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Effect of β-glucanase and β-xylanase enzyme supplemented barley diets on nutrient digestibility, growth performance and expression of intestinal nutrient transporter genes in finisher pigs L. C. Clarke a , T. Sweeney b , E. Curley c , V. Gath b , S. K. Duffy a , S. Vigors b , G. Rajauria a and J. V. O’Doherty a * a School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland b School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland c Department of Botany and Plant Science, National University of Ireland, Galway, Ireland. *Correspondence J. V. O’Doherty, Tel: +35317167128; Fax: +35317161103; Email: [email protected] 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

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Page 1: researchrepository.ucd.ie · Web viewThe higher β-glucan content in the high quality barley may be attributed to the higher level of nitrogen that this crop received (Oscarsson et

Effect of β-glucanase and β-xylanase enzyme supplemented barley diets on nutrient

digestibility, growth performance and expression of intestinal nutrient transporter

genes in finisher pigs

L. C. Clarkea, T. Sweeneyb, E. Curleyc, V. Gathb, S. K. Duffya, S. Vigorsb, G. Rajauriaa

and J. V. O’Dohertya*

a School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4,

Ireland

b School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland

c Department of Botany and Plant Science, National University of Ireland, Galway, Ireland.

*Correspondence J. V. O’Doherty, Tel: +35317167128; Fax: +35317161103; Email:

[email protected]

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Page 2: researchrepository.ucd.ie · Web viewThe higher β-glucan content in the high quality barley may be attributed to the higher level of nitrogen that this crop received (Oscarsson et

Abstract

The study investigated the effect of dietary supplementation of an enzyme mix (β-glucanase

and β-xylanase) to barley based diet that had different chemical compositions achieved

through different agronomical conditions on growth performance, nutrient digestibility and

intestinal nutrient transporters. Ninety-six pigs (44.7 kg (SD 4.88)) were assigned to one of

four dietary treatments. The treatments were as follows: (T1) low quality barley diet, (T2)

low quality barley diet supplemented with β-glucanase and β-xylanase enzyme supplement,

(T3) high quality barley diet and (T4) high quality barley diet supplemented with β-glucanase

and β-xylanase enzyme supplement. The inclusion of barley was 500 g/kg. There was an

interaction between barley type and enzyme supplementation on average daily gain (ADG)

and average daily feed intake (ADFI) (P<0.05). Pigs offered the low quality barley diet

supplemented with enzymes had an increase in both ADG and ADFI compared to the low

quality barley diet only. However, there was no response to enzyme inclusion in the high

quality barley diet. Pigs offered the low quality barley diet with enzymes had a higher

coefficient of apparent total tract digestibility (CATTD) of gross energy (GE) compared to

the low quality barley diet only (P<0.05). However, the increase in the high quality barley

diet with enzyme supplementation was not as great as with the low quality barley diet. Pigs

offered the low quality barley had an upregulation in the expression of the ghrelin gene

(GHRL) in the jejunum compared to pigs offered the high quality barley diet (P<0.05). There

was a barley × enzyme interaction observed for the expression of the cluster of differentiation

gene (CD36) in the duodenum and the peptide transporter 1 gene (PEPT1/SLC15A1) and

sodium-glucose linked transporter 1 gene (SGLT1/SLC5A1) in the ileum (P<0.01). Pigs

offered the high quality barley diet with enzymes had increased expression of CD36,

PEPT1/SLC15A1 and SGLT1/SLC5A1 compared to the high quality barley diet alone.

However the low quality barley diet with enzymes down regulated the expression of CD36,

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Page 3: researchrepository.ucd.ie · Web viewThe higher β-glucan content in the high quality barley may be attributed to the higher level of nitrogen that this crop received (Oscarsson et

PEPT1/SLC15A1 and SGLT1/SLC5A1 compared to the low quality barley diet alone. In

conclusion, offering a low quality barley diet supplemented with an enzyme mix improved

ADG, ADFI and nutrient digestibility as well as modifying the expression of CD36,

PEPT1/SLC15A1 and SGLT1/SLC5A1. The inclusion of an enzyme mix to the high quality

barley diet improved nutrient digestibility and caused an upregulation in the expression of

CD36, PEPT1/SLC15A1 and SGLT1/SLC5A1 but it did not improve animal performance.

Keywords: barley; enzyme; performance; nutrient digestibility; gene expression; nutrient

transporters

Abbreviations:

DM, dry matter; CP, crude protein; NDF, neutral detergent fibre; ADF, acid detergent fibre;

CF, crude fibre; GE, gross energy; EE, ether extract; NE, net energy; OM, organic matter; N,

nitrogen; GE, gross energy; DE, digestible energy; AOAC, Association of Official

Agricultural Chemists; ADG, average daily gain; FCR, feed conversion ratio; ADFI, average

daily feed intake; CATTD, coefficient of apparent total tract digestibility; CAID, coefficient

of apparent ileal digestibility; GIT, gastrointestinal tract; VFA, volatile fatty acids; PBS,

phosphate buffered saline; TGW, thousand grain weight; CCK, cholecystokinin; GLP,

glucagon-like peptide; PYY, peptide tyrosine tyrosine; MCT, monocarboxylate transporter;

dT, oligo-deoxy-thymine; GHRL, ghrelin; PEPT, peptide transporter; SGLT, sodium-glucose

linked transporter; CD, cluster of differentiation; FABP2, fatty acid-binding protein; GLUT,

glucose transporter; SLC, sodium-coupled monocarboxylate transporter; CCK,

cholecystokinin; GAST, gastrin; NPY, neuropeptide Y

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1. Introduction

Cereal grains such as barley and wheat vary in nutrient composition due to different cultivars

and agronomic conditions which may in turn affect nutrient digestibility and growth

performance in animals (Ovenell-Roy et al., 1998; Ball et al., 2013). Barley is one of the

major feed ingredients used in swine diets primarily as an energy source. The energy value of

barley is less than that of wheat and maize due to its greater dietary fibre content (NRC,

2012). Adverse agronomic conditions may influence the composition of barley, resulting in a

predicted high quality barley grain turning into a lower quality barley grain with greater fibre

content and lower digestible energy and net energy contents (Fairbairn et al., 1999; van

Barneveld, 1999). Dietary fibre can increase the digesta viscosity in the gastrointestinal tract

(GIT) which prolongs the presence of nutrients in the GIT. This action affects the release of

intestinal appetite regulating peptides such as cholecystokinin (CCK), glucagon-like peptide

1 (GLP-1) and peptide tyrosine tyrosine (PYY) (Cummings and Overduin, 2007; Wanders et

al., 2011) and ultimately reduces feed intake.

Exogenous fibre degrading enzymes are included in swine diets to mitigate the negative

effects associated with feeding dietary fibre to pigs (Kerr and Shurson, 2013). Among the

feed enzymes, carbohydrase enzymes such as β-glucanase and β-xylanase, are long

recognized to be effective in hydrolysing dietary fibre (Li et al., 2004). These enzymes have

the potential to overcome the limitation of using fibrous ingredients in swine diets (Kiarie et

al., 2007) and are likely to increase the availability of free nutrients such as glucose and

amino acids. Nutrient transporters are expressed on the apical membrane of the intestinal

absorptive cells. Nutrient digestibility can be improved by increasing the number of nutrient

transporters such as mono saccharide transporters (sodium-glucose-linked transporter

(SGLT/SLC5A) and glucose transporter (GLUT/SLC2A) (Wood and Tyrnan 2003)), fatty acid

binding proteins (FABP) (Hajri and Abumrad, 2002; Glatz and van der Vusse, 1996) and

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small-peptide (di and tri-peptide) transporters (PEPT/SLC15A) (Daniel, 2004). However, the

expression of these nutrient transporters may fluctuate through the mechanism of nutrient

sensing, depending on the availability of nutrients (Dyer et al., 2005). With the inclusion of

exogenous enzymes, more nutrients should become available, affecting the expression of

nutrient transporters (Wang et al., 2005; Woyengo et al., 2011). Although exogenous

enzymes have been shown to improve nutrient digestibility (Kiarie et al., 2007; Woyengo et

al., 2008) and growth performance in pigs (Yin et al., 2001; Omogbenigun et al., 2004), the

physiological response of intestinal nutrient transporters to β-glucanase and β-xylanase still

requires further characterisation.

Therefore, the objective of the present study was to examine the effect of dietary

supplementation of a β-glucanase and β-xylanase enzyme mix to barley based diets that had

different chemical composition achieved through different agronomical conditions. The

parameter categories which were assessed included growth performance, nutrient

digestibility, nutrient transporters and appetite hormones. The hypothesis of this experiment

is that the negative effects associated with feeding a low quality barley based diet to finisher

pigs would be removed with the addition of a β-glucanase and β-xylanase enzyme mix,

resulting in an improvement in growth performance, nutrient digestibility and an upregulation

of nutrient transporter genes involved in carbohydrate, peptide and fatty acid transport.

2. Materials and methods

All experimental procedures described in this work were approved under University College

Dublin animal research ethics committee (AREC-13-56-O’Doherty) and conducted under

experimental license from the Department of Health in accordance with the cruelty to animal

act 1876 and the European Communities (Amendments of Cruelty to Animal Act, 1876)

Regulations (1994).

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2.1. Grain management

The barley grain (cv. Sebastian) was sourced from Howard Farms Ltd. Mallow, Co. Cork,

Ireland. To obtain barley grain of different levels of quality, two blocks (A and B) of barley

were established and harvested in the 2014 season. To yield a high quality barley grain,

Block A had an early sowing date (3rd April 2014) and subsequently followed the

recommended barley husbandry practices (2 spray fungicide programme and a nitrogen (N)

application rate of 140 kg N/ha). Nitrogen was applied in a two-split programme (40:60

between a seedbed application and a mid-tillering application). Harvest was conducted under

ideal conditions on 24th August 2014 (15.5% moisture content). Block B had a delayed

sowing date (16th April 2014) and subsequently delayed husbandry practices and harvest date

(9th September 2014 at 26% moisture content). A nitrogen application rate of 120 kg N/ha

was used in a two-split programme (40:60 between a seedbed application and a mid-tillering

application). The harvest conditions were challenging given the lateness of the crop to ripen.

As such, the growing background for Block B represents a season of adverse agronomic

conditions. Due to the high grain moisture content harvested from Block B, drying and

cooling was necessary prior to storage to maintain grain integrity. All grain was ventilated

until its use in diet formulations. Prior to diet formulation, the quality of the barley samples

was assessed using density (hectolitre weight), grain screenings (% grain <2.5 mm) and

thousand grain weight (TGW) from subsamples of grain obtained on the combine at harvest.

Grain density and moisture content was determined using a DICKEY-john GAC 2500-

UGMA electric moisture meter (Illinois, USA). Grain screenings was determined by

weighing a 100 g grain sample and sieving the sample using a Herbst Sortimat while TGW

was determined using a Pfeuffer Contador (Kitzingen, Germany) seed counter and recording

the weight of 1,000 grains. After grain drying, the analysis of the barley grain was carried out

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on a total of 19 subsamples from both the high and the low quality barley. The analysis

included dry matter (DM), crude protein (CP), ash, neutral detergent fibre (NDF), acid

detergent fibre (ADF), crude fibre (CF), gross energy (GE), ether extract (EE) and lysine. The

concentration of β-glucans, xylose and starch in the barley were determined using a

Megazyme assay kits (Megazyme Int., Wicklow, Ireland). Reveal Q+ test kits were used to

analyse aflatoxin (Lot No. 222213), deoxynivalenol (Lot No. 217926), ochratoxin (Lot No.

223177), T-2 Toxin (Lot No.219165) and zearalenone (Lot No. 223094) mycotoxins in

barley samples (Chauhan et al., 2016). AccuScan Gold reader was used for measuring the

intensity of band developed on Reveal Q+ mycotoxins test strips (Neogen Corporation,

USA). The assay is based on single-step lateral flow immunocharomatographic principle with

competitive immunoassay format (Neogen Corporation, USA). The chemical characteristics

of both the low quality and the high quality barley are presented in Table 1.

2.2. Experimental diets

A 2 × 2 factorial design was used comprising of four dietary treatments. The dietary

treatments were as follows: (T1) low quality barley diet, (T2) low quality barley diet

containing a β-glucanase and β-xylanase enzyme supplement, (T3) high quality barley diet,

(T4) high quality barley diet containing a β-glucanase and β-xylanase enzyme supplement.

The diets were formulated to contain similar levels of net energy (NE) (9.25 MJ/kg) and

standard ileal digestible lysine (8.5 g/kg) (Sauvant et al., 2004). All other amino acids

requirements were met relative to lysine according to the ideal protein concept (NRC, 2012).

The inclusion rate of barley was 500 g/kg. The enzyme was derived from Trichoderma

reesei. Diets were supplemented with the enzymes endo-1,4-β-glucanase (EC/IUB No.

3.2.1.4) with an activity of 800 U/g, endo-1,3(4)-β-glucanase (EC/IUB No. 3.2.1.6) with an

activity of 700 U/g, and endo-1,4-β-xylanase (EC/IUB No. 3.2.1.8) with an activity of 2700

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U/g (DSM, Nutritional Products Limited, UK) and included at a rate of 0.15 g/kg. All diets

were milled on site and offered in meal form. Celite (500 mg/kg) was added to the feed

during the milling process in order to measure the coefficient of apparent total tract

digestibility (CATTD) and the coefficient of apparent ileal digestibility (CAID) of nutrients

using the acid insoluble ash technique (McCarthy et al., 1977). The composition of the diet is

presented in Table 2 and the chemical analyses of the dietary treatments are presented in

Table 3.

2.3. Growth performance and total tract digestibility

A total of ninety-six pigs (48 males, 48 females) (Meatline boars × (Large White × Landrace

sows) Hermitage, Co. Kilkenny, Ireland) with an initial live weight of 44.7 kg (SD 4.88 kg)

were used in this study. The pigs were blocked according to live weight and sex and within

each block assigned to one of four dietary treatments (n = 24). The pigs were grouped in

mixed gender (50:50) groups of 12 in 8 pens with a space allowance of 0.75 m2 per pig. The

diets were offered ad libitum for 4 weeks. Fresh water was also provided ad libitum. The

house was mechanically ventilated and temperature was maintained at 20°C.

Each pen had a solid floor lying area with access to slats at the rear. The pens were equipped

with single space computerised electronic feeders (Mastleistungsprufung MLP-RAP; Schauer

Agrotronic AG, Sursee, Switzerland) as described by Varley et al. (2011), which allowed

individual ad libitum feeding and daily recording of dietary intake. Each animal was fitted

with a uniquely coded ear tag transponder and the identification circuit recorded the animal’s

number. When the animal entered the feeder, it was recognized by the electronic system

(MLP-Manager 1.2; Schauer Maschinenfabrik Ges.m.b.H and CoKG, Prambachkirchen,

Austria). When the animal finished feeding and withdrew from the trough, the electronic

system recorded the difference between the pre and post visit trough weight and the data was

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stored in a file with the pen number, the animal’s identification number, the date and the time

of entry and exit. The recorded data were used to calculate the individual dietary intake. The

body weight of each animal was measured at the start of the experiment and subsequently on

day 14 and 28, while dietary intake was monitored daily. Feed samples were retained for

chemical analysis and faecal samples were taken from 8 pigs per treatment (four pigs from

each pen) on day 14 to day 17 in order to measure nutrient digestibility.

2.4. Post slaughter sample collection

Eight pigs per treatment (four from each pen, 2 male and 2 female) remained on their

respective dietary treatments until slaughter. Digesta samples were removed aseptically from

the ileum in a section approximately 50 cm in length from the ileo-caecal valve, in order to

measure the CAID of nutrients. Digesta samples were collected from the second loop of the

ascending colon, using sterile instruments and stored at -20°C for further volatile fatty acids

(VFA) analysis. Tissue samples from the duodenum (10 cm from the stomach), jejunum (60

cm from the stomach), ileum (10 cm from the ileo-ceacal valve) and colon (second loop)

were collected to analyse the gene expression of nutrient transporter, appetite regulator and

short chain fatty acid transporter genes. Tissue samples were emptied and cleaned by

dissecting along the mesentery and rinsing using sterile phosphate buffered saline (PBS)

(Oxoid Limited, UK) as described previously (Sweeney et al., 2012; Heim et al., 2014).

Sections measuring 1 cm2, which had been stripped of the overlying smooth muscle were cut

from the tissue and stored in RNAlater solution (Applied Biosystems, Foster City, CA)

overnight at 4°C. The RNAlater was removed and tissue sample was stored at -70°C until

RNA extraction.

2.5. Laboratory analyses

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Faecal samples were analysed for N, DM, organic matter (OM), ash, GE and NDF. Digesta

samples were analysed for DM, N, GE and OM. Following collection, faecal and digesta

samples were dried at 55°C for 72 h. The feed, dried faecal and dried digesta samples were

milled through a 1mm screen (Christy and Norris Hammer Mill, Chelmsford, England). The

DM content of feed, faeces and digesta was determined after drying overnight at 104°C.

Crude ash content was determined after ignition of a weighed sample in a muffle furnace

(Nabertherm, Bremen, Germany) (AOAC.942.05, 2005) at 550°C for 6 h. The GE content

was determined using an adiabatic bomb calorimeter (Parr Instruments, Moline, IL USA).

The N content was determined using the LECO FP 528 instrument (Leco Instruments UK

LTD., Cheshire, UK) (AOAC.990.03, 2005). The dietary concentration of lysine, threonine,

tryptophan, methionine and cysteine were determined by HPLC (Iwaki et al., 1987). The

NDF and ADF content was determined according to the method of Van Soest et al. (1991)

using the Ankom 220 Fibre Analyser (Ankom TM Technology, Macedon, NY, USA) and for

crude fibre the AOAC, (AOAC. 962.09, 2005) method was used. The EE concentration of the

diets was determined using light petroleum ether and Soxtec instrumentation (Tecator,

Sweden). The concentration of acid-insoluble ash was determined according to the method of

McCarthy et al. (1977). Diet samples were also analysed for mycotoxins (aflatoxin,

deoxynivalenol, ochratoxin, T-2 Toxin and zearalenone) as previously described (Chauhan et

al., 2016). The activities of β-glucanase and β-xylanase were determined using Megazyme

kits (Megazyme Int., Wicklow, Ireland). The activity of β-glucanase and β-xylanase were as

expected. The NE content of the diet was predicted using equation 4 from Noblet et al.

(1994).

NE= 0.703 DE + 0.066 EE + 0.020 starch – 0.041 CP – 0.041 CF

where NE and digestible energy (DE) values are expressed in MJ/kg DM and the chemical

constituents are expressed as % of DM.

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2.6. Volatile fatty acid analysis

Digesta samples were collected to measure total VFA concentration. The VFA concentrations

in the digesta were determined using gas liquid chromatography according to the method

described by Pierce et al. (2006). A 1 g sample was diluted with distilled water (2.5 x weight

of sample) and centrifuged at 1400 x g for 10 min (Sorvall GLC-2B laboratory centrifuge,

DuPont, Wilmington, DE, USA). One ml of the subsequent supernatant and 1 ml of internal

standard (0.05% 3-methyl-n-valeric acid in 0.15 M oxalic acid dihydrate) were mixed with 3

ml of distilled water. The reaction mixture was centrifuged at 500 g for 10 min and the

supernatant was filtered through 0.45 PTFE (Polytetrafluoroethylene) syringe filter into a

chromatographic sample vial. An injection volume of 1 µl was injected into a Varian 3800

GC equipped with a ECTM 1000 Grace column (15 m × 0.53 mm I.D) with 1.20 µm film

thickness. The temperature programme set was: 75°C - 95°C increasing by 3°C/minute, 95-

200 increasing by 20°C/ minute, which was held for 0.50 minutes. The detector and injector

temperature was 280°C and 240°C respectively while the total analysis time was 12.42

minutes.

2.7. RNA extraction and real-time RT-PCR

Total RNA was extracted from three regions of the small intestine (duodenum, jejunum and

ileum) and the colon using TRIreagent (Sigma-Aldrich, St. Louis, MO) according to the

manufacturer’s instructions. The crude RNA extract was further purified using the GenElute

Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) according to the manufacturer’s

instructions. A DNase removal step was included using an on-Column DNase 1 Digestion Set

(Sigma-Aldrich). The total RNA was quantified using a Nanodrop-ND1000

Spectrophotometer (Thermo Scientific) and the purity was assessed by determining the ratio

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of the absorbance at 260 nm and 280 nm. In addition, RNA integrity was established on the

Agilent RNA 6000 NanoChip Bioanalyser Kit (Agilent Technologies, Santa Clara, CA).

Total RNA (1 µg) was reverse-transcribed using a First Strand cDNA Synthesis Kit,

(Fermentas Waltham, Massachusetts, USA) using oligo (dT) primers in a final reaction

volume of 20 µl according to the manufacturer’s instructions. The final reverse transcription

product was adjusted to a volume of 120 µl using nuclease-free water. The mRNA expression

profiles of selected candidate genes were evaluated in duplicate using QPCR on the ABI

Prism 7500 Fast Sequence Detection System (Applied Biosystems). Oligonucleotide primers

were designed with Primer Express Software, version 2.0 (Applied Biosystems) and

synthesised by MWG Biotech (Ebersberg, Germany). All primers for selected genes: cluster

of differentiation (CD36), fatty acid-binding protein (FABP2), glucose transporter

(GLUT1/SLC2A1, GLUT2/SLC2A2 and GLUT7/SLC2A7), peptide transporter

(PEPT1/SLC15A1), sodium-glucose-linked transporter (SGLT1/SLC5A1), sodium-coupled

monocarboxylate transporter (SLC16 and SLC5A), cholecystokinin (CCK), gastrin (GAST),

ghrelin (GHRL), glucagon-like peptide (GLP-1 and GLP-2), neuropeptide Y (NPY) and

peptide tyrosine tyrosine (PYY) are presented in Table 4. PCR amplification was performed in

a total volume of 20 µl containing 10 µl of master mix (Fast SYBR PCR Master Mix,

Applied Biosystems), 1.0 µl of forward and reverse primers (300 pM final), 6.5 µl of RNAse-

free water, and 2.5 µl of DNA (5.0 ng of RNA equivalents). The two-step PCR programme

was as follows: 95°C for 10 min for one cycle, followed by 95°C of 15s and 60°C for 1 min

for forty cycles. All reactions were performed in duplicate and minus-RT and no template

controls were included. The specificity of all assays were confirmed using dissociation curve

analysis. Primer efficiencies were determined from the slope of a curve derived from the Cts

of a 1:4 dilution series of cDNA. The optimal reference genes were identified using the

geNorm algorithm within the qbasePLUS software package (Biogazelle, Gent, Belgium) and

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confirmed for the present study (geNorm V<0.15). The normalisation factor was calculated

for each tissue type from the geometric mean of the reference targets. Beta actin (ACTB) and

hydroxymethylbilane synthase (HMBS) were the most optimal for the duodenum,

glyceraldhyde 3-phosphate dehydrogenase (GAPDH) and tyrosine

3-mono-oxygenase/tryptophan 5-mono-oxygenase activation protein (YWHAZ) for the

jejunum, HMBS and YWHAZ for the ileum and YWHAZ and ACTB for the colon. Normalised

relative quantities were generated using the qbasePLUS package (Biogazelle) with efficiency

correction incorporated.

2.8. Statistical analysis

The data was initially checked for normality using the UNIVARIATE procedure of SAS

(SAS, 2006). The growth performance data was analysed as a 2 × 2 factorial by repeated

measures analysis using the PROC MIXED procedure of SAS (Littell et al., 1996). The

model included the fixed effects of barley type, enzyme inclusion and the associated two way

interaction. Initial live weight was included as a covariate in the model and day of weighing

was regarded as a repeated variable with pen and animal within pen as the experimental unit.

The data on nutrient digestibilities, nutrient transporter gene expression, appetite hormones

gene expression and VFA concentrations were analysed as a 2 × 2 factorial using the PROC

MIXED procedure of SAS (Littell et al., 1996). The model included the fixed effects of

barley type, enzyme inclusion and the associated two way interactions while the random

effect was the pen and animal within pen. The probability level that denoted significance was

P<0.05, while P values between 0.05 and 0.1 are considered numerical tendencies. Data are

presented as least-square means with their standard errors of the mean.

3. Results

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3.1. Barley type

The chemical analysis of the barley used is presented in Table 1. The level of aflatoxin B1

(<2 µg/kg), deoxynivalenol (<300 µg/kg), ochratoxin (<2.0 µg/kg), T-2 Toxin (<50 µg/kg)

and zearalenone (<25 µg/kg) were below detectable levels for both the high and the low

quality barley. The low quality barley had a lower hectolitre weight, TGW, starch content and

β-glucan content, along with a higher screening percentage and higher contents of DM, ash,

CP, lysine and ADF compared to the high quality barley. The concentration of crude protein

and lysine in the experimental diets are higher in the low quality barley diets compared to the

high quality barley diets (Table 3).

3.2. Performance

The effect of barley type and β-glucanase and β-xylanase enzyme inclusion on the growth

performance of the pigs is presented in Table 5. There was a barley × enzyme interaction on

average daily gain (ADG) and average daily feed intake (ADFI) (P<0.05). Pigs offered the

low quality barley diet with enzyme supplementation had increased ADG and ADFI

compared to the low quality barley diet only. However, there was no response to enzyme

inclusion on ADG or ADFI in pigs offered the high quality barley diet. There was a time ×

barley interaction on feed conversion ratio (FCR) (P<0.05). Pigs offered the low quality

barley diet had a poorer FCR on day 28 compared with pigs offered the high quality diet.

However, there was no effect of quality on FCR on day 14 (2.64 vs 2.44 and 2.15 vs 2.23,

SEM 0.061 kg/kg respectively).

3.3. Coefficient of apparent ileal digestibility

The effect of barley type and β-glucanase and β-xylanase enzyme inclusion on the CAID of

nutrients is presented in Table 6. Pigs offered the enzyme supplemented diets had increased

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(P<0.05) CAID of DM (0.79 vs 0.76, SEM 0.009), OM (0.84 vs 0.81, SEM 0.008) and GE

(0.80 vs 0.76, SEM 0.010) compared to the unsupplemented enzyme diets. There was a

barley × enzyme interaction on the CAID of N (P<0.01). Pigs offered the high quality barley

diet with enzymes had a higher CAID of N compared with the high quality barley diet only.

However, there was no response to enzyme supplementation in the low quality barley diet on

the CAID of N.

3.4. Coefficient of apparent total tract digestibility

The effect of barley type and β-glucanase and β-xylanase enzyme inclusion on the CATTD of

nutrients is presented in Table 6. Pigs offered the low quality barley had decreased (P<0.05)

CATTD of N (0.72 vs 0.76, SEM 0.007) and NDF (0.28 vs 0.45, SEM 0.021) compared with

pigs offered the high quality barley. The inclusion of an exogenous enzyme increased

(P<0.001) CATTD of N (0.80 vs 0.69, SEM 0.007) and NDF (0.49 vs 0.25, SEM 0.021)

compared to the unsupplemented enzyme groups. There was a barley × enzyme interaction

for the CATTD of DM, OM and GE, as well as on DE and predicted NE values (P<0.05).

Pigs offered the low quality barley diet with enzymes had a higher CATTD of DM, OM, GE,

DE and predicted NE compared with the low quality barley diet only. However, the increase

in the high quality barley diet with enzyme supplementation was not as great as with the low

quality barley diet. There was a barley × enzyme interaction on the CATTD for ash (P<0.01).

Pigs offered the high quality barley diet with enzymes had a higher CATTD of ash compared

with the high quality barley diet only. However, the increase in the low quality barley diet

with enzyme supplementation was not as great as with the high quality barley diet.

3.5. Volatile fatty acids

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The effect of barley type and β-glucanase and β-xylanase enzyme inclusion on total VFA

concentrations is presented in Table 7. Pigs offered the enzyme supplemented diets had

decreased (P<0.05) concentrations of total VFA (180 vs 213, SEM 6.75 mmol/g), acetic acid

(126 vs 150, SEM 5.32 mmol/g) and propionic acid (25 vs 31, SEM 1.21 mmol/g) compared

to the unsupplemented diets. Pigs offered the low quality barley diet had a lower

concentration of valeric acid (2.64 vs 3.17, SEM 0.166 mmol/g) (P<0.05) compared to the

high quality barley diet. There was a barley type × enzyme interaction observed for isobutyric

acid, isovaleric acid and butyric acid concentrations (P<0.05). Pigs offered the high quality

barley diet with enzymes had a decreased concentration of butyric acid compared with the

high quality barley diet only. However, there was no response to enzyme supplementation

with the low quality barley diets. Pigs offered the high quality barley diet with enzymes had

increased concentrations of the isobutyric acid and isovaleric acid compared to pigs offered

the high quality barley diet only. However, there was no response to enzyme supplementation

with the low quality barley diets.

3.6. Nutrient and short chain fatty acid transporters and appetite hormone gene expression

The effects of barley type and β-glucanase and β-xylanase inclusion on nutrient transporter

gene expression, appetite regulator gene expression and short chain fatty acid transporter

gene expression are presented in Table 8.

3.6.1. Duodenum

There was a barley × enzyme interaction on CD36 expression (P<0.001). Pigs offered the low

quality barley with enzymes had a down regulation in CD36 expression compared to the low

quality diet only. However, pigs offered the high quality barley diet with enzymes had an

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upregulation in CD36 compared to the high quality barley diet only. There were no other

effects in the expression of nutrient transporter genes observed in the duodenum.

3.6.2. Jejunum

Pigs offered the diets containing enzymes had a down regulation of GLUT7/SLC2A7 (0.92 vs

1.93, SEM 0.281 P<0.05) in the jejunum compared to pigs offered diets without enzyme

supplementation. There was an effect of barley quality on GHRL expression (P<0.05). Pigs

offered the low quality barley diets had increased expression of GHRL (1.18 vs 0.87, SEM

0.095) compared to the high quality barley diets.

3.6.3. Ileum

There was a barley × enzyme interaction on PEPT1/SLC15A1 and SGLT1/SLC5A1

expression (P<0.01). Pigs offered the low quality barley with enzymes had a down regulation

in PEPT1/SLC15A1 and SGLT1/SLC5A1 expression compared to the low quality diet only.

However, pigs offered the high quality barley diet with enzymes had an upregulation in

PEPT1/SLC15A1 and SGLT1/SLC5A1 expression compared to the high quality barley diet

only.

3.6.4. Colon

There was no effect on the expression of short chain fatty acid transporter genes observed in

the colon between dietary treatments (P>0.05).

4. Discussion

The hypothesis of the current experiment was that the negative effects associated with

feeding a low quality barley diet to finisher pigs would be removed with the addition of a β-

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glucanase and β-xylanase enzyme mix, resulting in an improvement in growth performance

as a consequence of enhanced nutrient digestibility. The response observed in animals offered

the low quality barley diet supplemented with β-glucanase and β-xylanase enzymes, such as

the increase in dietary intake, body weight gain and CATTD of nutrients supports the

experimental hypothesis.

The nutrient composition of barley is subject to a degree of variation and adverse agronomic

conditions might turn an expected high quality barley grain into low quality with greater fibre

content and lower DE and NE values (Fairbairn et al., 1999; van Barneveld, 1999). Defining

the ideal feed barley is complicated, as nutritional requirements differ not only among species

but even for different age groups of the same animal species (Bleidere and Gaile, 2012). The

husbandry and management of a barley crop can also affect the end quality. In the current

study, the delayed sowing date and subsequent delayed husbandry practices of one block was

carried out in order to achieve a lower quality grain with less starch and higher NDF and

ADF contents. As expected the high quality barley had a higher hectolitre weight, TGW, GE

and starch contents along with a lower screening percentage and ADF content compared to

the low quality barley, therefore this barley was identified as “high quality” barley. The high

quality barley had a lower crude protein and lysine contents compared to the low quality

barley. Generally as the starch content increases in grain, other nutrient components of the

grain decrease (Oscarsson et al., 1998). The higher β-glucan content in the high quality barley

may be attributed to the higher level of nitrogen that this crop received (Oscarsson et al.,

1998; Paynter and Harasymow, 2011).

In the current study, the depression in nutrient digestibility in the low quality barley diet was

expected and is most likely attributed to the higher levels of NDF and ADF and lower content

of starch in the low quality barley diet. The reduced CATTD of DM and GE in the low

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quality barley diet may be associated with the higher NDF and ADF content (Fairbairn et al.,

1999). The increase in the CATTD of NDF due to enzyme supplementation shows that the

anti-nutritional effects associated with fibre in barley are somewhat alleviated through

enzyme supplementation. This is most likely due to the enzyme mix reducing the digesta

viscosity and the partial removal of the nutrient encapsulation effect of the cell wall

components. These results are consistent with results from O’Connell et al. (2005) who

reported improvements in the CATTD of DM, OM and N in finisher pigs offered barley diets

supplemented with a similar β-glucanase and β-xylanase enzyme mix. There was an

interaction in the CAID of N. Pigs offered the high quality barley diet with enzymes had a

higher N digestibility compared with pigs offered the high quality barley diet alone.

However, there was no response to enzyme supplementation in the low quality barley diet.

Nonstarch polysaccharides such as β-glucans can cause the physical enclosure of dietary

nutrients like N and this prevents the diffusion of nutrients and digestive enzymes (De Lange,

2000). The increase in the CAID of N is a result of the high quality barley diet having higher

level of β-glucan content and the addition of a β-glucanase and β-xylanase enzyme mix

hydrolysed these β-glucans and made more N available.

During the overall experimental period there was an interaction between barley type and

enzyme inclusion on ADFI where the inclusion of a β-glucanase and β-xylanase enzyme mix

increased ADFI in the low quality barley diet. However, there was no response to enzyme

supplementation in the high quality diet. These results are in agreement with Garry et al.

(2007) who reported an increase in ADFI in grower pigs (45 kg) offered a barley based diet

with β-glucanase and β-xylanase enzyme mix. The inclusion of a β-glucanase and β-xylanase

enzyme ameliorated the negative effect of feeding low quality barley on ADG. The increase

in ADFI observed in pigs offered the low quality barley diet contributed to the increase in

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ADG. The GIT synthesises appetite inducing hormones which are suppressed following the

initiation of feeding and appetite suppressing hormones which are increased in circulation

following food consumption (Suzuki et al., 2010). In the current study there was an increase

in the expression of GHRL in the jejunum of pigs offered the low quality diet. Previous

studies in rodents and humans suggest that ghrelin may provide a peripheral signal to the

brain to stimulate food intake (Wren et al., 2000; Wren et al., 2001). In response to food

intake, the hypothalamus receives neural and endocrine signals from the gut. These signals

are interpreted and directed to other centres in the brain and peripheral organs to orchestrate

energy homeostasis. The increased expression of GHRL may potentially be considered as the

body’s physiological attempt to increase energy intake. This theory is supported by the

reduced energy intake observed in the low quality barley diet compared to the high quality

barley diet (30.39 vs 34.01 MJ DE on an as fed basis). Furthermore, the increase in ADG

may also be related to the increased CATTD of GE of the low quality barley diet with the

addition of β-glucanase and β-xylanase. Despite the increase in the CAID and CATTD of N

and GE in pigs offered the high quality barley diet with enzyme supplementation, there was

no effect on ADG. The lack of response in ADG may be attributed to the fact that pigs

offered the high quality diet matched their nutritional requirements for energy and protein for

this particular genotype of pig. Therefore the response to enzyme supplementation may be

more evident in low quality raw materials (Bedford et al., 1998). This is also verified by the

barley × enzyme interaction in the CATTD of GE, as well as the DE and NE values. The

increase in the CATTD of GE is higher in the low quality barley diet compared to the high

quality barley diet. This may be due to the higher NDF and ADF content in the low quality

diet and the addition of a β-glucanase and β-xylanase enzyme mix hydrolysed the NDF and

ADF contents and made more nutrients available.

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Following nutrient breakdown by digestive enzymes, nutrients are then transported into the

blood stream by nutrient transporters (Vander Heiden et al., 2009). Intestinal enterocytes

respond to fluctuations in intestinal nutrients by modifying the gene expression of intestinal

nutrient transporters (Dyer et al., 2005). This indicates that intestinal enterocytes can

upregulate the gene expression of nutrient transporters in response to increased nutrient

availability. In the present study there was an interaction observed for CD36 (duodenum),

PEPT1/SLC15A1 and SGLT1/SLC5A1 expression (ileum) where pigs offered the high quality

barley diet with enzyme supplementation had an upregulated expression of CD36,

PEPT1/SLC15A1 and SGLT1/SLC5A1 compared with pigs offered the high quality barley

diet only. However the inclusion of the enzyme to the low quality barley diet down regulated

the expression of CD36, PEPT1/SLC15A1 and SGLT1/SLC5A1. There was also a similar

tendency observed for GLUT2/SLC2A2 in the ileum. The glucose nutrient transporters coded

for by SGLT1/SLC5A1 and GLUT2/SLC2A2 are effectively responsible for glucose

absorption while the peptide transporter coded for by PEPT1/SLC15A1 is responsible for

peptide absorption. The increase in expression of SGLT1/SLC5A1 and PEPT1/SLC15A1

along with the numerical increase in GLUT2/SLC2A2 in the ileum of pigs offered the high

quality barley diet with enzymes would suggest that there was increased glucose and amino

acid availability. This may have been due to the enzyme mix hydrolysing the higher β-glucan

content present in the high quality barley, which encapsulates starch and protein thereby

reducing their availability (De Lange, 2000). The increase in both the CAID and CATTD of

N and GE would support this theory. The expression of PEPT1/SLC15A1 follows the

observed pattern of the CAID for N. These results are consistent with results observed by

Vigors et al. (2014), where the addition of a phytase enzyme significantly increased the

expression of PEPT1/SLC15A1 with accompanying changes in the ileal digestibility of

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nitrogen. Hosseini et al. (2017) also reported increases in the expression of SGLT1/SLC5A1

and PEPT1/SLC15A1 in the jejunum of broilers with the addition of xylanase to their diets.

Surprisingly the opposite affect occurred in pigs offered the low quality barley diet with

enzyme supplementation, despite the fact that the inclusion of the β-glucanase and β-xylanase

enzyme mix increased the CAID and CATTD of GE, N and NDF. The inclusion of a β-

glucanase and β-xylanase enzyme mix was associated with a downregulation in the

expression of CD36, PEPT1/SLC15A1 and SGLT1/SLC5A1. Several luminal factors such as

gut peptide hormones (e.g. GAST, GHRL, GLP2 and PYY) can modulate the expression of

SGLT1/SLC5A1 (Ferraris and Diamond, 1989; Bird et al., 1996). In the current study pigs

offered the low quality barley diet had an increased expression of CD36, PEPT1/SLC15A1

and SGLT1/SLC5A1 and a numerical increase in GAST compared with the low quality barley

diet supplemented with enzymes. As dietary fiber can increase the production of these gut

peptide hormones (Sánchez et al., 2012) it may be possible that the higher expression of

SGLT1/SLC5A1 in pigs offered the low quality barley diet may be due to the higher fibre

content. These results concur with research carried out by Agyekum et al. (2015) who

observed an increase in SGLT1/SLC5A1 expression in the jejunum of pigs that were offered a

high fibre diet (NDF 22.2%) without a β-glucanase and β-xylanase enzymes mix compared to

the control diet (NDF 11%) and the high fibre diet supplemented with enzymes (NDF

21.9%). However, the level of fibre in these diets was greater than that in the current study.

Additionally, it may be possible that the increase in expression of CD36, PEPT1/SLC15A1

and SGLT1/SLC5A1 in pigs offered the low quality barley diet may be an adaptive

mechanism to increase the absorptive capacity in these pigs. Similar results were previously

observed in hay supplemented calves compared with calves supplemented with concentrates

or corn silage (Klinger et al., 2013).

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The inclusion of a β-glucanase and β-xylanase enzyme mix reduced the total VFA

concentration, acetic acid, propionic acid and butyric acid concentrations in pigs offered the

high quality barley diet as there was a higher level of β-glucans present in the high quality

barley diet. This is consistent with the theory that the β-glucanase hydrolyses the β-glucan

fraction of the diet in the small intestine, resulting in a reduction in carbohydrate fermentation

in the large intestine, thereby reducing total VFA production. As discussed previously, the

enzyme supplemented pigs had improved CAID of GE compared to the non enzyme

supplemented pigs suggesting there may be more material available for fermentation in the

colon of the non supplemented pigs. This would explain the increased production of VFA in

the colon of non enzyme supplemented pigs. There was a barley × enzyme interaction

observed for isobutyric acid and isovaleric acid. Pigs offered the high quality barley diet with

enzyme supplementation had an increase in the concentration of isobutyric acid and

isovaleric acid compared to the high quality barley. However, there was no response to

enzyme supplementation in the low quality barley diet. The increase in isobutyric and

isovaleric acid is most likely due to the enzyme mix degrading the β-glucan content in the

high quality diet and therefore limiting the provision of fermentable carbohydrate. When this

occurs the population of fibre degrading bacteria falls and this results in less nitrogen being

used for protein synthesis. This causes an increase in the availability of proteinaceous

substrate for fermentation, with consequential increases in branch-chain fatty acid production

(Hobbs et al., 1995). The monocarboxylate transporter 1 (MCT1/SLC16A1) and the sodium

coupled monocarboxylate transporter 1 (SMCT1/SLC5A8) are involved in the absorption of

short chain fatty acid (Halestrap and Meredith, 2004). Despite the change in concentration of

the VFA with the inclusion of a β-glucanase and β-xylanase enzyme mix there was no

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difference between treatments in both SMCT1/SLC5A8 and MCT1/SLC16A1 expression in

the colon.

5. Conclusion

In conclusion, the addition of a β-glucanase and β-xylanase enzyme to a low quality barley

based diet increased animal performance mainly through an increase in ADFI and nutrient

digestibility. This indicates that low quality barley supplemented with β-glucanase and β-

xylanase enzyme may potentially be offered to finisher pigs to achieve equivalent growth

performance as high quality barley. The enzyme mix also increased the CAID and the

CATTD of N, GE and NDF in both the high and low quality barley diets. The inclusion of β-

glucanase and β-xylanase enzyme influenced the expression of CD36, PEPT1/SLC15A1 and

SGLT1/SLC5A1, however, more research is needed in this area to elucidate the effect of

enzyme supplementation on nutrient gene expression, especially with poor quality barley.

Conflict of interest

There is no conflict of interest.

Acknowledgments

This work was funded by the Irish Government under the National Development Plan 2007-

2013 through the Department of Agriculture Food and the Marine Research Stimulus Fund;

11/S/122: Feed Evaluation for Accurate Nutrition. Additionally the authors acknowledge the

contribution of the farm and laboratory staff at University College Dublin Lyons Farm.

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Table 1

The chemical analysis of experimental barley (g/kg) on a DM basis; (unless otherwise

indicated).

Low quality barley High quality barley

Chemical characteristics

DM 888.8 844.8

Ash 25.6 20.1

GE (MJ/kg) 18.2 18.2

EE 19.3 18.4

CP 127.0 105.8

CF 40.9 27.4

NDF 196.7 186.1

ADF 68.9 57.8

Starch 578.0 602.7

β-glucans 30.4 36.1

Xylose 22.9 23.0

Lysine 4.4 3.4

Physical characteristics- on a fresh weight basis

Hectolitre weight (kg/hL) 58.3 61.1

TGW (g) 35.6 50.2

Screenings 67.9 12.1

DM, dry matter; GE, gross energy; EE, ether extract; CP, crude protein; CF, crude fiber; NDF, neutral detergent fiber; ADF, acid detergent fiber; TGW, thousand grain weight.

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Table 2Composition of experimental diets on an as-fed basis.

a β-glucanase and β-xylanase enzyme was added to the diets in order to achieve different levels of dietary treatment (T1) low quality barley diet with no enzyme supplement, (T2) low quality barley diet containing 0.15 g/kg β-glucanase and β-xylanase enzyme supplement, (T3) high quality barley diet with no enzyme supplement and (T4) high quality barley diet containing 0.15 g/kg β-glucanase and β-xylanase enzyme supplement.b The premix provided vitamins and minerals (per kg diet) as follows:0.01g/kg of retinol acetate, 0.16 g/kg of alpha tocopherol acetate, 0.007 g/kg of menadione, 0.00125 g/kg of thiamine mononitrate, 0.005 g/kg of riboflavin, 0.0025 g/kg of pyridoxine HCL, 0.003 g/kg of cyanocobalamin, 0.0229 g/kg of nicotinamide, 0.0138 g/kg of calcium-D-pantohenate, 0.06 g/kg of copper as copper sulphate, 0.4167 g/kg of iron as iron sulphate, 0.0806 g/kg of manganese as manganese oxide, 0.0032 g/kg of iodine as calcium iodate, 0.1389 g/kg of zinc as zinc oxide, 0.0056 g/kg selenium, 1.24 g/kg of calcium.

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Ingredients (g/kg)a

Barley 500.00

Flaked maize 208.70

Soy bean meal 180.00

Sugar beet pulp ground 80.00

Limestone 13.00

Dicalcium phosphate 7.50

Salt 5.00

Minerals and vitaminsb 2.50

DL-Methionine 0.85

L-Threonine 0.70

L- rytophan 0.25

L-Lysine HCl 1.00

Celite 0.50

608609

610611612613614616617618619620621622623

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Table 3Analysed composition of experimental diets on an as fed basis (g/kg) (unless otherwise stated).

Dietary treatments

Quality Low Low High High

Enzyme No Yes No Yes

Analysed composition, g/kg

β-glucanase activity (units/kg) 0 1500 0 1600

Xylanase activity (units/kg) 0 2700 0 2600

DM 887.10 889.11 857.01 867.77

CP 178.52 171.48 162.74 167.74

CF 530.00 530.00 480.00 480.00

EE 271.00 271.00 246.00 246.00

Ash 45.30 44.30 39.99 42.27

NDF 126.21 127.84 111.73 122.43

GE (MJ/kg) 16.20 16.19 15.92 15.95

Starch 436.10 436.35 460.64 460.23

Β-glucans 14.43 14.43 16.93 16.93

Xylose 14.74 14.74 14.71 14.71

Lysine 10.13 10.19 9.98 10.01

Methionine and cysteine 5.30 5.39 5.61 5.64

Threonine 6.81 6.71 6.65 6.66

Trytophan 1.91 1.89 1.81 1.83

Calciuma 7.84 7.84 7.84 7.84

Phosphorousa 5.80 5.80 5.80 5.80

DM, dry matter; CP, crude protein; CF, crude fiber; EE, ether extract; NDF, neutral detergent fiber; ADF, acid detergent fiber; GE, gross energy; DE, digestible energy. a Calculated for the tabulated nutritional composition (Sauvant et al., 2004).

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Table 4

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Porcine oligonucleotide primers used for real-time PCR. Gene Accession

numberForward and reverse primers (5′-3′) Meltin

g temp (̊C)

Product length (bp)

Reference targetsACTB AY550069.1 F: CAAATGCTTCTAGGCGGACTGT 60.9 75

R: TCTCATTTTCTGCGCAATTAGG 59.5GAPDH AF017079.1 F: CAGCAATGCCTCCTGTACCA 60 72

R: ACGATGCCGAAGTTGTCATG 58.9YWHAZ XM_001927228

.1F: GGACATCGGATACCCAAGGA 58.5 71

R: AAGTTGGAAGGCCGGTTAATTT 58.7HMBS NM_001097412

.1F: CTGAACAAAGGTGCCAAGAACA 58.4 74

R: GCCCCGCAGACCAGTTAGT 61Nutrient transportersSGLT1/SLC5A1 NM_001164021

.1F: GGCTGGACGAAGTATGGTGT 59.4 153

R: ACAACCACCCAAATCAGAGC 57.3GLUT1/SLC2A1

XM_003482115.1

F: TGCTCATCAACCGCAATGA 54.5 61

R: GTTCCGCGCAGCTTCTTC 58.2GLUT2/SLC2A2

AF054835.1 F: CCAGGCCCCATCCCCTGGTT 65.5 96

R: GCGGGTCCAGTTGCTGAATGC 63.7GLUT7/SLC2A7

XM_003127552.3

F: ACATCGCCGGACATTCCATA 57.3 75

R: GCGAGGACTGCAGGAAGATC 61.4FABP2 NM_001031780

.1F: TCGGGATGAAATGGTCCAGACT 62.4 102

R: TGTGTTCTGGGCTGTGCTCCA 61.8CD36 NM_001044622

.1F:GGAGAAAAGATCACTACCATCATGAG

61.6 78

R: CTCCTGAAGTGCAATGTACTGACA

61

PEPT1/SLC15A1

NM_214347.1 F: GGATAGCCCTGTACCCCAAGCT 61.8 73

R: CATCCTCCACGTGCTTCTTGA 59.8SMCT1/SLC5A8 NM_001291414 F: CCTTCTTGGTGTGGGACTACGT 62.1 63

R: TGCCAATGACCGCAGAGA 56.0MCT1/SLC16A1

NM_001128445.1 F: GCAGCCCTGTGTTCCTCTCT 61.4 65

R: CCAGCCGTAGATACCGAAGAAA 60.3

Appetite regulatorsCCK NM_214237.2 F: GGACCCCAGCCACAGAATAA 59.4 61

R: GCGCCGGCCAAAATC 56.3GAST NM_001004036

.2F: TCCCAGCTCTGCAGTCAAGA 59.4 65

R: CCAGAGCCAGCACATGGAT 58.8GHRL XM_005669746 F: AAGCTGGAAATCCGGTTCAA 55.3 64

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.1R: CGGACTGAGCCCCTGACA 60.5

GLP-1 NM_001256594.1

F: CAGTGCAGAAATGGCGAGAA 57.3 61

R: GGTGGAGCCTCAGTCAGGAA 61.4GLP-2 NM_001246266

.1F: TCCCGGTGCTCTTTGTTGTC 59.4 68

R: TACCCAGCACCCTGTGTTCTC 61.8NPY NM_001256367

.1F: CAGGCAGAGATACGGAAAACG 59.1 71

R: TCCGTGCCTCTCTCATCAAG 59.2PYY XM_005668763

.1F: CTCCTGATTCGGTTTGCAGAA 57.9 61

R: GGACAGGAGCAGCAGGAAGA 61.4ACTB, actin beta; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; YWHAZ, tyrosine 3-mono-oxygenase/tryptophan 5-monooxygenase activation protein; HMBS, hydroxymethylbilane synthase; SGLT, sodium-glucose-linked transporter; FABP, fatty acid-binding protein; CD, cluster of differentiation; PEPT, peptide transporter; SLC5A8, sodium-coupled monocarboxylate transporter; SLC16A1, monocarboxylate transporter 1; CCK, Cholecystokinin; GAST, Gastrin; GHRL, Ghrelin; GLP, glucagon-like peptide; NPY, Neuropeptide Y; PYY, peptide YY.

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Table 5Effect of dietary treatment on average daily feed intake (ADFI), average daily gain (ADG) and feed conversion ratio (FCR) (least square means and SEM).

Dietary treatments SignificanceQuality Low Low High HighEnzyme No Yes No Yes SEM Quality Enzyme Quality × enzymeADFI (kg/d)d 0-28 2.33w 2.63x 2.63x 2.63x 0.046 0.0009 0.0011 0.0015

ADG (kg/d)d 0-28 1.02w 1.11x 1.13x 1.14x 0.027 0.0116 0.0996 0.0401

FCR (kg/kg)a

d 0-28 2.37 2.42 2.33 2.34 0.061 0.3291 0.7051 0.3377SEM, standard error of the mean.D, day.w,xMean values within a row with unlike superscript letters were significantly different (P<0.05)aThere was a time × barley interaction on FCR (P<0.05).

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Table 6The effect of dietary treatment on the coefficient of apparent ileal digestibility (CAID) and the coefficient of apparent total tract digestibility (CATTD) of dry matter (DM), organic matter (OM), nitrogen (N), ash and gross energy (GE), as well as digestible energy (DE) and net energy (NE) concentrations (least square means and SEM).

Dietary treatments SignificanceQuality Low Low High High

Enzyme No Yes No Yes SEM Quality Enzyme Quality x enzyme

Digestibility coefficientsCAIDDM 0.769 0.776 0.758 0.809 0.0137 0.4384 0.0493 0.1194OM 0.813 0.822 0.808 0.850 0.0123 0.3697 0.0500 0.1955N 0.701w 0.717w 0.705w 0.828x 0.0186 0.2339 0.0077 0.0014GE 0.763 0.780 0.759 0.811 0.0161 0.3867 0.0344 0.2715CATTDDM 0.745w 0.834x 0.797y 0.862z 0.0064 0.0001 0.0001 0.0413OM 0.774w 0.851x 0.826y 0.879z 0.0062 0.0001 0.0001 0.0417N 0.728 0.776 0.710 0.816 0.0100 0.0002 0.0001 0.6663Ash 0.444w 0.581x 0.449w 0.678y 0.0186 0.0155 0.0001 0.0067GE 0.712w 0.814x 0.774y 0.844z 0.0755 0.0001 0.0001 0.0424NDF 0.145 0.421 0.349 0.556 0.0300 0.0001 0.0001 0.2642DE (MJ/kga) 13.02w 14.61x 14.52y 15.50z 0.138 0.0001 0.0001 0.0181NE (MJ/kgb) 9.40w 10.42x 10.47x 11.13y 0.097 0.0001 0.0001 0.0090SEM, standard error of the mean. w,x,y,zMean values within a row with unlike superscript letters were significantly different (P<0.05).aCalculated from the tabulated nutritional composition. DE = GE x GE digestibility. bCalculated using equation 4 from Noblet et al.,(1994). NE= 0.703 DE + 0.066 EE + 0.020 starch – 0.041 CP – 0.041 CF. Where NE and DE Values are expressed in MJ/kg DM and the chemical constituents are expressed as % of DM.

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Table 7The effect of dietary supplementation on total volatile fatty acids (VFA) concentrations (mmol/g digesta) in the colonic digesta (least square means and SEM).

Dietary treatments Significance

Quality Low Low High High

Enzyme No Yes No Yes SEM Quality Enzyme Quality × enzyme

Total VFA 209.0 189.9 217.4 171.1 9.551 0.5916 0.0022 0.1668

Acetic acid 151.7 134.1 148.6 118.3 7.531 0.2198 0.0040 0.4092

Propionic acid 29.6 26.3 33.4 24.2 1.712 0.6276 0.0013 0.0938

Isobutyric acid 2.3w 1.9w 1.9w 4.0x 0.191 0.0002 0.0001 0.0001

Butyric acid 25.3wx 23.0x 28.1w 18.0y 1.605 0.4951 0.0007 0.0230

Isovaleric acid 2.3w 1.9w 2.0w 3.7x 0.170 0.0002 0.0006 0.0001

Valeric acid 2.7 2.6 3.4 3.0 0.235 0.0342 0.3291 0.4648

Acetate:Propionate ratio 5.1 5.1 4.5 4.9 0.146 0.0053 0.1685 0.1713

SEM, standard error of the mean. w,x,y Mean values within a row with unlike superscript letters were significantly different (P<0.05).

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Table 8The effect of dietary treatment on the normalised relative abundance of genes involved in nutrient transporter in the duodenum, jejunum and ileum and appetite regulators in the duodenum and jejunum and short chain fatty acid transporters in the colon of finisher pigs (least square means and SEM).

Dietary treatments SignificanceQuality Low Low High HighEnzyme No Yes No Yes SEM Quality Enzyme Quality

× enzyme

DuodenumCD36 1.17w 0.65x 0.83x 1.44w 0.130 0.0966 0.7103 0.0002FABP2 1.88 1.44 1.47 1.49 0.487 0.7123 0.6606 0.6312GLUT1/SLC2A1 1.24 1.11 0.77 1.00 0.216 0.1969 0.8240 0.4222GLUT2/SLC2A2 2.38 1.89 1.66 2.11 0.795 0.7565 0.9826 0.5559GLUT7/SLC2A7 2.11 2.47 2.02 1.22 0.859 0.4410 0.7982 0.5068PEPT1/SLC15A1

1.59 1.18 1.15 1.66 0.451 0.9631 0.9167 0.3194

SGLT1/SLC5A1 1.35 1.35 1.49 1.40 0.428 0.8253 0.9199 0.9186CCK 1.16 1.11 0.94 1.08 0.195 0.5303 0.8111 0.6230GAST 1.54 1.31 1.33 1.10 0.260 0.4272 0.3915 0.9994GHRL 1.28 1.28 1.23 0.99 0.236 0.4749 0.6155 0.6108GLP-1 1.28 1.43 0.86 1.03 0.259 0.1261 0.5401 0.9542GLP-2 1.39 0.79 1.03 1.16 0.205 0.9967 0.2555 0.0867NPY 1.84 0.74 1.60 1.24 0.484 0.7947 0.1485 0.4494PYY 1.11 0.92 1.17 1.19 0.167 0.3258 0.6207 0.5414

JejunumCD36 1.29 1.12 0.96 1.21 0.222 0.6134 0.8510 0.3550FABP2 1.27 1.15 1.15 1.07 0.212 0.6365 0.6460 0.9150GLUT1/SLC2A1 1.44 0.96 1.02 0.88 0.174 0.1580 0.0880 0.3450GLUT2/SLC2A2 1.13 1.15 1.53 0.90 0.217 0.7354 0.1646 0.1486GLUT7/SLC2A7 1.23 0.89 2.62 0.95 0.398 0.0785 0.0182 0.1074PEPT1/SLC15A1

1.05 1.12 1.48 1.16 0.248 0.3542 0.6328 0.4477

SGLT1/SLC5A1 1.41 1.25 1.63 1.33 0.321 0.6335 0.4792 0.8378CCK 0.70 1.05 1.33 1.03 0.196 0.1305 0.8840 0.1085GAST 1.16 1.31 0.80 1.04 0.166 0.0711 0.2485 0.7822GHRL 1.05 1.31 0.78 0.95 0.137 0.0298 0.1314 0.7396GLP-1 1.30 1.06 0.89 1.06 0.160 0.2071 0.8457 0.2132GLP-2 1.08 1.04 1.14 0.98 0.205 0.9954 0.6291 0.7995NPY 0.98 0.94 1.58 1.35 0.305 0.1151 0.6689 0.7588PYY 1.29 1.40 1.08 1.58 0.222 0.9670 0.1808 0.3891

IleumCD36 1.17 0.99 0.94 1.26 0.200 0.9185 0.7536 0.2226FABP2 1.06 0.86 0.90 2.45 0.598 0.2419 0.2665 0.1559GLUT1/SLC2A1 1.09 1.09 1.00 0.90 0.080 0.1027 0.5299 0.5299GLUT2/SLC2A2 1.40 0.85 1.05 1.52 0.290 0.5948 0.8857 0.0920GLUT7/SLC2A7 1.49 1.34 1.69 1.78 0.578 0.5877 0.9561 0.8441PEPT1/ 1.56w 0.89x 1.00x 1.66w 0.228 0.9807 0.6436 0.0080

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SLC15A1SGLT1/SLC5A1 1.70w 0.85x 0.72x 1.50w 0.208 0.8697 0.4336 0.0007

ColonMCT1/SLC16A1 1.23 1.19 0.97 1.20 0.201 0.5324 0.6378 0.5054SMCT1/SLC5A8 0.77 1.23 1.10 1.14 0.230 0.6096 0.2856 0.3778SEM, standard error of the mean. w,xMean values within a row with unlike superscript letters were significantly different (P<0.05).CD, cluster of differentiation; FABP, fatty acid-binding protein; PEPT, peptide transporter; SGLT, sodium-glucose-linked transporter; SLC5A8, sodium-coupled monocarboxylate transporter; SLC16A1, monocarboxylate transporter 1; CCK, Cholecystokinin; GAST, Gastrin; GHRL, Ghrelin; GLP, glucagon-like peptide; NPY, Neuropeptide Y; PYY, peptide YY.

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