Effect of Non-Starch Polysaccharide (NSP) of Wheat and Barley Supplemented with Exogenous Enzyme Blend on Growth Performance, Gut Microbial, Pancreatic Enzyme Activities, Expression of Glucose Transporter (SGLT1) and Mucin Producer (MUC2) Genes of Broiler Chickens

ABSTRACT

The experiment was carried out in a completely randomized design (CRD) with 625 broiler chicks (Ross 308) for 5 repetitions (25 birds per each replicated) on the 5 treatments diet. Treatments included two different types of cereal grains (wheat, and barley) with or without an enzyme supplementation along with a corn-based diet as control group. The experimental diets were formulated to have similar contents of crude protein, metabolizable energy, total non-starch polysaccharides (NSP) and were fed in two periods of starter and grower. Experimental traits were consisted growth performance, ileal flora numeration, villus morphology in 3 parts of the intestine, digesta viscosity and pancreatic enzyme activity, and determining the gene expression level of glucose transporter (SGLT1) and mucin producer (MUC2) in the jejunum. Results indicated that inclusion of wheat and barley to corn-soy based diet with or without exo-enzymes blend on growth performance traits were significant (p<0.01). Feed intake and average daily gain in wheat diet was lower, conversely FCR was higher than other groups (p<0.01). Maximum microbial count were observed in wheat and barley diets and minimum were observed in enzyme supplemented diets respectively (p<0.01). Control group and enzyme supplemented diets had minimum counting of gram negative, coliform and clostridium, but maximum counting of lactobacilli and bifidobacter were observed in enzyme supplemented diets (p<0.01). Viscosity and activities of pancreatic a-amylase and lipase were significantly increased in chicks fed wheat and barley when compared to the control group fed on corn (p<0.01). Feeding wheat and barley diets reduced villus height in different parts of the small intestine when compared to those fed on a corn diet (p<0.01). Gene expression level of glucose transporter (SGLT1) and mucin producer (MUC2) in jejunum was significantly affected by the inclusion of wheat and barley to corn-soy based diet with or without exo-enzymes blend (p<0.01). In conclusion, the inclusion of wheat and barley to corn-soy based diet without enzyme supplementation has an adverse effect on growth, ileal microflora villi morphology, digesta viscosity, pancreatic enzyme activity, and gene expression level of nutrient transporters. However, enzyme supplemented to wheat and barley diets significantly improved those traits, and restored the negative effects.

Keywords: Broilers; Enzyme activity; Gene expression; Micro flora; NSP

INTRODUCTION

Wheat and barley as alternative cereals can be successfully replace with corn in poultry diets. These grains could locally grow in many areas of the world and have lower water requirements than corn (Ravindran et al. 1999; Lin et al. 2010). The major components of wheat and barley are starch and proteins, though they have considerable content of non-starch polysaccharides (NSP), derived from the cell walls (Olukosi et al. 2007; Mirzaie et al. 2012).

NSP as anti-nutritional factors affected physiological characteristics of broiler chickens (Jamroz et al. 2002). The major portion of NSP in wheat is arabinoxylan polymers, whereas NSP in barley are polymers of (1®3) (1®4)-b- glucans (Choct 1997; Yin et al. 2000; Jamroz et al. 2002). A review of literature indicates that the presence of anti-nutritive compounds in the digestive tract of birds, cause predominant negative changes in the gut such as increasing the product shelf life, physicochemical properties of digesta (viscosity and pH), and impede the action of indigenous enzymes through the gut (Choct & Annison, 1992 a,b; Hetland et al., 2004), along with some changes in the intestinal environment and structure of the lining surface with the impact on the quantity and quality of relevant gene expression (Ferraris, 2001; Smirnov et al., 2004; Tanabe et al., 2005). Associate gene transporting nutrients through the gut enterocytes such as glucose, amino acids, peptides and genes involved in production and secretion of mucin as an important ingredient of mucus coating the intestinal tissue are the most effective factors in the intestinal environment and structural changes (Smirnov et al., 2006; Gilbert et al., 2008; Horn et al., 2009; Gilbert et al., 2010).

Consumption level and composition of carbo-hydrates consumed well over several days increases the concentration of glucose transporters in the cell membrane of the intestine and this increases along with increasing levels of mRNA in these cells, related to the gene SGLT1 (Ferraris, 1997 and 2001). Because high-fiber feeds increase mucin excretion through the gut wall, high consumption of fiber increases the level of gene expression and increases mucin synthesis rate in the intestine (Smirnov et al., 2004; Tanabe et al., 2005; Smirnov et al., 2006; Mott et al., 2008). These negative impacts would eliminate use of exogenous enzymes, but the combination of different types of enzymes has a better influence and results in more efficacy (Cowan & Hastrupt, 1995; Ravindran et al., 1999; Slominski, 2011). Due to the lack of information on wheat and barley inclusion to applying diets and effects on physiological characteristics of chickens, this study was conducted and for this purpose, equal fractions of NSP from wheat and barley were included in broiler diets with or without multi-carbohydrates to compare the negative effects on growth performance, intestinal bacterial population, villus morphology, digesta viscosity, and pancreatic a-amylase and lipase activities along with impact on expression of glucose transporter and mucin production genes in the intestine.

MATERIALS AND METHODS

Animals, management and treatments

The experiment was carried out in a completely randomized design (CRD) with 625 broiler chicks (Ross 308) for 5 repetitions (25 birds per each replicated) on the 5 treatments diet. Each replicate was a floor pen of 1.5×2.2 m². Three experimental diets based on corn, wheat and barley were formulated for the starter (1 to 3 wk of age) and grower (3 to 6 wk of age) stages. Two additional diets were prepared by supplementing exogenous multi-carbohydrates to the wheat and barley based diets (Table 1). The enzyme cocktail contained 180 U/g multi-glycanase and 1000 U/g phytase and were used in the experimental diets at 1 mg/kg. The compositions of the experimental diets during the starter and grower stages are shown in Table 1. All diets were formulated to have the same contents of metabolizable energy, crude protein and dietary electrolyte balance. Wheat and barley samples were analyzed before the experiment for NSP constituents. Total, insoluble and soluble dietary fiber, were determined by using Megazyme assay kits (Megazyme International Ireland Ltd, Wicklow, Ireland) according to Approved Methods of the American Association of Cereal Chemists (AACC, 2000; Table 2).

Table 1
The experimental diets and their chemical composition fed to broilers during the starter (1 to 21 days of age) and grower (22 to 42 days of age) stages.

Diets	Starter (1- 21 days)					Grower (22- 42 days)
Ingredients(%)/Treatment	C	W	B	WE	BE	C	W	B	WE	BE
Yellow corn	56	44.55	45	44.55	45	58	40	42.41	40	42.41
Soy bean meal	36.8	35.1	33.9	35.1	33.9	32	30.5	29.6	30.5	29.6
Soy oil	2	1.35	2	1.35	2	2.9	2.85	3.47	2.85	3.47
Wheat	-	15	-	15	-	-	20	-	20	-
Barley	-	-	15	-	15	-	-	20	-	20
DCP	1.83	1.78	1.78	1.78	1.78	1.81	1.74	1.71	1.74	1.71
Calcium Carbonate	1.12	1.14	1	1.14	1	1.13	1.14	1.13	1.14	1.13
Sodium Chloride	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3	0.3
Potassium Carbonate	0.1	0.13	0.13	0.13	0.13	0.12	0.12	0.11	0.12	0.11
DL-Methionine	0.17	0.15	0.15	0.15	0.15	0.25	0.25	0.05	0.25	0.05
L-Lysine HCL	0.1	-	0.1	-	0.1	0.15	0.1	0.05	0.1	0.05
Vitamin Premix**	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25
Mineral Premix**	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25
Inert*	1.08	-	0.14	-	0.14	2.84	2.5	0.67	2.5	0.67
Total	100	100	100	100	100	100	100	100	100	100
Calculated Values
MEn (MJ/Kg)	12.13	12.13	12.13	12.13	12.13	12.34	12.34	12.34	12.34	12.34
Protein (%)	21	21	21	21	21	19	19	19	19	19
Met + Cys (%)	0.86	0.84	0.82	0.84	0.82	0.85	0.85	0.84	0.85	0.84
Lysine (%)	1.2	1.19	1.18	1.19	1.18	1.2	1.19	1.11	1.19	1.11
Calcium (%)	0.95	0.94	0.92	0.94	0.92	0.95	0.95	0.87	0.95	0.87
AvailablePhosphorus (%)	0.43	0.43	0.45	0.43	0.45	0.45	0.42	0.43	0.42	0.43
Sodium (%)	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15
Chloride (%)	0.22	0.23	0.24	0.23	0.24	0.22	0.23	0.23	0.23	0.23
Potassium (%)	0.95	0.95	0.96	0.95	0.96	0.87	0.87	0.88	0.87	0.88
(Na+K)-Cl (meq/kg)	247.85	247.9	247.81	247.9	247.81	231.23	231.56	231.54	231.56	231.54
Total NSP (%)	12.49	12.92	12.95	12.92	12.95	11.73	12.18	12.19	12.18	12.19
Soluble NSP (%)	2.73	2.78	2.77	2.78	2.77	2.62	2.67	2.67	2.67	2.67
Non-Soluble NSP (%)	9.76	10.14	10.18	10.14	10.18	9.11	9.50	9.52	9.51	9.52

• *Inert contained sand plus enzyme and enzyme supplementation included 1 kg per 1000 kg of diet for all treatments and contained 1000 active units of Phytase and 480 active units of Multi-glycanase units per gram. **Each Kg of premix which was used in this experiment contained vitamins: 44000 IU A, 7200 IU D3, 440 mg E, 40 mg K3, 70 mg B12, 65 mg B1, 320 mg B2, 290 mg Pantothenic acid, 1220 mg Niacin, 65 mg B6, 22 mg Biotin, 270 mg Choline Chloride, and Minerals: 950 mg Mn, 450 mg Zn, 320 mg Fe, 100 mg Cu, 65 mg Se, 68 mg I and 45 mg Co.
• C: corn, W: wheat, B: barley, WE: wheat+enzyme BE: barley+enzyme.

Wheat and barley-based diets had also equal fractions of non-starch polysaccharides (NSP). Feed and water were provided adlibitum. Environmental conditions including temperature and lighting schedule followed the management guideline of the Ross-308 strain. The experimental animals were kept, maintained and treated in accepted standards for the human treatment of animals.

Growth performance and Intestinal micro-bial Numeration

Feed intake, weight gain, and feed to gain ratio were calculated as gram per bird throughout the period (1 to 42-days). At the day 42, three chicks per pen (15 chickens from each treatment) were randomly selected and slaughtered by cervical dislocation and ileum contents were collected. Gut flora was enumerated according to Langhout, (1999) with some modifications. Briefly, aqueous digesta samples (3 mL) were taken from the distal segment of ileum and immediately transferred to sterile bottles containing 15 mL anaerobic transport medium (TRM; pH=7.0). The samples were weighted and stored at 4^oC for further examination. Samples were homogenized and 1 mL of each sample serially diluted 10- fold. An aliquot (0.1 mL) of each diluted sample was then cultivated on specific media and transferred to an incubator set at 37^oC for 24 hours. At the end of the incubation period, bacterial colonies were counted. Specific media as described below were used to culture different types of bacteria including Nutrient Agar (NA) for total bacterial count, Eosin Methylene Blue (EMB) Agar for Gram-negative bacteria, MacConkey Agar (MCA) for coliforms, Rogosa Agar (RA) for lactic acids, Eugon Agar (EA) for bifidobacteria and Reinforced Clostridial Agar (RCA) for clostridium bacteria, respectively.

Intestinal Villi Morphology

Morphometric characteristics of small intestinal including villus height and width, crypt depth, Villus Height to Villus Width ratio (H/W), and villus height to crypt depth ratio (H/CD) were determined in the duodenum, jejunum, and ileum. Segments of 2-3 cm from the midpoint of the duodenum, the midpoint between the bile duct entry and Meckel’s diverticulum (jejunum), and the distal end of the ileum (ileum) were dissected. The segments were flushed with ice-cold phosphate buffered saline (PBS, pH=7.2) and fixed in 10% neutral buffered formalin solution for further histological study. Formalin-fixed tissues were dehydrated, cleared and impregnated in paraffin wax and cut into 6-µm sections with a LEICA RM 2145 microtome. These sections were mounted on 10% Poly-L-Lysine coated slides (Langhout, 1999). The slides were stained with hematoxylin and eosin. Histological indices were determined by use of a computer aided light microscopic image analyzer (Sigma Scan, San Rafael, and CA. USA) according to the method reported by Saki (2011). The mean of 10 measures per section was used for the analysis.

Table 2
Proximate composition of wheat and barley ingredients (%).

Composition	Wheat	Barley
Dry Matter	95.95	95.67
Crude Protein	14.69	10.92
Ether Extract	1.25	0.96
Crude Ash	1.66	2.84
Neutral Detergent Fiber	13.20	30.20
Acid Detergent Fiber	0.80	6.40
Acid Detergent Lignin	1.00	2.00
Total carbohydrate	82.40	85.28
Cellulose	1.80	4.40
Hemicelluloses	10.40	23.62
Total Dietary Fiber	17.22	30.72
Soluble Dietary Fiber	1.01	4.45
Un-Soluble Dietary Fiber	16.21	25.72
non-starch polysaccharides	18.80	35.17
Non Fiber Carbohydrate	69.20	55.08

• Soluble Dietary Fiber (SDF), Unsoluble Dietary Fiber (USDF)
• Total carbohydrate (CHO): [100 - (protein + fat + moisture + ash)].
• Cellulose content was calculated by difference: ADF - ADL.
• Hemicelluloses content was calculated by difference: NDF - ADF.
• Non Fiber Carbohydrate is calculated (NFC) by difference [100-(%NDF + %CP + %Fat + Ash)].

Intestinal viscosity and pancreatic enzyme activity

Measuring digesta viscosity was performed by samples and were obtained from selected birds. Ileal digesta was individually collected, homogenized at 4^°C and immediately measured for viscosity (Brookfield viscometer, Model DV-II, MA, USA) according to Langhout et al. (1999), with some modifications. For the pancreatic enzyme activity assay, sample sections (3 cm in length) were taken from the middle of the pancreas, rinsed with 0.01 M PBS (pH 7.2), and stored in liquid nitrogen at -80ºC until analysis. The pancreatic samples were homogenized in ice-cold 0.2 mol/L Tris-Hcl buffer, containing 0.05 mol/L NaCl as described by Li et al (2004). The homogenates were centrifuged at 3000 rpm for 15 min at 4ºC and the supernatants were saved for ulterior spectrophotometric measure-ment. The activity of a-amylase (EC 3.2.1.1) was determined using a validated kit from Parsazmun Chemical Company (PARSAZMUN Co., KARAJ, IRAN TS.M.91.4.5). The activity of lipase (EC 3.1.1.3) was measured by a validated kit from ZiestChem Chemical Company (ZIESTCHEM Co., TEHRAN, IRAN) according to the manufacturer’s instruction. The activities of amylase and lipase are expressed as unit per milligram of pancreatic crude protein.

Gene Expression of SGLT1 and MUC2

Fragments of the jejunum tissue of the slaughtered chickens were isolated and immediately placed in sterile aluminum foil and were transferred to a freezer with a temperature of -80^oC to subsequent RNA analyzing. Extraction of the RNA from the jejunum samples were done via the standard RNA purification kit (CINNAPURE RNA Purification Kit, Sina gene Co, Catalogue number 50k30341, TEHRAN, IRAN) according to the manufacturer´s directions. Synthesis of cDNA from mRNA samples was done using transcription kit (REVERSE TRANSCRIPTION KIT, QIAGEN Co, Catalogue number 2050311) according to the manufacturer´s protocol. RT-PCR processes were performed by ABI device model 7500, USA using specific primers of b-actin as reference gene and specific primers of poultry as target genes (METABION, CO, GERMANY) and Quanti-Fast produced by Qiagen Co, TM SYBR (Catalogue number 204052). Primers for SGLT1, MUC2 and b-actin were designed by Primer-Blast, publically available in the National Center for Biotechnology Information (NCBI). Details of primers depicted in table 3. To measure the gene expression of the experimental groups, first the Ct index was calculated and then the Ct index for the target and reference genes were compared. Gene expression levels were determined by 2 ^-∆∆Ct criterion (Formula 1), according to the method of Livak & Shcmittgen (2001).

∆∆C_t = (C_t mean target gene - C_t mean ß-Actin) - (C_t mean control gene - C_t mean ß-Actin) (Formula 1)

Table 3
Genes and Primers specifications

Target Gene	Gene code	Gene action	Primers	PCR Product (base pair)
SGLT1	XM_415247	Na+-dependent glucose and galactose transporter	5’-ATACCCAAGGTCATAGTCCCAAAC-3’ 5’-TGGGTCCCTGAACAAATGAAA-3’	169
MUC2	XM_421035	Intestinal mucin2; Gallus Gallus	5’-TCACCCTGCATGGATACTTGCTCA-3’ 5’-TGTCCATCTGCCTGAATCACAGGT-3’	228
b-actin	L-8165	Reference Gene	5’-GTCCACCGCAAATGCTTCTAA-3’ 5’-TGCGCATTTATGGGTTTTGTT-3’	270

Statistical Analysis

All data was statistically analyzed by GLM procedure of SAS software (SAS Institute Inc., 2004). Statistical scheme was based on a completely randomized design (CRD). Samples within pens (3 per each unit) were subjected to analysis. Logarithmic transformation was applied for microbial colony forming unit (CFU). The statistical model used for normal data was Y_ij= µ+ T_i + e_ij and for sampling observation within pens was Y_ijk = µ+ T_i + e_ij + Se_ijk. In these models, Y_ij and Y_ijk are observations; µ is the overall mean; T_i is the effect of treatments (different diets); e_ij is random error, and Se_ijk is the effect of sampling error. Duncan’s multiple range tests was used to separate the means.

RESULTS

Growth performance

Table 4 depicts the results of feed intake, body weight, and feed conversion ratio of chickens throughout the trial. Results indicated that birds fed with wheat significantly (p<0.01) reduced their feed intake compared to other groups. Supplementing multiple carbohydrates to the wheat diet remarkably restored feed intake so that the difference was not significant with the control fed on corn. On the other hand, birds fed barley had numerically higher feed intake than other treatments. Supplementing the barley diet with multiple carbohydrates significantly (p<0.01) increased feed intake compared to the wheat and corn diets. Nevertheless, birds fed on barley and wheat diets had significantly lower body weight and higher feed conversion ratio (FCR) than counterparts fed on corn. Enzyme supplementation of barley and wheat diets improved FCR (p<0.01).

Table 4
Effect of different types of cereal grains and enzyme supplementation on broiler growth performance during 1 to 42 days of age

Dietary treatments	Feed intake (g/b)	Weight gain (g/b)	Feed conversion ratio
C	4280b	2251a	1.90c
W	4164c	2015b	2.06a
WE	4253b	2119b	2.01ab
B	4333ab	2107b	2.05a
BE	4392a	2199ab	1.99b
SEM	23.58	22.30	0.02
p-value	< 0.001	< 0.001	< 0.001

• *^abc Means with different superscript letters within columns have significant difference (p<0.01). *C: corn, W: wheat, B: barley, WE: wheat+enzyme BE: barley+enzyme.

Intestinal microbial Numeration

Total counts of bacteria and Gram negative including E. coli and clostridia in the intestinal content were higher in birds fed wheat and barley than the control (p<0.01; Table 5). On the other hand, the number of lactic acid bacteria and bifidobacteria were significantly lower (p<0.01) in birds that received wheat and barley diets compared to the control. Inclusion of the multiple carbohydrates to the wheat and barley diets restored the situation so that no significant difference was found between the wheat and barley diets supplemented with the enzyme cocktail and the corn diet.

Table 5
Effect of different types of cereal grains and enzyme supplementation on ileal bacterial population in broiler chickens (log CFU/g digesta)

Dietary treatments	Total bacterial count	Total gram negative	E. Coli	Lactic acid bacteria	Bifido bacteria	Clostridia
C	6.67b	5.31b	5.07bc	4.91b	5.40b	4.86b
W	7.13a	6.33a	6.32a	3.87c	4.07c	6.29a
WE	6.32b	5.21bc	5.22b	5.20a	5.67a	4.83b
B	7.17a	6.24a	6.28a	4.90b	3.51d	6.65a
BE	6.75b	5.27b	4.56d	5.41a	5.76a	4.50bc
SEM	0.17	0.13	0.12	0.10	0.11	0.17
p-value	< 0.0001	< 0.001	< 0.001	< 0.0001	< 0.0001	< 0.001

• * ^abc Means with different superscript letters within columns have significant difference (p<0.01).
• *C: corn, W: wheat, B: barley, WE: wheat+enzyme BE: barley+enzyme.

Intestinal villi morphology

The intestinal villi morphometric indices including villus height, villus width, crypt depth and the related ratio at three parts of the duodenum, jejunum, and ileum were given in Table 6. The height of villus at three parts of the gut was lower, in contrary, villus width, and crypt depth was higher in birds fed wheat and barley than the control or enzyme supplemented diets (p<0.01). On the other hand, the villus height: width and villus height: crypt depth ratios were significantly lowers (p<0.01) in birds that received wheat and barley diets compared to the control or enzyme supplemented diets. The inclusion of the multiple carbohydrates to the wheat and barley diets restored the situation so that no significant differences were found between the wheat and barley diets supplemented with the multi-enzyme and the corn diet.

Table 6
Effect of different types of cereal grains and enzyme supplementation on intestinal morphology of chickens

Dietary treatments
	VH (µm)	VW (µm)	CD (µm)	H/W ratio	H/CD ratio
C	1530.33ab	115c	108.33c	13.31a	14.39a
W	1360 c	120b	114.67b	11.33b	11.86b
WE	1668 a	123.67ab	108c	13.49a	15.44a
B	1440.04b	117c	119 a	12.31ab	12.1b
BE	1589.82ab	125.33a	109c	12.68ab	14.59a
SEM	63.80	2.18	2.39	0.54	0.52
p-value	<0.001	<0.003	<0.001	<0.0001	<0.0001
	Jejunum
C	1273.67a	138.67b	109.67b	9.18a	11.61ab
W	1190ab	150a	116a	7.93b	10.26ab
WE	1297a	136b	108b	9.54a	12.01a
B	1025.23b	125c	110.33b	8.25ab	9.29b
BE	1191.33ab	135b	95c	8.83ab	12.54a
SEM	69.62	3.14	4.72	0.61	0.97
p-value	<0.001	<0.001	<0.005	<0.0001	<0.0001
	Ileum
C	1285.33ab	87d	105b	14.94 a	12.5a
W	1140.67b	128.33b	115a	8.68c	9.88b
WE	1408a	102.67c	109.67ab	13.33ab	12.61a
B	1172b	142.33a	114.33a	8.23 c	10.25b
BE	1308a	107c	108ab	12.22 b	12.12a
SEM	67.67	3.88	3.14	0.71	0.69
p-value	<0.001	<0.001	<0.002	<0.0001	<0.0001

• *^abcMeans with different superscript letters within columns have a significant difference (p<0.01).
• *C: corn B: barley, BE: barley + enzyme; VH: Villus Height, VW: Villus Width, CD: Crypt Depth,
• H/W: Villus Height to Villus Width ratio, H/CD: Villus Height to Crypt Depth ratio.

Intestinal viscosity and pancreatic enzyme activity

The intestinal digesta viscosity and pancreatic a-amylase and lipase activity of chicks fed wheat and barley (p<0.01) increased significantly compared to the control group fed on corn. Inclusion of carbohydrate enzyme mixture to the wheat and barley diets significantly reduced the digesta viscosity and activities of a-amylase and lipase (Table 7).

Table 7
Effect of different types of cereal grains and enzyme supplementation on digesta viscosity and specific activities of pancreatic amylase and lipase in chickens

Dietary treatments	Digesta Viscosity (cP) 1	Amylase (U/mg CP) 2	Lipase (U/mg CP) 2
C	1.59c	0.71c	0.24b
W	2.17a	1.37a	0.42a
WE	1.60b	0.89b	0.28b
B	1.95a	1.43a	0.42a
BE	1.60b	0.91b	0.26b
SEM	0.04	0.06	0.03
p-value	<0.001	<0.002	<0.001

• *^abcMeans with different superscript letters within columns have significant difference (p<0.01)
• ¹
  cP: centipoises; ²U/mg CP: Units of enzymes per one milligram of pancreatic crude protein.
• *C: corn, W: wheat, B: barley, WE: wheat enzyme, BE: barley+enzyme

Gene Expression of SGLT1 and MUC2

In Table 8, expression of SGLT1 and MUC2 of chicks fed wheat and barley were significantly (p<0.01) higher than other groups. But, expression of SGLT1 and MUC2 of chicks fed enzyme supplemented diets were significantly lower than wheat and barley diets and higher than control group fed on corn (p<0.01).

Table 8
Effect of different types of cereal grains and enzyme supplementation on expression of SGLT1 and MUC2 genes in jejunum of chickens

Dietary treatments	SGLT1	MUC2
C	1.00c	1.00c
B	1.85a	1.38a
BE	1.23b	1.05b
W	1.78a	1.45a
WE	1.19bc	1.13b
SEM	0.01	0.03
p-value	<0.001	<0.001

• *^abcMeans with different superscript letters within columns have a significant difference (p<0.01).
• *C: corn, B: barley, BE: barley + enzyme.

DISCUSSION

The present research has consistently elicited the negative effects of soluble NSP of wheat and barley, as also demonstrated by other researchers (Yin et al., 2000; Olukosi et al., 2007; Mirzaie et al., 2012). However, the comparative effects of different types of NSP on broiler performance and physiological responses have not been adequately cleared. Results reported herein indicate that arabinoxylan polymers of wheat’s NSP have a more deleterious impact on voluntary feed intake of broiler chickens than (1®3)(1®4)-b- glucans of barley’s NSP. Birds fed on wheat diet consumed lower feed than those on the barley, corn or enzyme supplemented diets throughout the trial (Table 4). Consequently, birds fed on wheat diet had lower body weight gain compared to those fed barley, corn or enzyme supplemented diets, primarily due to the differences in NSP structure, the size of molecules and the degree of digestion which can affect digesta viscosity and passage rate of gut content (Choct, 1997; 2006). The growth performance data are consistent with the digesta viscosity and pancreatic enzyme activity as depicted in Table 7. The viscosity of intestinal digesta in birds fed on wheat showed the highest value, which was significantly (p<0.01) greater than the control group fed on corn. In fact, NSP of both wheat and barley increased digesta viscosity in the intestine. These observations indicated that every change in the gut environment due to different dietary NSP sources could affect the physicochemical properties of the intestine and consequently the performance and other physiological responses of the birds.

Increased viscosity per se creates an ideal environment for maximal proliferation of anaerobic and Gram-negative bacteria as observed in this experiment. This mode leads to the increased production of volatile fatty acids (VFA) which could result in decreased digesta pH due to the production of a great amount of short chain fatty acid in the lumen. These observations are in line with Jaroni et al. (1999), and Langhout et al. (1999). Sedentary intestinal digesta and low oxygen condition due to indigestible NSP provide an appropriate environment for fermentative anaerobic bacteria proliferation (Langhout et al., 1999). The decrease in nutrient availability and production of detrimental by-products resulted in microbial changes throughout the gut (Choct et al., 2006). Water soluble fraction of barley NSP has a deleterious effect on intestinal physicochemical characteristics and microbial proliferation of chickens (Choct, 1997; 2006). Results of this study also indicate that NSP polymers of wheat and barley decreased the count of lactic acid bacteria and Bifidobacteria in the gut digesta. These bacteria are related to beneficial effects on birds and are known as probiotics growth promotants. The impaired live performance of birds fed on wheat and barley can partly be explained by reduced population of Gram-positive bacteria including Lactobacilli and Bifidobacteria. The probiotic-type micro flora modulates innate immune system of the host animal (Christensen et al., 2002) and they are necessary for the development of gut-associated lymphoid tissue (GLUT) (Rhee et al., 2005).

The negative effects of NSP on the proliferation of bacteria in the intestine were removed significantly after supplementation of wheat and barley diet with exogenous enzymes blend (especially on probiotic-type bacteria). These results are consistent with previous reports. (Yin et al., 2000; Choct et al., 2006; Mirzaie et al., 2012). Digest NSP of wheat and barley by carbohydrases has been successful and promising in broilers (Olukosi et al., 2007; Slominski, 2011). Glycanase enzymes including xylanases and b- glucanases release the encapsulated nutrients and decrease digesta viscosity. These events are further facilitated with action of phytases (Ravindran et al., 1999; Olukosi et al., 2007). Due to the fact that birds don’t possess adequate endogenous enzymes to digest NSP components, the addition of exogenous NSp-degrading enzymes seems to be necessary when corn is replaced by wheat and barley.

Results presented in Table 6 cleared that reduced villus height, and vice versa increased villus width and crypt depth can result from an increase in digesta viscosity which leads to quick changes in the intestinal mucosa due to the close proximity of the mucosal surface to the intestinal viscose materials (Saki et al., 2011). The crypt can be acted as villus factory and a large crypt depth indicates fast tissue turnover and a high demand for new tissue. Therefore, the addition of a viscose ingredient (NSP) to the diet can result in deeper crypts with a high rate of cell proliferation and tissue renewal (Iji et al., 2001). Hence, it is concluded that the shorter villus height induced by wheat and barley diets are related to NSP viscosity. Shorter villus height is associated with a reduction in absorptive potential throughout the intestine, further growth efficiency, and normal physiological conditions (Saki et al., 2011).

Pancreas a-amylase and lipase activities of broiler chickens were significantly increased in birds fed on wheat and barley diets as compared to those fed on corn or diets supplemented with enzymes. These results reflect the fact that water-soluble NSP of wheat and barley impede pancreatic a-amylase and lipase activities (Li et al., 2004). This mode may indicate needs for greater secretion of pancreatic enzymes (Williams, 1996; Denbow, 2000). Based on relevant research findings intestinal enzyme activity depends on the original of dietary nutrient and quantity or quality of anti-nutrients in the gut (Li et al., 2004; Mirzaie et al., 2012). Also diet issue affects the magnitude of secretion in the pancreas. Diets with high fat or carbohydrates content increase the pancreas secretion and serum concentration of amylase and lipase (Brenes et al., 1993; Zhao et al., 2007; Lin et al., 2010). Amylase is secreted in saliva, intestinal fluid, and pancreatic juices. As well as lipase is secreted in stomach and pancreatic juices (Denbow, 2000). In normal conditions, pancreas-derived amylase and lipase constitued only a small portion of serum enzymes, but in abnormal conditions and changing in diet cereal type with high amounts of anti-nutritional factors, acute pancreatitis and leakage of enzymes, total serum concentration of enzymes rise significantly (Williams, 1996).

The results of Table 8 indicated that the decrease in expression of SGLT1 and MUC2 in the jejunum of chickens at the age of 42 days fed on enzyme supplemented diets, may reflect modularly effects of exogenous enzyme blend on gut cell wall, villus structure and restoring the remodeled nutrient transporters which resulted in neutralization of negative effects. In the small intestine, the absorption of nutrients is mediated by transporter proteins expressed in the enterocyte. The nutrients passed through the epithelium of the small intestine and into the blood stream via special transporters. For example, amino acids are transported into the intestinal epithelial cells as di- or tri-peptides by the H+-dependent peptide transporter 1, PepT1 (Chen et al., 2015). Since the nutrient transporters have different gene expression patterns and actions, thus it can be concluded that it is necessary to understand the developmental patterns of intestinal absorptive capacity because of its key function in nutrient intake (Poncet & Taylor, 2013).

According to the literatures, amount and right combination of foods in the intestine is essential for the development of intestinal tissue and the words. Inversely, abundance of anti-nutrients in the gut causes mystification to tissue development or growth and increment of its properties for the sufficient intestinal cell proliferation (Uni et al., 2003a; Tako et al., 2004; Uni & Ferket, 2004). Increasing the level and composition of carbohydrates consumed well over several days increases the density of glucose transmitters in the intestinal cell membranes and this increase along with an increase in mRNA levels of genes in these cells is called SGLT1 (Ferraris, 1997 and 2001). The increase in peptide transmitters and increase in the level of mRNA for the gene MUC2 have also been reported (Gilbert et al., 2008; Mott et al., 2008). Plentitude of nutrient transmitters in the chicken´s intestine well affected by several factors such as genetic selection, the type and amount of feed intake, intestinal tissue growth and development, quality and quantity of dietary protein, as well as dietary fiber level (Chen et al., 2005; Mott et al., 2008; Gilbert et al., 2007, 2008, 2010).

Food deprivation or presence of anti-nutrients such as NSPs especially water-soluble NSP compounds causing delay and disruption of intestinal tissue growth, increased synthesis and secretion of mucin and eventually changing the structure of the intestinal mucus layer (Noy & Sklan, 1996; Uni et al., 2000 Uni et al., 1998, 2003a). The supply of nutrients and absorption of carbohydrates in the early growth period of the chickens has an important role in the normal development of intestinal tissue, mucus layer of the intestinal absorptive cells and other relevant events in the intestine (Uni et al., 1998; Tako et al., 2004; Uni & Ferket, 2004; Moran, 2007).

All of the incidents through the gut are set by regulating the gene expression of nutrient transmitters (glucose, amino acids and peptides) from lumen and through the mucus layer into enterocytes of lining surface at the absorptive area, which created in the intestine and are the main factors limiting further chicken growth (Ferraris, 1997 and 2001; Gilbert et al., 2007, 2010). In this experiment, the barley diet was increased MUC2 gene expression. Because high-fiber food or plentiful NSP content led to the increased excretion of mucin, it is obvious that, the increase in the level of food items containing these substances increases the production of mucin gene expression and mucin synthesis rate (Smirnov et al., 2004, 2006; Montagne et al., 2004; Mott et al., 2008).

Also the lining of the intestinal epithelial mucin plays a vital role to the protection of tissues, including protection of the gut against acidic stomach chyme and hydrolyzing pancreatic enzymes, slippery surface to facilitate the transportation of materials such as roughage and fiber, protection against invasion and establishment of pathogenic microbes as well as the treatment of intestinal absorption and facilitation of the transfer of nutrients to the cells (Smirnov et al., 2004, 2006; Tanabe et al., 2005). MUCIN layers are divided in two parts, including loose and tight layers, which action and composition are different. Tight layers have several membrane conjunction sites to catch and transmit nutrients into enterocyte cells (Montagne et al., 2004; Horn et al., 2009).

Various factors, including the type, quantity and composition of the diet (phytate, fiber and NSP), the number of invasive bacteria, disease, lack of amino acids and environmental factors can change mucin physicochemical properties and its integration. Finally, the mentioned factors can alter mucin synthesis and mucin disposal rate (Montagne et al., 2003, 2004; Cowieson et al., 2004; Tanabe et al., 2005).

CONCLUSION

Results of the present study suggest that wheat and barley based diet, containing high levels of NSP, due to its contribution to digesta viscosity and impede of endogenous digestive enzymes play an important role to intestinal health and subsequent transmission of hydrolyzed products to the enterocyte cells and nutrient absorption efficiency, as well as villi morphology, bacterial population, and level of nutrient transmitter gene expression in broilers. In conclusion, wheat and barley based diets without exo-enzyme blend supplementation has adverse effects on productive traits, however such changes are remarkably restored by supplementing NSp-degrading enzymes to broiler diets.

ACKNOWLEDGMENT

The authors appreciate the financial support of the deputy of presidency of Iran National Science Foundation (INSF).

REFERENCES

• AOAC - Association of Official Analytical Chemists. Official methods of analysis of the Association of Analytical Chemists International. 18th ed. Gaithersburg; 2005.
• Brenes A, Smith M, Guener W, Marquardt RR. Effect of enzyme supplementation on the performance and digestive tract size of broiler chickens fed wheat and barley based diets. Poultry Science 1993;72:1731-1739.
• Chen H, Pan Y, Wong EA, Webb JR KE. Dietary protein level and stage of development effect expression of an intestine peptide transporter (cPepT1) in chickens. Journal of Nutrition 2005;135:193-198.
• Chen M, Li X, Yang J, Gao C, Wang B, Wang X, et al . Growth of embryo and gene expression of nutrient transporters in the small intestine of the domestic pigeon (Columba livia). Journal of Zhejiang University-Science B (Biomedicine & Biotechnology) 2015;16(6):511-523.
• Choct M, Annison G. The inhibition of nutrient digestion by wheat Pentosans. British Journal of Nutrition 1992a;67:123-132.
• Choct M, Annison G. Aniti-Nutritive activity of wheat Arabinoxylans: role of viscosity. British Poultry Science 1992b;33:821-834.
• Choct M. Feed non-starch polysaccharides:chemical structures and nutritional significance. Journal of Feed Milling International 1997;191:13-26.
• Choct M, Sinlae M, Al-Jassim RAM, Pettersson D. Effects of xylanase supplementation on between-bird variation in energy metabolism and the number of Clostridium perfringens in broilers fed a wheat-based diet. Australian Journal of Agriculture Research 2006;57:1017-1021.
• Cowan WD, Hastrupt T. Application of Xylanases and b-Glucanases to the feed of turkeys and ducks. Proceedings of the 10th European Symposium Poultry Nutrition; 1995 Oct 15-19; Antalya. Turkey; 1995. p 320.
• Cowieson AJ, Acamovic T, Bedford MR. The effects of phytase and phytic acid on the loss of endogenous amino acids and minerals from broiler chickens. British Poultry Science 2004;45:101-108.
• Christensen HR, Frokiar H, Pestka JJ. Lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells. Journal of Immunology 2002;168:171- 178.
• Denbow DM. Gastrointestinal anatomy and physiology. In: Whittow G, editor. Sturkie's avian physiology 5th ed. San Diego: Academic Press; 2000. p.299-325.
• Ferraris RP, Diamond J. Regulation of intestinal sugar transport. Physiological Reviews 1997;77:257-302.
• Ferraris RP. Dietary and developmental regulation of intestinal sugar transport. Journal of Biochemistry 2001;360:265-275.
• Garcia M, Lazaro R, Latorre MA, Gracia MI, Mateos, GG. Influence of enzyme supplementation and heat processing of barley on digestive traits and productive performance of broilers. Poultry Science 2008;87:940-948.
• Gilbert ER, Li H, Emmerson DA, Webb JR KE, Wong EA. Developmental regulation of nutrient transporter and enzyme mRNA aboundance in the small intestine of broilers. Poultry Science 2007;86:1739-1753.
• Gilbert ER, Li H, Emmerson DA, Webb JR KE, Wong EA. Development regulation of nutrient transporter and enzyme mRNA abundance in the small intestine of broilers. Poultry Science 2007;86:1739-1753.
• Gilbert ER, Wong EA, Webb JR KE. Peptide absorption and utilization: Implications for animal nutrition and health. Animal Science 2008;86:2135-2155.
• Gilbert ER, Li H, Emmerson DA, Webb JR KE, Wong EA. Dietary protein quality and feed restriction influence abundance of nutrient transporter mRNA in the small intestine of broiler chicks. Journal of Nutrition 2008;138:262-271.
• Gilbert ER, Li H, Emmerson DA, Webb JR KE, Wong EA. Dietary protein composition influences abundance of peptide and amino acid transporter messenger ribonucleic acid in the small intestine of 2 lines of broiler chicks. Poultry Science 2010;89:1663-1676.
• Hetland H, Choct M, Svihus B. Role of insoluble non-starch polysaccharides in poultry nutrition. World's Poultry Science Journal 2004;60:415-422.
• Horn NL, Donkin SS, Applegate TJ, Adeola O. Intestinal mucin dynamics: response of broiler chickens and white Pekin duckling to dietary threonine. Poultry Science 2009;88:1906-1914.
• Iji PA, Saki AA, Tivey DR. Intestinal development and body growth of broiler chicks on diets supplemented with non-starch polysaccharides. Journal of Animal Feed Science and Technology 2001;89:175-188. Available from: http://www.sciencedirect.com/science/article/pii/S0377840100002236
  » http://www.sciencedirect.com/science/article/pii/S0377840100002236
• Jaroni D, Scheideler SE, Beck MM, Wyatt C. The effect of dietary wheat middling and enzyme supplementation. II:Apparent nutrient digestibility, digestive tract size, guts viscosity and gut morphology in two strains of leghorn hens. Poultry Science 1999;78:1664-1674.
• Langhout DJ, Schutte JB, Van Leeuwen P, Wiebenga J, Tamminga S. Effect of dietary high-and low-methylated citrus pectin on the activity of the ileal micro flora and morphology of the small intestinal wall of broiler chicks. Journal of British Poultry Science 1999;40:340-347.
• Li WF, Feng J, Xu ZR, Yang CM. Effects of non-starch polysaccharides enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley. World Journal of Gastroenterology 2004;10:856-859.
• Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-??Ct method. Journal of Methods 2001;25:402-408.
• Lin PH, Shih BI, Hsu JC. Effects of different source of dietary non-starch polysaccharides on the growth performance, development of digestive tract and activities of pancreatic enzymes in goslings. British Poultry Science 2010;51:270-277.
• Mirzaie S, Zaghari M, Aminzadeh S, Shivazad M, Mateos GG. Effect of wheat inclusion and xylanase supplementation of the diet on productive performance, nutrient retention and endogenous intestinal enzyme activity of laying hens. Poultry Science 2012;91:413-425.
• Montagne L, Crevieu-Gabriel I, Toullec R, Lalles JP. Influence of dietary protein level and source on the course of protein digestion along the small intestine of the veal calf. Journal of Dairy Science 2003;86:934-943.
• Montagne L, Piel C, Lalles JP. Effect of diet on mucin kinetics and composition:Nutrition and health implications. Nutrition Review 2004;62:105-114.
• Moran JR ET. Nutrition of the developing embryo and hatchling. Poultry Science 2007;86:1043-1049.
• Mott CR, Siegel PB, Webb Jr RE, Wong EA. Gene expression of transporters in the small intestine of chickens from lines divergently selected for high or low Junvenile body weight. Poultry Science 2008;87:2215-2224.
• Noy Y, Sklan D. Uptake capacity in vitro for glucose and methionine and in-sito for oleic acid in the proximal small intestine of posthatch chicks. Poultry Science 1996;75:998-1002.
• NRC- National Research Council. Nutrient requirements of poultry. Washington: National Academy of Science; 1994. 176 p.
• Saki AA, Hemati Matin HR, Tabatabai MM, Zamani P, Naseri Harsini R. Microflora population, intestinal condition and performance of broilers in response to various rates of pectin and cellulose in the diet. European Poultry Science 2010;74(3):183-188.
• Smirnov A, Sklan D, Uni Z. Mucin dynamics in the small intestine are altered by starvation. Journal of Nutrition 2004;134:738-742.
• Smirnov A, Tako E, Ferket PR, Uni Z. Mucin gene expression and mucin content in the chicken intestinal goblet cells are affected by in ovo feeding of carbohydrates. Poultry Science 2006;85:669-673.
• Olukosi OA, Cowieson AJ, Adeola O. Age-related influence of a cocktail of xylanase, amylase, and protease or phytase individually or in combination in broilers. Poultry Science 2007;86:77-86.
• Poncet N, Tylor PM. The role of amino acid transporters in nutrition. Current Opinion Clinical Nutrition & Metabolic Care 2013;16(1):57-65.
• Ravindran V, Selle PH, Bryden WL. Effects of phytase supplementation, individually and in combination, with glycanase, on the nutritive value of wheat and barley. Poultry Science 1999;78:1588-1595.
• Rhee KJ, Jasper PJ, Sethupathi P, Shanmugam N, Lanning D, Knight KL. Positive selection of the peripheral B cell repertoire in gut-associated lymphoid tissues. Journal of Experimental Medicine 2005;201:55-62.
• Saki AA, Hematti Matin HR, Zamani P, Tabatabai MM, Vatanchian M. Various ratios of pectin to cellulose affect intestinal morphology, DNA quantitation, and performance of broiler chickens. Journal of Livestock Science 2011;139:237-244.
• SAS - Statistical Analyzing System. SAS procedure guide for personal computers. STAT user's guide, atatistics. Version 9.1. Cary: SAS Institute; 2004.
• Slominski BA. Recent advances in research on enzymes for poultry diets. Poultry Science 2011; 90:2013-2023.
• Tanabe H, Sugiyama K, Matsuda T, Kiriyama S, Morita T. Small intestine mucins are secreted in proportion to the setting volume in water if dietary indigestible components in rats. Journal of Nutrition 2005;135:2431-2437.
• Tako E, Ferket PR, Uni. Effects of in ovo feeding of carbohydrates and beta-hydroxy-beta-methylbutyrate on the development of chicken intestine. Poultry Science 2004;83:2023-2028.
• Uni Z, Ganot S, Sklan D. Posthatch development of mucosal function in the broiler small intestine. Poultry Science 1998;77:75-82.
• Uni Z, Geyra a, Ben Hur H, Sklan D. Small intestine development in the young chick:Crypt formation and enterocyte proliferation and migration. British Poultry Science 2000;41:544-551.
• Uni Z, Smirnov A, Sklan D. Pre- and posthatch development of goblet cells in the broiler small intestine:Effect of delayed access to feed. Poultry Science 2003;82:320-327.
• Uni Z, Ferket P. Methods for early nutrition and their potential. World's Poultry Science Journal 2004;60:101-111.
• Williams DA. The pancreas. In: Guilford WG. Strombeck's small animal gastroenterology. 3rd ed. Philadelphia: WB Saunders; 1996. p.381-410.
• Yin YL, Baidoo SK, Boychuk JLL. Effect of enzyme supplementation on the performance of broilers fed maize, wheat, barley or micronized dehulled barley diets. Journal of Animal Feed Science and Technology 2000;9:493-504.
• Zhao F, Hou SS, Zhang HF, Zhang ZY. Effects of dietary metabolizable energy and crude protein content on the activities of digestive enzymes in jejunal fluid of Peking ducks. Poultry Science 2007;86:1690-1695.

Publication Dates

• Publication in this collection
  Oct-Dec 2017

History

• Received
  03 Dec 2016
• Accepted
  28 July 2017