Post on 23-Jul-2019
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:
john.vodoherty@ucd.ie
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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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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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594595596597598599600601602603604605606607
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.
26
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
624625626627
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).
27
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631632633
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
29
661
.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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662663664665666667668669
670
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).
31
638639640
641642643644645646647648649650651652653654655
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.
32
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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).
33
664665666667
668669670
671
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
34
669670671672
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.
35
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