Oat bran and wheat bran impact net energy by shaping microbial communities and fermentation products in pigs fed diets with or without xylanase

Zhiqian Lyu, Li Wang, Jinrong Wang, Zhenyu Wang, Shuai Zhang, Junjun Wang, Jinlong Cheng, Changhua Lai

► To cite this version:

Zhiqian Lyu, Li Wang, Jinrong Wang, Zhenyu Wang, Shuai Zhang, et al.. Oat bran and wheat bran impact net energy by shaping microbial communities and fermentation products in pigs fed diets with or without xylanase. Journal of Animal Science and Biotechnology, 2020, 11 (1), 10.1186/s40104-020-00505-7. hal-03132508

HAL Id: hal-03132508

https://hal.inrae.fr/hal-03132508

Submitted on 5 Feb 2021

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
RESEARCH

Open Access

<figure><img src="image_2.png" /></figure>

Check for
updates

# Oat bran and wheat bran impact net energy by shaping microbial communities and fermentation products in pigs fed diets with or without xylanase

Zhiqian Lyu¹,², Li Wang¹, Jinrong Wang³, Zhenyu Wang¹, Shuai Zhang¹, Junjun Wang¹, Jinlong Cheng⁴ and Changhua Lai¹*

## Abstract

**Background:** Dietary fiber can be fermented in gut of pigs and the end products of fermentation were short-chain fatty acids (SCFA). The SCFA had positive effects on gut bacteria and host immune system. In addition, SCFA can provide a part of available energy for pigs. However, there were limited reports on the relationship between dietary fiber, gut bacteria, and energy metabolism. Therefore, this study investigated how dietary fiber and enzyme addition impacted energy metabolism by acting on the microbial community and SCFA.

**Methods:** Wheat bran (WB) was added to the corn-soybean meal-based diet at the levels of 12% and 27%, and oat bran (OB) at 15% and 36%. One of each diet was supplemented with or without 5000 U/kg feed of xylanase, so a total of 10 diets were allotted to 60 growing pigs (initial body weight: 27.2 ± 1.2 kg) using a randomized complete block design. The experiment was conducted in 10 consecutive periods using 6 similar open-circuit respiration chambers. Each pig was used for one 20-day period. During each period, six pigs were allowed 14 d to adapt to the diets in metabolic cages followed by 6 d (from d 15 to d 20) in respiration chambers to measure heat production (HP).

**Results:** Pigs fed 36% OB diets had greater (*P* < 0.05) nutrient digestibility and net energy (NE) values compared to those fed 27% WB diets. Apparent digestibility coefficients of dry matter (DM) and crude protein (CP) were lower (*P* < 0.05) in pigs fed 27% WB diets compared with those fed 12% WB diets. Enzyme addition improved (*P* < 0.05) the NE values (11.37 vs. 12.43 MJ/kg DM) in diets with 27% WB. Supplementation of xylanase did not affect NE values for basal diets, OB diets and 12% WB diets. Compared with diets with 36% OB, pigs fed 27% WB-based diets excreted more total SCFA, acetate and propionate (expressed as g/kg feed DM) in fecal samples of pigs (*P* < 0.05). Pigs in the WB diets had greater proportion of phylum Bacteroidetes while phylum Firmicutes were greater in pigs fed OB diets (*P* < 0.05). Pigs fed WB diets had greater (*P* < 0.05) abundance of Succinivibrio and *Prevotella*, which were associated with fiber degradation and SCFA production.

(Continued on next page)

<footnote>* Correspondence: laichanghua999@163.com
¹State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China
Full list of author information is available at the end of the article</footnote>
(Continued from previous page)

**Conclusion:** Our results indicated diets supplied by high level of OB or WB promote the growth of fiber-degrading bacteria. The differences in fiber composition between WB and OB led to differences in nutrient digestibility and bacterial communities, which were ultimately reflected in energy metabolism. Enzyme supplementation improved nutrient digestibility as well as NE values for 27% WB diets but not for other diets, which indicated that effects of enzyme were related to type and level of dietary fiber in diets.

**Keywords:** Bacterial community, Bacterial metabolites, Dietary fiber, Exogenous enzyme, Net energy, Pig

## Background

Dietary fiber (DF) means carbohydrate polymers with ten or more monomeric units which are not hydrolyzed by the endogenous enzymes in the small intestine of humans [1]. The microbial conversions of DF to mono-saccharides in the gut involves a number of principal reactions mediated by the carbohydrate-active enzymes. These enzymes are from specific members of the gut microbiota and have the function to cleave the glycosidic bonds between sugar monomers or between carbohydrate and non-carbohydrate structures. Major end products from these fermentations are the short-chain fatty acids (SCFA) [2], mainly acetate, propionate, and butyrate. Recent studies show that SCFA from microbial fermentation generates 11% to 24% of the total digestible energy (DE) as wheat bran (WB) level increases from 10% to 30% when fed to pigs [3]. It suggests that DF has a final impact on energy metabolism by shaping microbes and regulating SCFA production [4]. However, there is little information on the relationship between DF, gut bacteria, SCFA, and energy metabolism [5].

Oat bran (OB) and WB are rich in DF that has negative effects on nutrient digestibility due to their anti-nutritive properties, but they may act as substances and energy sources to stimulate the intestinal microbiota [6]. Dietary fiber in OB is easier fermentable energy source for bacteria in the large intestine due to their lower proportion of lignified fiber types and higher proportion of soluble fiber types compared with fiber in WB. Specially, OB is rich in mixed linkages β-glucan which to a high degree is soluble and insoluble arabinoxylan [7]. By contrast, WB contained rich insoluble arabinoxylan. In the small intestine, β-glucan in OB is partly digested while arabinoxylan in OB or WB is hardly digested [7, 8]. Accordingly, arabinoxylan is considered as one of the main components for DF in OB and WB to resist fermentation by bacteria in gastrointestinal tract. By adding xylanase to feeds, xylose backbone of arabinoxylan can be cleaved, thereby improving accessibility and utilization of nutrients in WB or OB diets and providing some fermentable oligosaccharides degraded from indigestible polysaccharides to intestinal bacteria [9]. Substrates available to intestinal bacteria and physiology of lumen change along gastrointestinal tract, and consequently

intestinal bacteria are altered. Therefore, DF and exogenous enzymes, independently or in combination interaction are important means of regulating intestinal bacteria and ultimately affecting energy metabolism [4–6].

Therefore, we hypothesize that the diets supplemented with different fiber sources, with or without xylanase, could exert different impacts on nutrient digestibility and NE values of diets, and these differences were related to the activity of certain bacteria. Specially, our objectives are to determine effects of dietary characteristics and xylanase addition on nutrient digestibility, NE values of diets, fecal SCFA concentrations, and fecal bacterial communities of growing pigs.

## Methods

### Experimental diets and enzyme

As shown in Table 1, WB diets contained 12% or 27% WB which was added at the expense of corn and soybean meal. Oat bran diets contained 15% or 36% OB which was added at the expense of corn and soybean meal. Enzyme was added to one half of each diet at the rate of 40 g/1000 kg diet to supply enzyme activity of 5000 U/kg complete feed (as-fed basis; Table 1). The enzyme was first mixed in the vitamin-mineral premix and then mixed with other ingredients. A total of 10 diets were fed to 60 growing pigs with a randomized complete block design. Diets were formulated on NE basis to meet estimated AA requirements for growing pigs [10]. Diets were formulated based on total dietary fiber (TDF) content in OB and WB. Specially, DF content (calculated values) between 36% OB diets and 27% WB diets or between 15% OB diets and 12% WB diets was similar. The xylanase used in the current study was endo-1, 4-β-xylanase in granular form. The xylanase was provided by Bestzyme Co. Ltd. (Jinan, China) with enzyme activity of 200,000 U/g.

### Animals and experimental procedure

The animal procedures used in this experiment, including animal care and use, were approved by the Animal Care and Use Ethics Committee of China Agricultural University (Beijing, China). Sixty growing barrows (initial BW of 27.2 ± 1.2 kg) were allotted to 10 diets with 6 replicate pigs per diet in a randomized block design. The
<table><caption>**Table 1** Ingredients of experimental diets (as-fed basis)<fnref n="1" /></caption><thead><tr><th rowspan="2">Item</th><th rowspan="2">Basal diet</th><th colspan="4">Test diets</th></tr><tr><th>36% OB</th><th>27% WB</th><th>15% OB</th><th>12% WB</th></tr></thead><tbody><tr><td>Ingredients, %</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Corn</td><td>71.60</td><td>45.12</td><td>51.74</td><td>60.57</td><td>62.77</td></tr><tr><td>Soybean meal</td><td>24.96</td><td>15.73</td><td>18.04</td><td>21.11</td><td>21.88</td></tr><tr><td>Oat bran</td><td>–</td><td>36.00</td><td>–</td><td>15.00</td><td>–</td></tr><tr><td>Wheat bran</td><td>–</td><td>–</td><td>27.00</td><td>–</td><td>12.00</td></tr><tr><td>Dicalcium phosphate</td><td>0.90</td><td>0.90</td><td>0.90</td><td>0.90</td><td>0.90</td></tr><tr><td>Limestone</td><td>0.90</td><td>0.90</td><td>0.90</td><td>0.90</td><td>0.90</td></tr><tr><td>Salt</td><td>0.35</td><td>0.35</td><td>0.35</td><td>0.35</td><td>0.35</td></tr><tr><td>Premix<sup>2</sup></td><td>0.50</td><td>0.50</td><td>0.50</td><td>0.50</td><td>0.50</td></tr><tr><td>Lys HCl</td><td>0.50</td><td>0.32</td><td>0.36</td><td>0.42</td><td>0.44</td></tr><tr><td>Met</td><td>0.07</td><td>0.04</td><td>0.05</td><td>0.06</td><td>0.06</td></tr><tr><td>Thr</td><td>0.16</td><td>0.10</td><td>0.12</td><td>0.14</td><td>0.14</td></tr><tr><td>Val</td><td>0.06</td><td>0.04</td><td>0.04</td><td>0.05</td><td>0.05</td></tr></tbody></table>

experiment was conducted in 10 consecutive periods using 6 similar open-circuit respiration chambers as described by Lyu et al. [11]. Each pig was used for one 20-d period. During each period, 6 pigs were allowed 14 d to adapt to diets in metabolic cages followed by 6 d in respiration chambers to measure heat production (HP). Pigs were fed their assigned diets at 2.3 MJ ME/(kg BW)⁰.⁶d) on basis of BW measured on d 0, 7, and 14 [12]. Pigs were fed equal-sized dried meals twice daily at 08:30 and 15:30 h in mash form and had free access to water via a low-pressure nipple drinker. Urine was collected in buckets containing 50 mL of 6 mol/L HCl. From d 15 to 19 of each period, total feces and urine were collected from the chambers once daily at 8:30 h and then stored at –20 °C. On d 19 (from 7:30 to 9:00 h) of each period, six freshly voided fecal samples from each dietary treatment were collected to measure SCFA concentrations and bacterial community. Fecal samples were acquired using a 5-mL centrifuge tube, put in liquid nitrogen and then were stored at –80 °C. Diet samples were collected in each collection period and then stored at –20 °C. At the end of experiment, diet samples were mixed and subsampled based on treatments to measure xylanase activity. From d 19 to 20 of each period, pigs were fasted.

The design of 6 open-circuit respiration chambers were reported by van Milgen et al. [13] and Lyu et al. [11]. Volume of each chamber was approximately 7.8 m³, and relative humidity in chamber was controlled at 70%. During the feeding period, the temperature was

22 °C, and it was increased to 24 °C on the fasted day. The measurement range of analyzers was 19.5% to 21% for O₂, 0 to 1% for CO₂, 0 to 0.1% for CH₄, and 0 to 0.1% for NH₃ with a sensitivity of 0.2% within the measurement range.

## Sample preparation and chemical analyses

Diets were collected during each feeding period to measure chemical composition. Fecal samples were oven-dried for 72 h at 65 °C and were ground through a 1-mm screen for further chemical analysis. Urine samples were pooled separately within pig.

Xylanase activity in diets was analyzed using spectrophotometric method as described by Lærke et al. [14]. The chemical analyses of ingredients and diets included dry matter (DM, method 930.15) [15], crude protein (CP, method 984.13) [15], ether extract (EE) [16], ash (method 942.05) [15], Ca (method 968.08) [15], and P (method 946.06) [15]. Gross energy (GE) of ingredients, diets, feces, and urine (UE) was determined using an isoperibol calorimeter (Parr 6300 Calorimeter, Moline, IL USA). The neutral detergent fiber (NDF) and acid detergent fiber (ADF) in ingredients, diets, and feces were determined using a fiber analyzer (model A220 fiber analyzer; Ankom Technology, Macedon, NY, USA) following a procedure by Van Soest et al. [17]. The combination of enzymatic and gravimetric procedures reported by Prosky et al. [18] was used to measure TDF, insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) in ingredients and diets. Non-starch

<footnote n="1">¹OB Oat bran, WB Wheat bran. The xylanase was provided by Bestzyme Co. Ltd. (Jinan, China). Enzyme was added to one half of each diet at the rate of 40 g/ 1000 kg diet to supply enzyme activity of 5000 U/kg complete feed (as-fed basis)</footnote>

<footnote n="2">²Vitamin-mineral premix supplied the following per kg of complete diet: vitamin A as retinyl acetate, 5512 IU; vitamin D₃ as cholecalciferol, 2200 IU; vitamin E as DL-alpha-tocopheryl acetate, 30 IU; vitamin K₃ as menadione nicotinamide bisulfite, 2.2 mg; vitamin B₁₂, 27.6 µg; riboflavin, 4 mg; pantothenic acid as DL-calcium pantothenate, 14 mg; niacin, 30 mg; choline chloride, 400 mg; folacin, 0.7 mg; thiamin as thiamine mononitrate, 1.5 mg; pyridoxine as pyridoxine hydrochloride, 3 mg; biotin, 44 µg; Mn as MnO, 40 mg; Fe as FeSO₄·H₂O, 75 mg; Zn as ZnO, 75 mg; Cu as CuSO₄·5H₂O, 25 mg; I as KI, 0.3 mg; Se as Na₂SeO₃, 0.3 mg</footnote>
polysaccharides (NSP) and their constituent sugars in
OB and WB were determined based on alditol acetates
by gas-liquid chromatography (Aglilent GC 6890, USA)
with a flow of 20 mL/min and split 40:1. The column
temperature was 220 °C and the injector and detector
temperature were 250 °C.

Fecal SCFA concentrations were measured using the method reported by Liu et al. [19]. About 0.5 g of fecal sample was placed in a centrifuge tube with 8 mL of distilled water. The mixture was centrifuged at 13,000 r/ min for 10 min to obtain the supernatant. One milliliter of supernatant was diluted 1:50 with water and filtered through a 0.22-mm nylon membrane filter. Filtered supernatant was analyzed using high-performance ion exchange chromatograph (ICS 3000 Dionex, USA).

Analysis for bacterial microbiota by 16S RNA sequences

Bacterial DNA was extracted from fecal samples using
the DNA Kit (Omega Bio-tek, Norcross, GA, USA).
NanoDrop 2000 UV-VIS spectrophotometer (Thermo
Scientific, Wilmington, USA) was used to determine
DNA concentration and purification. Quality of DNA
was assessed by 1% agarose gel electrophoresis. The V3-
V4 hypervariable regions of the bacteria 16S rRNA gene
were amplified with primers 338F (5'-ACTCCTACGG
GAGGCAGCAG-3') and 806R (5'-GGACTACHVGGG
TWTCTAAT-3') by thermocycler PCR system (GeneAmp
9700, ABI, USA). The PCR products were extracted from a
2% agarose gel and then purified using the AxyPrep DNA
Gel Extraction Kit (Axygen Biosciences, Union City, CA,
USA) and quantified using QuantiFluor™-ST (Promega,
USA).

Pooled and purified amplicons in equimolar and
paired-end were sequenced (2 × 300 bp) on an Illumina
MiSeq platform (Illumina, San Diego, USA) according to
the standard protocols introduced by Majorbio Bio-
Pharm Technology Co. Ltd. (Shanghai, China). Raw fastq
files were quality-filtered by Trimmomatic and merged
by FLASH.

Operational taxonomic units (OTU) were clustered
with 0.97 similarity cutoff using UPARSE (version 7.1
http://drive5.com/uparse/). The taxonomy of each 16S
rRNA gene sequence was analyzed by Ribosomal Data-
base Project (RDP) Classifier algorithm (http://rdp.cme.
msu.edu/) against the Silva (SSU123) 16S rRNA database
using confidence threshold of 0.70.

Calculations

The apparent total tract digestibility (ATTD) of nutri-
ents, total heat production (THP), retained energy (RE),
and NE values in diets were calculated using the follow-
ing equations [12, 20]: ATTD = [(Fi – Ff)/Fi] × 100%,
where Fi was total intake of energy (kJ) or nutrients (g),
and Ff was the total fecal output of energy (kJ) or

nutrients (g); THP = 16.18 × O₂ + 5.02 × CO₂−2.17 × CH₄−5.99 × urinary nitrogen excretion, where THP was in kilojoule, O₂ was oxygen consumption in liters, CO₂ was carbon dioxide production in liters, and CH₄ was methane production in liters. Urinary nitrogen excretion was in grams. The data collection for O₂, CO₂, and CH₄ used the standard procedure as described by Lyu et al. [11]. In addition, to eliminate the effects of activity of pigs, FHP was calculated using the equation for THP with gas data obtained from 22:30 h (d 19) to 06:30 h (d 20). To calculate the FHP throughout the day, the 8-h FHP was extrapolated to a 24-h period; RE = (ME intake – THP), where RE was in kilojoule, and ME intake and THP were in kilojoule per day. The retained energy as protein (RE<sub>P</sub>) was calculated based a recognized formula (nitrogen × 6.25 × 23.84, kJ/g), whereas RE as lipids (RE<sub>L</sub>) was calculated as the difference between RE and RE<sub>P</sub> [21]; NE = (RE + FHP)/DM intake, where NE was in kilojoule per kilogram DM; RE and FHP were in kilojoule per day and DM intake was in kilograms per day. The DE of diets was calculated by subtracting energy lost in the feces from GE. The ME of diets was calculated by subtracting energy lost in the urine and methane from DE. Energy lost as methane (CH₄E) was calculated assuming 1 L of CH₄ contained 39.54 kJ energy [20]. The DE, metabolizable energy (ME) and NE content of OB and WB in diets containing 36% OB and 27% WB were calculated using the difference method, respectively [22].

Statistical analysis

All data of the experiment were averaged per pig and
were analyzed using the MIXED procedure of SAS (SAS
Institute Inc., Cary, NC, USA). The experimental unit
was the individual pig. Diet, enzyme and their interac-
tions were treated as the fixed effects and collection,
period and chamber as random effects. The LSMEANS
statement with Tukey's adjustment was used to separate
mean values. To eliminate the effect of ME intake, the
THP and RE data were adjusted for each collection
period by covariance analysis for ME intake of 2322 kJ/
(kg BW<sup>0.60</sup>.d) (mean value for the experiment). A mix-
ture of two fecal samples was used to analyze bacterial
communities. Bacterial diversity was analyzed using
standardized OTU reads with the help of R software.
Relative abundance of bacteria at phylum and family
levels were showed as bar plots. Heatmap was used to
analyze bacterial communities at genus level. The linear
discriminant analysis (LDA) effect size (LEfSe) algorithm
was adopted to classify abundances of differential bac-
teria from phylum to genus. The logarithmic LDA value
of 2.0 was used to be the criterion. The comparative
analysis between OB-based diets (n = 12) and WB-based
(n = 12) diets or between high-fiber diets (36% OB diets
and 27% WB diets; n = 12) and low-fiber diets (15% OB diets and 12% WB diets; n = 12), was conducted based on the method of Welch's t-test. If *P* < 0.05, it was considered significant difference. The contrast for data obtained in the enzyme-supplemented group to those obtained in enzyme-free group was also calculated using same method to validate the enzyme effect.

## Results

### Chemical composition of ingredients and diets

Wheat bran contained more content of NDF, ADF, hemicellulose, and IDF but lower content of SDF than OB (Supplementary Table S1). For NSP composition, OB contained more soluble NSP (7.8% vs. 3.9%), especially glucose. By contrast, WB contained more insoluble NSP (28.7% vs. 15.6%), mainly arabinose and xylose. In addition, OB contained more content of CP (22.0% vs. 15.1%), starch (28.1% vs. 8.2%) and EE (7.6% vs. 3.2%) than WB. Dietary fiber content was similar between 36% OB diets and 27% WB diets or between 15% OB diets and 12% WB diets, which was consistent to the study design (Table 1 and Table 2).

### Nutrient digestibility, nitrogen balance, and energy metabolism

There were interactions (*P* < 0.05) between diet and enzyme supplementation on ATTD of DM, GE, CP and ADF (Table 3). In the enzyme-free treatments, 27% WB diets had lower (*P* < 0.05) ATTD of DM, GE, CP, ADF and OM than OB diets and basal diets. Pigs fed 27% WB diets had lower (*P* < 0.05) ATTD of DM, CP and ADF than those fed 12% WB diets. By contrast, there were no differences on the ATTD of nutrients between 36% OB diets and 15% OB diets. The nitrogen output from feces

was greater (*P* < 0.05) in 27% WB diets than that in basal diets and 15% OB diets. Total nitrogen retention was not affected by dietary characteristics. Supplementation of enzyme improved (*P* < 0.05) ATTD of DM, CP and ADF in 27% WB diets, and no differences were observed for the enzyme effects in other dietary treatments.

No interactions between diet and enzyme on the energy retention were found (Supplementary Table S2). The THP, FHP, and RE data were not affected by dietary characteristics. Enzyme supplementation improved (*P* < 0.05) ME intake in pigs. There were interactions (*P* < 0.05) between diet and enzyme supplementation on ME and NE content of diets (Table 4). The NE:ME ratios and ME:DE ratios were not affected by diets and enzyme supplementation. The DE content was lower (*P* < 0.05) in pigs fed WB-based diets compared with those fed OB-based diets and basal diets. In enzyme-free dietary groups, 27% WB diets had lower (*P* < 0.05) ME and NE values than other diets. There were no differences for the ME and NE values between 36% OB diets and 12% OB diets. Enzyme addition improved the NE values (*P* < 0.05) in pigs fed 27% WB diets. Enzyme addition improved (*P* < 0.05) ME and NE content of WB from 11.75 to 13.02, and 8.77 to 10.04 (MJ/kg of feed DM) (Table 5).

### Short-chain fatty acids in feces

The total SCFA were considered as the sum of lactate, formate, acetate, propionate, isobutyrate, butyrate, isovalerate, and valerate, but the lactate and formate were not listed because of their minor concentrations (Table 6). The concentrations of SCFA were expressed in mg/kg feed DM. There were interactions (*P* < 0.05) between diet and enzyme supplementation on concentrations of

<table><caption>**Table 2** Chemical composition of experimental diets (DM basis)<fnref n="1" /></caption><thead><tr><th rowspan="2">Item<sup>2</sup></th><th colspan="2">Basal diet</th><th colspan="2">36% OB</th><th colspan="2">27% WB</th><th colspan="2">15% OB</th><th colspan="2">12% WB</th></tr><tr><th>-</th><th>+</th><th>-</th><th>+</th><th>-</th><th>+</th><th>-</th><th>+</th><th>-</th><th>+</th></tr></thead><tbody><tr><td>Analyzed composition</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>GE, MJ/kg DM</td><td>18.21</td><td>18.37</td><td>19.00</td><td>19.10</td><td>18.62</td><td>18.65</td><td>18.66</td><td>18.58</td><td>18.60</td><td>18.45</td></tr><tr><td>CP, % DM</td><td>19.5</td><td>19.7</td><td>21.1</td><td>21.6</td><td>18.5</td><td>18.9</td><td>20.3</td><td>20.4</td><td>19.8</td><td>19.0</td></tr><tr><td>Ether extract, % DM</td><td>3.6</td><td>3.5</td><td>5.9</td><td>5.6</td><td>3.8</td><td>4.1</td><td>4.6</td><td>4.8</td><td>3.9</td><td>4.0</td></tr><tr><td>NDF, % DM</td><td>10.3</td><td>10.9</td><td>17.9</td><td>15.7</td><td>19.4</td><td>19.6</td><td>12.1</td><td>11.6</td><td>14.2</td><td>14.5</td></tr><tr><td>ADF, % DM</td><td>3.9</td><td>4.1</td><td>5.3</td><td>5.1</td><td>6.1</td><td>6.4</td><td>4.4</td><td>4.2</td><td>5.0</td><td>5.0</td></tr><tr><td>TDF, % DM</td><td>14.9</td><td>14.6</td><td>23.6</td><td>24.3</td><td>24.1</td><td>23.7</td><td>18.4</td><td>19.0</td><td>18.9</td><td>18.3</td></tr><tr><td>SDF, % DM</td><td>2.9</td><td>2.7</td><td>7.8</td><td>7.7</td><td>2.8</td><td>2.9</td><td>4.9</td><td>5.0</td><td>2.8</td><td>2.5</td></tr><tr><td>IDF, % DM</td><td>12.0</td><td>11.9</td><td>15.8</td><td>16.6</td><td>21.3</td><td>20.7</td><td>13.6</td><td>14.1</td><td>16.1</td><td>15.8</td></tr><tr><td>Ash, % DM</td><td>4.8</td><td>4.9</td><td>5.5</td><td>6.0</td><td>5.6</td><td>5.6</td><td>5.1</td><td>5.2</td><td>5.1</td><td>5.3</td></tr><tr><td>Enzyme activity, U/kg</td><td>-</td><td>5325</td><td>-</td><td>5120</td><td>-</td><td>4876</td><td>-</td><td>4795</td><td>-</td><td>5145</td></tr></tbody></table>

<footnote n="1">¹Data are the means of two replicates of analyzed values</footnote>

<footnote n="2">²OB Oat bran; WB Wheat bran; GE Gross energy; DM Dry matter; CP Crude protein; NDF Neutral detergent fiber; ADF Acid detergent fiber; TDF Total dietary fiber; SDF Soluble dietary fiber; IDF Insoluble dietary fiber. The - and + symbol represents enzyme-free diets and enzyme addition diets, respectively</footnote>
<table><caption>**Table 3** Effect of dietary characteristics and xylanase addition on nutrient digestibility and nitrogen balance of growing pigs (DM basis)<fnref n="1" /></caption><thead><tr><th rowspan="2">Item</th><th colspan="3">Xylanase-free diets</th><th colspan="10">Xylanase supplementation diets</th><th colspan="3">SEM</th></tr><tr><th>Basal diet</th><th>36% OB</th><th>27% WB</th><th>15% OB</th><th>12% WB</th><th>Basal diet</th><th>36% OB</th><th>27% WB</th><th>15% OB</th><th>12% WB</th><th>P-value</th><th>Diet</th><th>Xylanase</th><th>Dxx<fnref n="2" /></th></tr></thead><tbody><tr><td>Average BW</td><td>35.0</td><td>35.5</td><td>35.0</td><td>35.6</td><td>36.5</td><td>36.6</td><td>38.2</td><td>35.9</td><td>35.1</td><td>35.6</td><td>0.96</td><td>0.572</td><td>0.212</td><td>0.313</td></tr><tr><td>DMI, kg/d</td><td>1.21</td><td>1.23</td><td>1.28</td><td>1.25</td><td>1.28</td><td>1.26</td><td>1.37</td><td>1.34</td><td>1.25</td><td>1.28</td><td>0.04</td><td>0.295</td><td>0.051</td><td>0.421</td></tr><tr><td>ATTD, %</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>DM</td><td>91.0<sup>a</sup></td><td>86.8<sup>bc</sup></td><td>80.7<sup>d</sup></td><td>88.8<sup>abc</sup></td><td>85.2<sup>c</sup></td><td>89.9<sup>ab</sup></td><td>86.0<sup>bc</sup></td><td>85.3<sup>c</sup></td><td>89.4<sup>abc</sup></td><td>87.2<sup>abc</sup></td><td>1.00</td><td>&lt; 0.001</td><td>0.043</td><td>0.041</td></tr><tr><td>GE</td><td>90.2<sup>a</sup></td><td>86.0<sup>b</sup></td><td>79.6<sup>c</sup></td><td>87.9<sup>ab</sup></td><td>84.2<sup>bc</sup></td><td>89.1<sup>ab</sup></td><td>85.4<sup>b</sup></td><td>81.6<sup>bc</sup></td><td>88.4<sup>ab</sup></td><td>86.4<sup>ab</sup></td><td>1.08</td><td>&lt; 0.001</td><td>0.082</td><td>0.048</td></tr><tr><td>CP</td><td>89.6<sup>a</sup></td><td>86.9<sup>a</sup></td><td>80.2<sup>c</sup></td><td>88.2<sup>a</sup></td><td>84.8<sup>ab</sup></td><td>88.5<sup>a</sup></td><td>84.7<sup>ab</sup></td><td>85.7<sup>ab</sup></td><td>89.0<sup>a</sup></td><td>87.0<sup>a</sup></td><td>1.44</td><td>&lt; 0.001</td><td>0.045</td><td>0.043</td></tr><tr><td>NDF</td><td>63.5<sup>ab</sup></td><td>70.0<sup>a</sup></td><td>53.3<sup>b</sup></td><td>63.4<sup>ab</sup></td><td>57.5<sup>ab</sup></td><td>61.0<sup>ab</sup></td><td>63.5<sup>ab</sup></td><td>58.9<sup>ab</sup></td><td>65.2<sup>ab</sup></td><td>60.7<sup>ab</sup></td><td>2.74</td><td>0.003</td><td>0.861</td><td>0.215</td></tr><tr><td>ADF</td><td>65.2<sup>a</sup></td><td>58.1<sup>ab</sup></td><td>44.9<sup>c</sup></td><td>61.4<sup>a</sup></td><td>56.2<sup>ab</sup></td><td>63.8<sup>a</sup></td><td>53.7<sup>ab</sup></td><td>53.5<sup>ab</sup></td><td>62.1<sup>a</sup></td><td>60.4<sup>a</sup></td><td>3.29</td><td>0.022</td><td>0.025</td><td>0.036</td></tr><tr><td>OM</td><td>92.1<sup>a</sup></td><td>88.8<sup>ab</sup></td><td>82.6<sup>c</sup></td><td>90.3<sup>ab</sup></td><td>86.7<sup>bc</sup></td><td>91.1<sup>a</sup></td><td>88.1<sup>ab</sup></td><td>86.6<sup>bc</sup></td><td>90.8<sup>ab</sup></td><td>88.5<sup>b</sup></td><td>0.93</td><td>&lt; 0.001</td><td>0.122</td><td>0.064</td></tr><tr><td>UE/DE</td><td>1.80</td><td>1.97</td><td>2.47</td><td>1.85</td><td>1.77</td><td>1.54</td><td>2.02</td><td>1.63</td><td>1.47</td><td>1.54</td><td>0.26</td><td>0.412</td><td>0.118</td><td>0.353</td></tr><tr><td>CH<sub>4</sub>E/DE</td><td>0.58</td><td>0.40</td><td>0.57</td><td>0.52</td><td>0.45</td><td>0.51</td><td>0.31</td><td>0.34</td><td>0.55</td><td>0.51</td><td>0.08</td><td>0.142</td><td>0.243</td><td>0.385</td></tr><tr><td>Nitrogen balance, g/d</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Intake</td><td>36.8<sup>b</sup></td><td>40.4<sup>ab</sup></td><td>40.1<sup>ab</sup></td><td>39.0<sup>b</sup></td><td>39.3<sup>ab</sup></td><td>38.3<sup>b</sup></td><td>45.0<sup>a</sup></td><td>41.7<sup>ab</sup></td><td>39.0<sup>b</sup></td><td>39.1<sup>b</sup></td><td>1.27</td><td>0.002</td><td>0.075</td><td>0.353</td></tr><tr><td>FN</td><td>4.0<sup>c</sup></td><td>5.4<sup>abc</sup></td><td>7.7<sup>a</sup></td><td>4.8<sup>bc</sup></td><td>6.0<sup>abc</sup></td><td>4.7<sup>bc</sup></td><td>7.1<sup>ab</sup></td><td>7.2<sup>ab</sup></td><td>4.5<sup>bc</sup></td><td>5.2<sup>abc</sup></td><td>0.58</td><td>&lt; 0.001</td><td>0.702</td><td>0.181</td></tr><tr><td>UN</td><td>7.1</td><td>7.0</td><td>6.3</td><td>7.7</td><td>6.5</td><td>6.2</td><td>7.6</td><td>7.0</td><td>7.7</td><td>8.7</td><td>0.80</td><td>0.544</td><td>0.298</td><td>0.395</td></tr><tr><td>RN</td><td>25.6</td><td>28.0</td><td>26.1</td><td>26.5</td><td>26.8</td><td>27.5</td><td>30.3</td><td>27.5</td><td>26.7</td><td>25.2</td><td>1.28</td><td>0.324</td><td>0.142</td><td>0.571</td></tr></tbody></table>

a,b,c,d Values with different characters in superscripts were different (P < 0.05) in the same row

<footnote n="1">¹Treatment means are reported as least squares means and are based on 6 observations per treatment. OB Oat bran, WB Wheat bran; BW Body weight (mean values in collection period); DM Dry matter intake; ATTD Apparent total tract digestibility, GE Gross energy; CP Crude protein; NDF Neutral dietary fiber; ADF Acid detergent fiber; OM Organic matter; UE Urinary energy; CH4E Methane energy; FN Nitrogen output from feces; UN Urinary nitrogen excretion; RN Total retained nitrogen; SEM Standard error of the mean</footnote>

<footnote n="2">²Dxx is the interaction between diet and xylanase supplementation</footnote>
<table><caption>**Table 4** Effect of dietary characteristics and enzyme addition on energy utilization and energy values of experimental diets (DM basis)<fnref n="1" /></caption><thead><tr><th rowspan="2">Item</th><th colspan="3">Xylanase-free diets</th><th colspan="9">Xylanase supplementation diets</th><th colspan="2">SEM</th><th colspan="2">P-value</th></tr><tr><th>Basal diet</th><th>36% OB</th><th>27% WB</th><th>15% OB</th><th>12% WB</th><th>Basal diet</th><th>36% OB</th><th>27% WB</th><th>15% OB</th><th>12% WB</th><th>Diet</th><th>Xylanase</th><th>Dxx<fnref n="2" /></th></tr></thead><tbody><tr><td colspan="15">Energy utilization, %</td></tr><tr><td>ME/DE</td><td>97.6</td><td>97.6</td><td>96.9</td><td>97.6</td><td>97.7</td><td>97.9</td><td>97.7</td><td>98.0</td><td>98.0</td><td>97.6</td><td>0.28</td><td>0.801</td><td>0.073</td><td>0.241</td></tr><tr><td>NE/ME</td><td>82.8</td><td>82.7</td><td>79.2</td><td>82.2</td><td>81.5</td><td>83.4</td><td>81.6</td><td>83.3</td><td>81.7</td><td>82.6</td><td>1.56</td><td>0.924</td><td>0.186</td><td>0.709</td></tr><tr><td colspan="15">Energy values, MJ/kg DM</td></tr><tr><td>DE</td><td>16.40<sup>a</sup></td><td>16.38<sup>a</sup></td><td>14.81<sup>c</sup></td><td>16.43<sup>a</sup></td><td>15.55<sup>bc</sup></td><td>16.36<sup>a</sup></td><td>16.32<sup>a</sup></td><td>15.22<sup>c</sup></td><td>16.40<sup>a</sup></td><td>16.07<sup>ab</sup></td><td>0.16</td><td>&lt; 0.001</td><td>0.130</td><td>0.232</td></tr><tr><td>ME</td><td>16.01<sup>a</sup></td><td>15.99<sup>a</sup></td><td>14.35<sup>d</sup></td><td>16.04<sup>a</sup></td><td>15.20<sup>bc</sup></td><td>16.02<sup>a</sup></td><td>15.94<sup>ab</sup></td><td>14.92<sup>c</sup></td><td>16.07<sup>a</sup></td><td>15.68<sup>abc</sup></td><td>0.16</td><td>&lt; 0.001</td><td>0.049</td><td>0.034</td></tr><tr><td>NE</td><td>13.26<sup>a</sup></td><td>13.23<sup>a</sup></td><td>11.37<sup>c</sup></td><td>13.19<sup>a</sup></td><td>12.38<sup>b</sup></td><td>13.36<sup>a</sup></td><td>13.00<sup>a</sup></td><td>12.43<sup>b</sup></td><td>13.13<sup>a</sup></td><td>12.95<sup>ab</sup></td><td>0.27</td><td>&lt; 0.001</td><td>0.043</td><td>0.041</td></tr></tbody></table>

<footnote>a,b,c Values with different characters in superscripts were different (P < 0.05) in the same row</footnote>

<footnote n="1">¹Treatment means are reported as least squares means and are based on 6 observations per treatment. O8 Oat bran, W8 Wheat bran; DE Digestible energy; ME Metabolizable energy; NE Net energy; SEM Standard error of the mean</footnote>

<footnote n="2">²Dxx is the interaction between diet and xylanase supplementation</footnote>
<table><caption>**Table 5** Nutrient digestibility and energy values of the oat bran and wheat bran<fnref n="1" /></caption><thead><tr><th rowspan="2">Item<sup>2</sup></th><th colspan="2">Ingredient<sup>3</sup></th><th colspan="2">Wheat bran</th><th rowspan="2">SEM</th><th rowspan="2">P-value</th></tr><tr><th>Oat bran</th><th>+</th><th>Oat bran</th><th>+</th></tr></thead><tbody><tr><td>ATTD, %</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Gross energy</td><td>83.0<sup>a</sup></td><td>81.5<sup>a</sup></td><td>63.3<sup>c</sup></td><td>71.8<sup>b</sup></td><td>6.3</td><td>0.045</td></tr><tr><td>Crude protein</td><td>83.1<sup>a</sup></td><td>79.5<sup>ab</sup></td><td>52.4<sup>c</sup></td><td>77.5<sup>b</sup></td><td>6.8</td><td>0.034</td></tr><tr><td>Neutral detergent fiber</td><td>73.6<sup>a</sup></td><td>65.3<sup>ab</sup></td><td>47.0<sup>c</sup></td><td>62.8<sup>b</sup></td><td>7.8</td><td>0.020</td></tr><tr><td>Acid detergent fiber</td><td>52.2<sup>a</sup></td><td>43.8<sup>b</sup></td><td>28.0<sup>c</sup></td><td>52.8<sup>a</sup></td><td>4.7</td><td>0.031</td></tr><tr><td>Organic matter</td><td>83.7<sup>a</sup></td><td>83.4<sup>a</sup></td><td>58.0<sup>c</sup></td><td>75.0<sup>b</sup></td><td>5.0</td><td>0.044</td></tr><tr><td>Energy utilization, %</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ME/DE</td><td>97.7</td><td>97.2</td><td>98.4</td><td>96.3</td><td>3.6</td><td>0.405</td></tr><tr><td>NE/ME</td><td>77.5</td><td>79.6</td><td>74.7</td><td>77.1</td><td>5.4</td><td>0.223</td></tr><tr><td>Energy values, MJ/kg DM</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>DE</td><td>16.80<sup>a</sup></td><td>16.50<sup>a</sup></td><td>11.93<sup>b</sup></td><td>13.53<sup>ab</sup></td><td>0.72</td><td>0.122</td></tr><tr><td>ME</td><td>16.41<sup>a</sup></td><td>16.04<sup>a</sup></td><td>11.75<sup>c</sup></td><td>13.02<sup>b</sup></td><td>0.80</td><td>0.033</td></tr><tr><td>NE</td><td>12.72<sup>a</sup></td><td>12.76<sup>a</sup></td><td>8.77<sup>c</sup></td><td>10.04<sup>b</sup></td><td>0.95</td><td>0.046</td></tr></tbody></table>

acetate, propionate, butyrate, and isovalerate. In the enzyme-free dietary groups, pigs fed WB-based diets excreted more (P < 0.05) acetate and propionate and total SCFA in feces than those fed a basal diet. The acetate and propionate concentrations in feces were greater (P < 0.05) in pigs fed 27% WB diets compared with those fed 36% OB diets and a basal diet. The acetate concentrations in feces were greater (P < 0.05) in pigs fed 27% WB diets compared with those fed 12% WB diets. By contrast, the increase of fiber level in OB diets did not affect the acetate and propionate concentrations in fecal samples. Enzyme supplementation increased (P < 0.05) acetate, propionate, isobutyrate and total SCFA excretion in pigs fed 12% WB diets.

## The richness and biodiversity of bacterial communities

The indices of Chao and Shannon at the OTU level were used to elevate bacterial richness and diversity. The two indices were not affected by fiber source, fiber level or enzyme addition (Fig. 1). The bacterial composition was presented at the phylum, family, and genus levels, respectively (Fig. 2 and Supplementary Fig. S1). Firmicutes and Bacteroidetes, were the two main phyla of bacteria in the fecal samples of growing pigs (Supplementary Fig. S1A). The predominant families in Firmicutes consisted of Ruminococcaceae, Lachnospiraceae, Veillonellaceae, and Christensenellaceae, while Prevotellaceae, Bacteroidales_S24-7_group, and Erysipelotrichaceae were the predominant families in Bacteroidetes (Supplementary Fig. S1B).

Cladogram of LEfSe showed all differential bacteria from phylum to genus level (Fig. 3 and Fig. 4). The relative abundances of main kinds of bacteria were greater (P < 0.05) in high-fiber diets than those in low-fiber diets (Fig. 3a), including *Marvinbryantia*, [*Eubacterium_hallii_group*, *Lachnospiraceae_FCS020_group* and *Lachnospiraceae_ND3007_group*], which belonged to family Lachnospiraceae, and *Bifidobacterium*, which belonged to family Bifidobacteriaceae. The abundances of *Senegalimassilia* and *norank_fPeptococcaceae* were decreased in high-fiber diet than those in low-fiber diets (P < 0.05).

The abundance of *Anaerotruncus*, *Ruminococcaceae_ UCG_014*, and *Solobacterium* was greater (P < 0.05) in pigs fed OB diets than pigs fed WB, which resulted in an increase (P < 0.05) in the population of phylum Firmicutes (Fig. 3b). Pigs fed the WB diets had greater (P < 0.05) proportion of phylum Bacteroidetes, family Prevotellaceae and order Bacteroidia compared to those fed the OB diets, which was due to increased relative abundances of *unclassified_f_Prevotellaceae*, *Prevotella_1*, and *dgA_11_gut_group* (P < 0.05). The increased abundance of *Succinivibrio* (P < 0.05) led to greater proportion of Spirochaetae (P < 0.05) in pigs fed WB diets compared with those fed OB diets.

When pigs were fed diets with 36% OB (Fig. 4a), enzyme addition decreased (P < 0.05) the relative abundances of *unclassified_f_Lachnospiraceae*, *Lachnospiraceae_NK4A136_group*, *Lachnospiraceae_FCS020_group*, *Coprococcus_3*, *Marvinbryantia*, *Eubacterium_hallii_group*, and *Blautia* belonging to family Lachnospiraceae. Meanwhile, xylanase addition decreased (P < 0.05) the relative abundances of

<footnote>a,b,c:Values with different characters in superscripts were different (P < 0.05) in the same row</footnote>

<footnote n="1">¹Treatment means are reported as least squares means and are based on 6 observations per treatment</footnote>

<footnote n="2">²The apparent total tract digestibility (ATTD) of nutrient and energy values of oat bran and wheat bran were calculated using difference method in diets containing 36% oat bran or 27% wheat bran. DE Digestible energy; ME Metabolizable energy; NE Net energy; SEM Standard error of the mean</footnote>

<footnote n="3">³The – and + symbol represents enzyme-free dietary treatment and enzyme addition dietary treatment, respectively</footnote>
<table><caption>**Table 6** Effect of dietary characteristics and xylanase addition on fecal SCFA in growing pigs<fnref n="1" /></caption><thead><tr><th rowspan="2">Item<sup>2</sup></th><th colspan="4">Xylanase-free diets</th><th colspan="4">Xylanase supplementation diets</th><th rowspan="2">SEM</th><th colspan="2">P-value</th></tr><tr><th>Basal diet</th><th>36% OB</th><th>27% WB</th><th>15% OB</th><th>Basal diet</th><th>36% OB</th><th>27% WB</th><th>15% OB</th><th>12% WB</th><th>Diet</th><th>Xylanase</th><th>Dxx<sup>3</sup></th></tr></thead><tbody><tr><td>Acetate</td><td>0.68<sup>e</sup></td><td>1.23<sup>cde</sup></td><td>2.22<sup>ab</sup></td><td>1.24<sup>cde</sup></td><td>1.59<sup>cd</sup></td><td>1.07<sup>de</sup></td><td>1.66<sup>bcd</sup></td><td>1.73<sup>bc</sup></td><td>1.22<sup>cde</sup></td><td>2.50<sup>a</sup></td><td>0.123</td><td>&lt; 0.001</td><td>&lt; 0.001</td></tr><tr><td>Propionate</td><td>0.36<sup>e</sup></td><td>0.81<sup>cde</sup></td><td>1.28<sup>ab</sup></td><td>0.74<sup>cde</sup></td><td>0.99<sup>bcd</sup></td><td>0.62<sup>de</sup></td><td>1.15<sup>abc</sup></td><td>0.92<sup>bcd</sup></td><td>0.76<sup>cde</sup></td><td>1.54<sup>a</sup></td><td>0.096</td><td>&lt; 0.001</td><td>0.015</td></tr><tr><td>Isobutyrate</td><td>0.04<sup>c</sup></td><td>0.08<sup>abc</sup></td><td>0.09<sup>abc</sup></td><td>0.09<sup>abc</sup></td><td>0.08<sup>abc</sup></td><td>0.06<sup>bc</sup></td><td>0.13<sup>ab</sup></td><td>0.14<sup>a</sup></td><td>0.07<sup>abc</sup></td><td>0.15<sup>a</sup></td><td>0.016</td><td>&lt; 0.001</td><td>0.003</td></tr><tr><td>Butyrate</td><td>0.19<sup>b</sup></td><td>0.35<sup>b</sup></td><td>0.59<sup>ab</sup></td><td>0.40<sup>ab</sup></td><td>0.45<sup>ab</sup></td><td>0.26<sup>b</sup></td><td>0.44<sup>ab</sup></td><td>0.35<sup>b</sup></td><td>0.39<sup>ab</sup></td><td>0.67<sup>a</sup></td><td>0.065</td><td>&lt; 0.001</td><td>0.521</td></tr><tr><td>Isovalerate</td><td>0.09<sup>c</sup></td><td>0.17<sup>abc</sup></td><td>0.24<sup>a</sup></td><td>0.18<sup>abc</sup></td><td>0.11<sup>bc</sup></td><td>0.11<sup>bc</sup></td><td>0.22<sup>ab</sup></td><td>0.23<sup>ab</sup></td><td>0.17<sup>abc</sup></td><td>0.26<sup>a</sup></td><td>0.025</td><td>&lt; 0.001</td><td>0.009</td></tr><tr><td>Valerate</td><td>0.09<sup>b</sup></td><td>0.20<sup>ab</sup></td><td>0.17<sup>ab</sup></td><td>0.23<sup>ab</sup></td><td>0.15<sup>ab</sup></td><td>0.15<sup>ab</sup></td><td>0.28<sup>a</sup></td><td>0.14<sup>ab</sup></td><td>0.18<sup>ab</sup></td><td>0.24<sup>ab</sup></td><td>0.037</td><td>0.023</td><td>0.184</td></tr><tr><td>Total SCFA</td><td>1.48<sup>e</sup></td><td>2.88<sup>cde</sup></td><td>4.65<sup>ab</sup></td><td>2.90<sup>cd</sup></td><td>3.41<sup>bcd</sup></td><td>2.29<sup>cde</sup></td><td>3.95<sup>bc</sup></td><td>3.58<sup>bcd</sup></td><td>2.83<sup>cde</sup></td><td>5.42<sup>a</sup></td><td>0.295</td><td>&lt; 0.001</td><td>0.012</td></tr></tbody></table>

<footnote><sup>a,b,c,d,e</sup>Values with different characters in superscripts were different (P < 0.05) in the same row</footnote>

<footnote n="1"><sup>1</sup>Treatment means are reported as least squares means and are based on 6 observations per treatment. O<sup>B</sup> Oat bran; W<sup>B</sup> Wheat bran; SCFA Short-chain fatty acid; SEM Standard error of the mean</footnote>

<footnote n="2"><sup>2</sup>SCFA concentration (mg/g Feed DM) = SCFA concentration (mg/g feces DM) × feces DM (g)/feed DM (g)</footnote>

<footnote n="3"><sup>3</sup>Dxx is the interaction between diet and xylanase supplementation</footnote>
<figure><img src="image_3.png" /></figure>

Ruminococcaceae_UCG_005, Ruminococcus_1, Faecalibacterium, and Candidatus_Saccharimonas and increased (*P* < 0.05) the abundances of Escherichia_Shigella and Olsenella. When pigs were fed the diet with 27% WB (Fig. 4b), enzyme addition increased (*P* < 0.05) the relative abundances of Christensenellaceae_R-7_group and Ruminococcaceae_UCG_002, thereby increasing (*P* < 0.05) the abundances of families Christensenellaceae and Ruminococcaceae. Enzyme addition decreased (*P* < 0.05) the relative abundances of genrea Eubacterium_ruminantium_group and Lactobacillus and decreased (*P* < 0.05) the abundances of families Lachnospiraceae, Bacteroidales, and Lactobacillaceae when pigs were fed diet with 27% WB. When pigs were fed the diet containing 12% OB (Fig. 4c), enzyme addition improved (*P* < 0.05) the relative abundances of Blautia, Catenibacterium, and Prevotella_1. When pigs were fed diet with 12% WB (Fig. 4d), enzyme addition increased

(*P* < 0.05) the relative abundances of Faecalibacterium, Ruminococcaceae_UCG_013, Ruminococcaceae_UCG_014, thereby increasing (*P* < 0.05) the abundances of family Ruminococcaceae. In addition, enzyme addition in diet with 12% WB decreased (*P* < 0.05) the abundance of Chlamydia.

Discussion

The high inclusion levels of DF in 36% OB diets and 27% WB diets decreases ATTD of DM and GE compared with basal diets. The results are consistent with the previous studies that report DF negatively affects nutrient digestibility due to their anti-nutritive effects [23–25]. The differences in chemical composition of OB and WB lead to different results on nutrient digestibility, SCFA concentrations and abundance of bacteria in feces, and energy values in diets. Wheat bran contains a higher proportion of insoluble lignified fiber and is therefore
<figure><img src="image_4.png" /></figure>

less degraded than DF in OB. As a result, pigs fed 36%
OB diets have greater nutrient digestibility compared
with those fed 27% WB diets. Meanwhile, 27% WB diets
contain lower nutrient (DM and CP) digestibility than
12% WB diets. However, nutrient digestibility is not

affected by inclusion levels of OB. The results confirm
DF in WB has greater negative effects on nutrient di-
gestibility than that in OB. In addition, OB contains
more starch, CP, and EE content, which is the main rea-
son why OB has greater NE values than WB. Wheat
<figure><img src="image_5.png" /><figcaption>**Fig. 3** Effects of fiber level and type on differential bacteria from genus to phylum. **a** Differential bacteria (*P* < 0.05) between HFG and LFG. **b** Differential bacteria (*P* < 0.05) between oat bran and wheat bran treatments. HFG, diet containing 36% oat bran and diet containing 27% wheat bran (without enzyme); LFG, diets containing 15% oat bran or 12% wheat bran (without enzyme); oat bran, oat bran-based diets; wheat bran, wheat bran-based diets</figcaption></figure>

bran has a typical NSP composition of gramineous grain by-products that mainly consists of insoluble arabinoxylan and cellulose [26], whereas OB is rich in mixed linkages β-glucan which to a high degree is soluble and insoluble arabinoxylan. In addition, only trace levels of cellulose are found in OB [7]. In the small intestine, β-glucan in OB is partly digested while arabinoxylan in OB or WB is hardly digested [7, 8]. In pigs, fiber in OB was readily fermented by bacteria along gastrointestinal tract

[27]. By contrast, most of DF in WB enters hindgut to contribute to microbial fermentation and SCFA generation. Therefore, supplementation of diets with high levels WB rather than OB caused fermentation of fiber and undigested nutrients to shift from the small intestine to hindgut, thereby contributing to growth of fiber-degrading bacteria in hindgut of pigs [27, 28]. In the current study, the abundances of Firmicutes in pigs fed OB diets are greater while the abundances of Bacteroidetes in WB diets
<figure><img src="image_6.png" /></figure>

are greater. Similarly, Liu et al. [19] showed that corn bran-based diets rich in IDF (mainly insoluble arabinoxylan) increased the population of Bacteroidetes while reducing the abundances of Firmicutes. Bacteroidetes has special polysaccharide utilization loci, which generates numerous carbohydrate enzymes to cleave the linkages that existed in complex polysaccharide structures [29]. Compared with OB diets, WB diets increase the abundance of Succinivibrio which is correlated positively with propionate and butyrate concentrations in feces [30]. The Prevotellaceae are a dominant family within Bacteroidetes phylum and produces numerous enzymes, including xylanases and β-glucanases, that are reported to be positively associated with arabinoxylan

degradation in the gastrointestinal tract of animals [31, 32]. *Prevotella* has a strong ability to generate acetate [33]. The greater abundances of *Prevotella* in WB groups compared to OB groups is also reported by Zhang et al. [34] who observes that digestion of NSP and xylose in ileal digesta is correlated positively with abundance of *Prevotella*. Meanwhile, the concentrations of acetate, propionate and total SCFA are greater in pigs fed 27% WB diets than those fed 36% OB diets, which is consistent with the results about fecal bacteria. Our findings support the hypothesis that differences in chemical composition for OB diets and WB diets affect the abundance of the gut bacteria and concentrations of SCFA, and is ultimately reflected in energy metabolism.
Inconsistent results regarding the effects of NSP-
degrading enzyme on nutrient digestibility based on
cereal by-products have been reported widely [28, 35,
36]. In the current study, the addition of enzyme im-
proves ATTD of nutrients and NE values of diets in
pigs fed 27% WB diets. By contrast, the addition of
enzyme to the basal diets or OB diets does not im-
pact the ATTD of nutrients and NE values of diets.
This observation suggests that the effect of enzymes
on nutrient digestibility is related to type and concen-
tration of substrates. Hemicellulose in WB consists
predominantly of xylose and arabinose residues, and
the concentration of arabinoxylan in WB is double
than that in OB [37]. Consequently, diets containing
27% WB have the greatest arabinoxylan concentration
among all diets. According to the theory of enzymatic
kinetics, the higher the substrate concentration is, the
faster the enzymatic reaction within a certain concen-
tration range [38]. Yin et al. [24] reported that en-
zyme supplementation had a greater effect on ATTD
of nutrients in pigs fed high NSP wheat bran-based
diets compared with those fed low NSP wheat or
wheat flour diets. Zeng et al. [28] also reported sup-
plementation of carbohydrate increased ATTD of DM
(74.5% vs. 78.0%) and CP (75.4% vs. 79.7%) in pigs
fed wheat-wheat bran (10% wheat and 20% WB) diets,
but not in wheat (10% wheat) diets. It confirms that
effects of enzyme are highly related to type and con-
centration of the substrates. Additionally, another
possible explanation is that OB contains significant
quantities of insoluble arabinoxylans along with soluble
β-glucans [26], so the use of xylanase does not eliminate
the anti-nutritional effects of β-glucans. Soluble β-glucans
increase viscosity of digesta, which, in turn, prevent arabi-
noxylans to contact with xylanase. The explanation is con-
firmed by a previous study that reports that xylanase is
more efficient in degrading arabinoxylan in wheat than in
rye. In addition, Yin et al. [39] reported that β-glucanase
or an enzyme cocktail (β-glucanase, xylanase and prote-
ase) added to barley diets (higher proportion of β-glucans)
improves ATTD of NDF (60.9%, 68.1%, 66.6% for control
diets, β-glucanase addition diets and cocktail addition di-
ets, respectively) but adding xylanase alone has no effect
on the ATTD of NDF although values varied greatly
(60.9% vs. 67.1%). It indicates that the effects of enzyme
depend on whether the type of enzyme matches the diet-
ary composition. Furthermore, the enzyme addition not
only has an impact on digestion of specific NSP but also
other nutrients by improving probability for nutrients to
contact with endogenous enzymes [14]. In the current
study, the enzyme addition improves the NE values only
for 27% WB diets. Because DF provides a negligible net
amount of DE to growing pigs [40], the explanation for
this result is that the enzyme supplementation improves

ATTD of CP and DM, resulting in more energy sources
available to pigs. Exogenous enzymes influenced the intes-
tinal microbiota by enhancing nutrient delivery to the
host, providing fermentable oligosaccharides degraded
from indigestible polysaccharides, and affecting the fer-
mentation site and viscosity and flow rate of digesta [9,
14]. In general, addition of carbohydrate enzymes influ-
enced more bacterial abundances in pigs fed high-fiber di-
ets compared to pigs fed low-fiber diets. The results were
consistent with those of digestibility, SCFA, and energy
metabolism in high-fiber diets (mainly 27% WB diets) and
low-fiber diets. Zhao et al. [41] reports that SCFA concen-
trations in feces are positively correlated with the IDF di-
gestibility in diets. Enzyme addition increases SCFA
concentrations in feces when pigs are fed 12% WB diets,
which may be attributed to increased fiber digestibility
(57.5% vs. 60.7% for NDF and 56.2% vs. 60.4% for ADF).
However, there is an inconsistent result for effects of en-
zyme on 27% WB diets. This maybe because enzyme
addition hydrolyzed cell wall components, promoting the
digestion of nutrients in the foregut, resulting in a de-
crease in the substrate available for hindgut fermentation.
Enzyme supplementation decreases abundance of Lach-
nospiraceae, Bacteroidales, and Lactobacillaceae for 27%
WB-fed pigs, which seems to support this explanation.
However, comprehensive explanation is not available be-
cause SCFA concentrations in feces do not represent the
SCFA production in intestinal tract.

Conclusions

In conclusion, diets supplied by high level of OB or WB
promote the growth of fiber-degrading bacteria. The dif-
ferences in chemical composition between WB and OB
lead to differences in nutrient digestibility and bacterial
communities, which were ultimately reflected in energy
metabolism. Enzyme supplementation improves nutrient
digestibility as well as NE values for 27% WB but not for
other diets, which indicates that effects of enzyme are
determined by type and level of NSP in diets. However,
the SCFA production and absorption is not measured in
this study, leading to an inability to comprehensively
explain the correlation between fatty acids and energy
metabolism. Therefore, the effect of dietary structure on
digestive properties in different intestinal segments
deserves further study.

**Supplementary information**

**Supplementary information** accompanies this paper at https://doi.org/10.1186/s40104-020-00505-7.

<footnote>**Additional files 1: Table S1.** Chemical composition of fibrous ingredients (%, as-fed basis)¹. **Table S2.** Effect of dietary characteristics and xylanase addition on heat production and energy retention of growing pigs¹. **Figure S1.** Effects of xylanase on fecal microbial community structure in growing pigs.</footnote>
**Abbreviations**

AA: Amino acid; ADF: Acid detergent fiber; ATTD: Apparent total tract digestibility; BSNE: The basal diet without xylanase; BW: Body weight; CH4E: Energy lost as methane; CP: Crude protein; DE: Digestible energy; DF: Dietary fiber; DM: Dry matter; EE: Ether extract; FG: Wheat bran diets and oat bran diets without xylanase; FHP: Fasting heat production; GE: Gross energy; HFG: Diet containing 36% oat bran and diet containing 27% wheat bran (without enzyme); HP: Heat production/DFinsoluble dietary fiber; LDA: Linear discriminant analysis effect size; LEFSe: Linear discriminant analysis effect size; LFG: Diets containing 15% oat bran or 12% wheat bran (without enzyme); ME: Metabolizable energy; NDF: Neutral detergent fiber; NE: Net energy; NSP: Non-starch polysaccharide; OB: Oat bran; OBNE: Oat bran diets without xylanase; OUT: Operational taxonomic units; OM: Organic matter; RDP: Ribosomal database project; RE: Retained energy; REl: Retained energy as lipids; REp: Retained energy as protein; SCFA: Short-chain fatty acids; SDF: Soluble dietary fiber; TDF: Total dietary fiber; THP: Total heat production; UE: Gross energy of urine; WB: Wheat bran; WBNE: Wheat bran diet without xylanase

**Acknowledgements**

Not applicable.

**Authors' contributions**

ZQL, SZ and CHL designed the experiment. ZYW and CHL gave advice on the experiment design. ZQL analyzed the study data and prepared the manuscript. The manuscript was edited by LW, CHL, JJW. All the authors have read and approved the final manuscript.

**Funding**

This project was financially supported by the National Key Research and Development Program of China (2018YFD0500405), National Natural Science Foundation of China (31630074), National Natural Science Foundation of China (U1604106) and the 111 Project (B16044).

**Availability of data and materials**

The data were shown in the main manuscript and supplemental materials.

**Ethics approval and consent to participate**

The experimental protocols used in this experiment, including animal care and use, were reviewed and approved by the Animal Care and Use Ethics Committee of China Agricultural University (Beijing, China).

**Consent for publication**

Not applicable.

**Competing interests**

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

**Author details**

¹State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China. ²INRA-Agrocampus, UMR PEGASE1348, Saint-Gilles, 35000 Rennes, France. ³College of Bioengineering, Henan University of Technology, Zhengzhou 450001, China. ⁴Jiangsu Youshi Biotechnology Development Co. Ltd., Suqian 223800, Jiangsu, China.

Received: 23 March 2020 Accepted: 14 August 2020

Published online: 08 October 2020

**References**

1. Phillips GO, Cui SW. An introduction: evolution and finalisation of the regulatory definition of dietary fibre. Food Hydrocoll. 2011;25:139-43.

2. Morrison DJ, Preston T. Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism. Gut Microbes. 2016;7: 189-200.

3. Iyayi AE, Adeola O. Quantification of short-chain fatty acids and energy production from hindgut fermentation in cannulated pigs fed graded levels of wheat bran. J Anim Sci. 2015;93:4781-7.

4. Besten GD, van Eunen K, Groen AK, Venema K, Beijingoud DJ, Bakker BM. The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism. J Lipid Res. 2013;54:2325-40.

5. Sommer F, Ståhlman M, Ilkayeva O, Arnemo JM, Kindberg J, Josefsson J, Newgard CB, Fröbert O, Bäckhed F. The gut microbiota modulates energy metabolism in the hibernating brown bear Ursus arctos. Cell Rep. 2016;14: 1655-61.

6. Metzler-Zebeli BU, Zijlstra RT, Mosenthal R, Gänzle MG. Dietary calciumphosphate content and oat β-glucan influence gastrointestinal microbiota, butyrate-producing bacteria and butyrate fermentation in weaned pigs. FEMS Microbiol Ecol. 2011;75:402-13.

7. Bach Knudsen KE, Jensen BB, Hansen I. Digestion of polysaccharides and other major components in the small and large intestine of pigs fed diets consisting of oat fractions rich in β-D-glucan. Br J Nutr. 1993;70:537-56.

8. Bach Knudsen KE, Canibe N. Breakdown of plant carbohydrates in the digestive tract of pigs fed on wheat- or oat-based rolls. J Sci Food Agric. 2000;80:1253-61.

9. Bedford MR, Cowieson AJ. Exogenous enzymes and their effects on intestinal microbiology. Anim Feed Sci Technol. 2012;173:76-85.

10. NRC. Nutrient requirements of swine. 10th rev. ed. Natl. Acad. Press, Washington, DC. 2012.

11. Lyu ZQ, Li YK, Liu H, Li EK, Li PL, Zhang S, et al. Net energy content of rice bran, defatted rice bran, corn gluten feed, and corn germ meal fed to growing pigs using indirect calorimetry. J Anim Sci. 2018;96:1877-88.

12. Noblet J, Fortune H, Shi XS, Dubois S. Prediction of net energy value of feeds for growing pigs. J Anim Sci. 1994;72:344.

13. van Milgen J, Noblet J, Dubois S, Bernier JF. Dynamic aspects of oxygen consumption and carbon dioxide production in swine. Br J Nutr. 1997;78: 397-410.

14. Lærke HN, Arent S, Dalsgaard S, Bach Knudsen KE. Effect of xylanases on ileal viscosity, intestinal fiber modification, and apparent ileal fiber and nutrient digestibility of rye and wheat in growing pigs. J Anim Sci. 2015;93: 4323-35.

15. Hortwitz W, Latimer GW. Official methods of analysis of AOAC international. 18th ed. Rev. 2. ed. AOAC Int., Gaithersburg. 2007.

16. Thiex NJ, Anderson S, Gildemeister B. Crude fat, diethyl ether extraction, in feed, cereal grain, and forage (Randall/Soxtec/submersion method): collaborative study. J AOAC Int. 2003;86:888-98.

17. Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci. 1991;74:3583-97.

18. Prosky L, Asp NG, Schweizer TF, DeVries JW, Furda I. Determination of insoluble and soluble in foods and food-products-collaborative study. J AOAC Int. 1992;75:360-7.

19. Liu P, Zhao JB, Wang W, Guo PT, Lu WQ, Wang CL, et al. Dietary corn bran altered the diversity of microbial communities and cytokine production in weaned pigs. Front Cell Infect Microbiol. 2018;9:2090.

20. Brouwer E. Report of sub-committee on constants and factors. In: Proc. 3rd EAAP Symp. Energy Metab., Troonn, Publ. 11. London, UK: Academic Press; 1965. p. 441-3.

21. van Milgen J, Noblet J, Dubois S. Energetic efficiency of starch, protein and lipid utilization in growing pigs. J Nutr. 2001;131:1309-18.

22. Adeola O. Digestion and balance techniques in pigs. In: Lewis J, Southern LL, editors. Swine nutrition. Washington, DC: CRC Press; 2001. p. 903-16.

23. He BB, Bai Y, Jiang LL, Wang W, Li TT, Liu P, et al. Effects of oat bran on nutrient digestibility, intestinal microbiota, and inflammatory responses in the hindgut of growing pigs. Int J Mol Sci. 2018;19:2407.

24. Yin YL, McEvoy JDG, Schulze H, Hennig U, Souffrant WB, McCracken KJ. Apparent digestibility (ileal and overall) of nutrients and endogenous nitrogen losses in growing pigs fed wheat (var. Soissons) or its by-products without or with xylanase supplementation. Liv Produc Sci. 2000;62:119-32.

25. Choi H, Sung JY, Kim BG. Neutral detergent fiber rather than other dietary fiber types as an independent variable increases the accuracy of prediction equation for digestible energy in feeds for growing pigs. Asian-Australas J Anim Sci. 2020;33:615-22.

26. Bach Knudsen KE. Carbohydrate and lignin contents of plant materials used in animal feeding. Anim Feed Sci Technol. 1997;67:319-38.

27. Johansen HN, Bach Knudsen KE, Wood PJ, Fulcher RG. Physico-chemical properties and the degradation of oat bran polysaccharides in the gut of pigs. J Agric Food Chem. 1997;73:81-92.
28. Zeng ZK, Li QY, Tian QY, Xu YT, Piao XS. The combination of carbohydrates and phytase to improve nutritional value and non-starch polysaccharides degradation for growing pigs fed diets with or without wheat bran. Anim Feed Sci Technol. 2018;235:138-46.

29. Sonnenburg ED, Zheng HJ, Joglekar P, Higginbottom SK, Firbank SJ, Bolam DN, et al. Specificity of polysaccharide use in intestinal bacteroides species determines diet-induced microbiota alterations. Cell. 2010;141:1241-52.

30. Iqbal MW, Zhang Q, Yang YB, Zou CX, Li LL, Liang X, et al. Ruminal fermentation and microbial community differently influenced by four typical subtropical forages in vitro. Anim Nutr. 2018;4:100-8.

31. Flint HJ, Bayer EA, Rincon MT, Lamed R, White BA. Polysaccharide utilization by gut bacteria: potential for new insights from genomic analysis. Nat Rev Microbiol. 2008;6:121-31.

32. Okeke BC, Lu J. Characterization of a defined cellulolytic and Xylanolytic bacterial consortium for bioprocessing of cellulose and hemicelluloses. Appl Biochem Biotechnol. 2011;163:869-81.

33. Rey FE, Faith JJ, Bain J, Muehlbauer MJ, Stevens RD, Newgard CB, et al. Dissecting the in vivo metabolic potential of two human gut acetogens. J Biol Chem. 2010;285:22082-90.

34. Zhang J, Chen XY, Liu P, Zhao JB, Sun J, Guan WY, et al. Dietary Clostridium butyricum induces a phased shift in fecal microbiota structure and increases the acetic acid-Producin Bacteria in a weaned piglet model. J Agric Food Chem. 2018;66:5157-66.

35. Nørgaard JV, Pedersen TF, Blaabjerg K, Bach Knudsen KE, Lærke HN. Xylanase supplementation to rye diets for growing pigs. J Anim Sci. 2016;94: 91-4.

36. Tiwari UP, Chen HY, Kim SW, Jha R. Supplemental effect of xylanase and mannanase on nutrient digestibility and gut health of nursery pigs studied using both in vivo and in vitro models. Anim Feed Sci Technol. 2018;245: 77-90.

37. Yu CY, Zhang SH, Yang Q, Peng Q, Zhu JL, et al. Effect of high fibre diets formulated with different fibrous ingredients on performance, nutrient digestibility and faecal microbiota of weaned piglets. Arch Anim Nutr. 2016; 4:263-77.

38. Truhlar DG. Transition state theory for enzyme kinetics. Arch Biochem Biophys. 2015;582:10-7.

39. Yin YL, Baidoo SK, Jin LZ, Liu YG, Schulze H, Simmins PH. The effect of different carbohydrate and protease supplementation on apparent (ileal and overall) digestibility of nutrients of five hulless barley varieties in young pigs. Livest Prod Sci. 2001;71:109-20.

40. Noblet J, Le Goff G. Effect of dietary fibre on the energy value of feeds for pigs. Anim Feed Sci Technol. 2001;90:35-52.

41. Zhao JB, Bai Y, Zhang G, Liu L, Lai CH. Relationship between dietary fiber fermentation and volatile fatty acids' concentration in growing pigs. Animals. 2020;10:26.

Ready to submit your research? Choose BMC and benefit from:

* fast, convenient online submission

* thorough peer review by experienced researchers in your field

* rapid publication on acceptance

* support for research data, including large and complex data types

* gold Open Access which fosters wider collaboration and increased citations

* maximum visibility for your research: over 100M website views per year

At BMC, research is always in progress.

Learn more biomedcentral.com/submissions

<figure><img src="image_7.png" /></figure>