Use of wheat dilution to improve digestive function in broilers: application in low protein diets

ABSTRACT

1. This study evaluated the effect of wheat dilution increasing in particle size in low crude protein diets on growth performance, digestive tract, nitrogen efficiency and litter quality in broiler chickens.

2. Ross 308 male broiler chicks (n = 336) were allocated to one of four dietary treatments (each with 7 pens, 12 chicks per pen); Control (CON; commercial pellet diet with standard crude protein, CP: 22.50%), W578 (CON +20% wheat of geometric mean diameter (GMD) of 578 µm; CP: 20.25%), W1326 (CON +20% wheat of GMD 1326 µm; CP: 20.25%) and WW (CON +20% whole wheat, CP: 20.25%), from d 0 to 21 of age.

3. Body weight gain was increased (P < 0.05) for birds fed CON compared to the low crude protein diets. However, WW increased (P < 0.05) body weight gain compared to W578 and W1326, while feed intake and feed conversion ratio on CON and WW were similar (P > 0.05). Birds fed W1326 showed the poorest (P < 0.05) FCR compared to CON, W578 and WW.

4. Gizzard relative weight (g/kg body weight) was increased (P < 0.05) on WW compared to CON on d 14 and 21, whereas gastric isthmus diameter was significantly reduced on W1326 and WW compared with CON and W578.

5. There were no differences (P > 0.05) in the depth of gizzard mucosa of lamina propria between CON and WW at d 14 and 21. Birds fed WW increased (P < 0.05) gizzard tensile strength compared to W578 and W1326, whereas no difference was observed between WW and CON on d 14.

6. No significant differences were seen for ileum villus height and mucosal layer between CON and WW on d 21, however, feeding CON increased the extent of the mucosal layer compared to W578 and W1326.

7. Nitrogen excretion (g/kg BWG) was significantly lower (P > 0.05) on W1326 and WW compared with CON and W578. Litter nitrogen, moisture, and footpad scores significantly decreased (P < 0.05) for birds fed WW compared with CON.

8. Diluting dietary protein content from 22.50 to 20.25% resulted in lower body weight gain in broilers. However, dilution with whole wheat resulted in comparable FCR, reduced nitrogen excretion, litter moisture and footpad dermatitis compared with a standard protein diet.

KEYWORDS:

• Whole wheat
• particle size
• low protein
• food security
• sustainability
• digestive tract

Introduction

Protein ingredients used for poultry diets are expensive (Jahan et al. 2006). In addition to these economic considerations, plant protein sources are being used by both animals and humans, thus creating competition between them (Liu et al. 2015). Reducing the protein content of poultry diets can substantially improve environmental sustainability by lowering the excretion of nitrogenous compounds, thereby reducing the environmental footprint of poultry meat production (Greenhalgh et al. 2020). Providing lower crude protein diets to poultry could be employed to achieve these improvements. However, studies have shown impaired growth performance associated with diets where protein content is decreased without the addition of single, crystalline amino acids (Incharoen et al. 2010; Laudadio et al. 2012; Lemme et al. 2019). Therefore, supplementing lower protein diets with a single amino acid to ensure no loss in growth performance is becoming an established approach for lowering protein levels in poultry diets (Bennett and Classen 2003; Chrystal et al. 2020; Ullrich et al. 2018; van Harn et al. 2019). However, little consideration has been given to bird-focussed approaches to lowering protein levels without additional amino acid supplementation or losses in broiler performance. Previous work has shown that dilution of a high protein pelleted feed down to standard levels with whole wheat did not affect the time to reach market bird weight but did negatively affect feed conversion efficiency (Bennett et al. 1995). Contrary to this, if protein content is lowered from the recommended levels for poultry, then reduced growth performance of the broilers has been observed (Lemme et al. 2019).

Increasing particle size for cereal grains in poultry diets affects feed costs due to reduced grinding energy costs associated with feed production (Reece et al. 1986). It has been observed that the energy required for grinding corn can be reduced by 35% when the screen size of the hammer mill is increased from 4.76 to 7.94 mm (Reece et al. 1986). Alongside this benefits, is has been reported that feed with increased particle size is linked with increased gizzard weights (Engberg et al. 2004; Gabriel et al. 2003; Nir et al. 1994; Plavnik et al. 2002) and improved FCR (Plavnik et al. 2002; Preston et al. 2000). This improvement is thought to be directly related to the increased development of the gizzard muscular wall (Preston et al. 2000). However, other researchers have suggested that increased particle size may increase the energy required by the birds to grind feed and can lead to more indigestible coarse particles entering into the small intestine (Lentle et al. 2006). Recently, in extensive research, particle size has been reported to improve the performance of poultry by improving gizzard development which plays a vital role in the digestion and utilisation of nutrients (Amerah et al. 2008; Amerah et al. 2009; Lv et al. 2015; Zaefarian et al. 2016).

Lowering dietary protein is important for reducing the environmental impact of poultry production via less reliance on soya and reducing nitrogenous compounds in manure. Current methods to lower protein without negatively impacting broiler chickens involve additions of synthetic amino acids to the diet, such as increased crystalline amino acid content or the use of digestive enzymes. However, little work to date has focused on enhancing the natural digestive efficiency of broiler chickens as an adaptation to low-protein diets.

The present study was conducted to determine whether the inclusion of medium ground (GMD 578 µm), coarse (GMD 1326 µm) or whole-wheat in low-protein diets could meet or outperform a broiler diet formulated with recommended protein levels. The main objective of the current study was to compare the effect of a standard diet and three modified low-protein diets but iso-caloric on growth performance, digestive tract development, and litter characteristics of broiler chickens from d 1 to 21.

Materials and methods

Ethical statement

The experiment was approved by the Ethical Review Committee of the School of Animal, Rural and Environmental Sciences, Nottingham Trent University (NTU) (Approval Code: ARE 894) and carried out at the Poultry Research Unit of Nottingham Trent University.

Bird husbandry

A total of 336 male broiler chicks (Ross 308) were obtained from PD Hook Hatchery (Cote, UK) and allocated to 28 pens with 7 pens per treatment containing 12 chicks each. Dietary treatments were randomly assigned to experimental pens evenly distributed around the room. The experimental pens (each with a floor area of 0.64 m²) were bedded with softwood shavings approximately 3 cm in depth. All the chicks had ad libitum access to feed and water throughout the 21 d experimental period. Broiler chickens were fed starter crumbs for the first week (d 0–7) and then grower pellets for the following two weeks (d 7–21). On chick placement, a 1 h dark period was provided which was increased each day by 1 h up to 6 h on d 6, which was then maintained for the duration of the trial. The temperature was set at 32°C at placement which was reduced by approximately 0.5°C every day up to 21.5°C on d 21. Temperature and minimum ventilation requirements were maintained and recorded twice daily throughout the experimental period according to Ross 308 guidelines (Aviagen 2018).

Dietary treatments

Commercially formulated and manufactured starter crumbs (d 0–7) and grower pellets about 8-10 mm in length and 3.5 mm diameter (d 8–21) were obtained from a local feed mill (GLW Feeds, Loughborough, UK) as basal diets. Three additional diets were prepared by replacing 20% of the basal diets with wheat of increasing particle size. Post-pellet inclusion of wheat was chosen to eliminate any confounding effect of pelleting (heat or additional grinding during pelleting) on wheat particle size. Locally produced whole wheat was obtained from a local feed mill. The medium ground wheat was obtained by passing whole wheat through a 3.75 mm screen of a knife mill (Retsch, Germany) which produced a particle size of 578 µm geometric mean diameter (GMD). Coarse wheat was obtained by passing whole wheat through a roller mill with a roller gap of 3.75 mm which produced a particle size of 1326 µm GMD. The inclusion of wheat resulted in a total of four dietary treatments: CON (control – commercial diet, crude protein 22.50%), W578 (CON +20% wheat of GMD 578 µm, crude protein 20.25%), W1326 (CON +20% wheat of GMD 1326 µm, crude protein 20.25%) and WW (CON +20% whole wheat, crude protein 20.25%). The composition of basal diets is given in Table 1.

Table 1. Ingredients (%) and calculated nutritional composition of basal starter and grower with analysed nutrient values given in brackets where available.

Ingredient	Basal stater (days 0–7)	Basal grower (days 7–21)
Wheat	42.94	46.50
Soybean meal	23.30	34.50
Rapeseed (whole)	–	6.60
Maize	15.00	–
Maize DDGS	–	4.76
Wheat feed	5.00	–
Barley	5.00	–
Sunflower meal	4.00	–
Soybean oil	1.20	3.45
Calcium carbonate	0.97	0.85
Monocalcium phosphate	0.97	–
Dicalcium phosphate	–	1.85
Salt	0.27	0.30
Lysine-HCl	0.37	0.39
Methionine-DL	0.26	0.30
Premix^1	0.50	0.50
Sodium bicarbonate	0.10	–
Elancoban G200	0.05	–
L-Threonine	0.03	–
Roxazyme G2G liquid 35.7%	0.03	–
Hiphos (L) 10000 (Liquid)	0.01	–
Calculated Nutrients
DM (%)	88 (87.32)	88 (88)
CP (%)	23 (22.50)	22 (22.50)
ME	12MJ/kgDM	12.5MJ/kgDM
Crude fat (%)	3.60	(5.02)
Fibre (%)	4.63	(2.78)
Ash (%)	5 (4.49)	5 (4.74)
Lysine digestible (%)	1.32	1.32
Methionine digestible (%)	0.49	0.65
Methionine + Cystine	0.95	0.87
Threonine digestible (%)	0.86	0.77
Isoleucine digestible (%)	0.86	0.78
Valine digestible (%)	0.96	0.86
Arginine digestible (%)	1.37	1.23
Tryptophan digestible (%)	0.20	0.18
Leucine digestible (%)	1.41	1.27
Sodium (%)	0.14	(0.13)
Total phosphorus (%)	0.62	(0.44)
Calcium (%)	0.81	(0.64)

¹Premix per kg of the diet: Vitamins; A: 8000IU; D3: 5000IU; E:0.075 g; Trace elements; Caalcium iodate: 3.1 mg (iodine:2.0 mg); Copper sulphate: 80 mg (copper 3b405:27 mg); Iron sulphate: 83.5 mg (Iron 3b103).

Zootechnical additives; Axtra PHY 10 000 L: 0.01%; Ronozyme cereal WX-MG MP E1607 in cat N of directive 70/524/EEC: 0.029%; Maxiban G160 (80 mg/kg Narasin and 80 mg/kg Nicarbazin to provide 50 mg/kg Narasin and 50 mg/kg Nicarbazin in final feed as an aid in the prevention of coccidiosis in broiler chickens).

Growth performance, nitrogen utilisation, footpad dermatitis and litter quality

The weekly growth performance of each pen was calculated by measuring the feed consumption, weight gain and calculating feed conversion ratio (FCR) each week. Any mortality and weight of dead chickens were recorded daily. Nitrogen efficiency and excretion were calculated following a method described by Belloir et al. (2017). Footpad dermatitis (FPD) scoring was performed following the guidance of Aviagen Ltd. on footpad dermatitis (de Jong and van Harn 2012). Within each pen, a litter sample (approximately 50 g) was taken from five points; corners and centre and mixed well to create a homogenous sample for moisture and nitrogen analysis.

Bird sampling and digestive organs measurements

Two birds, closest to the average bird weight per pen, were removed and killed by cervical dislocation on d 14 and 21 of age. Immediately post-mortem, the digestive organs from each bird were excised and digesta contents were removed from the proventriculus, gizzard and intestinal sections to record their empty weights. Gastric isthmus diameter and lengths of intestinal sections were recorded as well. Gizzard tensile strength, digesta weight and particle size were recorded.

Particle size analysis

Particle size analysis of wheat, final feeds, residual feeds and freeze-dry gizzard digesta contents was performed by following a method developed by Baker and Herrman (2002). Briefly, particle size distribution was performed using the sieving method and a vibratory shaker (Retsch®, Germany). The vibratory shaker was set with an amplitude of 1.5 mm/g for 3 min duration to sieve wheat, diets, and freeze-dry gizzard digesta. Sieving was performed using a six-sieve set stacked from top to bottom in order; 4.0 mm, 2.0 mm, 0.5 mm, 0.25 mm, 0.125 mm and 0.063 mm.

Geometric mean diameter (GMD) was calculated using the following formulae.

Average diameter of the two sieves $\left({\mathrm{d}}_{\mathrm{i}}\right)={\left({\mathrm{d}}_{\mathrm{u}}\times {\mathrm{d}}_{\mathrm{o}}\right)}^{1/2}$

$\begin{array}{rl}& \mathrm{Geometric}\mathrm{Mean}\mathrm{Diameter}\left(\mathrm{GMD}\right)\\ & =\mathrm{lo}{\mathrm{g}}^{-1}\left[\sum \mathit{\hspace{0.17em}}\left({\mathrm{W}}_{\mathrm{i}}\log {\mathrm{d}}_{\mathrm{i}}\right)÷\sum {\mathrm{W}}_{\mathrm{i}}\right]\end{array}$

Where d_i was the diameter of the i^th sieve in the stack, d_u the diameter opening through which particles will pass (sieve proceeding i^th), d_o the diameter opening through which particles will not pass (i^th sieve) and W_i the weight of the sample retained on each sieve.

Histology of gizzard and ileum

Cross sections of the gizzard wall were excised from the right cranioventral thick muscle of the gizzard wall adjacent to the pylorus junction to maintain consistency. Ileum cross sections were excised from the distal 5 cm of the ileum preceding the ileo-caecal junction. All sections were washed with distilled water and fixed in Bouin’s fixative for 6 h then transferred to 70% ethanol for processing. All fixed sections were embedded with paraffin wax on a tissue processor (HistoCore, Leica Microsystems, Milton Keynes, UK) and embedding station (HistoCore, Leica Microsystems, Milton Keynes, UK). Samples were dehydrated through graded alcohols (70%, 90% and 100%) at ambient temperature, cleared in a xylene bath and carefully infiltrated with paraffin wax at 56°C. Embedded samples were processed using a rotary microtome processer (Leica 1215, Leica Microsystems, Milton Keynes, UK) with disposable blades cutting 8 µm thick slices. Slices were stained with haematoxylin and eosin and examined under a light microscope. Histological measurements of the gizzard and ileum were taken using an arbitrary line tool of the Olympus Soft Image Solution program (Tokyo, Japan) with calibration performed with an Olympus microscope (BX52). Measurements were taken from the two thickest and two thinnest layers from cross sections of the gizzard wall and the average was used for data analysis, whereas the villus height, villus width, crypt depth and mucosal depth were measured from six healthy and unbroken villi from each cross section on the slides and the average is used for data analysis.

Tensile strength

The tensile strength of the gizzard was measured using TA.XT plus Texture Analyzer 100 (Stable Micro Systems, Surrey, UK). The tearing or shearing force in Newtons (N) was measured using a 100 kg load cell at a speed of 25 mm per minute, following the method developed by Abdollahi et al. (2019).

Chemical analysis

Chemical analysis of diets and litter was performed by the standard procedures of the Association of Official Analytical Chemists (AOAC 1990). Dry matter (method 930.15), and ash content (method 942.05) of the feed were determined by standard methods. Nitrogen content was determined by the combustion method (Dumas) using a nitrogen analyser (Gerhardt, Germany) following a standard method (method 968.06). Crude protein was calculated by multiplying nitrogen with a conversion factor of 6.25.

Statistical analysis

Analysis of variance was used to analyse data according to the following model.

${\mathrm{Y}}_{\mathrm{ij}}=\mathrm{M}+\mathrm{TR}{\mathrm{T}}_{\mathrm{i}}+{\mathrm{B}}_{\mathrm{j}}+{\mathrm{E}}_{\mathrm{ij}}$

Where Y_ij was the response variable, M the overall mean, TRT_i the effect of dietary treatment and B_j the effect of block and E_ij is the residual. Data were analysed using the analytical software JMP Statistical Discovery 2020 by SAS Institute, NC, U.S.A. Means were compared by protected LSD test at P ≤ 0.05.

Results

The particle size distribution of wheat included in the diets is given in Figure 1. All the whole wheat particle size distribution (100%) was between 2.0 and 4.0 mm, whereas medium and coarse wheat fell in the distribution range of 2.0 mm and 0.063 mm. None of the wheat particle size distributions exceeded 4.0 mm.

Figure 1. Particle size distribution of wheat included in the diets.

Growth performance

The results (Table 2) showed that dietary treatments significantly affected (P < 0.001) the final body weight and body weight gain of broilers. All the low protein diets diluted either with medium (W578), coarse (W1326) or whole wheat (WW) showed decreased final body weight by 13.34 to 45.86% and body weight gain by 13.93 to 47.76%, compared to CON on d 21. Furthermore, final body weight and body weight gain differed (P < 0.001) between the diluted protein diets. However, W578 showed significantly lower (26.78%, P = 0.024) feed intake compared to CON, and only W1326 showed significantly poorer (68.34%, P = 0.008) FCR than birds in the CON group.

Table 2. Effect of standard protein and wheat diluted diets on growth performance of broilers (day 0–21).

Variable	Treatment^1				SEM^2	P-value
	CON	W578	W1326	WW
Initial body weight (g)	40.04	39.82	40.00	40.26	0.197	0.477
Final body weight (g)	1004^a	681.6^c	543.6^d	870.1^b	25.56	<0.001
Body weight gain (g)	964.1^a	641.7^c	503.6^d	829.8^b	25.58	<0.001
Feed intake (g)	1223^a	895.5^b	1039^ab	1146^a	70.84	0.024
Feed conversion ratio (g/g)	1.273^b	1.408^b	2.143^a	1.387^b	0.172	0.008

¹CON: Commercial pellet diet with standard crude protein, CP: 22.50%; W578: CON + 20% wheat of GMD 578 µm; CP: 20.25%; W1326: CON + 20% wheat of GMD 1326 µm; CP: 20.25%; WW: CON + 20% whole wheat, CP: 20.25%.

²SEM: Standard error of means.

^abc:Means within a row with different superscripts differ significantly (P ≤ 0.05).

Digestive organ relative weights and measurements

The results in Table 3 show there was no difference in proventriculus relative weight between treatments on d 14, but, by d 21, broilers fed W578 or W1326 had significantly heavier proventriculi than either CON or WW fed birds. In contrast, on d 14 and day 21, broilers fed any of the three wheat-diluted diets had a significantly heavier gizzard relative to body weight (19.87 to 31.10% and 12.66 to 27.26%, respectively) than the CON-fed broilers.

Table 3. Effect of standard protein and wheat diluted diets on digestive organs relative weights to body weight (BW) and measurements.

Variable	Treatment^1				SEM^2	P-value
	CON	W578	W1326	WW
Day 14
Proventriculus (g/kg BW)	6.049	7.211	6.878	6.590	0.320	0.104
Gizzard (g/kg BW)	24.31^b	31.87^a	31.52^a	29.14^a	0.988	<0.001
Small intestine (g/kg BW)	43.26^b	53.25^a	50.22^ab	50.84^a	2.373	0.045
Gastric isthmus diameter (mm)	7.020^a	6.588^a	5.362^b	5.775^b	0.264	0.001
Duodenum length (cm)	24.14^a	20.81^b	20.50^b	22.51^ab	0.867	0.029
Jejunum length (cm)	59.14^a	54.14^b	51.37^b	53.86^b	1.563	0.018
Ileum length (cm)	52.47	49.36	46.60	50.01	2.349	0.392
Day 21
Proventriculus (g/kg BW)	4.580^b	5.670^a	5.831^a	5.018^b	0.182	<0.001
Gizzard (g/kg BW)	19.04^b	24.23^a	23.53^a	21.45^a	1.095	0.015
Small intestine (g/kg BW)	27.78^b	30.18^ab	33.83^a	29.24^b	1.436	0.047
Gastric isthmus diameter (mm)	7.515^a	7.257^a	6.469^b	6.300^b	0.148	<0.001
Duodenum length (cm)	27.63^a	24.97^ab	23.79^b	27.39^a	0.991	0.035
Jejunum length (cm)	63.16^a	59.43^ab	55.97^b	62.53^a	1.452	0.010
Ileum length (cm)	62.00	60.21	54.51	60.50	1.971	0.070

¹CON: Commercial pellet diet with standard crude protein, CP: 22.50%; W578: CON + 20% wheat of GMD 578 µm; CP: 20.25%; W1326: CON + 20% wheat of GMD 1326 µm; CP: 20.25%; WW: CON + 20% whole wheat, CP: 20.25%.

²SEM: Standard error of means.

^abc:Means within a row with different superscripts differ significantly (P ≤ 0.05).

BW: Body weight.

Small intestine relative weight was significantly increased by 23.09% on W578 and 17.52% on WW compared with CON on d 14, whereas on d 21, it was significantly increased by 8.64% and 21.78% on W578 and W1326, respectively, compared with birds fed CON. However, no significant difference was observed between birds fed CON or WW. Gastric isthmus diameter significantly reduced on CON and W578 compared with W1326 and WW on d 14 and 21. The W578 and W1326 significantly decreased duodenum length by 13.79% and 15.08%, respectively, compared to birds fed CON on d 14 whereas feeding W1326 decreased this by 13.90% on d 21. However, no difference in duodenum length was observed between CON and WW. All protein-diluted diets decreased jejunum length by 8.45 to 13.14% compared with CON on d 14. However, no difference in jejunum length was observed between CON and WW on d 21. The treatments did not affect ileum length significantly on d 14 and 21.

Gizzard histology, gizzard digesta relative weight and particle size

The results, given in Table 4, showed no difference (P > 0.05) in the mucosa of lamina propria depth on d 14 and 21 between birds fed WW or CON. Similarly, no significant difference (P > 0.05) was observed in the extent of tunica muscularis between birds fed CON or WW on d 21. Microscopic view of gizzard layers is provided in Figure 2.

Table 4. Effect of standard protein and wheat diluted diets on gizzard histological measures (µm) and gizzard digesta relative weight (g/kg BW) and particle size (GMD in µm).

Variable	Treatment^1				SEM^2	P-value
	CON	W578	W1326	WW
Day 14
Koilin	373.1	383.0	379.3	373.8	38.23	0.997
Mucosa of Lamina Propria	538.4^a	444.7^b	404.8^b	473.9^ab	30.75	0.042
Submucosa	181.8	156.8	141.4	162.1	23.06	0.671
The extent of Tunica Muscularis	1093	984.5	925.5	1010	75.06	0.479
Gizzard digesta weight	15.92	16.19	17.45	16.51	0.910	0.662
Gizzard digesta GMD	1572^a	1483^a	1309^b	1442^ab	54.16	0.023
Gizzard tensile strength	0.968^a	0.795^b	0.788^b	1.032^a	0.052	0.007
Day 21
Koilin	401.6	400.6	349.9	418.0	33.07	0.512
Mucosa of Lamina Propria	528.8^a	468.3^ab	356.6^b	480.2^a	39.32	0.039
Submucosa	177.6	131.2	113.5	192.3	24.00	0.099
The extent of Tunica Muscularis	1108^a	1000^ab	820.0^b	1090^a	62.70	0.017
Gizzard digesta weight	9.978^b	13.66^a	15.07^a	12.32^a	0.949	0.009
Gizzard digesta GMD	1640^a	1527^ab	1399^b	1674^a	51.43	0.006
Gizzard tensile strength	1.089	1.130	1.207	1.030	0.080	0.476

¹CON: Commercial pellet diet with standard crude protein, CP: 22.50%; W578: CON + 20% wheat of GMD 578 µm; CP: 20.25%; W1326: CON + 20% wheat of GMD 1326 µm; CP: 20.25%; WW: CON + 20% whole wheat, CP: 20.25%.

²SEM: Standard error of means.

^abc:Means within a row with different superscripts differ significantly (P ≤ 0.05).

Extent of Tunica Muscularis: Koilin + Mucosa of Lamina Propria + Submucosa.

BW: Body weight; GMD: Geometric mean diameter.

Figure 2. Microscopic view of gizzard layers (example image from study samples): Koilin (a), Mucosa of Lamina Propria (b), Submucosa (c), Tunica Muscularis (d).

The treatment effect on gizzard digesta particle was almost aligned with the effect on the mucosa of lamina propria and the extent of tunica muscularis on both age points; d 14 and 21. Feeding W1326 reduced gizzard digesta particle size by 16.73% and 14.70% on d 14 and 21, respectively, compared with CON. Feeding the protein-diluted diets (W578, W1326 and WW) increased (P < 0.05) gizzard digesta weight on day 21 by 23.47 to 51.03%, compared with birds fed CON.

Ileum histology

The results (Table 5) showed that the treatments significantly affected certain ileum histological measures. On d 14, birds fed W1326 had significantly increased crypt (29.72%) and mucosal depth (35.62%) compared with the W578 group, whereas no difference (P > 0.05) in crypt depth and mucosal depth was observed between feeding CON or WW on d 14. With age, the treatment effect was limited to villus height and the extent of the mucosal layer on d 21. Feeding W578 and W1326 significantly decreased villus height by 18.60% and 14.46% respectively, compared with CON on d 21. However, no significant difference was observed in villus height between the birds fed WW or CON.

Table 5. Effect of standard protein and wheat diluted diets crude protein diets on ileum histological measures.

Variable	Treatment^1				SEM^2	P-value
	CON	W578	W1326	WW
Day 14
Villus height (µm)	545.8	559.1	546.4	592.5	44.34	0.864
Crypt depth (µm)	151.5^ab	132.9^b	172.4^a	137.3^b	9.639	0.041
Mucosal depth (µm)	186.3^a	142.6^b	193.4^a	166.6^ab	10.54	0.014
Villus: crypt	3.693	4.272	3.258	4.579	0.450	0.199
The extent of the mucosal layer (µm)	697.2	692.0	718.8	729.7	45.00	0.924
Day 21
Villus height (µm)	697.2^a	567.5^c	596.4^bc	665.1^ab	32.72	0.042
Crypt depth (µm)	123.0	131.1	121.7	121.6	9.429	0.871
Mucosal depth (µm)	287.4	252.7	227.6	247.4	23.26	0.358
Villus: crypt	5.799	4.566	5.072	5.633	0.574	0.434
The extent of the mucosal layer (µm)	820.2^a	698.7^b	718.0^b	786.8^ab	29.74	0.031

¹CON: Commercial pellet diet with standard crude protein, CP: 22.50%; W578: CON + 20% wheat of GMD 578 µm; CP: 20.25%; W1326: CON + 20% wheat of GMD 1326 µm; CP: 20.25%; WW: CON + 20% whole wheat, CP: 20.25%.

²SEM: Standard error of means.

^abc:Means within a row with different superscripts differ significantly (P ≤ 0.05).

The extent of mucosal layer: Villus height + crypt depth; D: Day.

Feeding W578 and W1326 significantly decreased the extent of the mucosal layer by 14.81% and 12.46% respectively, compared with CON on d 21. However, no difference was seen in the extent of the mucosal layer between birds fed WW or CON on d 21.

Nitrogen utilisation, litter analysis and FPD scores

The results, (Table 6), showed that nitrogen efficiency was similar (P > 0.05) between birds fed CON, W578 or WW. However, feeding W578 resulted in the poorest nitrogen efficiency, which was decreased by 26.16% compared with birds fed CON. Feeding WW significantly decreased litter nitrogen by 11.22%, litter moisture by 42.18% and FPD scores by 99.72%, compared with birds fed CON.

Table 6. Effect of standard protein and wheat diluted diets on nitrogen utilisation (day 0–21), litter characteristics (day 20), and FPD scores (day 21).

Variable	Treatment^1				SEM^2	P-value
	CON	W578	W1326	WW
Nitrogen utilisation
N Efficiency (%)	63.91^a	63.97^a	47.19^b	65.49^a	3.992	0.013
N-excretion (g/kg BWG)	45.83^a	45.62^a	69.43^b	44.93^b	5.594	0.015
Litter N (%)	2.931^a	2.665^ab	2.739^ab	2.602^b	0.092	0.021
Litter moisture (%)	15.40^a	10.18^ab	8.144^b	8.905^b	2.039	0.022
FPD scores^3	0.357^a	0.286^ab	0.143^ab	0.001^b	0.119	0.048

¹CON: Commercial pellet diet with standard crude protein, CP: 22.50%; W578: CON + 20% wheat of GMD 578 µm; CP: 20.25%; W1326: CON + 20% wheat of GMD 1326 µm; CP: 20.25%; WW: CON + 20% whole wheat, CP: 20.25%.

²SEM: Standard error of means.

^ab:Means within a row with different superscripts differ significantly (P ≤ 0.05).

³FPD scores: Sum of all birds’ scores within a pen.

Discussion

The study hypothesised that the growth performance of broilers fed low-protein diets may be mitigated by the inclusion of structural components e.g., coarse or whole grains, to develop the digestive tract of broilers efficiently from the start which could influence nutrient utilisation efficiently. Lower growth performance was expected, as most of the studies on low protein reported reduced body weight. The current findings were in agreement with those of Incharoen et al. (2010) who observed decreased weight gain on low-protein diets when no extra amino acids were supplemented. Laudadio et al. (2012) lowered the crude protein content in diets for Hubbard female broilers aged d 14 to 49 and found that body weight was significantly reduced on low protein diets but feed intake or FCR was not affected. Comparable FCR to a control (without whole wheat) was reported by Truong et al. (2017) with 18% post-pellet inclusion of whole wheat to broiler diets. However, Leeson et al. (1991) reported that broilers used 40% more feed, when diets were diluted with rice hulls, in order to meet their nutritional requirements (Zubair and Leeson 1994). When Zubair and Leeson (1994) diluted diets with 50% oat hulls, they found significantly reduced body weight of broilers. These researchers reported increased feed intake which they speculated was an attempt to maintain nutrient intake by the broilers. The current study did not show increased feed intake contrary to those previous findings. This may have been due to differences in feed ingredients, as, in the current study, wheat was used to dilute diets which had better energy and protein value than rice hulls or oat hulls. Bennett and Classen (2003) suggested that the dilution of poultry diets with whole grains only exhibits a beneficial effect on performance when undiluted diets are formulated to have nutrient levels in excess, so that the diluted diets should not have any deficiency of nutrients.

The reduced body weight or body weight gain in birds fed W1326 led to poor FCR compared with other treatments, which may have been due to the inability of broilers to digest coarse grains efficiently compared to fine ground or whole grain. The current study did not use a standard diet with excessive or increased levels of nutrients. However, the inclusion of whole wheat performed better than the medium or coarse wheat in the reduced crude protein diets and even out-competed the standard-protein formulated diet in terms of feed conversion ratio. In agreement with the current findings, Mahdavi Sadati et al. (2022) reported improved FCR with the inclusion of whole wheat compared with ground or pelleted whole wheat, although the dietary crude protein was not reduced or diluted. The improvement in FCR with whole wheat inclusion in protein-diluted diets may mitigate the difference in body weight gain at a later age. However, impaired growth performance has been reported when diets were diluted with ground or whole grains at a higher inclusion rate at a later age in broilers (Bennett and Classen 2003). The reason for lower body weight on a low protein diet may be because it was reduced too much (10%, 20.25 vs. 22.50%) at an early age, in agreement with previous studies (Incharoen et al. 2010; Laudadio et al. 2012; Lemme et al. 2019).

In the current study, gizzard relative weight from birds fed WW was increased by 19.87% on d 14 and 12.66% on d 21 compared to those fed CON. The tensile strength of the gizzard on d 14, the depth of mucosa of lamina propria of the gizzard on d 14 and 21, and the extent of tunica muscularis on d 21 were similar to standard protein and whole wheat reduced protein diets. When broilers are fed diets containing whole wheat, a more developed gizzard has been observed, which has increased the frequency of contractions to reduce the size of whole grains to fine particles during grinding (Singh et al. 2014). Similarly, a significant increase in gizzard weight was observed in the broiler chickens fed diets with WW at inclusion levels of 200 g/kg either pre- or post-pelleting, compared with broilers fed ground wheat diets previously (Singh et al. 2019). In a recent study on the partial grain inclusion, gizzard weight was increased by 12.5% when whole wheat was included in the post-pellet diets at 12.5% (Moss et al. 2017). Mahdavi Sadati et al. (2022) observed significantly improved relative weight, length and width of gizzard and proventriculus and gizzard muscular thickness with the inclusion of whole wheat. Relative gizzard weight was increased by 26.1% when WW was included post-pelleting, however, only a 13.0% increase in the relative weight of gizzard was observed in the same study when WW was included in the pre-pellet diets (Truong et al. 2017). This indicated that WW inclusion or pelleting diets has a significant impact on the relative gizzard weights due to changes in the particle size of feed. Improved feed efficiency has been associated with increased gizzard weights in broilers fed whole wheat diets compared with those fed on conventional pellet diets (Plavnik et al. 2002; Preston et al. 2000). The improvement in FCR and reduced nitrogen excretion on the WW can be attributed to a well-developed and stronger gizzard compared to standard and medium or coarse wheat-diluted diets.

The gizzard mucosa of lamina propria, a layer made up of glands, secretes koilin which plays a role in grinding (Akester 1986; Svihus 2011). The koilin layer, known as the keratinoid layer, cuticle or gizzard lining, is a solid layer of a carbohydrate and protein complex that covers the mucosa of the gizzard. Proteins make up around 80% of koilin, with high quantities of glutamic and aspartic acids, as well as leucine and arginine (Pivnenko et al. 1998). It was expected that on a whole wheat diet, koilin weight may be increased, however, it might have worn due to grinding of whole grains (Akester 1986). On d 14 and 21, the increased depth mucosa of lamina propria may have been due to the high protein content (10% higher) in the diet as the gizzard layers are made up of lipo-proteins which may play a role in building these layers (Akester 1986). The current results showed that large particle sizes developed the gizzard mucosal layer efficiently, especially when whole wheat was included in the diet rather than coarse ground wheat.

The gastric isthmus diameter was reduced in birds fed WW and W1326 compared to CON and W576. An increased isthmus diameter results in dilatation of the proventriculus and both the stomach parts (gizzard and proventriculus) act as transit organs, instead of performing grinding and mixing functions. The dilation of the proventriculus, a condition where it cannot be well distinguished from the gizzard, causes condemnations at processing as this results in carcass contamination (Amer 2021). These findings were in agreement with those of Taylor and Jones (2004) who showed that feeding coarsely ground diets to poultry can reduce proventricular dilatation and give better mixing and grinding of the feed material in the upper digestive tract.

The comparable FCR and reduced nitrogen excretion for birds fed WW compared to CON can be attributed to intestinal integrity, which is defined as the intactness of the intestine to maintain its structure and function (Dharne 2008). Increased length or weight of the small intestinal sections may play a role in providing increased surface area for the absorption of nutrients (Ravindran et al. 2006). No difference was observed for mucosal depth on d 14, and villus height and extent of mucosal layer on d 21, between those fed the WW diluted diet and CON. Increased surface area and intestinal mass supports nutrient supply function (Ravindran et al. 2006) and play a role in overall gut integrity by increasing the absorption of nutrients (Kheravii et al. 2018).

The higher gizzard digesta relative weight on d 21 of the whole wheat low protein diet compared to the standard protein diet may be a contributing factor for improved nutrient utilisation, observed as reduced nitrogen excretion for birds fed WW and comparable FCR for those fed CON and WW, since higher digesta weight is associated with more retention time and more exposure time to digestive enzymes in the digestive tract (Svihus 2011). In the current study, gizzard digesta contents from birds fed WW increased by 23.47% compared to CON. However, Amerah and Ravindran (2008) found a three-fold increase in gizzard content (9.5 versus 3.0 g/kg body weight) in broilers fed whole wheat rather than ground wheat diets. This difference may have been due to the protein content of the diets, as they used recommended levels, while in the current trial protein level was reduced. The assessment of gizzard digesta particle size revealed that gizzards worked efficiently to get a similar particle size of gizzard digesta compared to a standard protein diet (Hetland et al. 2002). However, the final feed particle size was the highest in the whole wheat diluted protein diet compared to a standard protein diet.

Nitrogen efficiency was similar between birds fed CON, W578 and WW while nitrogen excretion was significantly lower for the W1326 and WW groups, compared with CON and W578. Litter nitrogen and moisture, and footpad scores were significantly decreased in birds fed WW compared with CON. Reducing crude protein in diets can diminish nitrogen excretion and ammonia emissions in the environment, which is an increasingly critical issue in animal production (Liu et al. 2021). The study showed that feeding a whole wheat low crude protein diet decreased litter moisture and nitrogen by 42.18% and 11.22%, respectively, compared to the standard diet. These findings agreed with those which reported decreased nitrogen in the litter due to the reduced protein (Francesch and Brufau 2004; Lemme et al. 2019). There are some explanations for this. To begin with, whole grains contain a huge quantity of water in their matrix compared to ground meal, and this capacity varies depending on the type of grain employed (Brachet et al. 2015). The water-holding capacity of wheat grain increases linearly with grinding screen size (Brachet et al. 2015). Longer digesta retention combined with a higher water-holding capacity in the gut promotes water reuse in the caeca and decreases water excretion, which affects litter quality.

Additionally, in broilers fed the coarse diet, the gizzard operates as a pace-setter for nutrient digestion and absorption, regulating the release of digesta and water absorption (Kheravii et al. 2017). This further reduces the quantity of water excreted in the litter by reducing the superfluous impulse of broilers to drink excessively. High crude protein content in diets has been associated with high water intake and excretion of water through urination, due to the need for extra water to dilute excess nitrogenous compounds (Hilliar et al. 2020; James and Wheeler 1949). Low moisture content in litter has been linked to lower ammonia emissions and fewer welfare issues, such as hock burns and foot pad dermatitis (Miles et al. 2011). Therefore, poor litter quality and the problems associated with it are major welfare concerns (Dunlop et al. 2016). Significantly lower footpad scores in birds fed the whole wheat diluted crude protein diet can be explained by the lower moisture content of the litter and excreta. Lowering the dietary CP improves litter quality and reduces the frequency of footpads and other lesions, improving welfare (de Jong et al. 2012; Lemme et al. 2019; van Emous et al. 2019).

Furthermore, the particle size before and after feeding did not provide statistically relevant outcomes and conclusive information about selection or flicking behaviour of the broilers because mixing residual diets from each replicate could not be analysed. Numerically, the particle size difference was +100.7, +90.83, +112.6, +93.43% at the end of the first week, +4.338, −17.51, +18.88, and +17.08% at the end of the second week, and +0.681, −17.51, +1.274, and +14.00% at the end of the third week between new and residual feeds for CON, W578, W1326 and WW, respectively. The numerical increase in particle size of residual feeds revealed that broiler chickens tended to select smaller rather than bigger particles in the starter phase of feeding. With age, broiler chickens were more capable of handling larger feed particles efficiently. The results showed that the difference in gizzard digesta particle size evened out at the end of the experimental period on d 21. This supported the previous finding that particles need to be ground to a certain critical size before exiting the gizzard can be sustained as the majority of the digesta coming through was of constant particle size (Hetland et al. 2002).

Due to the lack of data on growth performance beyond d 21, it could not be confirmed if there was a positive effect of low protein diets in body weight in later periods due to compensatory growth. Additionally, the feed flicking behaviour of broilers has not been examined statistically regarding selection based on particle size. It was assumed that, with the increase of particle size, the GIT would develop linearly. However, the results showed that very coarse ground wheat diluted diets resulted in lower growth performance of broilers, although the mechanism behind the difference between coarse and whole wheat needs elucidating to see if there is any role of particle shape or if feed flicking behaviour is involved or not.

Conclusions

This study demonstrated the significance of optimising digestive tract development for enhanced efficiency of meat poultry. Utilising whole wheat as a diluent for protein content, particularly in reduced crude protein diets, proved advantageous over medium or coarse ground wheat. This approach minimised nitrogen excretion, litter issues and footpad scores and offered a viable means to curtail feed costs in chicken meat production by reducing grinding costs and dependence on protein-rich resources. Despite compromised weight gain without supplemental synthetic amino acids, the use of whole wheat maintained comparable digestive efficiency. Future trials need to investigate broiler feed particle size selection behaviour and explore strategies for gradually adjusting protein content and feed particle size to bolster sustainable poultry meat production and food security.

Acknowledgments

British Poultry Science (BPS) Ltd. is thankfully acknowledged for providing a small research grant to conduct this study. Special thanks are due to Dr George Mieras (BPS) for arranging the grant and NTU Poultry Research Unit technicians; Ben Gadsby, Ashley Maxey, Kate Wilshaw, Agne Cerniauskaite and John Olifent, for helping in running this study.

Disclosure statement

No potential conflict of interest was reported by the author(s).