaccharidases, selected peptidases, and AP is shown in Table 7. The activities of
lactase are analyzed separately for each segment due
to the large differences in activity between the SI50
and SI90. In the SI50, the fiber concentration in the
diet affected lactase activity (P = 0.04), whereas no
effect of diet was observed in the SI90 (P > 0.1). Pigs
fed HFH and HFP had a greater maltase activity than
pigs fed MHF and MFP (P = 0.05). The pectin-containing diets increased the sucrase activity (P = 0.05).
Feeding high fiber diets increased the specific activity
of APN and dipeptidylpeptidase especially in the SI90.
The γ-glutamyl transpeptidase activity was unaffected
by the experimental diets. The activity of AP was significantly affected by the experimental diets, but the
effect could not be attributed to pectin or the fiber concentration of the diets. No interaction was found between fiber concentration and fiber source for any of
the enzyme activities.
Except for APN, the activity of the measured enzymes
differed between SI50 and SI90. The disacchararidases
exhibited the greatest activities at SI50, whereas the
peptidases and AP showed the greatest activities at
SI90.
DISCUSSION
It is widely established that postweaning feed intake
is an important factor in the digestive development of
pigs (Pluske et al., 1996; van Beers-Schreurs et al.,
1998; Marion et al., 2002). In the current study, feed
intake depended on fiber composition with pectin-containing diets having a lower feed intake. The inclusion
of pectin in the diets resulted in increased luminal viscosity and water binding capacity (J. E. Lindberg, unpublished data), which may have slowed down digesta
passage and increased the satiety of the pigs, leading
to lower feed intakes. Additionally, the citrus pectin
included in the diets may have had a negative impact
on taste, mouth feel of the diets, or both.
A period of low feed intake is likely to result in damaged gut architecture (Pluske et al., 1997), and several
authors have found that the effect of diet composition
on mucosal integrity in the small intestine has been
shown to be overridden by diminished feed intake (van
Beers-Schreurs et al., 1998; Spreeuwenberg et al., 2001;
Vente-Spreeuwenberg et al., 2003). Marion et al. (2002)
showed that 56% of the variation in the villous height
in the proximal small intestine was explained by the
level of feed intake. The association between feed intake
31.6
54.2
13.0
(7.2 to 23.1)
16.5
(11.5 to 23.7)
SI50
SI90
SI50
36.3
36.2
155
108
148
162
Aminopeptidase N, mU/mg of protein
46.4
29.9
HFH
23.6
3.28
180
174
Sucrase, ␮U/mg of protein
195
114
Maltase, ␮U/mg of protein
20.3
3.18
Lactase, ␮U/mg of protein
7
MFP
73.8
100
36.4
40.911
35.0
69.1
6.5
(3.7 to 11.8)
14.6
(10.2 to 21.0)
9.5
(5.4 to 17.2)
15.5
(10.8 to 22.4)
Alkaline phosphatase,12 U/mg of mucosa
7.2
(4.0 to 12.8)
14.9
(10.2 to 21.8)
34.8
56.8
γ-Glutamyl transpeptidase, mU/mg of protein
68.6
73.4
Dipeptidylpeptidase IV, mU/mg of protein
61.4
67.9
133
148
36.0
34.9
163
133
19.6
3.48
MFH
16.6
(8.8 to 31.3)
20.9
(14.6-30.1)
38.610
63.6
81.6
99.7
180
182
62.9
43.4
210
178
32.7
3.49
HFP
6.3
7.2
9.2
12.3
14
20
6.9
7.4
26
27
4.1
0.6
SEM3
0.04
0.15
0.06
0.04
0.02
0.08
0.09
0.98
Diet
0.01
0.001
0.06
0.74
0.01
0.01
Segment
P-value4
0.22
0.79
0.49
0.55
0.05
0.49
0.21
0.42
Pectin
0.70
0.11
0.005
0.03
0.10
0.05
0.04
0.42
Fiber
0.66
0.61
0.86
0.71
0.14
0.75
0.28
0.33
P×F
P-value5
0.25
0.001
0.06
0.56
0.06
0.03
Segment
2
Values are least square means and SEM (n = 10).
LF = low fiber; MFH = medium fiber hulls; MFP = medium fiber pectin; HFH = high fiber hulls; HFP = high fiber pectin.
3
Pooled SEM.
4
P-values obtained using diet as the main effect and analyzing data from SI50 and SI90 as repeated measurements.
5
P-values obtained when excluding LF and separating the effect of fiber concentration (medium or high) and fiber source (pectin or no pectin) and analyzing data from SI50 and SI90 as
repeated measurements. P = pectin; F = fiber.
6
SI50 and SI90 = samples taken at 50 and 90% of the small intestinal length, respectively.
7
Lactase activity was analyzed segment by segment.
8
n = 9.
9
SEM = 0.8 (n = 6).
10
SEM = 6.5 (n = 9).
11
SEM = 7.5 (n = 9).
12
Values are back-transformed least square means (lower and upper limits of the 95% confidence interval).
1
SI90
69.3
75.2
152
151
41.3
24.6
150
113
19.1
3.58
LF
SI50
SI90
SI50
SI90
SI50
SI90
SI50
SI90
SI506
SI90
Segment
Diet2
Table 7. Activity of lactase, maltase, sucrase, aminopeptidase N, dipeptidylpeptidase IV, γ-glutamyl transpeptidase, and alkaline phosphatase at 50 and 90%
of the small intestinal length of pigs fed different fiber concentrations and sources postweaning1
Dietary fiber and gut function in weaned pigs
1383
1384
Hedemann et al.
and villous height has been suggested to determine
postweaning weight gain (Pluske et al., 1996), which
is supported by the current study.
The pectin-fed pigs that had the lowest feed intake
in the present experiment also had the shortest villi.
Apart from the effect on feed intake, pectin per se may
also have influenced the intestinal architecture. Feeding a diet containing carboxymethylcellulose, which increased the intestinal viscosity, reduced the villous
height and increased the crypt depth (McDonald et al.,
2001). However, feeding a low viscosity carboxymethylcellulose resulted in longer villi (McDonald et al., 2001).
Conflicting results on the effect of soluble fiber on the
intestinal morphology also exists in growing pigs, rats,
and chickens (Glitsø et al., 1998; Iji et al., 2001; Kim,
2002). Feeding pectin-containing diets resulted in the
shortest crypts; this is in contrast to the literature
where it has been shown that the consequence of low
feed intake or intake of viscous diets is crypt elongation
(Pluske et al., 1996; McDonald et al., 2001). The crypt
depth was correlated to the mitotic counts in SI50 in
the current study, and because the crypt depth is an
indication of the cell production in the crypts (Hedemann et al., 2003) this indicates that the low feed intake
of the pectin-fed piglets reduced the production of new
cells in the intestine.
In the current study, the fiber concentration had no
influence on the morphological characteristics, and the
mitotic counts in the small intestine were lower in pigs
fed high fiber diets. In contrast, the inclusion of 10%
wheat straw to a low fiber diet resulted in deeper crypts
in the jejunum and ileum and augmented cell division
in growing pigs (Jin et al., 1994). The discrepancies in
the present experiment may be because the pigs were
newly weaned, and the microflora was not adapted to
the dietary fiber (Jensen, 1998).
Free AA were added to the medium and high fiber
diets to balance the reduced amount of whey protein
concentrate in these diets. The calculated content of
AA in the diets all exceed the recommendations given
by NCPP (NCPP, 2005). In a recent study it was shown
that feeding low-protein, AA-supplemented diets did
not result in any morphological changes (Nyachoti et
al., 2006). Hence the morphological differences observed in the present investigation cannot be attributed
to the use of crystalline AA in some of the diets.
The villous height/crypt depth ratio is a useful criterion for estimating the digestive capacity in the small
intestine (Montagne et al., 2003). In this study, the
maintenance of the villous height/crypt depth ratio suggests that a reduction in villous height is less deleterious when it is not accompanied by increased crypt
depth. Furthermore, the number of villi and crypts per
millimeter of intestine was increased in pectin-fed pigs,
which increased the absorptive surface. In rats, pectincontaining diets have been shown to either increase
(Tasman-Jones et al., 1982) or decrease (Brunsgaard
and Eggum, 1995) the villi density.
Villous atrophy is generally assumed to be associated
with reduced mucosal enzyme activity. As the cells lost
are the mature enterocytes where the digestive enzymes are located, and decreased mucosal enzyme activity has generally been observed in association with
weaning (Hampson and Kidder, 1986; Hedemann et al.,
2003). In rats and pigs, it has been shown that soluble
fiber sources increase the brush border enzyme activity
(Chun et al., 1989; Lizardo et al., 1997). However, in
this study, the greatest enzyme activities were observed
in the pigs with the shortest villi that were fed the HFP
diet. It has been observed that pectin increases the
plasma glucagon-like peptide-2 concentration (Fukunaga et al., 2003), an intestinotrophic hormone that
reduces mucosal atrophy by decreasing apoptosis and
proteolyses (Burrin et al., 2000). This led the authors
to hypothesize that the negative effect of the low feed
intake in the pectin-fed pigs is counteracted by a lower
level of apoptosis in the intestine of these pigs. This
aspect, however, warrants further investigations. In
the current study, pectin (or feed intake) exerted main
effects on the intestinal morphology, whereas the fiber
concentration mainly affected the enzyme activities.
Hence in agreement with other studies, it may be concluded that the effects of dietary fiber on epithelial
structure are not clearly associated with changes in
enzymatic activities (Montagne et al., 2003).
The number of goblet cells in the small intestine has
been shown to increase after weaning, whereas no
changes were observed in the hindgut (Brown et al.,
1988), and dietary fiber modifies the nature of the mucins secreted (More et al., 1987). The lower mucin staining area in the small intestinal crypts of the pectin-fed
pigs implies a lower production and secretion of mucin.
This effect was primarily seen for the acidic and sulfated subtypes, which indicates immaturity of the mucins secreted (Koninkx et al., 1988). Because acidic mucins, particularly sulfomucins, protect against bacterial
translocation (Deplancke and Gaskins, 2001), the pectin-fed pigs may be more susceptible to infections.
The mucin staining area in both the small intestine
and the colon was considerably lower than that observed in slaughter pigs (Brunsgaard, 1998; Hedemann
et al., 2005) implying a decreased susceptibility to certain intestinal infections as the pig proceeds from the
weaning through the fattening period (Brunsgaard,
1997).
Lectins are carbohydrate-binding proteins that can
be used to characterize glucoconjugates covering the
epithelium, which are essential to provide a barrier to
invading pathogens (Neutra and Forstner, 1987). Age
influences the lectin-binding characteristics (Jaeger et
al., 1989), and differences in lectin binding can potentially serve as a marker for maturational status of the
intestine. Mucosal lectin binding is affected by dietary
changes, but none of the observed differences were specific to a particular diet. This may be due to the age of
the pig and its gut maturity.
Dietary fiber and gut function in weaned pigs
In conclusion, the morphological changes observed in
the current study could be explained by the reduced
feed intake in the pigs fed pectin-containing diets or as
a direct effect of pectin. In contrast the mucosal enzyme
activity was mainly affected by the fiber concentration.
The mucin staining characteristics suggest that pigs
fed pectin-containing diets may be more susceptible to
pathogenic bacteria. Overall the results of the present
investigation showed that pigs fed HFH had the best
morphological and enzymatic characteristics 9 d postweaning.
IMPLICATIONS
Different types and amounts of fiber seem to be useful
in optimizing the function of the gut of a newly weaned
pig. Of particular importance may be the improvement
in gut morphology and enzymatic activity when an insoluble fiber source is utilized, whereas the possible
negative impact of soluble fiber sources on the feed
intake should be considered. Additional investigations
are necessary to determine if fiber may be useful in
newly weaned pigs in lieu of antibiotics.
LITERATURE CITED
AOAC. 1990. Official Methods of Analysis. 15th ed. Assoc. Off. Anal.
Chem., Arlington, VA.
Bach Knudsen, K. E. 1997. Carbohydrate and lignin contents of plant
materials used in animal feeding. Anim. Feed Sci. Technol.
67:319–338.
Bach Knudsen, K. E., B. B. Jensen, J. O. Andersen, and I. Hansen.
1991. Gastrointestinal implications in pigs of wheat and oat
fractions. 2. Microbial activity in the gastrointestinal-tract. Br.
J. Nutr. 65:233–248.
Brown, P. J., B. G. Miller, C. R. Stokes, N. B. Blazquez, and F. J.
Bourne. 1988. Histochemistry of mucins of pig intestinal secretory epithelial cells before and after weaning. J. Comp. Path.
98:313–323.
Brunsgaard, G. 1997. Morphological characteristics, epithelial cell
proliferation, and crypt fission in cecum and colon of growing
pigs. Dig. Dis. Sci. 42:2384–2393.
Brunsgaard, G. 1998. Effects of cereal type and feed particle size on
morphological characteristics, epithelial cell proliferation, and
lectin binding patterns in the large intestine of pigs. J. Anim.
Sci. 76:2787–2798.
Brunsgaard, G., and B. O. Eggum. 1995. Small intestinal tissue structure and proliferation as influenced by adaptation period and
indigestible polysaccharides. Comp. Biochem. Physiol.
112A:365–377.
Burrin, D. G., B. Stoll, R. Jiang, Y. Petersen, J. Elnif, R. K. Buddington, M. Schmidt, J. J. Holst, B. Hartmann, and P. T. Sangild.
2000. GLP-2 stimulates intestinal growth in premature TPNfed pigs by suppressing proteolysis and apoptosis. Am. J. Physiol.
279:G1249–G1256.
Callesen, J. 2004. Effects of termination of AGP-use on pig welfare
and productivity. Pages 57–60 in Working Papers from the International Symposium: Beyond Antimicrobial Growth Promoters
in Food Animal Production. DIAS report Animal Husbandry no.
57, Tjele, Denmark.
Chun, W., T. Bamba, and S. Hosoda. 1989. Effect of pectin, a soluble
dietary fiber, on functional and morphological parameters of the
small-intestine in rats. Digestion 42:22–29.
Dahlqvist, A. 1968. Assay of intestinal disaccharidases. Anal. Biochem. 22:99–107.
1385
Deplancke, B., and H. R. Gaskins. 2001. Microbial modulation of
innate defense: Goblet cells and the intestinal mucus layer. Am.
J. Clin. Nutr. 73:1131S–1141S.
Freire, J. P. B., A. J. G. Guerreiro, L. F. Cunha, and A. Aumaitre.
2000. Effect of dietary fibre source on total tract digestibility,
caecum volatile fatty acids and digestive transit time in the
weaned piglet. Anim. Feed Sci. Technol. 87:71–83.
Fukunaga, T., M. Sasaki, Y. Araki, T. Okamoto, T. Yasuoka, T. Tsujikawa, Y. Fujiyama, and T. Bamba. 2003. Effects of the soluble
fibre pectin on intestinal cell proliferation, fecal short chain fatty
acid production and microbial population. Digestion 67:42–49.
Gibson, G. R., and X. Wang. 1994. Regulatory effects of bifidobacteria
on the growth of other colonic bacteria. J. Appl. Bacteriol.
77:412–420.
Glitsø, L. V., G. Brunsgaard, S. Højsgaard, B. Sandstrøm, and K. E.
Bach Knudsen. 1998. Intestinal degradation in pigs of rye dietary fibre with different structural characteristics. Br. J. Nutr.
80:457–468.
Goodlad, R. A. 1994. Microdissection-based techniques for the detection of cell proliferation in gastrointestinal epithelium: Application to animal and human studies. Pages 205–216 in Cell Biology: A Laboratory Handbook. Academic Press Inc., San Diego,
CA.
Hampson, D. J., and D. E. Kidder. 1986. Influence of creep feeding
and weaning on brush border enzyme activities in the piglet
small intestine. Res. Vet. Sci. 40:24–31.
Hedemann, M. S., S. Højsgaard, and B. B. Jensen. 2003. Small intestinal morphology and activity of intestinal peptidases in piglets
around weaning. J. Anim. Physiol. Anim. Nutr. 87:32–41.
Hedemann, M. S., L. L. Mikkelsen, P. J. Naughton, and B. B. Jensen.
2005. Effect of feed particle size and feed processing on morphological characteristics in the small and large intestine of pigs
and on adhesion of Salmonella enterica serovar Typhimurium
DT12 in the ileum in vitro. J. Anim. Sci. 83:1554–1562.
Iji, P. A., A. A. Saki, and D. R. Tivey. 2001. Intestinal development
and body growth of broiler chicks on diets supplemented with
non-starch polysaccharides. Anim. Feed Sci. Technol. 89:175–
188.
Jaeger, L. A., C. H. Lamar, and J. J. Turek. 1989. Lectin binding to
small intestinal goblet cells of newborn, suckling, and weaned
pigs. Am. J. Vet. Res. 50:1984–1987.
Jensen, B. B. 1998. The impact of feed additives on the microbial
ecology of the gut in young pigs. J. Anim. Feed Sci. 7:45–64.
Jin, L., L. P. Reynolds, D. A. Redmer, J. S. Caton, and J. D. Crenshaw.
1994. Effects of dietary fiber on intestinal growth, cell proliferation, and morphology in growing pigs. J. Anim. Sci. 72:2270–
2278.
Johansen, H. N., K. E. Bach Knudsen, P. J. Wood, and R. G. Fulcher.
1997. Physico-chemical properties and the degradation of oat
bran polysaccharides in the gut of pigs. J. Sci. Food Agric.
73:81–92.
Kiernan, J. A. 1990. Histological and Histochemical Methods. Theory
and Practice. Pergamon Press, Oxford, UK.
Kim, M. 2002. The water-soluble extract of chicory affects rat intestinal morphology similarly to other non-starch polysaccharides.
Nutr. Res. 22:1299–1307.
Koninkx, J. F., A. F. Stemerdink, M. H. Mirck, H. J. Egberts, J. E.
van Dijk, and J. M. Mouwen. 1988. Histochemical changes in
the composition of mucins in goblet cells during methotrexateinduced mucosal atrophy in rats. Exp. Pathol. 34:125–132.
Larsson, K., and S. Bengtsson. 1983. Bestämning av lättilgängelig
kolhydrater i växtmaterial (Determination of readily available
carbohydrates in plant material). Chemistry Methods Report
No. 22. Natl. Lab. Agric. Chem., Uppsala, Sweden.
Littell, R. C., G. A. Milliken, W. W. Stroup, and R. D. Wolfinger.
1996. SAS System for Mixed Models. SAS Inst. Inc., Cary, NC.
Lizardo, R., J. Peiniau, and A. Aumaitre. 1997. Inclusion of sugarbeet pulp and change of protein source in the diet of the weaned
piglet and their effects on digestive performance and enzymatic
activities. Anim. Feed Sci. Technol. 66:1–14.
1386
Hedemann et al.
Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951.
Protein measurement with the folin phenol reagent. J. Biol.
Chem. 193:265–275.
Marion, J., M. Biernat, F. Thomas, G. Savary, Y. Le Breton, R. Zabielski, I. Huerou-Luron, and J. Le Dividich. 2002. Small intestine
growth and morphometry in piglets weaned at 7 days of age.
Effects of level of energy intake. Reprod. Nutr. Dev. 42:339–354.
McDonald, D. E., D. W. Pethick, B. P. Mullan, and D. J. Hampson.
2001. Increasing viscosity of the intestinal contents alters small
intestinal structure and intestinal growth, and stimulates proliferation of enterotoxigenic Escherichia coli in newly-weaned pigs.
Br. J. Nutr. 86:487–498.
Montagne, L., J. R. Pluske, and D. J. Hampson. 2003. A review of
interactions between dietary fibre and the intestinal mucosa,
and their consequences on digestive health in young non-ruminant animals. Anim. Feed Sci. Technol. 108:95–117.
More, J., J. Fioramonti, F. Benazet, and L. Bueno. 1987. Histochemical characterization of glycoproteins present in jejunal and colonic goblet cells of pigs on different diets. A biopsy study using
chemical methods and peroxidase-labelled lectins. Histochemistry 87:189–194.
NCPP. 2005. Nutrients Standards. National Committee for Pig Production, Copenhagen, Denmark. Available: http://www.danskeslagterier.dk/view.asp?ID=1753 Accessed Jan. 11, 2006.
Neutra, M. R., and J. F. Forstner. 1987. Gastrointestinal mucus:
Synthesis, secretion, and function. Pages 975–1009 in Physiology of the Gastrointestinal Tract. L. R. Johnson, ed. Raven Press,
New York, NY.
Nyachoti, C. M., F. O. Omogbenigun, M. Rademacher, and G. Blank.
2006. Performance responses and indicators of gastrointestinal
health in early-weaned pigs fed low-protein amino acid-supplemented diets. J. Anim. Sci. 84:125–134.
Pluske, J. R. 2001. Morphological and functional changes in the small
intestine of the newly-weaned pig. Pages 1–27 in A. Piva, K. E.
Bach Knudsen, and J. E. Lindberg, ed. Gut Environment of Pigs.
Nottingham Univ. Press, Nottingham, UK.
Pluske, J. R., D. J. Hampson, and I. H. Williams. 1997. Factors
influencing the structure and function of the small intestine in
the weaned pig: A review. Livest. Prod. Sci. 51:215–236.
Pluske, J. R., I. H. Williams, and F. X. Aherne. 1996. Maintenance
of villous height and crypt depth in piglets by providing continous
nutrition after weaning. Anim. Sci. 62:131–144.
Spreeuwenberg, M. A. M., J. M. A. J. Verdonk, H. R. Gaskins, and
M. W. A. Verstegen. 2001. Small intestine epithelial barrier
function is compromised in pigs with low feed intake at weaning.
J. Nutr. 131:1520–1527.
Stoldt, W. 1952. Vorschlag zur Vereinheitlichung der Fettbestimmung in Lebensmittlen. F. Seif. Anst. 54:146–164.
Tasman-Jones, C., R. L. Owen, and A. L. Jones. 1982. Semipurified
dietary fiber and small-bowel morphology in rats. Dig. Dis. Sci.
27:519–524.
Theander, O., and P. Åman. 1979. Studies on dietary fibres. Swed.
J. Agr. Res. 9:97–106.
van Beers-Schreurs, H. M. G., M. J. A. Nabuurs, L. Vellenga, H. J.
Kalsbeek-van der Valk, T. Wensing, and H. J. Breukink. 1998.
Weaning and the weanling diet influence the villous height and
crypt depth in the small intestine of pigs and alter the concentrations of short-chain fatty acids in the large intestine and blood.
J. Nutr. 128:947–953.
Vente-Spreeuwenberg, M. A. M., J. M. A. J. Verdonk, A. C. Beynen,
and M. W. A. Verstegen. 2003. Interrelationships between gut
morphology and faeces consistency in newly weaned piglets.
Anim. Sci. 77:85–94.
Wang, X., and G. R. Gibson. 1993. Effects of the in-vitro fermentation
of oligofructose and inulin by bacteria growing in the human
large-intestine. J. Appl. Bacteriol. 75:373–380.