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Bioactive Constituents, Metabolites, and Functions

Dietary fiber increases the butyrate-producing bacteria and improves growth performance of weaned piglets Jinbiao Zhao, Ping Liu, Yi Wu, Pingting Guo, Ling Liu, Ning Ma, Crystal Levesque, Yiqiang Chen, Jinshan Zhao, Jie Zhang, and Xi Ma J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02545 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

ACS Paragon Plus Environment


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Dietary fiber increases the butyrate-producing bacteria and improves growth performance

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of weaned piglets

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Jinbiao Zhao1†, Ping Liu1†, Yi Wu†, Pingting Guo†, Ling Liu†, Ning Ma†, Crystal Levesque‡,

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Yiqiang Chen†, Jinshan Zhao∥, Jie Zhang§, and Xi Ma*†∥&

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†

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Agricultural University, Beijing 100193, China

8

‡

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sciences, South Dakota State University, Brookings, SD 57007, USA

State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China

Monogastric Nutrition Department of Animal Science, College of agriculture and biological

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§

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Beijing 102442, China

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Department of Animal Husbandry and Veterinary, Beijing Vocational College of Agriculture,

∥

College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109,

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China

14

&

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Southwestern Medical Center, Dallas, TX 75390, USA

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1

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* Correspondence: E-mail: [email protected] (X. Ma)

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Phone: 86-10-62733588. Fax: 86-10-62733688.

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ORCID

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Xi Ma: 0000-0003-4562-9331

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Jinbiao Zhao: 0000-0001-7900-0131

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Ping Liu: 0000-0002-8298-6576

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Jie Zhang: 0000-0001-5010-0833

Department of Internal Medicine, Department of Biochemistry, University of Texas

These authors have contributed equally to this work.

1


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Abstract

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The study investigated the impact of dietary fibers on performance, fecal short-chain fatty acids,

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nutrients digestibility and bacterial community in weaned piglets with control group (CON),

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dietary supplementation of 5% corn bran (CB), 5% wheat bran (WB) or 5% soybean hulls (SB).

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The piglets in CB and WB groups showed greater weight gain and feed efficiency (P < 0.05) in

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comparation with piglets in CON and SB groups. Fecal samples from piglets in CB, SB and WB

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groups contained greater (P < 0.05) butyrate levels than fecal samples from piglets in CON group.

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Fecal sample from piglets in CB or WB groups contained greater (P < 0.05) abundances of

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Actinobacteria and Firmicutes or Fibrobacteres than fecal sample from piglets in CON group,

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which could promote fiber degradation and the production of butyrate. In summary, dietary CB or

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WB may enhance growth performance of weaned piglets via altering gut microbiota and

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improving butyrate production, which shed light on the mechanism of dietary fibre in improving

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gut health.

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Keywords: bacterial community, bacterial metabolites, dietary fiber, gut health, weaned piglets

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Introduction

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Dietary fiber was divided into soluble (SDF) and insoluble fiber (IDF) according to its

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physicochemical properties and fermentation capacities in the gut and played the different

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impacts on the gastrointestinal physiological function and gut health of host.1 Recently, many

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studies reported dietary fiber exerted an important role on intestinal health of human and animals,

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since it not only promoted the intestinal epithelial barrier function,2 but also maintained the

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homeostasis of intestinal microenvironment in host by modulating gut microbial composition.3,4

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Dietary fiber can be degraded to generate short-chain fatty acids (SCFAs) by intestinal bacteria,

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which improve intestinal health by modifying gut microbiota and inducing the expression of

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porcine host defense peptides.5,6 A recent research showed that inulin, as a source of SDF,

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protected mice against the increment of adiposity via modulation of bacterial community structure

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and SCFAs concentration.7 Meanwhile, arabinoxylan, as an IDF, promoted the intestinal barrier

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function and induced changes of bacterial community.8 Fiber-deprived microbiota impaired

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barrier function of colonic mucosa and increased the susceptibility of pathogens.9 Overall, it is

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necessary to add moderate level of dietary fiber into the diets to sustain the normal physiological

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function of gastrointestinal tract and improve gut health of host.

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Weaned piglets are regarded as a relevant model for studying the development of infant

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immune system and the composition of gut microbiota.10 It is reported that the diets supplied with

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wheat bran, soybean hulls or oat hulls did not affect the populations of Lactobacillus and total

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bacteria in fecal samples of piglets.11 The result that arabinoxylan extracted from wheat bran

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could stimulate the colonization of Bifidobacteria had been reported in many previous

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researches.8,12 Therefore, we hypothesized that the diet supplemented with different fiber types

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would exert different impacts on growth performance, SCFAs production and bacterial

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community of weaned piglets. To verify our hypotheses, corn bran (CB), wheat bran (WB), and

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soybean hulls (SB) were respectively added to the diet to evaluate the impacts on performance,

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nutrients digestibility, SCFAs production and bacterial community of weaned piglets. 3


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Materials and methods

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The management and design of the experiment were kept to animal care rules approved by the

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department of China Agricultural University Animal Care and Use Ethics Committee (Beijing,

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CAU20150925-2). The experimental regulations and methods were approved and then performed

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according to relevant criterions. The chemical composition of CB, WB, and SB ingredients was

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analyzed in Table 1.

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Animals and dietary treatments

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One hundred and forty-four Duroc × (Landrace × Yorkshire) crossbred piglets were allotted into 4

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dietary groups randomly by considering sex and weight of pigs. The piglets were 28 days old

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(weaned at 21 d and fed creep feed after weanling for one week) and their initial body weight

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(BW) was 7.01 ± 0.39 kg. Each dietary group contained 6 replicate pens with 3 gilts and 3

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barrows per pen. Experimental diets included one control (CON) group and three additional tests

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dietary groups which contained different fiber ingredients including 5% CB, 5% WB and 5% SB,

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respectively (Supplementary Table S1). The contents of digestible energy and crude protein

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contents were equalized by adjusting the proportion of maize and soybean meal (SBM) in each

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diet. Piglets’ requirements for standardized ileal digestible amino acids (AAs) were satisfied by

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supplementing crystalline AAs to diets according to NRC.13 This experiment lasted 28 d. No

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antibiotics or medicines were used. Water and ration were provided ad libitum for piglets. The

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relative humidity and temperature of piglet house were monitored at 60% to 65% and 25°C to

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28°C, respectively.

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Sample collection and processing

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The BW of piglets and feed intake per pen were measured to measure average daily feed intake

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(ADFI), weight gain (ADG), and feed to gain ratio (F/G) of pigs on d 0, 14, and 28 of the trial.

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The diarrhea frequency of piglets was also estimated according to a previous report.11 To

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calculate nutrient digestibility, about 300g of fecal sample was collected in each pen at the last 2

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days of experiment and then placed at –18°C immediately. At the end of feeding trial, all fecal 4



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samples of piglets in each pen were thawed and then stoved at temperature of 65°C for 70 hours.

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Fecal samples of piglets were smashed and then sieved 1.0 mm screen for further analysis. The

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equation was used to determine the digestibility of dietary nutrients as follow: Nutrients

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digestibility = 1 - (TC×FN) / (FC×TN). In this equation, TC and TN were acid insoluble ash and

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nutrient contents in diets, respectively. FC and FN were acid insoluble ash and nutrient contents

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in feces, respectively.

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Six fresh fecal samples of piglets (1 sample per pen) in each dietary group were acquired to

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determine SCFAs contents. After mixing two samples in per group randomly, three fresh fecal

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samples of each group were analyzed for bacterial community. Samples of feces were obtained by

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using 5 mL centrifuge tubes and placed into liquid nitrogen, then were stored at temperature of -

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80°C.

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According to AOAC methods,14 crude protein, dry matter, starch, acid hydrolyzed ether

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extract, ash, calcium, phosphorous, IDF and total dietary fiber (TDF) in fiber ingredients and dry

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matter, TDF, acid hydrolyzed ether extract, crude protein and ash in diets and feces samples were

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analyzed. Acid detergent ﬁber and neutral detergent fiber in feed ingredients were determine by

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using a fiber analyzer according to a previous report.15 The content of acid detergent lignin was

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also determined according to the method of Ankom Technology. Automatic adiabatic oxygen-

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bomb calorimeter was used to determine gross energy.

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SCFAs concentrations in the feces were analyzed by using the method described as a previous

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report.16 About 1.0 g fecal samples were put into 10 mL centrifuge tube. The tube was handled by

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adding 2.0 mL 0.10% hydrochloric acid and placed into ice lasted 25 min, mixed and centrifuged

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at 15,000 rpm to get the supernate. The supernate was filtered using a 0.20 mm Nylon Membrane

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Filter (Millipore, Bedford, OH) and poured into a Gas Chromatograph System (Agilent HP 6890

115

Series, Santa Clara, CA, USA).

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Analysis for bacterial microbiota by 16S RNA sequences

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DNA Kit (Omega Bio-tek, Norcross, USA) was applied to extract bacterial DNA in fecal samples

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of piglets according to the manufacturer’s instruction. The genes of bacteria 16S ribosomal RNA

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in the region of V4-V5 were amplified by using polymerase chain reaction (PCR) with primers

120

(515F

121

CCGTCAATTCMTTTRAGTTT-3’). Electrophoresis was applied to analyze the integrity of PCR

122

amplicons by using a Tapestation Instruction (Agilent technologies, USA). AxyPrep DNA Gel

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Extraction Kit was chosen to extract and purify PCR amplicons using 2% agarose gels (Axygen

124

Biosciences, Union City, CA, USA) and then the production was quantified using QuantiFluor™

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-ST and sequenced on an Illumina MiSeq system. QIIME software was used to demultiplex and

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quality-filtered raw Illumina fastq files. And operational taxonomic units (OTUs) were defined as

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a similarity threshold of 0.97 using UPARSE. Then UCHIME was applied to identify and delete

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the unnormal gene sequences. RDP database (http://rdp.cme.msu.edu/) was also referenced to

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take the taxonomy-based analysis for OUTs using RDP classifier at a 90% confidence level.

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PICRUSt analysis for predicted metabolic functions of microbial communities

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The result of 16S rRNA gene sequencing was applied by Phylogenetic Investigation of

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Communities by Reconstruction of Unobserved States (PICRUSt) analysis in order to predict

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metabolic functions of bacterial community in the fecal samples of piglets.17 The resultant OTUs

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table was then used to predict metabolic functions by referencing the Kyoto Encyclopedia of

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Genes and Genome (KEGG) Orthology (KO) Database.18

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Statistical analysis

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Data for performance, the digestibility of nutrients and SCFAs concentration were obtained with

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each pen as an experimental unit. And GLM models of SAS and Turkey’s tests were used to

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analyze the experimental data, and the results were showed as mean values ± SEM. Data for

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bacterial community were analyzed with the mixture of two fecal samples in per group randomly.

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Standardized OTUs reads were applied to analyze bacterial diversity by the guidance of R

142

software. The populations of bacterial community in fecal samples of piglets at the phyla, families

5’-barcode-

GTGCCAGCMGCCGCGG)-3’

6


and

907R

5’-


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and genera levels were analyzed by the method of Kruskal-Wallis. The abundance of bacteria at

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the phylum and family levels were showed as bar plots. The abundances of differential bacteria

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were classified by using the procedure of linear discriminant analysis (LDA) effect size (LEfSe)

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algorithm, if the logarithmic LDA values of bacteria exceeded 2.0. The comparative analysis

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between two dietary groups was conducted by using the method of Welch’s t-test. It was

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considered as a significant difference if P < 0.05.

149 150

Results

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Growth performance

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The BW, ADFI and diarrhea frequency of piglets were not affected when fibrous ingredients were

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added in the CON diet (P > 0.05; Table 2). Piglets in CB and WB groups showed a greater ADG

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than in SB group, and a lower (P < 0.05) F/G than in CON and SB groups in the first two weeks

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and the whole experimental period (P < 0.05). However, no differences in ADFI, ADG, F/G and

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the diarrhea frequency of pigs were detected among all groups at the last two weeks.

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Nutrient digestibility

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Piglets in CB and WB groups had a less digestibility of gross energy, dry matter and organic

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matter (P < 0.05) in comparation with the piglets in the CON group. Nutrient digestibility was not

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different (P > 0.05) between piglets in CON and SB groups (Table 3). Piglets in SB group had a

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greater digestibility of TDF than in CON group (P < 0.05). Dietary fiber inclusion had no impacts

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(P > 0.05) on the digestibility of acid hydrolyzed ether extract and crude pretein. Furthermore,

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fiber sources did not alter the digestibility of nutrients (P > 0.05) except for dry matter in diets for

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weaned piglets.

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Microbial metabolites

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Fecal samples of piglets in fiber inclusion treatments showed a greater concentration of butyrate

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than fecal samples of piglets in the CON diet (P < 0.05; Table 4). Fecal samples from the piglets

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in CB group had a greater valerate concentration than (P < 0.05) fecal samples from the piglets in 7


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CON group, but no differences were found among the CON, SB, and WB groups (P > 0.05). No

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differences in acetate, propionate, and total SCFAs contents were found among all treatment

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groups (P > 0.05).

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Fecal bacterial community

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The fecal samples of piglets in CON, CB, SB and WB groups showed 567, 554, 593 and 600

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OTUs, respectively. Among them, 457 shared common bacteria and total 26 individual bacteria

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were isolated in 4 dietary treatments (Figure 1A). At the genera level, the abundance of top 30

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bacterial communities was presented in the heatmap (Figure 1B). The abundance of

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Megasphaera increased, but not significantly, when piglets received the 3 treatments including

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CB, SB and WB compared with the CON treatment. The piglets in CB dietary treatment showed a

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higher relative abundance of Lachnospiraceae-AC2044-group in comparation with the CON

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group. The alpha-diversity of bacterial communities was evaluated for the species richness and

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evenness to analyze the bacterial alteration. Both of Chao and Shannon indexes were not different

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among 4 groups (Table 5). At the phylum level, the population of bacteria in fecal samples of

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pigs showed that 13 phyla were detected in 4 dietary treatments (Figure 2A). Firmicutes and

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Bacteroidetes were the dominant bacteria and the total abundance accounted for about 95%. The

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abundance of Actinobacteria and Firmicutes showed an increment in the CB group in

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comparation with the CON group, but Bacteroidetes abundance was lower (P < 0.05). Especially,

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Fibrobacteres which belonged to the cellulolytic bacteria were found even with a small proportion

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in fecal sample, and their relative abundance in CB, SB and WB dietary groups increased, but not

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significantly, compared to CON group. The numerical composition of bacterial community on the

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phylum level was analyzed (Supplementary Table S2). The predominant bacteria of Firmicutes

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were consisted of Peptostreptococcaceae, Veillonellaceae, Lactobacillaceae, Lachnospiraceae,

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Ruminococcaceae and Clostridiaceae_1 were the mainly family in phylum Firmicutes (Figure

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2B), while Bacteroidales_S24-7_group and Prevotellaceae were the predominant family in

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phylum Bacteridetes. The predominant bacterium of Actinobacteria was consisted of 8



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Coriobacteriaceae in the present study. However, relative richness of bacterial communities on

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the family level did not change (P ˃ 0.05) in all treatments. The specific abundance of bacterial

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community on the family level was analyzed (Supplementary Table S3).

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All differential bacteria were demonstrated from phylum to species level in cladogram of

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LEfSe between CON and 3 additional treatments (Figure 3). The circle from inner to outer

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represents distinct bacteria from phylum to genus level, respectively. The yellow dots inserted in

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circle suggest no significant difference in bacteria among different dietary treatments. Using the

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analysis methods of Metastats to compare microbial compositions on the genera level among 4

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groups, Prevotellaceae was only significant bacterium on the relative abundance of bacteria

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among the 4 dietary groups (Figure 3A). The difference in bacterial community between the

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CON group and individual fibrous treatment was further performed. The population of

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Anaerofilum, norank_f_Ruminococcaceae and Eubacterium_ventriosum_group which belonged

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to class Clostridia and phylum Firmicutes significantly decreased (P < 0.05) in SB group

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comparing with CON group (Figure 3B). When piglets were fed the WB diet, the relative

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abundances

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norank_f_Ruminococcaceae and Ruminococcaceae were decreased, whereas Prevotella_7,

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Eubacterium_nodatum_group,

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Ruminococcaceae_UCG_002 and Ruminococcus_1, were increased (P < 0.05) (Figure 3C).

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Particularly, the abundance of Fibrobacter was remarkably increased in piglets fed diet

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supplemented with WB, resulting in an markedly increase in the proportion of phylum

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Fibrobacteres (P < 0.05). Piglets in the CB group had greater relative abundances of Prevotella

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and Bacteroidetes (P < 0.05) in comparation with the piglets in the CON group, which led to an

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obvious increase in the relative abundance of Bacteroidetes (Figure 3D). However, piglets in the

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CB group showed greater relative abundance of Bacilli, a class of phylum Firmicutes, compared

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with the piglets in the CON group (P < 0.05). In addition, the relative abundances of phylum

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Tenericutes and Actinobacteria also were increased in CB group than CON group (P < 0.05).

of

Prevotellaceae,

Veillonella,

Roseburia,

Ruminococcaceae-NK4A214_group

Lachnospiraceae,

9


Oribacterium,

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Predicted functional profiles of microbial communities using PICRUSt

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To predict the potential function of gut bacteria on nutrient metabolism in piglets after feeding

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different dietary fiber, KEGG pathways were analyzed by PICRUSt program. The result showed a

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total of 295 KEGG pathways. Six pathways related to carbohydrate metabolism were focused

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(Figure 4), including energy metabolism, carbohydrate digestion and absorption, fructose and

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mannose metabolism, galactose metabolism, starch and sucrose metabolism, and fatty acids

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biosynthesis. The number of the genes related to energy metabolism pathway in CB treatment

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showed an increment, but not significantly, compared with the other 3 treatments. In addition, the

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number of the genes involved in fatty acid biosynthesis in the CB group increased (P < 0.05)

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comparing to WB and SB groups. The results showed there were no differences in the number of

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the gene tags associated with energy metabolism, carbohydrate digestion and absorption, fructose

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and mannose metabolism, galactose metabolism, starch and sucrose metabolism pathways.

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Discussion

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In our study, fiber inclusions had no effects on the ADFI and BW of piglets, which was consistent

235

with some previous studies.11,19 The ADFI was affected by body weight and health status of pigs,

236

palatability and nutritive level of ration, thus fiber supplementation did not affect the ADFI of

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piglets in our study. However, previous studies reported that diet added insoluble fibrous

238

ingredients increased feed intake in weaned piglets since IDF decreased the retention time of

239

digesta.20,21 Moreover, Hopwood et al. reported inclusion of sugar beet pulp which contained

240

most of SDF could decrease ADFI by increasing digesta viscosity and prolonging satiety time.22

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These diverse results mentioned above were associated with different fiber sources supplemented

242

in diets. In this study, piglets in the CB and WB groups had a lower F/G than CON and SB groups

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at the first 2 weeks, which was consistent a previous study reported moderate level of insoluble

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fiber sources were beneficial for growth performance of pigs at the first 2 weeks after weanling.23

245

This positive effect on growth performance of piglets at the first 2 weeks might be considered that 10



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arabinoxylan in CB and WB enhanced the barrier function and health of gut in piglets by

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regulating the production of butyrate.8,12 However, Yu et al. reported the diet supplemented with 5%

248

SB had negative effects on growth performance for weaned pigs on account of the concentration

249

of anti-nutritional factors, especially trypsin inhibitors and α-galactosides.11 Therefore, the

250

different fiber sources exerted various effects on growth performance, physiological function of

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gastrointestine and gut health of pigs depending on their physicochemical properties and chemical

252

components.

253

Fiber inclusions had no effect on the diarrhea frequency of piglets in our study, which was

254

consistent with a previous study reported there were no differences in diarrhea frequency between

255

the CON and WB or SB treatments after weaning.11 In contrast, some previous studies showed

256

that fiber inclusion in diets had a negative effect on post-weaning diarrhea of piglets.24,25 However,

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Molist et al. reported that insoluble fiber added into rice based diet reduced post-weaning diarrhea

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and improved growth performance in weaned pigs, but there was no effect on the pigs fed on

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maize based diet.23 The results indicated that the effects of fiber supplementation on growth

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performance and diarrhea frequency after weaning might be relative to the fiber sources and types

261

of basal diet, as well as feeding environment. Piglets in CB and WB groups had lower

262

digestibilities of gross energy, dry matter and organic matter than the CON group, which were

263

consistent with other studies.19,25 The reason is that the majority IDF components of CB and WB

264

are characterized by low viscosity and slow rate of fermentation, resulting in decreased retention

265

time of digesta in the gut.1 In contrast, SB group showed a higher digestibility of TDF in weaned

266

piglets due to the high content of SDF in SB ingredient.

267

In the present study, fecal samples of piglets in CB and WB groups had greater levels of

268

butyrate and valerate compared with the piglets in the CON group, which was consistent with a

269

previous study reported the diet with 5% WB showed greater concentration of butyrate in the

270

cecum of piglets.8 The result for the increment of butyrate in fecal sample of piglets might be

271

caused by the gastrointestinal microflora colonization and improving the degradation of fiber 11


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components. An increased butyrate was considered beneficial to the host by promoting the

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proliferation of mucosal epithelial cells, the differentiation of intestinal epithelial cell and the

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function of colonic barrier.26,27 A previous study reported butyrate improved the biosynthesis of

275

defense peptides to promote the immunity of host and prevent infectious diseases.6 The previous

276

studies showed that SCFAs mediated glucose homeostasis by activating G protein-coupled

277

receptors 41 and 43 and stimulating enteroendocrine L-cells to produce glucagon-like peptide 1

278

and peptide YY,28 and then improved insulin sensitivity.29 Overall, those results indicate that

279

dietary fiber plays a crucial role in gut health of weaned piglets by increasing butyrate

280

concentrations. In addition, using PICRUSt program to predict functional profiles of microbial

281

communities, the number of gene tags involved in fatty acid biosynthesis in CB treatment were

282

significantly increased compared with WB and SB treatments. The result suggests that fiber

283

supplementation may be involved in lipid metabolism by improving SCFAs production, but it is

284

necessary to conduct the further study about the relationship of fiber supplementation and lipid

285

metabolism.

286

The gut microbiota are beneficial to host by regulating physiological process and mucosal

287

immunity, such as strengthening integrity of intestinal barrier on epithelial cells,30,31 suppressing

288

colonization of enteric pathogens,32 harvesting energy33 and producing antimicrobial peptides in

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mucus layer.34 Similar to the previous report,35 Firmicutes and Bacteriodetes represented

290

approximately 95% of total gene sequences in fecal samples of weaned piglets. Samples of feces

291

in the WB group had greater relative abundance of Fibrobacteres compared with the fecal samples

292

in the CON group, whereas piglets in the CB group showed greater population of Actinobacteria.

293

Fibrobacteres was characterized due to its high cellulolytic activity and the capability of

294

fermenting complex polysaccharides. Fibrobacteres exerted an important impact on fiber

295

digestion and energy metabolism in the intestine.36 In addition; Firmicutes and Actinobacteria

296

were predominant butyrate-producing bacteria, e.g. Eubacterium rectale or Eubacterium. Hallii,

297

whereas propionate is mainly produced by Bacteroidetes.26,37 In our study, CB group showed the 12



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highest abundance of Firmicutes that should be explanation for the increased concentration of

299

butyrate in fecal samples. Bacillus is mainly consisted of Streptoccus and Latobacillus, and their

300

abundances in CB group were greater than CON group. The previous study reported that Bacillus

301

species, as a probiotic, could improve ADG and feed efficiency of piglets.38 Lactobacillus is one

302

of the most commonly lactic acid bacteria and utilized as probiotics in human and animals.39

303

Furthermore, Clostridium was reported as SCFAs producer for polysaccharide fermentation.40

304

Lachnospiraceae and Rumininococcaceae were two of the most abundant bacteria on family level

305

from Clostridia among the 4 groups, and they were considered as fibrolytic bacteria to ferment the

306

complex component of plant cell wall. Fiber inclusion in the diet increased the relative abundance

307

of Megasphaera, which degraded lactic acid into propionic acid and acetic acid.41 However, our

308

results showed the concentration of acetic acid was not discrepant among the 4 dietary treatments,

309

which might be caused by the low relative abundant of Megasphaera. In addition, Prevotella in

310

WB and CB groups were lower than CON group by using the LEfSe algorithm. However,

311

previous studies reported that Prevotella was more prevalent than Bacteroides when the subject

312

was served diets supplemented with plant food, which are rich in soluble carbohydrates, such as

313

fruit and bread.42,43 Therefore, the diverse results for effects of dietary fiber on the abundance of

314

Prevotella should be associated with the chemical composition and properties of different fibrous

315

ingredients.

316

In summary, corn bran and wheat bran added in the diet with an appropriate level were

317

beneficial for growth performance and could shape the bacterial community of piglets. This

318

finding would contribute to a better understanding how different fibers modulate gut health via

319

nutrient intervention. The underlying mechanism on the interplay between fiber, gut microbiota

320

and health is needed further studies.

321 322

Abbreviations

13


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ADFI, average daily feed intake; ADG, average daily gain; BW, body weight; F/G, feed to gain

324

ratio; IDF, insoluble dietary fiber; LEfSe, linear discriminant analysis effect size; OTUs,

325

operational taxonomic units; PICRUSt, phylogenetic investigation of communities by

326

reconstruction of unobserved states; SCFAs, short-chain fatty acids; SDF, soluble dietary fiber;

327

TDF, total dietary fiber.

328 329

Author contributions

330

JZ, PL and XM designed the research. JZ, PL, PG, LL and MN conducted the research. JZ, PL,

331

LL and YW analyzed the data. The manuscript was mainly written by JZ and PL, and edited by

332

XM, CL and JZ. All the authors have read and approved the final manuscript.

333 334

Funding

335

This work was supported by the National Key R&D Program of China (2018YFD0500601,

336

2017YFD0500501), the National Natural Science Foundation of China (31722054, 31472101 and

337

31528018), College of Animal Science and Technology "Young Talents Program" in China

338

Agricultural University (2017DKA001), the 111 Project (B16044), the developmental fund for

339

animal science by Shenzhen Jinxinnong Feed Co., Ltd., and the Beijing Nova Programme

340

Interdisciplinary Cooperation Project (xxjc201804).

341 342

Notes

343

The authors declare that the research was conducted in the absence of any commercial or financial

344

relationships that could be construed as a potential conflict of interest.

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Table legends

Table 1 Chemical composition of fibrous ingredients (g/kg, as-fed basis) a. items

corn bran wheat bran soybean hulls dry matter 921 905 913 crude protein 128 166 174 starch 311 168 64 neutral detergent fiber 454 423 430 acid detergent fiber 132 124 302 acid detergent lignin 18 29 12 cellulose 114 95 290 hemicellulose 322 299 128 total dietary fiber 541 434 549 soluble dietary fiber 60 45 102 insoluble dietary fiber 481 389 448 insoluble non-starch polysaccharides 379 265 483 rhamnose 15 8 17 arabinose 73 61 34 xylose 109 100 63 mannose 18 6 36 galactose 11 5 15 glucose 139 76 283 uronic acids 14 9 35 soluble non-starch polysaccharides 56 41 84 rhamnose 2 1 1 arabinose 9 7 2 xylose 15 13 1 mannose 5 3 7 galactose 2 1 3 glucose 19 14 42 uronic acids 4 3 30 total non-starch polysaccharides 434 306 567 water holding capacity, g/g 4.18 4.32 4.94 a Data are the means of two replicates of analyzed values. The soluble dietary fiber was calculated as the difference between total dietary fiber and insoluble dietary fiber. The hemicellulose was calculated as the difference in neutral detergent fiber and acid detergent fiber. Additionally, cellulose was calculated as the difference in acid detergent fiber and acid detergent lignin.

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Table 2 Effect of fiber inclusions on growth performance and diarrhea incidence in weaned piglets a. items BW, kg 1d 14 d 28 d 1-14 d ADFI, g ADG, g F/G diarrhea rate, % 14-28 d ADFI, g ADG, g F/G diarrhea rate, % 1-28 d ADFI, g ADG, g F/G diarrhea rate, % a Data were shown as

diets CON

CB

WB

SB

7.18 ± 0.15 11.6 ± 0.20 17.5 ± 0.21

7.18 ±0.16 12.1 ± 0.20 18.1 ± 0.29

7.18 ± 0.16 11.9 ± 0.21 18.2 ± 0.26

7.19 ± 0.16 11.4 ± 0.24 16.8 ± 0.33

502 ± 32 318 ± 17ab 1.59 ± 0.06a 2.18 ± 0.78

501 ± 40 351 ± 19a 1.43 ± 0.07b 1.98 ± 0.91

500 ± 28 340 ± 14a 1.47 ± 0.03b 2.38 ± 1.02

471 ± 52 301 ± 22b 1.57 ± 0.02a 2.88 ± 1.11

785 ± 28 419 ± 16 1.88 ± 0.07 3.85 ± 0.17

755 ± 60 432 ± 40 1.75 ± 0.06 3.21 ± 0.08

797 ± 29 445 ± 21 1.79 ± 0.04 2.14 ± 0.08

707 ± 78 387 ± 48 1.85 ± 0.13 1.97 ± 0.11

644 ± 23 368 ± 12ab 1.75 ± 0.05a 2.88 ± 0.77 mean ± SEM (n =

628 ± 44 391 ± 27a 1.61 ± 0.04b 2.38 ± 0.69 6). The result was

648 ± 25 592 ± 56 a 393 ± 12 344 ± 29b 1.65 ± 0.02b 1.72 ± 0.05a 2.18 ± 0.82 2.29 ± 1.05 analyzed by one-way ANOVA with

Turkey’s test and the variant alphabetical superscript in the same row indicated significant difference when P < 0.05. Data were analyzed by one-way ANOVA with Turkey’s test. ADFI, average daily feed intake; ADG, average daily gain; F/G, the ratio of ADFI to ADG; CB, corn bran; CON, control; SB, soybean hulls; WB, wheat bran.

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Table 3 Effects of fiber inclusions on nutrients digestibility in weaned piglets a. diets items, %

CON

CB

GE 87.9 ± 0.5a CP 82.5 ± 1.0 DM 88.2 ± 0.7a OM 90.0 ± 0.5a AEE 75.7 ± 3.6 TDF 66.5 ± 1.9b a Data were shown as mean ± SEM

WB

SB

86.8 ± 0.4bc 87.0 ± 0.6bc 87.6 ± 0.4ab 81.5 ± 1.6 81.7 ± 1.1 81.7 ± 1.0 b b 86.9 ± 0.5 86.8 ± 0.6 87.8 ± 0.4a 89.1 ± 0.4b 89.2 ± 0.5b 89.7 ± 0.4ab 74.2 ± 2.7 75.3 ± 4.8 73.8 ± 4.0 ab ab 67.4 ± 1.6 68.0 ± 1.6 69.2 ± 1.1a (n = 6). The result was analyzed by one-way ANOVA with

Turkey’s test and the variant alphabetical superscript in the same row indicated significant difference when P < 0.05. AEE, acid hydrolyzed ether extract; CB, corn bran; CON, control; CP, crude protein; DM, dry matter; GE, gross energy; OM, organic matter, SB, soybean hulls; TDF, total dietary fiber; WB, wheat bran.

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Table 4 Effects of fiber inclusions on fecal short-chain fatty acids in weaned piglets a. diets CON CB WB SB acetate 3.18 ± 0.75 2.97 ± 0.66 3.19 ± 0.82 3.74 ± 0.98 propionate 1.33 ± 0.52 1.55 ± 0.67 1.67 ± 0.80 1.65 ± 0.48 isobutyrate 0.15 ± 0.08 0.17 ± 0.06 0.14 ± 0.04 0.12 ± 0.05 c a ab butyrate 0.53 ± 0.06 0.83 ± 0.09 0.76 ± 0.08 0.76 ± 0.09ab isovalerate 0.08 ± 0.04 0.12 ± 0.05 0.12 ± 0.04 0.10 ± 0.03 b a ab valerate 0.14 ± 0.05 0.30 ± 0.11 0.21 ± 0.08 0.19 ± 0.02ab total SCFAs 5.42 ± 0.92 5.94 ± 1.21 6.09 ± 1.15 6.38 ± 1.38 a Data were shown as mean ± SEM (n = 6). The result was analyzed by one-way ANOVA with items, mg/g

Turkey’s test and the variant alphabetical superscript in the same row indicated significant difference when P < 0.05. CB, corn bran; CON, control; SB, soybean hulls; WB, wheat bran; SCFAs, short-chain fatty acids.

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Table 5 Effects of fiber inclusions on alpha-diversity of fecal bacterial community in weaned piglets a diets

items

CON CB WB SB shannon 4.42 ± 0.34 4.18 ± 0.43 4.59 ± 0.06 4.43 ± 0.32 chao 469 ± 57 467 ± 63 524 ± 19 498 ± 50 a Alpha-diversity analysis of Chao and Shannon indexes for bacterial community determined by 16s RNA gene sequencing. Data were analyzed by Kruskal-Wallis H test and shown as mean ± SEM (n = 3). CB, corn bran; CON, control; SB, soybean hulls; WB, wheat bran.

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Figure legends Figure 1. The bacterial OTU community composition and bacterial community heatmap on the genera level in weaned piglets. (A) Venn diagrams for bacterial OTU in 4 dietary treatments. The results were analyzed by Kruskal-Wallis H test and presented as mean percentage of different bacteria. OTU, operational taxonomic units. (B) Microbial community heatmap of top 30 bacteria on genus level. The different colors represented the relative abundance of bacteria in 4 dietary treatments. CON, control group; CB, corn bran; SB, soybean hulls; WB, wheat bran.

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Figure 2. Fecal microbial diversity on the phylum and family levels in piglets fed the 4 dietary treatments. (A) Microbial community barplot on the phylum level with the abundance higher than 0.01%. Kruskal-Wallis method was applied to identify the differences in the relative abundant of gut microbiota on phyla level. (B) Microbial community barplot on the family level with the proportion higher than 0.1%. The data were analyzed by Kruskal-Wallis H test. CON, control; CB, corn bran; SB, soybean hulls; WB, wheat bran.

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Figure 3. Analysis for differential bacteria among 4 dietary treatments. (A) Differential bacteria among 4 treatments. (B) Differential bacteria between CON and SB treatments. (C) Differential bacteria between CON and WB treatments. (D) Differential bacteria between CON and CB treatments. CON, control; CB, corn bran; SB, soybean hulls; WB, wheat bran.

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Figure 4. Prediction on carbohydrate metabolism of bacterial communities using PICRUSt program. (A) Energy metabolism. (B) Fatty acid biosynthesis. (C) Carbohydrate digestion and absorption. (D) Starch and sucrose metabolism. (E) Fructose and mannose metabolism. (F) Galactose metabolism. CB, corn bran; CON, control; SB, soybean hulls; WB, wheat bran.

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