Effects of two native Iranian wheat cultivars, processing method, and enzyme supplementation on performance, carcass, intestinal morphology, and microbiota activity in broiler chickens

ABSTRACT

A 2 × 2 × 2 factorial arrangement of treatments was used to investigate the effects of two Iranian wheat cultivars (Alvand or Atrak), two processing methods (dry heating or microwave), and two enzyme (Kemzyme) levels on the performance, microbiota activity, and intestinal morphology of broiler chickens. A total of 320 broiler chickens were assigned to 8 treatments with 4 replicate pens of 10 broiler chickens per pen. The results showed that birds that received Alvand cultivar with microwave processing and enzyme supplementation had greater weight gain and feed conversion ratio (FCR) than those that received Atrak cultivar (P < .05). The total bacteria count in ileo-caecum was greater in broilers fed wheat-based diets with enzyme and dry heating process (P < .05). Atrak cultivar, microwave processing, and enzyme supplementation increased the population of Lactobacilli in broiler chickens (P < .05). Besides, the inclusion of enzyme decreased the population of E. coli in the ileo-caecum of broiler chickens (P < .05). The morphological traits of the intestine were greater in birds that received Atrak cultivar with enzyme supplementation and microwave treatment (P < .05). In conclusion, the positive effects of microwave processing and enzyme supplementation on the growth performance and intestinal morphology were clearly evident for broiler chickens.

KEYWORDS:

• Wheat
• enzyme
• broiler
• intestinal morphology
• microflora

1. Introduction

Wheat grain is an important ingredient which can be included in the diets of broiler chickens as a suitable alternative for corn grain in Iran. Compared with corn grain, wheat is lower in energy but greater in other nutrients, such as protein and many amino acids, including lysine, methionine, arginine, phenylalanine, and tryptophan (Wang et al. 2005). In this regard, wheat cultivars are different with respect to their metabolizable energy content, which is the reason for the variation in wheat quality for broiler chickens (Józefiak et al. 2007). Moreover, some wheat cultivars considered to be of high quality have produced unexpectedly low broiler performance (del Alamo et al. 2008). It is well documented that wheat cultivars affect nutrient digestibility and metabolizable energy content of the diet, which could in part be explained by the difference in the non-starch polysaccharides (NSP) content of the wheat (Smeets et al. 2015).

Heating cereal grains is a common procedure for improving the performance of broiler chickens (Teitge et al. 1991; Gracia et al. 2003; Afsharmanesh et al. 2008). Heat processing of cereals increases starch gelatinization to some extent, facilitating endogenous enzymatic degradation (Afsharmanesh et al. 2008). More recent reports suggest that starch gelatinization during pelleting is generally small and is of minor importance (Zaefarian et al. 2015). However, it is found that during expander and extrusion processing, high temperatures under pressure and water addition to the feed increase starch gelatinization and its digestibility (Svihus et al. 2005). Microwave processing is an alternative for heat treatment to increase nutrient bioavailability in cereal grains for poultry. Microwave energy significantly affects all the physical and biochemical properties of the wheat grain (Walde et al. 2002). In microwave processing, heat is generated throughout the material, leading to faster heating rates and shorter processing time compared to conventional heating (Sadeghi & Shawrang 2006). The effects of conventional heat treatments on the nutritional value of wheat grain have been reported (Teitge et al. 1991; Afsharmanesh et al. 2008), but the effects of microwave processing of wheat grain on the growth performance, intestinal morphology, and microflora population have not been evaluated. Recently, Zeller et al. (2015) suggested that microwave treatment of wheat in broiler diets can increase inositol phosphate hydrolysis. These authors indicated that this increase can be explained by changes in the structure of the wheat grain.

The beneficial effects of exogenous enzyme supplementation, such as xylanase, to wheat-based diets are well documented (Engberg et al. 2004; Kiarie et al. 2014). The presence of NSP, especially arabinoxylans in wheat grain, can increase the viscous conditions in the small intestine and decrease the contact between digestive enzymes and substrates (Wang et al. 2005). It is well accepted that the addition of enzymes is effective in diets based on wheat grain through the mechanism of gut viscosity reduction (Kiarie et al. 2014). Much less information is available about the effects of xylanase supplementation in wheat-based diets on intestinal morphology and microbiota activity of broiler chickens.

Therefore, the objective of this research was to investigate the effects of wheat cultivars, processing method, and enzyme supplementation in broiler chicken diets on growth performance, carcass characteristics, intestinal morphology, and microflora population.

2. Materials and methods

2.1. Birds and dietary treatments

All animal care and use procedures were approved by the Department of Animal Science, Islamic Azad University (Qaemshahr Branch, Qaemshahr, Iran). The study was conducted at a commercial broiler chicken farm (Qaemshahr, Iran).

Three hundred and twenty 1-d-old broiler chickens (Ross 308) were obtained from a commercial hatchery (Zarbal Company, Amol, Iran) and randomly allocated into 8 treatments with 4 replicate pens of 10 broiler chickens per pen. The broiler chickens were raised in floor pens (1.0 × 1.7 m) for the experimental period of 42 d. Each pen was equipped with a separate feeder and a manual drinker. The house temperature was maintained at 35°C during the first week, and it was reduced by 2°C per week until reaching the temperature of 23°C. The broiler chickens were provided access to feed and water ad libitum.

The experiment used a completely randomized design with a 2 × 2 × 2 factorial arrangement of treatments, including two Iranian wheat cultivars (Alvand or Atrak), two methods of processing (dry heating or microwave), and two levels of enzyme supplementation (0 or 0.5 kg/ton). The two wheat cultivars utilized in this study were purchased from the agriculture and natural resources centre of Mazandaran province. Nutrient analysis of the wheat cultivars used in this experiment is shown in Table 1. Briefly, wheat grains were analysed for dry matter by oven drying procedure (934.01), ash by muffle furnace (942.05), crude protein by Kjeldahl method (954.01), ether extract by Soxhlet analysis (920.39), starch by glucosidase method (996.11), and soluble and non-soluble NSP (method 991.43) as described by the Association of official analytical chemists (AOAC) (1995). A batch of each wheat cultivar was split into two fractions. The first part was subjected to dry heating (toasting). Briefly, the wheat grains were spread thinly in a pan placed in an oven (110°C) for 10 min. It was stirred several times to maintain uniform heating. Then, the sample was ground through a 2-mm hammer mill. The second fraction was treated in portion of 5 kg for 5 min (1000 W) within a rotating vessel using a microwave system (Samsung, Model SAMI11W, Samsung Electronics Inc., Tehran, Iran) and then ground as the first fraction. The maximum temperature measured in the ground wheat after microwave treatment was 107°C. The exogenous enzyme was a commercial powdered preparation (Kemzyme, Toekomstlaan 42, 2200 Herentals, Belgium) containing xylanase (100,000 units/g), beta-glucanase (11,750 units/g), cellulase (20,000 units/g), protease (5000 units/g), phytase (10,000 units/g), and alpha-amylase (2000 units/g) derived from Trichoderma viride, Aspergillus aculeatus, Trichoderma longibrachiatum, Bacillus amyloliquefaciens, Trichoderma reesei, and Bacillus amyloliquefaciens, respectively. The commercial powdered enzyme was added 50 g/t (on top), as recommended by the supplier. The experimental diets were formulated to meet or exceed the energy and nutrient requirements . The composition of the experimental diets is presented in Table 2.

Table 1. Nutrient analysis of the two Iranian wheat cultivars used in this study.

Item	Wheat cultivar
	Alvand	Atrak
Dry matter (DM)	90.32	90.01
Ash	2.23	2.11
Crude protein (CP)	12.03	11.96
Ether extract (EE)	1.51	1.45
Crude fibre (CF)	3.54	3.27
Nitrogen-free extract^a (NFE)	71.01	71.22
Starch	66.39	67.54
Soluble NSP^b	1.51	1.82
Non-soluble NSP	9.86	10.12
Total NSP	11.37	11.94

^aNitrogen-free extract = 100 − (%humidity + %EE + %ash + %CP + %CF).

^bNon-starch polysaccharides.

Table 2. Composition of basal diets (as-fed basis).^a

Ingredient (g/kg unless stated otherwise)	Starter	Grower	Finisher
	d 0 to 10	d 11 to 24	d 25 to 42
Wheat grain	564.1	596.1	631.3
Soybean meal (440 g CP/kg)	361.8	331.4	285.6
Soybean oil	27.10	34.40	47.30
Sodium bicarbonate	1.50	1.50	1.10
Oyster shell	13.10	10.30	10.30
Dicalcium phosphate	18.60	16.00	14.80
Common salt	3.00	2.80	2.50
Vitamin premix^b	2.50	2.50	2.50
Mineral premix^c	2.50	2.50	2.50
DL-Met	3.30	2.30	2.00
L-Lys.HCl	2.00	0.20	0.10
L-Thr	0.50	–	–
Chemical composition (calculated)
ME (MJ/kg)	12.14	12.47	12.81
CP	220	208	191
Ca	10.20	8.50	8.20
Available P	4.90	4.30	4.00
Na	1.90	1.80	1.60
Lys	13.90	11.80	10.50
Met + Cys	10.40	9.10	8.30
Thr	9.10	8.10	7.40
Arg	14.8	13.91	12.50
Met	6.50	5.41	4.80

^aKemzyme, Toekomstlaan 42, 2200 Herentals, Belgium, was added to experimental diets at levels of 0 or 500 g/ton.

^bProvides per kilogram of diet: 9000 IU vitamin |A; 2000 IU vitamin D₃; 18 IU vitamin E; 2 mg menadion; 1.8 mg thiamine; 6.6 mg riboflavin; 30 mg niacin; 3 mg pyridoxine; 15 µg vitamin B₁₂; 100 mg D-pantothenic acid; 1 mg folic acid; 0.1 mg biotin; 500 mg choline chloride; and 100 mg antioxidant.

^cProvides per kilogram of diet: 100 mg Mn; 84.7 mg Zn; 50 mg Fe; 10 mg Cu; 1 mg I; and 0.2 mg Se.

2.2. Growth performance and carcass characteristics

The broiler chickens were fed the 8 experimental diets until 42 d of age. Feed intake and body weight gain of each pen were measured at the end of each phase. FCR for each pen was calculated by dividing the feed intake by body weight gain. Mortality was recorded and weight gain and feed consumption data were corrected accordingly.

At the end of the study (42 d of age), one broiler chicken from each pen, which was close to the mean weight of the pen, was selected and killed by cervical dislocation for the assessment of carcass characteristics, intestinal morphology, and microflora population. pH values of the different parts of the digestive tract were measured using a pH meter as described by Chaveerach et al. (2004). After removing the viscera manually, carcass characteristics, including the weight of the breast, thigh, liver (without gallbladder), pancreas, heart, gizzard, proventriculus, crop, and abdominal fat, was recorded. All the carcass data are presented based on the percent of live weight of each broiler chicken.

2.3. Jejunum morphology

After removing the intestinal contents, 3 cm lengths of the jejunum (mid-point of jejunum) and ileum (5 cm after Meckel’s diverticulum) were removed for morphological measurements. The segments were flushed clean with phosphate-buffered saline to avoid damage to the tissues. Then, the samples were fixed in Clark solution for 1 h. The samples were then transferred in 50% ethanol solution. A 0.5-cm section was processed, embedded in paraffin, stained with eosin blue, and examined under a light microscope. The villus height and crypt depth were measured with linear scaled graticule. The number of goblet cells was measured by 25 squared graticule. Ten microscopic fields per bird were measured, and the average value was expressed as the morphological value for each broiler chicken (Eftekhari et al. 2015).

2.4. Microbial enumeration

At 42 d of age, 4 broiler chickens (1/pen) per treatment were selected, weighed, and killed by cervical dislocation. The intestinal tract of each broiler chicken was removed and samples of fresh digesta (1–2 g) from the ileum (Meckel’s diverticulum to 1 cm proximal to the ileo-caecal junction) and caeca were collected and both samples were mixed together and gently placed in sterile sampling tubes. The samples were put on ice until they were transported to the laboratory for enumeration of microbial populations. The populations of total aerobic bacteria, Escherichia coli, and Lactobacilli were estimated as the log 10 of colony-forming units (cfu) per gram of caecal and ileal digesta contents. The total aerobic bacteria population was measured by using the brain heart infusion agar. Escherichia coli was cultured in MacConkey agar (Merck, Darmstadt, Germany) at 37°C for 24 h. Lactobacilli were enumerated on de Man–Rogosa–Sharpe agar (Merck, Darmstadt, Germany) after incubation for 48–72 h at 37°C (Eftekhari et al. 2015).

2.5. Statistical analysis

Statistical analysis was conducted using the general linear model procedure of statistical analytical system (SAS 1999) to evaluate the effects of treatments on growth performance, carcass traits, intestinal morphology, and microflora of broiler chickens. The model included the main effects of wheat cultivars (Alvand or Atrak), processing method (dry heating or microwave), and enzyme supplementation (with or without), and their interactions. An alpha level of 0.05 was used to determine significance.

3. Results

Results of the growth performance of broiler chickens are presented in Table 3. According to the results, the birds that received Alvand cultivar had greater weight gain (P < .05) than those fed diets with Atrak cultivar. On the other hand, the microwave processing method and enzyme supplementation improved weight gain and FCR in broiler chickens during 1–42 days of age (P < .05). However, the inclusion of 50 g/t enzyme negatively influenced feed intake in broiler chickens (P < .05). The effects of the dietary treatments on carcass characteristics and internal organ weights are shown in Table 4. Liver weight was greater in broiler chickens fed Atrak cultivar than those that received Alvand cultivar (P < .05). Microwave processing increased the weight of the thigh in broilers, while the liver weight was greater in birds fed with dry heating (P < .05). Inclusion of the enzyme improved the weights of breast, thigh, and liver in broiler chickens (P < .05).

Table 3. Effects of dietary treatments on the growth performance of broiler chickens (d 0 to 42).

WC	PM	Enzyme	Weight gain	Feed intake	FCR
			(g/bird)	(g/bird)	(g feed/g gain)
Alvand	Microwave	+	55.06	102.77	1.87
		−	50.42	102.89	2.04
	Heat	+	53.04	102.58	1.93
		−	48.39	100.98	2.09
Atrak	Microwave	+	53.06	99.94	1.88
		−	50.42	104.35	2.07
	Heat	+	50.65	97.54	1.93
		−	48.78	104.57	2.14
WC
Alvand			51.72	102.31	1.98
Atrak			50.73	101.59	2.00
PM
Microwave			52.24	102.48	1.96
Heat			50.21	101.41	2.02
Enzyme
+			52.95	100.71	1.90
−			49.50	103.19	2.08
			Probability
WC			*	NS	NS
PM			***	*	*
Enzyme			***	***	***
WC × PM			NS	NS	NS
WC × Enzyme			**	***	NS
PM × Enzyme			NS	NS	NS
WC × PM × Enzyme			NS	*	NS
SEM			0.67	0.72	0.03

Note: WC = wheat cultivar [Alvand or Atrak]; PM = processing method; Enzyme = Kemzyme [Toekomstlaan 42, 2200 Herentals, Belgium]; FCR = feed conversion ratio; NS = non-significant; and SEM = standard error of the mean.

*p < .05.

**p < .01.

***p < .001.

Table 4. Effects of treatments on carcass characteristics and internal organs of broiler chickens (g/100 g body weight of bird).^a

WC	PM	Enzyme	Breast	Thigh	Gizzard	Liver	AF	Pancreas	Crop
Alvand	Microwave	+	25.34	23.02	1.65	1.91	1.68	0.16	0.28
		−	22.78	20.88	1.71	1.82	1.72	0.17	0.30
	Heat	+	25.54	21.22	1.65	1.96	1.69	0.15	0.27
		−	22.32	20.73	1.72	2.00	2.20	0.17	0.30
Atrak	Microwave	+	26.35	24.38	1.71	2.01	1.92	0.17	0.30
		−	23.75	20.44	1.71	1.86	1.95	0.15	0.29
	Heat	+	24.74	22.65	1.73	2.14	1.85	0.17	0.30
		−	23.17	20.81	1.76	1.97	1.78	0.16	0.30
WC
Alvand			23.99	21.46	1.68	1.92	1.82	0.16	0.29
Atrak			24.50	22.07	1.72	1.99	1.87	0.16	0.30
PM
Microwave			24.55	22.18	1.69	1.90	1.82	0.16	0.29
Heat			23.94	21.35	1.71	2.01	1.88	0.16	0.29
Enzyme
+			25.49	22.82	1.68	2.00	1.79	0.16	0.29
−			23.05	20.71	1.72	1.91	1.91	0.16	0.30
					Probability
WC			NS	NS	NS	**	NS	NS	NS
PM			NS	*	NS	***	NS	NS	NS
Enzyme			***	***	NS	**	NS	NS	NS
WC × PM			NS	NS	NS	NS	NS	NS	NS
WC × Enzyme			NS	*	NS	*	NS	***	**
PM × Enzyme			NS	**	NS	NS	NS	NS	NS
WC × PM × Enzyme			NS	NS	NS	NS	NS	NS	NS
SEM			0.68	0.51	0.04	0.04	0.16	0.004	0.007

Note: WC: wheat cultivar [Alvand or Atrak]; PM: processing method; Enzyme: Kemzyme [Toekomstlaan 42, 2200 Herentals, Belgium]; NS: non-significant; AF: abdominal fat; and SEM: standard error of the mean.

*p < .05.

**p < .01.

***p < .001.

The results of microbiology and gut pH alteration in broiler chickens are shown in Table 5. The total bacteria count in the ileo-caecum was greater in broilers fed wheat-based diets with enzyme and dry heating process (P < .05). The results indicated that the population of Lactobacilli was increased in broiler chickens fed Alvand cultivar with microwave processing and enzyme supplementation (P < .05). On the other hand, the inclusion of enzyme decreased the population of E. coli in the ileo-caecum of broiler chickens (P < .05). On gut acidity, supplemental enzyme decreased the pH value of the proventriculus in broilers (P < .05). The pH values of the gizzard and jejunum were increased in birds fed Alvand cultivar diet (P < .05).

Table 5. Effects of treatments on viable cell counts of microflora in ileo-caecum and gut acidity (pH) of broiler chickens.

			Lactobacillus
WC	PM	Enzyme	Total	log10 cfu/g	E. coli	Crop	Proventriculus	Gizzard	Jejunum	Caecum
Alvand	Microwave	+	7.06	5.12	4.91	5.62	4.48	3.82	6.85	7.10
		−	7.00	4.90	5.78	6.13	4.57	3.95	6.43	6.74
	Heat	+	7.26	5.29	4.86	5.90	4.73	3.60	6.60	6.77
		−	7.16	4.26	5.58	5.90	4.78	3.93	6.82	6.97
Atrak	Microwave	+	7.16	5.84	4.94	5.82	4.50	3.52	6.63	6.77
		−	7.15	5.36	5.13	5.75	4.75	3.34	6.61	6.94
	Heat	+	7.19	5.60	5.01	5.75	4.45	3.48	6.44	6.97
		−	7.12	5.37	5.77	5.80	4.63	3.48	6.31	6.81
WC
Alvand			7.12	4.89	5.28	5.88	4.64	3.82	6.67	6.89
Atrak			7.15	5.54	5.21	5.78	4.58	3.45	6.50	6.87
PM
Microwave			7.09	5.30	5.19	5.83	4.57	3.66	6.63	6.89
Heat			7.18	5.13	5.31	5.84	4.65	3.62	6.54	6.88
Enzyme
+			7.17	5.46	4.93	5.77	4.54	3.61	6.63	6.90
−			7.11	4.97	5.56	5.89	4.68	3.67	6.54	6.87
					Probability
WC			NS	***	NS	NS	NS	**	*	NS
PM			**	*	NS	NS	NS	NS	NS	NS
Enzyme			*	***	***	NS	*	NS	NS	NS
WC × PM			**	NS	*	NS	*	NS	*	NS
WC × Enzyme			NS	NS	NS	NS	NS	NS	NS	NS
PM × Enzyme			NS	NS	NS	NS	NS	NS	NS	NS
WC × PM × Enzyme			NS	**	NS	NS	NS	NS	*	*
SEM			0.04	0.11	0.15	0.145	0.087	0.15	0.09	0.12

Note: WC = wheat cultivar [Alvand or Atrak]; PM = processing method; Enzyme = Kemzyme [Toekomstlaan 42, 2200 Herentals, Belgium]; NS = non-significant; and SEM = standard error of the mean.

*p < .05.

**p < .01.

***p < .001.

The effects of the experimental diets on jejunal and ileal morphology are shown in Table 6. The villus height, crypt depth, and epithelial thickness of jejunum were greater in birds that received Atrak cultivar than in those that received Alvand cultivar (P < .05). In the ileum, microwave processing increased the villus height and epithelial thickness of broiler chickens (P < .05). All intestinal morphology traits in the birds were improved by adding the enzyme supplementation in the diets (P < .05).

Table 6. Effects of treatments on jejunum and ileum morphology of broiler chickens.

WC	PM	Enzyme	Jejunum (µm)					Ileum (µm)
			VH	CD	VH/CD	ET	GC	VH	CD	VH/CD	ET	GC
Alvand	Microwave	+	875.7	159.7	5.48	34.32	7.62	821.2	113.7	7.23	33.02	7.45
		−	864.0	151.7	5.69	29.75	6.72	802.2	102.5	7.83	29.05	7.10
	Heat	+	866.1	157.7	5.49	33.15	6.75	803.2	107.7	7.46	32.75	6.36
		−	834.5	150.5	5.54	33.93	6.63	783.5	106.3	7.37	28.27	6.92
Atrak	Microwave	+	888.2	175.5	5.06	38.13	7.01	808.2	110.2	7.33	31.87	6.87
		−	865.7	150.0	5.78	36.18	6.57	798.5	107.2	7.45	31.77	6.40
	Heat	+	883.2	165.8	5.33	35.71	7.78	798.7	107.7	7.42	31.65	7.77
		−	868.7	155.6	5.58	34.26	6.38	789.7	106.0	7.45	27.25	6.15
WC
Alvand			860.1	154.9	5.55	32.78	6.93	802.5	107.5	7.47	30.77	6.95
Atrak			876.5	161.7	5.43	36.07	6.93	798.8	107.8	7.41	30.63	6.80
PM
Microwave			873.4	159.2	5.50	34.59	6.98	807.5	108.4	7.46	31.43	6.95
Heat			863.1	157.4	5.48	34.26	6.88	793.8	106.9	7.42	29.98	6.80
Enzyme
+			878.3	164.7	5.34	35.32	7.29	807.8	109.8	7.36	32.32	7.11
−			858.2	152.0	5.64	33.53	6.57	793.5	105.5	7.52	29.08	6.64
Probability
WC			*	***	NS	***	NS	NS	NS	NS	NS	NS
PM			NS	NS	NS	NS	NS	***	NS	NS	*	NS
Enzyme			**	***	***	**	***	***	**	*	***	*
WC × PM			NS	NS	NS	**	**	*	NS	NS	NS	*
WC × Enzyme			NS	**	**	NS	NS	*	NS	NS	NS	**
PM × Enzyme			NS	**	*	*	NS	NS	*	*	*	NS
WC × PM × Enzyme			NS	*	NS	*	**	NS	NS	NS	NS	***
SEM			9.7	1.83	0.08	0.78	0.18	3.1	1.8	0.11	0.76	0.26

Notes: WC = wheat cultivar [Alvand or Atrak]; PM = processing method; Enzyme = Kemzyme [Toekomstlaan 42, 2200 Herentals, Belgium]; NS = non-significant; and SEM = standard error of the mean. VH = Villus height; CD = Crypt depth; ET = Epithelial thickness; GC = Goblet cells.

*p < .05.

**p < .01.

***p < .001.

4. Discussion

The results of our experiment demonstrated that wheat cultivar type can influence the weight gain of broiler chickens. These results are supported by the findings of Scott et al. (1998) and del Alamo et al. (2008) who indicated that wheat cultivar type is an important factor that affects broiler performance. It is reported that the NSP content in wheat can be variable, depending on many factors such as the cultivar and growing conditions (del Alamo et al. 2008). Also, it is well recognized that soluble NSP-rich materials such as wheat result in poor broiler growth performance (Kiarie et al. 2014). In accordance with these results, our findings demonstrated that Atrak cultivar, which had more soluble NSP content than Alvand cultivar, negatively influenced growth performance.

In the present study, microwave processing and enzyme supplementation improved FCR, and increased the thigh and breast weights in broiler chickens. Very little research has been completed to investigate the effects of microwave treatment of wheat grain on broiler performance (Zeller et al. 2015). In contrast to our results, these researchers found that including microwave-treated wheat grain in broiler diets had no significant effect on growth performance. It is reported that the endogenous enzymes in wheat grain, such as xylanase, are heat labile (Amerah 2015), which may affect their importance in broiler nutrition. Besides, overheating can result in the resistance of protein and starch to digestion (Silversides and Bedford 1999) and increased intestinal viscosity in broiler chickens (Gracia et al. 2003). Therefore, in the present experiment, inactivation of endogenous enzymes in the heat treatment may be the reason for the reduction in growth performance in broilers compared to those that received microwave-treated diet. On the other hand, microwave processing may have caused changes in wheat characteristics such as starch gelatinization that may also have influenced broiler performance.

In accordance with our results, several studies well documented that xylanase supplementation improved the growth performance of broiler chickens fed wheat-based diets (Engberg et al. 2004; Ponte et al. 2004; Kiarie et al. 2014; Smeets et al. 2014). The beneficial role of NSP-degrading enzymes has been explained by many authors. In addition to the decrease in ileal viscosity, the mode of action of xylanase is explained by a reduced microbial activity in terms of short-chain fatty acids (SCFA) concentration (Józefiak et al. 2007). On the other hand, two main modes of actions for xylanase in wheat-based diets, including the viscosity theory and the so-called cage effect, were reported (Cowieson et al. 2006). The viscosity theory is associated with a reduction in viscosity with exogenous enzymes, which improved nutrient digestibility, while the cage effect theory is related to the effects of carbohydrases on cell walls, reducing their integrity and then releasing nutrients that were encapsulated (Bedford 2002). Therefore, it is concluded that NSP removal and viscosity reduction are two critical roles of xylanase when added to diets containing wheat grain.

In the present experiment, the count of Lactobacilli was increased in broilers that received Atrak cultivar. However, reports from a literature review show a lack of information about the effects of wheat cultivars on microbial population in broilers. In general, the rate of starch digestion was different between wheat cultivars (del Alamo et al. 2008) and may affect the composition of microflora in broiler chickens (Ball et al. 2013).

Lactobacilli population was increased in broilers fed diets based on microwave processing and enzyme supplementation. On the other hand, E. coli count was lower in birds that received enzyme supplementation than in those fed without enzyme. It is indicated that the microbial composition and activity in the broiler gastrointestinal tract were affected by the structure of the feed (Engberg et al. 2004). The NSP composition of feed ingredients has an important role in the microbial diversity of the gut (Kiarie et al. 2014). In particular, the presence of viscous polysaccharides has been found to increase intestinal microbial activity associated with poor broiler growth performance (Józefiak et al. 2007). It is likely that both mechanisms are involved in the responses to xylanase in broiler diets, leading to desirable changes in the microbial population in the distal gastrointestinal tract, a reduction in viscosity, and beneficial changes in nutrient retention and growth performance (Cowieson et al. 2006). It is found that the degradation products of sugars, such as xylose and xylo-oligomers, are fermented by caecal bacteria, thus stimulating the production of SCFA and the growth of specific beneficial bacteria (Bedford 2002).

The results of the present experiment showed that the enzyme supplementation decreased the pH value of the proventriculus in broiler chickens. In accordance with this result, it is found that enzyme supplementation to a rye-based diet resulted in a reduction of the pH of the crop content in broiler chickens (Józefiak et al. 2007). The addition of xylanase reduced the pH value of the contents of the duodenum and jejunum (Engberg et al. 2004). It is reported that carbohydrase enzymes, such as glucanase, indirectly stimulate the activity of the crop microorganisms in terms of greater lactic acid concentration and lower acidity value in broiler chickens (Józefiak et al. 2007). The low pH value of the proventriculus content from broilers that received xylanase supplementation may be due to an alteration in bacteria fermentation or an increased secretion of hydrochloric acid by the proventriculus.

In the present study, villus height, crypt depth, and epithelial thickness of jejunum were increased in broilers fed Atrak cultivar. This observation was unexpected because according to the results of Table 1 and authors Parsaie et al. (2006), Atrak cultivar had greater soluble NSP than Alvand cultivar. Information on the effect of wheat cultivars on intestinal morphology is scarce in broiler chickens. The effect of dietary fibre on intestinal morphology is variable, and depends on the physical and chemical characteristics of the dietary fibre (Montagne et al. 2003). The NSP content in cereal grains detrimentally increases digesta viscosity and negatively alters villus height, surface area, and shape in broiler chickens (Baurhoo et al. 2011). In this regard, it is understood that the high viscosity of the digesta may increase the rate of villus cell loss, leading to villus atrophy, associated with an increased crypt cell production, and generally increased crypt depth (Montagne et al. 2003).

In the present experiment, microwave processing of wheat increased the villus height and epithelial thickness of broiler chickens in the ileum segment. This effect may be due to the influence of microwave treatment on changes in the physico-chemical characteristics of carbohydrates, especially starch, in wheat grains. In this regards, the microwave energy significantly affected all the physical and biochemical properties of the wheat grain (Walde et al. 2002). Nevertheless, data on the effect of starch gelatinization on gut morphology are scarce. In the present experiment, differences in the impact of microwave processing and starch gelatinization on gut morphology indicate that the exact mechanism of this action has to be further investigated.

In this study, enzyme supplementation of the wheat-based diets resulted in improving the intestinal morphology parameters in jejunum and ileum. The inclusion of exogenous enzymes, including xylanase and glucanase, significantly altered the morphology of the digestive tract segments of broilers fed wheat-based diets (Wang et al. 2005). In a study with rye-based diets, it was found that xylanase supplementation improved gut morphology, including villus size and villus height ratio, in broiler chickens (Mathlouthi et al. 2002). In this regard, it is reported that enzyme supplementation increased intestinal morphology traits such as crypt depth and villus height in broilers (Viveros et al. 1994). In contrast to these results, Cowieson et al. (2006) indicated that enzyme addition had no overall effect on the gut morphology of growing broiler chicks. Poorer gut health in high NSP diets may arise from the alteration of the intestinal morphology in birds receiving a high NSP feed (Adeola & Cowieson 2011). Several studies have demonstrated that xylanase supplementation decreased the negative effects of high NSP diets in broiler chickens (Cowieson et al. 2005; Józefiak et al. 2007; Smeets et al. 2014; Zeller et al. 2015). Therefore, the positive effect of xylanase on intestinal morphology may be due to the reduction in viscosity (Smeets et al. 2014), which then reduces the deleterious effect of soluble NSP on the intestine mucosa (Montagne et al. 2003) in broiler chickens.

5. Conclusions

In summary, the results of the present experiment indicated that wheat cultivars did not affect the FCR of broilers. However, Lactobacilli count and intestinal morphology were altered by the wheat cultivars. Microwave processing of wheat grain and enzyme supplementation improved broiler growth performance and increased Lactobacilli population. Moreover, the villus height, crypt depth, and epithelial thickness of the jejunum and ileum were increased in broiler chickens fed with enzyme supplementation.

Disclosure statement

No potential conflict of interest was reported by the authors.