N U TR I TION RE S E ARCH 3 4 ( 2 0 14 ) 7 8 0–7 88
Available online at www.sciencedirect.com
ScienceDirect www.nrjournal.com
Dietary supplementation with soybean oligosaccharides increases short-chain fatty acids but decreases protein-derived catabolites in the intestinal luminal content of weaned Huanjiang mini-piglets☆ Xiao-Li Zhou a, b , Xiang-Feng Kong a, c,⁎, Guo-Qi Lian a , Francois Blachier d , Mei-Mei Geng a , Yu-Long Yin a,⁎ a
Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Provincial Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan 410125, China b Food and Pharmaceutical Engineering Institute, Guiyang University, Guiyang, Guizhou 550005, China c Research Center of Mini-Pig, Huanjiang Observation and Research Station for Karst Ecosystems, Chinese Academy of Sciences, Huanjiang, Guangxi 547100, China d INRA, CNRH-IdF, AgroParisTech, UMR 914 Nutrition Physiology and Ingestive Behavior, Paris 75005, France
ARTI CLE I NFO
A BS TRACT
Article history:
The improvement of gut health and function with prebiotic supplements after weaning is
Received 17 March 2014
an active area of research in pig nutrition. The present study was conducted to test the
Revised 14 August 2014
working
Accepted 22 August 2014
oligosaccharides (SBOS) can affect the gut ecosystem in terms of microbiota composition,
hypothesis
that
medium-term
dietary
supplementation
with
soybean
luminal bacterial short-chain fatty acid and ammonia concentrations, and intestinal Keywords:
expression of genes related to intestinal immunity and barrier function. Ten Huanjiang
Cytokine
mini-piglets, weaned at 21 days of age, were randomly assigned to 2 groups. Each group
Huanjiang mini-piglet
received a standard diet containing either dietary supplementation with 0.5% corn starch
Intestinal microbiota
(control group) or 0.5% SBOS (experimental group). The results showed that dietary
Soybean oligosaccharide
supplementation with SBOS increased the diversity of intestinal microflora and elevated
Tight junction
(P < .05) the numbers of some presumably beneficial intestinal bacteria (eg, Bifidobacterium sp,
Faecalibacterium
prausnitzii,
Fusobacterium
prausnitzii,
and
Roseburia).
Soybean
oligosaccharide supplementation also increased the concentration of short-chain fatty acid in the intestinal lumen, and it reduced (P < .05) the numbers of bacteria with pathogenic potential (eg, Escherichia coli, Clostridium, and Streptococcus) and the concentration of several protein-derived catabolites (eg, isobutyrate, isovalerate, and ammonia). In addition, SBOS supplementation increased (P < .05) expression of zonula occludens 1 messenger RNA, and it decreased (P < .05) expression of tumor necrosis factor α, interleukin 1β, and interleukin 8 messenger RNA in the ileum and colon. These findings suggest that Abbreviations: BCFA, branched-chain fatty acids; cDNA, complementary DNA; Ct, threshold cycle; IL, interleukin; mRNA, messenger RNA; SBOS, soybean oligosaccharide; SCFA, short-chain fatty acid; TNF, tumor necrosis factor; ZO, zonula occludens. ☆ Conflicts of interest: The authors have no conflicts of interest to declare. ⁎ Corresponding authors: Tel.: +86 731 84619763; fax: +86 731 84612685. E-mail addresses: [email protected] (X.-F. Kong), [email protected] (Y.-L. Yin). http://dx.doi.org/10.1016/j.nutres.2014.08.008 0271-5317/© 2014 Elsevier Inc. All rights reserved.
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N U T RI TI O N RE S E ARCH 3 4 ( 2 0 14 ) 7 8 0–7 88
SBOS supplementation modifies the intestinal ecosystem in weaned Huanjiang minipiglets and has potentially beneficial effects on the gut. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Previous reports focusing on gut microbiota and metabolism in relation to animal gastrointestinal physiology reveal a complex regulation of metabolism and physiology in the intestinal mucosa that involves bacterial compounds and metabolites, such as short-chain fatty acids (SCFAs) and ammonia [1]. Increasing attention has been paid to the use of functional dietary supplements, such as prebiotics, which modulate the microbial community composition and metabolism in a presumably beneficial way [2]. This is due, even more so, to their capacity to selectively stimulate the growth and/or activities of endogenous bacteria with potentially beneficial effects (including Lactobacillus and Bifidobacterium) in the colon [3]. One major aim of prebiotic supplementation during critical periods, such as pig weaning, is to avoid growth of bacterial species in the gut that could have potentially adverse effects on the host and to relieve stress-associated gut injury [4]. Soybean oligosaccharides (SBOSs) have been designated as “generally recognized as safe” material in the United States with a presumed, but not proven, “prebiotic identity.” Our previous studies indicated that SBOS can be selectively fermented by commensal bacteria in the colon, thus improving the balance of gut microflora while modulating metabolism in vitro [5]. Therefore, in view of the potential interest in SBOS as a prebiotic supplement, we developed our working hypothesis. In this study, we hypothesized that medium-term dietary supplementation with SBOS in weaned piglets can affect the gut ecosystem, both in bacterial metabolites and in microbiota composition and gene expression in the intestine. Therefore, we used the Huanjiang mini-piglet model to analyze the microbiota composition. Specifically, we examined the luminal composition of several bacterial metabolites (including bacterial SCFA and iso-SCFA) and the amount of ammonia in the distal part of the small intestine and the colon (where bacterial concentration is high). In addition, gene expression in relation to intestinal immunity (proinflammatory cytokines) and the barrier function of the epithelium (tight junction–associated proteins) were measured to determine if putative changes in the intestinal luminal content were associated with modifications of gene expression in the host.
2.
Methods and materials
2.1.
Source and composition of SBOSs
The SBOS (catalog no. 29389090) used in the present study was supplied by Nantong Sihai Plant Extract Co, Ltd, Jiangsu, China. The active ingredients in SBOS are stachyose (≥ 55%) and raffinose (≥ 25%), as detected by high performance liquid chromatography [6]. The moisture content was less than 5%.
2.2.
Animals, housing, and treatment
Ten Huanjiang mini-piglets who were weaned at 21 days of age (average body weight, 3.19 ± 0.36 kg) were obtained from 5 litters (1 male piglet and 1 female piglet per litter). The minipiglets were randomly assigned to 1 of 2 groups with all receiving the same standard diet and supplementation of either a 0.5% corn starch (control group) or 0.5% SBOS (experimental group). The diet supplementation was continued for a 14-day period to test the medium-term effect of oligosaccharide supplementation [7]. The basal diet was formulated to meet the nutrient requirements and physiological characteristics of Chinese piglets (Table 1). The piglets were individually housed in an environmentally controlled nursery (temperature, 25°C-28°C), with ad libitum access to feed and drinking water. All experimental procedures performed in the study were approved by the Animal Care and Use Committee of the Chinese Academy of Sciences.
2.3.
Sample collection and preparation
At the end of the 14-day experimental period and 12 hours after the last feeding, all piglets were euthanized using general anesthesia [8]. After ileum and colon recovery, luminal contents of the ileum (10 cm at anterior to the ileocecal valve) and colon (10 cm at posterior to the ileocecal
Table 1 – Ingredient and analyzed composition of the basal diet fed to mini-piglets Ingredient
g/kg
Corn Wheat bran Rice Soybean meal Fish meal Vitamin-mineral premix a Analyzed composition DE, MJ/kg CP, % Ca, % Av. P, % Neutral fibers, % Acid fibers, %
470.0 220.0 150.0 100.0 20.0 40.0 12.9 14.73 0.90 0.65 15.10 5.72
Note that soybean meal contains approximately 0.5% α-galactosides. a Providing the following nutrients by per kilogram premix: vitamin A, 7.5 mg; vitamin D, 8.8 mg; vitamin E, 0.02 mg; vitamin K3, 71 mg; vitamin B1, 30 mg; vitamin B2, 177 mg; vitamin B6, 32 mg; vitamin B12, 0.8 mg; nicotinic acid, 1073 mg; D-pantothenic acid, 540 mg; folic acid, 22 mg; biotin, 3.0 mg; choline, 8 g; Fe (as ferrous sulfate), 2.0 g; Cu (as copper sulfate), 1.0 g; Zn (as zinc sulfate), 3.5 g; Mn (as manganese sulfate), 1.3 g; I (as calcium iodide), 14 mg; Co (as cobalt chloride), 35 mg; Se (as sodium selenite), 8.3 mg; Ca (as calcium carbonate), 200 mg; and P (as calcium phosphate), 20 mg.
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valve) were collected and stored at −20°C. Then, ammonia (considered as the sum of NH3 and NH4+) and SCFA contents were analyzed, and the composition of gut microbiota was determined. Samples of the ileum and colon (approximately 2 cm), which were collected after washing with ice-cold phosphate-buffered saline, were immediately frozen in liquid nitrogen and stored at −80°C until they were used for extraction of total RNA.
2.4. Polymerase chain reaction–denaturing gradient gel electrophoresis analysis of gut microbiota Total bacterial DNA was extracted from the intestinal luminal contents, as described by Kong et al [9]. Briefly, after 3 washes in saline containing 0.1% Tween 80 and with vigorous manual shaking for 5 minutes per wash, the contents were precipitated by centrifugation (27 000g, 20 minutes) at 4°C. DNA from the precipitates was extracted and purified using a QIAamp DNA Stool Kit (Qiagen, Hilden, Germany) and then stored at − 80°C. For polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) analysis, each DNA sample was standardized to 20 μg/mL and then amplified using primers specific for conserved sequences flanking the variable V3
region of 16S rRNA [10]. Part of the 16S rRNA gene from bacterial genomic DNA was amplified by polymerase chain reaction by using eubacterial primers HAD1-GC (5-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG T-3) and HAD2 (5-GTA TTA CCG CGG CTG CTG GCA C-3). The amplification program was as follows: 94°C for 4 minutes, followed by 30 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 2 minutes, and then 10 minutes at 72°C. Denaturing gradient gel electrophoresis was performed, as described previously, using a Bio-Rad DCode System (Bio-Rad, Hercules, CA, USA) [11]. Denaturing gradient gel electrophoresis images were analyzed using Quantity One version 4.5.2 software (Bio-Rad). The software was configured to automatically detect bands on gels. Automatic band detection criteria with a 2% limit of detection were identical on all lanes for each gel. When gel imperfections were detected as bands by the software, these false bands were manually removed and not included in subsequent numerical analyses. Anomalous staining residues (spotting and peppering) were removed from the digital images of gels, as necessary [12]. Denaturing gradient gel electrophoresis profiles were also compared using Sorenson's index, a pairwise similarity coefficient Cs that was
Table 2 – Primer pairs for bacteria 16S rRNA genes Bacteria
Primer sequence
Product size (bp)
References
Bacteroidetes
Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
184
[13]
495
[14]
230
[15]
288
[16]
285
[16]
440
[14]
239
[14]
429
[17]
95
[18]
248
[19]
179
[13]
158
[20]
166
[21]
123
[13]
513
[14]
230
[22]
277
[5]
146
[5]
B fragilis Bifidobacterium sp B breve Bifidobacterium catenulatum C coccoides C leptum subgroup C coccoides–E rectale E coli F prausnitzii Firmicutes F prausnitzii Lactobacillus sp M smithii Prevotella Roseburia Streptococcus Total bacteria
5′-AGCAGCCGCGGTAAT-3′ 5′-CTAHGCATTTCACCGCTA-3′ 5′-ATAGCCTTTCGAAAGRAAGAT-3′ 5′-CCAGTATCAACTGCAATTTTA-3′ 5′-GATTCTGGCTCAGGATGAACGC-3′ 5′-CTGATAGGACGCGACCCAT-3′ 5′-CCGGATGCTCCATCACAC-3′ 5′-ACAAAGTGCCTTGCTCCCT-3′ 5′-CGGATGCTCCGACTCCT-3′ 5′-CGAAGGCTTGCTCCCGAT-3′ 5′-AAATGACGGTACCTGACTAA-3′ 5′-CTTTGAGTTTCATTCTTGCGAA-3′ 5′-GCACAAGCAGTGGAGT-3′ 5′-CTTCCTCCGTTTTGTCAA-3′ 5′-CGGTACCTGACTAAGAAGC-3′ 5′-AGTTTTATTCTTGCGAACG-3′ 5′-CATGCCGCGTGTATGAAGAA-3′ 5′-CGGGTAACGTCAATGAGCAAA-3′ 5′-GGCAGCATTTCAGTTTGCTTG-3′ 5′-ATTCCGCCTACCTCTGCACT-3′ 5′-GTCAGCTCGTGTCGTGA-3′ 5′-CCATTGTATACGTGTGT-3′ 5′-CCCTTCAGTGCCGCAGT-3′ 5′-GTCGCAGGATGTCAAGAC-3′ 5′-CTGATGTGAAAGCCCTCG-3′ 5′-GAGCCTCAGCGTCAGTTG-3′ 5′-CCGGGTATCTAATCCGGTTC-3′ 5′-CTCCCAGGGTAGAGGTGAAA-3′ 5′-CACRGTAAACGATGGATGCC-3′ 5′-GGTCGGGTTGCAGACC-3′ 5′-TACTGCATTGGAAACTGTCG-3′ 5′-CGGCACCGAAGAGCAAT-3′ 5′-GATGGACCTGCGTTGTATTAGCT-3′ 5′-CCCTTTCTGGTAAGATACCGTCAC-3′ 5′-CAGGATTAGATACCCTGGTAGT-3′ 5′-CCCGTCAATTCCTTTGAGTTT-3′
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determined by: Cs = [2j/(a + b)] × 100, where a is the number of DGGE bands in lane 1, b is the number of DGGE bands in lane 2, and j is the number of common DGGE bands [11].
2.5. Real-time polymerase chain reaction analysis of gut microbiota Real-time quantitative polymerase chain reaction analysis was used for measuring the expression levels of bacterial 16S rRNA by using 100-ng DNA as well as both sense and antisense primers (Table 2). This measurement was performed using 384well plates and the 7900 HT PCR instrument (Applied Biosystems, Marsiling Industrial Estate Road 3, Singapore). Duplicate sample analysis was routinely performed using SYBR Premix Ex Taq II kit (Takara, Bio, Inc, Sigma, Japan), following the protocol of the manufacturer. The amplification program was 95°C for 30 seconds and 40 cycles of 95°C for 5 seconds, followed by 60°C for 30 seconds. Data were analyzed using the ABI 7900 SDS software (version 2.3; Applied Biosystems, Foster City, CA, USA). Results were expressed as a proportion of total bacterial 16S rRNA according to the equation: relative quantification = 2 − (Ct, target − Ct, total bacteria) treatment group − (Ct, target − Ct, total bacteria) control group , where Ct represents threshold cycle [23].
2.6. Analysis of SCFA and ammonia concentrations in intestinal content Contents of the ileum and colon were recovered by expulsion, homogenized, and centrifuged at 1000g for 10 minutes. A mixture of supernatant fluid and 25% metaphosphoric acid solution (1 mL: 0.25 mL) was used to determine SCFA by gas chromatography [5,9]. Briefly, the supernatant portion was filtered through a 0.45-μm polysulfone filter and analyzed using an Agilent 6890 gas chromatograph with a flame ionization detector and a 1.82 m × 0.2 mm I.D. glass column that was packed with 10% SP-1200/1% H3PO4 on 80/100 Chromosorb W AW (HP, Inc, Boise, ID, USA). The concentration of ammonia in the supernatant fluid was measured at 550 nm by a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan) [7,9].
2.7. Analysis of cytokine messenger RNA and tight junction–associated protein expression Total RNA was extracted from the sampled ileum and colon tissues by using TRIzol reagent (Invitrogen-Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions and quantified by measuring optical density at 260 and 280 nm. Complementary DNA (cDNA) was reverse transcribed from 1 μg of eluted RNA by using a First Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada), according to the manufacturer's instructions. Real-time quantitative polymerase chain reaction analyses for tumor necrosis factor (TNF) α, interleukin (IL) 1β, IL-8, IL-10, zonula occludens (ZO) 1, occludin, and GAPDH (internal control) were performed with cDNA as well as both sense and antisense primers (Table 3). Real-time quantitative polymerase chain reaction for expansion program and reaction conditions was performed, as described previously. As described by Fu et al [24], the comparative Ct value was calculated to determine expression levels of target genes relative
Table 3 – Primer pairs for host genes Target gene
Primer Product size (bp) sequence
GAPDH
Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
IL-1β IL-8 IL-10 Occludin TNF-α ZO-1
5′-ACTCACTCTTCTACCTTTGATGCT-3′ 5′-TGTTGCTGTAGCCAAATTCA-3′ 5′-GAAAGATAACACGCCCACCC-3′ 5′-TCTGCTTGAGAGGTGCTGATGT-3′ 5′-ACTGGCTGT TGCCTTCTT-3′ 5′-CAGTT CTCTTCAAAAATATCTG-3′ 5′-GCATCCACTTCCCAACCA-3′ 5′-GCAACAAGTCGCCCATCT-3′ 5′-ATGCTTTCTCAGCCAGCG TA-3′ 5′-AAG GTTCCATAGCCTCGGTC-3′ 5′-CCACGCTCTTCTGCCTACTGC-3′ 5′-GCTGTCCCTCGGCTTTGAC-3′ 5′-GAGGATGGTCACACCGTGGT-3′ 5′-GGAGGATGCTGTTGTCTCGG-3′
149 165 278 108 176 168 169
to that of GAPDH. Data were expressed as the relative values from comparison with values obtained for control piglets.
2.8.
Statistical analyses
Values are expressed as means ± SE, together with the number of animals used. The analysis of power was performed based on data from previous experiments that focused on the effects of dietary supplementation on pig intestinal physiology and ecosystems, and this indicated the requirement of 5 animals per experiment [7,25]. Statistical analysis was performed by t test using SPSS 17.0 software (SPSS, Inc, Chicago, IL, USA). Differences with P values less than .05 were considered statistically significant.
3.
Results
3.1.
Diversity of intestinal microbiota
We analyzed the effect of dietary supplementation with SBOS on the DGGE profiles of V3 amplicons in the ileum and colon contents, which were obtained from the Huanjiang mini-piglets, to calculate similarity indices of DGGE (Figs. 1a and 2a) and then built a sketch map of the DGGE profiles (Figs. 1b and 2b). This showed that SBOS did not significantly increase profiles from the ileum (13.60 ± 1.57 bands in SBOS group vs 12.00 ± 2.88 bands in control group) and the colon (20.40 ± 2.04 bands in SBOS group vs 19.00 ± 1.38 bands in control group) microbiota.
3.2. Quantification of bacterial species in the microbiota in intestinal contents Table 4 summarizes the effect of dietary supplementation with SBOS on the 16S rRNA levels of different bacterial species in the intestinal contents from the Huanjiang mini-piglets. The Ct values of total bacteria in the ileum and colon were 10.78 ± 0.44 and 13.81 ± 0.43 in the control group and 16.26 ± 0.42 and 9.65 ± 0.64 in the SBOS group. When compared with the control group after a 14-day period of feeding, SBOS increased (P < .05) the numbers of Bifidobacterium sp,
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N U TR I TION RE S E ARCH 3 4 ( 2 0 14 ) 7 8 0–7 88
6 5 1 3 4 9 10 8 2 7
(a)
(b)
Fig. 1 – a, Similarity indices of the DGGE profiles of the ileal microflora recovered from the control group and the SBOSsupplemented group. b, Sketch map of the DGGE profiles of the ileum microflora recovered from the control group and the SBOS-supplemented group. n = 5 pigs in each group. Lanes 1 to 5, control group; lanes 6 to 10, SBOS-supplemented group.
Bifidobacterium breve, Faecalibacterium prausnitzii, Fusobacterium prausnitzii, and Roseburia in both the ileum and the colon as well as that of Prevotella in the ileum and that of Methanobrevibacter smithii in the colon. In addition, SBOS decreased (P < .05) the numbers of Clostridium coccoides and Clostridium leptum in both the ileum and the colon, those of Bacteroides fragilis and Eubacterium rectale in the ileum, and those of Streptococcus and Escherichia coli in the colon.
3.3. Concentrations of bacterial SCFA and ammonia in intestinal contents The effect of dietary supplementation with SBOS on the concentrations of SCFA and ammonia in the intestinal contents from Huanjiang mini-piglets is summarized in Table 5. Data indicate that when compared with the control group, SBOS increased (P < .05) the concentrations of propionate, butyrate,
and total SCFA in the ileum and colon as well as acetate and valerate in the ileum. However, it decreased (P < .05) the concentration of isobutyrate and isovalerate in the colon. When expressed as relative amounts of total SCFA, the results indicate that the proportion of different SCFAs in the ileum was not vastly different; but in the colon, the relative amounts of isobutyrate and isovalerate were markedly lower in the SBOS group as compared with the control group. Dietary supplementation with SBOS also decreased (P < .05) ammonia concentration in the colon, in comparison with the control group.
3.4. Intestinal cytokines and tight junction–associated protein messenger RNA expression As indicated in Table 6, when compared with the control group, dietary supplementation with SBOS decreased (P < .05) the messenger RNA (mRNA) expression levels of TNF-α, IL-1β,
6 3 8 10 2 4 1 7 5 9
(a)
(b)
Fig. 2 – A, Similarity indices of the DGGE profiles of the colon microflora recovered from the control group and the SBOS-supplemented group. B, Sketch map of the DGGE profiles of the colonic microflora recovered from the control group and the SBOS-supplemented group. n = 5 pigs in each group. Lanes 1 to 5, control group; lanes 6 to 10, SBOS-supplemented group.
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N U T RI TI O N RE S E ARCH 3 4 ( 2 0 14 ) 7 8 0–7 88
Table 4 – Relative abundance of bacterial groups or species in intestinal microbiota from Huanjiang mini-piglets fed SBOSs compared with controls Bacteria
Ileum SBOS
Bacteroidetes B fragilis Bifidobacterium sp B breve B catenulatum C coccoides C leptum subgroup E coli E rectale F prausnitzii Firmicutes F prausnitzii Lactobacillus sp M smithii Prevotella Roseburia Streptococcus
1.41 0.26 1.54 2.79 1.22 0.15 0.45 0.75 0.25 8.74 0.87 2.79 1.14 0.68 4.16 4.92 0.79
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.16 0.05 a 0.12 a 0.36 a 0.17 0.03 a 0.05 a 0.08 0.05 a 0.84 a 0.08 0.34 a 0.14 0.07 0.53 a 0.43 a 0.09
Colon Control
SBOS
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
1.40 0.75 1.52 1.86 1.18 0.26 0.58 0.64 0.74 1.96 0.81 5.93 0.98 1.64 0.87 2.84 0.72
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.08 0.21 0.12 0.13 0.15 0.15 0.11 0.10 0.13 0.14 0.11 0.12 0.12 0.18 0.10 0.16 0.15
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
P
0.15 0.13 0.16 a 0.17 a 0.12 0.05 a 0.05 a 0.07 a 0.09 0.25 a 0.07 0.68 a 0.11 0.12 a 0.11 0.28 a 0.06 a
Control
Ileum
Colon
1.00 ± 0.16 1.00 ± 0.22 1.00 ± 0.11 1.00 ± 0.15 1.00 ± 0.15 1.00±0.11 1.00 ± 0.11 1.00 ± 0.11 1.00 ± 0.15 1.00 ± 0.15 1.00 ± 0.09 1.00 ± 0.13 1.00 ± 0.13 1.00 ± 0.13 1.00 ± 0.09 1.00 ± 0.13 1.00 ± 0.09
.063 .016 .015 .004 .450 .002 .002 .067 .001 .000 .108 .002 .435 .130 .002 .000 .326
.116 .332 .026 .003 .405 .003 .008 .026 .182 .011 .358 .001 .874 .005 .371 .001 .027
Values are expressed as means ± SE and are derived from 5 pigs in each group. a Values within a row differ (P
Available online at www.sciencedirect.com
ScienceDirect www.nrjournal.com
Dietary supplementation with soybean oligosaccharides increases short-chain fatty acids but decreases protein-derived catabolites in the intestinal luminal content of weaned Huanjiang mini-piglets☆ Xiao-Li Zhou a, b , Xiang-Feng Kong a, c,⁎, Guo-Qi Lian a , Francois Blachier d , Mei-Mei Geng a , Yu-Long Yin a,⁎ a
Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Provincial Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan 410125, China b Food and Pharmaceutical Engineering Institute, Guiyang University, Guiyang, Guizhou 550005, China c Research Center of Mini-Pig, Huanjiang Observation and Research Station for Karst Ecosystems, Chinese Academy of Sciences, Huanjiang, Guangxi 547100, China d INRA, CNRH-IdF, AgroParisTech, UMR 914 Nutrition Physiology and Ingestive Behavior, Paris 75005, France
ARTI CLE I NFO
A BS TRACT
Article history:
The improvement of gut health and function with prebiotic supplements after weaning is
Received 17 March 2014
an active area of research in pig nutrition. The present study was conducted to test the
Revised 14 August 2014
working
Accepted 22 August 2014
oligosaccharides (SBOS) can affect the gut ecosystem in terms of microbiota composition,
hypothesis
that
medium-term
dietary
supplementation
with
soybean
luminal bacterial short-chain fatty acid and ammonia concentrations, and intestinal Keywords:
expression of genes related to intestinal immunity and barrier function. Ten Huanjiang
Cytokine
mini-piglets, weaned at 21 days of age, were randomly assigned to 2 groups. Each group
Huanjiang mini-piglet
received a standard diet containing either dietary supplementation with 0.5% corn starch
Intestinal microbiota
(control group) or 0.5% SBOS (experimental group). The results showed that dietary
Soybean oligosaccharide
supplementation with SBOS increased the diversity of intestinal microflora and elevated
Tight junction
(P < .05) the numbers of some presumably beneficial intestinal bacteria (eg, Bifidobacterium sp,
Faecalibacterium
prausnitzii,
Fusobacterium
prausnitzii,
and
Roseburia).
Soybean
oligosaccharide supplementation also increased the concentration of short-chain fatty acid in the intestinal lumen, and it reduced (P < .05) the numbers of bacteria with pathogenic potential (eg, Escherichia coli, Clostridium, and Streptococcus) and the concentration of several protein-derived catabolites (eg, isobutyrate, isovalerate, and ammonia). In addition, SBOS supplementation increased (P < .05) expression of zonula occludens 1 messenger RNA, and it decreased (P < .05) expression of tumor necrosis factor α, interleukin 1β, and interleukin 8 messenger RNA in the ileum and colon. These findings suggest that Abbreviations: BCFA, branched-chain fatty acids; cDNA, complementary DNA; Ct, threshold cycle; IL, interleukin; mRNA, messenger RNA; SBOS, soybean oligosaccharide; SCFA, short-chain fatty acid; TNF, tumor necrosis factor; ZO, zonula occludens. ☆ Conflicts of interest: The authors have no conflicts of interest to declare. ⁎ Corresponding authors: Tel.: +86 731 84619763; fax: +86 731 84612685. E-mail addresses: [email protected] (X.-F. Kong), [email protected] (Y.-L. Yin). http://dx.doi.org/10.1016/j.nutres.2014.08.008 0271-5317/© 2014 Elsevier Inc. All rights reserved.
781
N U T RI TI O N RE S E ARCH 3 4 ( 2 0 14 ) 7 8 0–7 88
SBOS supplementation modifies the intestinal ecosystem in weaned Huanjiang minipiglets and has potentially beneficial effects on the gut. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Previous reports focusing on gut microbiota and metabolism in relation to animal gastrointestinal physiology reveal a complex regulation of metabolism and physiology in the intestinal mucosa that involves bacterial compounds and metabolites, such as short-chain fatty acids (SCFAs) and ammonia [1]. Increasing attention has been paid to the use of functional dietary supplements, such as prebiotics, which modulate the microbial community composition and metabolism in a presumably beneficial way [2]. This is due, even more so, to their capacity to selectively stimulate the growth and/or activities of endogenous bacteria with potentially beneficial effects (including Lactobacillus and Bifidobacterium) in the colon [3]. One major aim of prebiotic supplementation during critical periods, such as pig weaning, is to avoid growth of bacterial species in the gut that could have potentially adverse effects on the host and to relieve stress-associated gut injury [4]. Soybean oligosaccharides (SBOSs) have been designated as “generally recognized as safe” material in the United States with a presumed, but not proven, “prebiotic identity.” Our previous studies indicated that SBOS can be selectively fermented by commensal bacteria in the colon, thus improving the balance of gut microflora while modulating metabolism in vitro [5]. Therefore, in view of the potential interest in SBOS as a prebiotic supplement, we developed our working hypothesis. In this study, we hypothesized that medium-term dietary supplementation with SBOS in weaned piglets can affect the gut ecosystem, both in bacterial metabolites and in microbiota composition and gene expression in the intestine. Therefore, we used the Huanjiang mini-piglet model to analyze the microbiota composition. Specifically, we examined the luminal composition of several bacterial metabolites (including bacterial SCFA and iso-SCFA) and the amount of ammonia in the distal part of the small intestine and the colon (where bacterial concentration is high). In addition, gene expression in relation to intestinal immunity (proinflammatory cytokines) and the barrier function of the epithelium (tight junction–associated proteins) were measured to determine if putative changes in the intestinal luminal content were associated with modifications of gene expression in the host.
2.
Methods and materials
2.1.
Source and composition of SBOSs
The SBOS (catalog no. 29389090) used in the present study was supplied by Nantong Sihai Plant Extract Co, Ltd, Jiangsu, China. The active ingredients in SBOS are stachyose (≥ 55%) and raffinose (≥ 25%), as detected by high performance liquid chromatography [6]. The moisture content was less than 5%.
2.2.
Animals, housing, and treatment
Ten Huanjiang mini-piglets who were weaned at 21 days of age (average body weight, 3.19 ± 0.36 kg) were obtained from 5 litters (1 male piglet and 1 female piglet per litter). The minipiglets were randomly assigned to 1 of 2 groups with all receiving the same standard diet and supplementation of either a 0.5% corn starch (control group) or 0.5% SBOS (experimental group). The diet supplementation was continued for a 14-day period to test the medium-term effect of oligosaccharide supplementation [7]. The basal diet was formulated to meet the nutrient requirements and physiological characteristics of Chinese piglets (Table 1). The piglets were individually housed in an environmentally controlled nursery (temperature, 25°C-28°C), with ad libitum access to feed and drinking water. All experimental procedures performed in the study were approved by the Animal Care and Use Committee of the Chinese Academy of Sciences.
2.3.
Sample collection and preparation
At the end of the 14-day experimental period and 12 hours after the last feeding, all piglets were euthanized using general anesthesia [8]. After ileum and colon recovery, luminal contents of the ileum (10 cm at anterior to the ileocecal valve) and colon (10 cm at posterior to the ileocecal
Table 1 – Ingredient and analyzed composition of the basal diet fed to mini-piglets Ingredient
g/kg
Corn Wheat bran Rice Soybean meal Fish meal Vitamin-mineral premix a Analyzed composition DE, MJ/kg CP, % Ca, % Av. P, % Neutral fibers, % Acid fibers, %
470.0 220.0 150.0 100.0 20.0 40.0 12.9 14.73 0.90 0.65 15.10 5.72
Note that soybean meal contains approximately 0.5% α-galactosides. a Providing the following nutrients by per kilogram premix: vitamin A, 7.5 mg; vitamin D, 8.8 mg; vitamin E, 0.02 mg; vitamin K3, 71 mg; vitamin B1, 30 mg; vitamin B2, 177 mg; vitamin B6, 32 mg; vitamin B12, 0.8 mg; nicotinic acid, 1073 mg; D-pantothenic acid, 540 mg; folic acid, 22 mg; biotin, 3.0 mg; choline, 8 g; Fe (as ferrous sulfate), 2.0 g; Cu (as copper sulfate), 1.0 g; Zn (as zinc sulfate), 3.5 g; Mn (as manganese sulfate), 1.3 g; I (as calcium iodide), 14 mg; Co (as cobalt chloride), 35 mg; Se (as sodium selenite), 8.3 mg; Ca (as calcium carbonate), 200 mg; and P (as calcium phosphate), 20 mg.
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valve) were collected and stored at −20°C. Then, ammonia (considered as the sum of NH3 and NH4+) and SCFA contents were analyzed, and the composition of gut microbiota was determined. Samples of the ileum and colon (approximately 2 cm), which were collected after washing with ice-cold phosphate-buffered saline, were immediately frozen in liquid nitrogen and stored at −80°C until they were used for extraction of total RNA.
2.4. Polymerase chain reaction–denaturing gradient gel electrophoresis analysis of gut microbiota Total bacterial DNA was extracted from the intestinal luminal contents, as described by Kong et al [9]. Briefly, after 3 washes in saline containing 0.1% Tween 80 and with vigorous manual shaking for 5 minutes per wash, the contents were precipitated by centrifugation (27 000g, 20 minutes) at 4°C. DNA from the precipitates was extracted and purified using a QIAamp DNA Stool Kit (Qiagen, Hilden, Germany) and then stored at − 80°C. For polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) analysis, each DNA sample was standardized to 20 μg/mL and then amplified using primers specific for conserved sequences flanking the variable V3
region of 16S rRNA [10]. Part of the 16S rRNA gene from bacterial genomic DNA was amplified by polymerase chain reaction by using eubacterial primers HAD1-GC (5-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG T-3) and HAD2 (5-GTA TTA CCG CGG CTG CTG GCA C-3). The amplification program was as follows: 94°C for 4 minutes, followed by 30 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 2 minutes, and then 10 minutes at 72°C. Denaturing gradient gel electrophoresis was performed, as described previously, using a Bio-Rad DCode System (Bio-Rad, Hercules, CA, USA) [11]. Denaturing gradient gel electrophoresis images were analyzed using Quantity One version 4.5.2 software (Bio-Rad). The software was configured to automatically detect bands on gels. Automatic band detection criteria with a 2% limit of detection were identical on all lanes for each gel. When gel imperfections were detected as bands by the software, these false bands were manually removed and not included in subsequent numerical analyses. Anomalous staining residues (spotting and peppering) were removed from the digital images of gels, as necessary [12]. Denaturing gradient gel electrophoresis profiles were also compared using Sorenson's index, a pairwise similarity coefficient Cs that was
Table 2 – Primer pairs for bacteria 16S rRNA genes Bacteria
Primer sequence
Product size (bp)
References
Bacteroidetes
Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
184
[13]
495
[14]
230
[15]
288
[16]
285
[16]
440
[14]
239
[14]
429
[17]
95
[18]
248
[19]
179
[13]
158
[20]
166
[21]
123
[13]
513
[14]
230
[22]
277
[5]
146
[5]
B fragilis Bifidobacterium sp B breve Bifidobacterium catenulatum C coccoides C leptum subgroup C coccoides–E rectale E coli F prausnitzii Firmicutes F prausnitzii Lactobacillus sp M smithii Prevotella Roseburia Streptococcus Total bacteria
5′-AGCAGCCGCGGTAAT-3′ 5′-CTAHGCATTTCACCGCTA-3′ 5′-ATAGCCTTTCGAAAGRAAGAT-3′ 5′-CCAGTATCAACTGCAATTTTA-3′ 5′-GATTCTGGCTCAGGATGAACGC-3′ 5′-CTGATAGGACGCGACCCAT-3′ 5′-CCGGATGCTCCATCACAC-3′ 5′-ACAAAGTGCCTTGCTCCCT-3′ 5′-CGGATGCTCCGACTCCT-3′ 5′-CGAAGGCTTGCTCCCGAT-3′ 5′-AAATGACGGTACCTGACTAA-3′ 5′-CTTTGAGTTTCATTCTTGCGAA-3′ 5′-GCACAAGCAGTGGAGT-3′ 5′-CTTCCTCCGTTTTGTCAA-3′ 5′-CGGTACCTGACTAAGAAGC-3′ 5′-AGTTTTATTCTTGCGAACG-3′ 5′-CATGCCGCGTGTATGAAGAA-3′ 5′-CGGGTAACGTCAATGAGCAAA-3′ 5′-GGCAGCATTTCAGTTTGCTTG-3′ 5′-ATTCCGCCTACCTCTGCACT-3′ 5′-GTCAGCTCGTGTCGTGA-3′ 5′-CCATTGTATACGTGTGT-3′ 5′-CCCTTCAGTGCCGCAGT-3′ 5′-GTCGCAGGATGTCAAGAC-3′ 5′-CTGATGTGAAAGCCCTCG-3′ 5′-GAGCCTCAGCGTCAGTTG-3′ 5′-CCGGGTATCTAATCCGGTTC-3′ 5′-CTCCCAGGGTAGAGGTGAAA-3′ 5′-CACRGTAAACGATGGATGCC-3′ 5′-GGTCGGGTTGCAGACC-3′ 5′-TACTGCATTGGAAACTGTCG-3′ 5′-CGGCACCGAAGAGCAAT-3′ 5′-GATGGACCTGCGTTGTATTAGCT-3′ 5′-CCCTTTCTGGTAAGATACCGTCAC-3′ 5′-CAGGATTAGATACCCTGGTAGT-3′ 5′-CCCGTCAATTCCTTTGAGTTT-3′
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determined by: Cs = [2j/(a + b)] × 100, where a is the number of DGGE bands in lane 1, b is the number of DGGE bands in lane 2, and j is the number of common DGGE bands [11].
2.5. Real-time polymerase chain reaction analysis of gut microbiota Real-time quantitative polymerase chain reaction analysis was used for measuring the expression levels of bacterial 16S rRNA by using 100-ng DNA as well as both sense and antisense primers (Table 2). This measurement was performed using 384well plates and the 7900 HT PCR instrument (Applied Biosystems, Marsiling Industrial Estate Road 3, Singapore). Duplicate sample analysis was routinely performed using SYBR Premix Ex Taq II kit (Takara, Bio, Inc, Sigma, Japan), following the protocol of the manufacturer. The amplification program was 95°C for 30 seconds and 40 cycles of 95°C for 5 seconds, followed by 60°C for 30 seconds. Data were analyzed using the ABI 7900 SDS software (version 2.3; Applied Biosystems, Foster City, CA, USA). Results were expressed as a proportion of total bacterial 16S rRNA according to the equation: relative quantification = 2 − (Ct, target − Ct, total bacteria) treatment group − (Ct, target − Ct, total bacteria) control group , where Ct represents threshold cycle [23].
2.6. Analysis of SCFA and ammonia concentrations in intestinal content Contents of the ileum and colon were recovered by expulsion, homogenized, and centrifuged at 1000g for 10 minutes. A mixture of supernatant fluid and 25% metaphosphoric acid solution (1 mL: 0.25 mL) was used to determine SCFA by gas chromatography [5,9]. Briefly, the supernatant portion was filtered through a 0.45-μm polysulfone filter and analyzed using an Agilent 6890 gas chromatograph with a flame ionization detector and a 1.82 m × 0.2 mm I.D. glass column that was packed with 10% SP-1200/1% H3PO4 on 80/100 Chromosorb W AW (HP, Inc, Boise, ID, USA). The concentration of ammonia in the supernatant fluid was measured at 550 nm by a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan) [7,9].
2.7. Analysis of cytokine messenger RNA and tight junction–associated protein expression Total RNA was extracted from the sampled ileum and colon tissues by using TRIzol reagent (Invitrogen-Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions and quantified by measuring optical density at 260 and 280 nm. Complementary DNA (cDNA) was reverse transcribed from 1 μg of eluted RNA by using a First Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada), according to the manufacturer's instructions. Real-time quantitative polymerase chain reaction analyses for tumor necrosis factor (TNF) α, interleukin (IL) 1β, IL-8, IL-10, zonula occludens (ZO) 1, occludin, and GAPDH (internal control) were performed with cDNA as well as both sense and antisense primers (Table 3). Real-time quantitative polymerase chain reaction for expansion program and reaction conditions was performed, as described previously. As described by Fu et al [24], the comparative Ct value was calculated to determine expression levels of target genes relative
Table 3 – Primer pairs for host genes Target gene
Primer Product size (bp) sequence
GAPDH
Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
IL-1β IL-8 IL-10 Occludin TNF-α ZO-1
5′-ACTCACTCTTCTACCTTTGATGCT-3′ 5′-TGTTGCTGTAGCCAAATTCA-3′ 5′-GAAAGATAACACGCCCACCC-3′ 5′-TCTGCTTGAGAGGTGCTGATGT-3′ 5′-ACTGGCTGT TGCCTTCTT-3′ 5′-CAGTT CTCTTCAAAAATATCTG-3′ 5′-GCATCCACTTCCCAACCA-3′ 5′-GCAACAAGTCGCCCATCT-3′ 5′-ATGCTTTCTCAGCCAGCG TA-3′ 5′-AAG GTTCCATAGCCTCGGTC-3′ 5′-CCACGCTCTTCTGCCTACTGC-3′ 5′-GCTGTCCCTCGGCTTTGAC-3′ 5′-GAGGATGGTCACACCGTGGT-3′ 5′-GGAGGATGCTGTTGTCTCGG-3′
149 165 278 108 176 168 169
to that of GAPDH. Data were expressed as the relative values from comparison with values obtained for control piglets.
2.8.
Statistical analyses
Values are expressed as means ± SE, together with the number of animals used. The analysis of power was performed based on data from previous experiments that focused on the effects of dietary supplementation on pig intestinal physiology and ecosystems, and this indicated the requirement of 5 animals per experiment [7,25]. Statistical analysis was performed by t test using SPSS 17.0 software (SPSS, Inc, Chicago, IL, USA). Differences with P values less than .05 were considered statistically significant.
3.
Results
3.1.
Diversity of intestinal microbiota
We analyzed the effect of dietary supplementation with SBOS on the DGGE profiles of V3 amplicons in the ileum and colon contents, which were obtained from the Huanjiang mini-piglets, to calculate similarity indices of DGGE (Figs. 1a and 2a) and then built a sketch map of the DGGE profiles (Figs. 1b and 2b). This showed that SBOS did not significantly increase profiles from the ileum (13.60 ± 1.57 bands in SBOS group vs 12.00 ± 2.88 bands in control group) and the colon (20.40 ± 2.04 bands in SBOS group vs 19.00 ± 1.38 bands in control group) microbiota.
3.2. Quantification of bacterial species in the microbiota in intestinal contents Table 4 summarizes the effect of dietary supplementation with SBOS on the 16S rRNA levels of different bacterial species in the intestinal contents from the Huanjiang mini-piglets. The Ct values of total bacteria in the ileum and colon were 10.78 ± 0.44 and 13.81 ± 0.43 in the control group and 16.26 ± 0.42 and 9.65 ± 0.64 in the SBOS group. When compared with the control group after a 14-day period of feeding, SBOS increased (P < .05) the numbers of Bifidobacterium sp,
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6 5 1 3 4 9 10 8 2 7
(a)
(b)
Fig. 1 – a, Similarity indices of the DGGE profiles of the ileal microflora recovered from the control group and the SBOSsupplemented group. b, Sketch map of the DGGE profiles of the ileum microflora recovered from the control group and the SBOS-supplemented group. n = 5 pigs in each group. Lanes 1 to 5, control group; lanes 6 to 10, SBOS-supplemented group.
Bifidobacterium breve, Faecalibacterium prausnitzii, Fusobacterium prausnitzii, and Roseburia in both the ileum and the colon as well as that of Prevotella in the ileum and that of Methanobrevibacter smithii in the colon. In addition, SBOS decreased (P < .05) the numbers of Clostridium coccoides and Clostridium leptum in both the ileum and the colon, those of Bacteroides fragilis and Eubacterium rectale in the ileum, and those of Streptococcus and Escherichia coli in the colon.
3.3. Concentrations of bacterial SCFA and ammonia in intestinal contents The effect of dietary supplementation with SBOS on the concentrations of SCFA and ammonia in the intestinal contents from Huanjiang mini-piglets is summarized in Table 5. Data indicate that when compared with the control group, SBOS increased (P < .05) the concentrations of propionate, butyrate,
and total SCFA in the ileum and colon as well as acetate and valerate in the ileum. However, it decreased (P < .05) the concentration of isobutyrate and isovalerate in the colon. When expressed as relative amounts of total SCFA, the results indicate that the proportion of different SCFAs in the ileum was not vastly different; but in the colon, the relative amounts of isobutyrate and isovalerate were markedly lower in the SBOS group as compared with the control group. Dietary supplementation with SBOS also decreased (P < .05) ammonia concentration in the colon, in comparison with the control group.
3.4. Intestinal cytokines and tight junction–associated protein messenger RNA expression As indicated in Table 6, when compared with the control group, dietary supplementation with SBOS decreased (P < .05) the messenger RNA (mRNA) expression levels of TNF-α, IL-1β,
6 3 8 10 2 4 1 7 5 9
(a)
(b)
Fig. 2 – A, Similarity indices of the DGGE profiles of the colon microflora recovered from the control group and the SBOS-supplemented group. B, Sketch map of the DGGE profiles of the colonic microflora recovered from the control group and the SBOS-supplemented group. n = 5 pigs in each group. Lanes 1 to 5, control group; lanes 6 to 10, SBOS-supplemented group.
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Table 4 – Relative abundance of bacterial groups or species in intestinal microbiota from Huanjiang mini-piglets fed SBOSs compared with controls Bacteria
Ileum SBOS
Bacteroidetes B fragilis Bifidobacterium sp B breve B catenulatum C coccoides C leptum subgroup E coli E rectale F prausnitzii Firmicutes F prausnitzii Lactobacillus sp M smithii Prevotella Roseburia Streptococcus
1.41 0.26 1.54 2.79 1.22 0.15 0.45 0.75 0.25 8.74 0.87 2.79 1.14 0.68 4.16 4.92 0.79
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.16 0.05 a 0.12 a 0.36 a 0.17 0.03 a 0.05 a 0.08 0.05 a 0.84 a 0.08 0.34 a 0.14 0.07 0.53 a 0.43 a 0.09
Colon Control
SBOS
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
1.40 0.75 1.52 1.86 1.18 0.26 0.58 0.64 0.74 1.96 0.81 5.93 0.98 1.64 0.87 2.84 0.72
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.08 0.21 0.12 0.13 0.15 0.15 0.11 0.10 0.13 0.14 0.11 0.12 0.12 0.18 0.10 0.16 0.15
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
P
0.15 0.13 0.16 a 0.17 a 0.12 0.05 a 0.05 a 0.07 a 0.09 0.25 a 0.07 0.68 a 0.11 0.12 a 0.11 0.28 a 0.06 a
Control
Ileum
Colon
1.00 ± 0.16 1.00 ± 0.22 1.00 ± 0.11 1.00 ± 0.15 1.00 ± 0.15 1.00±0.11 1.00 ± 0.11 1.00 ± 0.11 1.00 ± 0.15 1.00 ± 0.15 1.00 ± 0.09 1.00 ± 0.13 1.00 ± 0.13 1.00 ± 0.13 1.00 ± 0.09 1.00 ± 0.13 1.00 ± 0.09
.063 .016 .015 .004 .450 .002 .002 .067 .001 .000 .108 .002 .435 .130 .002 .000 .326
.116 .332 .026 .003 .405 .003 .008 .026 .182 .011 .358 .001 .874 .005 .371 .001 .027
Values are expressed as means ± SE and are derived from 5 pigs in each group. a Values within a row differ (P
