Impact of the particle size of wheat bran inclusions in feed on the precaecal gastrointestinal tract fibre particle size distribution, caecal digesta particle size and caecal fibre fermentation in young and growing broilers

Abstract

Including additional dietary fibre fractions in broiler feed has gained renewed interest due to its potential for stimulating (gut) health and performance. The beneficial effects of fibre are mainly due to its fermentation in the caecal lobes, but the influx of digesta into these lobes seems restricted to small particles and solubles. To boost caecal fermentation via fibre inclusions, more knowledge on how fibre particle size in feed can affect the fibre particle size distribution along the gastrointestinal tract (GIT), and hence the caecal influx and the fermentation of fibre, is required. To this end, 78 one-day-old male broilers (Ross 308, average initial weight of 43.96 ± 2.97 g) were divided into 3 treatment groups (n = 26 birds per treatment group, 1 replicate pen per treatment), receiving a wheat-based diet with a 10% replacement of the wheat fraction by wheat bran with an average particle size of 1026 (coarse bran), 506 (medium bran) or 106 μm (fine bran), respectively, over a period of d 1 to 24. Digesta of the gizzard, duodenum-jejunum and ileum was collected at d 7 and 24 to determine the fibre particle size distribution in it. The overall ileal and caecal digesta particle size distribution and short-chain fatty acid (SCFA) content were also determined. An increase in the relative content of arabinoxylan along the GIT in the size fractions corresponding to the included bran particle size in the feed was observed for each dietary treatment (P < 0.05), indicating that the particle size differences induced by the bran incorporations were conserved along the GIT. Bran particle size did not affect the gizzard's development or grinding action. The coarse bran treatment resulted in the smallest caecal digesta average particle size (50th percentile, D50) (P = 0.088) and the highest caecal SCFA contents at d 7 (P < 0.001). At d 24, no differences in caecal digesta D50 were observed (P = 0.298), and the fine bran treatment resulted in the highest caecal SCFA contents (P = 0.026). The different effects of bran size on caecal fermentation as a function of broiler age can be ascribed to the limitations of caecal influx that also change with age. The fibre particle size distribution along the GIT can be steered by including the desired fibre particle size in the feed, and the effect of broiler age on caecal influx should be considered when aiming to improve caecal fibre fermentation through fibre inclusion.

1. Introduction

The inclusion of dietary fibre fractions in broiler diets can improve (gut) health and growth performance, which makes it a potential strategy in the search for health- and production-stimulating alternatives to in-feed antibiotics. Such beneficial effects have been described for fibre inclusions originating from various cereal sources such as wheat bran, oat hulls, soy hulls, corn, triticale and rice (González-Alvarado et al., 2007; Józefiak et al., 2007; Paraskeuas and Mountzouris, 2019; Tejeda and Kim, 2021). The positive effects of specific fibre inclusions can be attributed to their role as structural components in diets on one hand and their fermentability on the other hand. Firstly, coarse fibre inclusions (>1 mm) can stimulate the development and functionality of the gizzard, leading to increased nutrient utilisation (Kheravii et al., 2018; Svihus, 2011). In this way, coarse fibre inclusions can also indirectly impact the caecal microbiota by increasing the digesta retention time in the gizzard and, consequently, the digestion of material before it reaches the small intestine. This has been shown to positively affect the caecal microbiota composition and thus possibly also hindgut fermentation (Jacobs et al., 2010; Kheravii et al., 2018; Singh et al., 2014). Secondly, fibre fractions smaller than 300 μm may directly affect the fermentation process and microbial composition in the caecal lobes (Boguslawska-Tryk et al., 2015; De Maesschalck et al., 2019; Vermeulen et al., 2016). These caecal lobes are two blind sacs at the end of the small intestine and are the hotspot for microbial fibre fermentation in broilers. This fermentation results in the production of short-chain fatty acids (SCFA), that can act as an immune stimulator, gut health promotor and additional energy source for beneficial bacteria, gut epithelial cells and the broiler itself (Singh and Kim, 2021; Vermeulen et al., 2016). Previous research has shown that only solubles and small particles, mostly smaller than 100 μm, can enter the caeca and thus potentially be fermented by the resident caecal microbiome (Svihus et al., 2013; Vanderghinste et al., 2025). The lack of consensus on the optimal particle size to stimulate caecal fibre fermentation can partly be due to these different effects of coarse and fine particles along the GIT. The effect of fibre inclusions on the caecal microbiota composition may also depend on the fibre particle size. For example, Jacobs et al. (2010) observed an increase in Lactobacilli but a decrease in Bifidobacteria with increasing corn particle size (557–1387 μm). On the other hand, a more substantial pathogen reduction in the caeca was observed for fine wheat bran inclusions (<280 μm) compared to coarser bran inclusions (520–1690 μm) (Vermeulen et al., 2016). Hence, multiple studies indicate that broilers can benefit from both coarse fibre inclusions, which mainly affect the development and digestion in the foregut, and fine (soluble) fibre inclusions, which may affect the fermentation processes in the hindgut. To stimulate either of these effects, fibre particle size seems to be one of the key factors.

Research on the evolution of the particle size of fibre along the GIT is scarce. There is consensus on the grinding action of the gizzard, which reduces particles to a size smaller than 1 to 2 mm before their exit towards the small intestine. The grinding properties of the gizzard can be stimulated by including coarse fibre particles (Amerah et al., 2007; Kheravii et al., 2018; Svihus, 2011). However, it is unclear if fibre fractions that differ in particle size are all ground to the same extent or if coarse particles are ground more extensively in the gizzard compared to finer particles. It has been shown that the inclusion of coarse wheat particles (>1 mm) in feed also increases the fraction of particles between 500 and 1000 μm in the small intestine and consequently increases the average digesta particle size found after passage through the gizzard, suggesting that the capacity of the gizzard to grind coarse particles is limited (Amerah et al., 2007; Hetland et al., 2002). In contrast, the inclusion of coarse particles has also been shown to result in an overall decrease in the digesta particle size along the tract due to the stimulation of the gizzard's grinding action (Svihus et al., 1997; Xu et al., 2015). Although particle size seems to be one of the determining factors for the final effect of fibre inclusions on the grinding action of the gizzard and the resulting digesta particle size distribution, there is no consensus on the optimal fibre particle size to stimulate nutrient utilisation, broiler performance or gut health (Kheravii et al., 2018). Either way, it is clear that the particle size of fibre inclusions in the feed can impact the fibre particle size distribution of the digesta in the small intestine after having passed through the gizzard, and thus possibly also affect the fibre particle size distribution in the hindgut and at the caecal entrance.

In addition to the contrasting results on particle size and fibre fermentation, broiler age can also be an important factor as the GIT development, the caecal influx of particles and the composition of the microbiome are known to evolve during the broiler's lifespan. It has been stated that the inclusion of coarse fibre particles is more beneficial for older birds (>7 d) than for younger ones, as the underdeveloped gizzard of the latter may not yet be able to cope with such large particles (Kheravii et al., 2018; Svihus, 2011). Studies on the effect of fibre particle size on caecal influx and fermentation as a function of broiler age are scarce. Finally, the inclusion level, chemical composition, solubility and source of fibre additions can also play an important role in the effect on diet structure, GIT development and fibre fermentation (Kheravii et al., 2018; Morgan, 2023; Svihus et al., 2013), but these factors are beyond the scope of this study.

To summarise, fibre particle size has been the subject of studies aiming to improve the functionality of the foregut, as well as studies targeting the fermentation capacity in the hindgut. A reoccurring knowledge gap is the effect of gizzard grinding and enzymatic actions along the GIT on the particle size of fibre fractions before they arrive at the caecal entrance and the subsequent consequences for their fermentation potential. In addition, consensus on the optimal fibre particle size to stimulate the fermentation processes in the hindgut of broilers is still lacking. Broiler age also plays an important but complex role in modulating the physical state of fibre fractions along the GIT and the fermentation capacity of the microbiome. Hence, this study aimed to understand the effect of the particle size of fibre inclusions in the feed on the fibre particle size distribution along the GIT, and consequently evaluate the potential of steering the digesta fibre particle size distribution. In addition, the particle size-reduction effect of the gizzard on fibres of three different sizes was investigated. Finally, the effect of the particle size of fibre inclusions on their caecal influx and fermentation as a function of broiler age was studied.

2. Materials and methods

2.1. Animal ethics statement

This broiler trial was approved by the ethical committee for the experimental use of animals of the KU Leuven under accession number P140/2020.

2.2. Milling, particle size distribution and arabinoxylan (AX) content of bran material

Commercial wheat bran (Triticum aestivum L.) was obtained from Dossche Mills (Deinze, Belgium). Unmilled bran was further referred to as coarse bran, and particle size reduced bran was obtained by Cyclotec milling (1093 Sample mill, Foss, Höganäs, Sweden) using sieves with a pore size of 500 μm, and 180 μm for the medium bran and fine bran, respectively. The particle size distribution of the bran fractions was determined by dry sieving using a Vibratory Sieve Shaker (AS 200 control, Retsch GmbH, Aartselaar, Antwerp, Belgium) with a sieve set with pore sizes indicated in Table S1. The sieves were shaken for 30 min at an amplitude of 1.5 mm and gently brushed in between to prevent clogging of the pores. The amount of bran left on each sieve was weighed afterwards to calculate the average particle size (d_av) using the following formula:

$$d_{av}=\mathrm{\Sigma }{d}_i\times m_i\text{,}$$

where d_i is the average particle size of the particle on sieve i; m_i is the mass percentage of the fraction of bran on sieve i. Coarse bran, medium bran, and fine bran had an average particle size of 1026, 506, and 106 μm, respectively. The particle size distributions are shown in Fig. S1.

The AX content of the bran fractions and the crumble feeds was determined by gas chromatography (GC) using a procedure based on Cummings and Englyst (1987) and adapted by Gebruers et al. (2009). In short, all polysaccharides in the sample were hydrolysed to monosaccharides in trifluoroacetic acid (TFA; 2 mol/L) for 1 h at 110 °C. The resulting aldoses were reduced to alditols in alkaline conditions using NaBH₄ (2 mol/L), after which the alditols were derivatised to alditol acetates using (CH₃CO)₂O and catalyst CH₃C₃H₃N₂. The concentration of the volatile alditol acetates was measured using a 6890 Series A GC (Agilent Technologies, Wilmington, DE, USA) fitted with a SP-2380 polar column (30 m × 0.32 mm, film thickness 0.2 μm; SU-24111, Merck Supelco, Bellefonte, PA, USA) using helium as carrier gas (flow rate 20 mL/min). Coarse bran, medium bran and fine bran had an average AX content of 23.43 ± 1.89%, 24.94 ± 1.11% and 21.32 ± 2.25%, respectively, which did not differ significantly between the bran sizes (P > 0.05).

2.3. Experimental diets

The composition of the 3 experimental diets in this broiler trial are shown in Table 1. The used formulation is a wheat-based one-phase grower-like crumble, fed over the complete trial period and formulated to meet the nutrition requirements of Ross 308 broilers (Aviagen, 2022). No fibre-degrading enzymes were included, but phytase was. For all dietary treatments, 10% of the wheat fraction was replaced with coarse bran, medium bran or fine bran for the coarse bran treatment, medium bran treatment and fine bran treatment, respectively. Crude fat and crude protein levels were slightly adapted to the nutritional recommendations for Ross 308 broilers to sustain broilers' standard growth despite replacing 10% of the wheat fraction with wheat bran (Aviagen, 2022). Dry matter (thermo-gravimetry; European Comission, 2009), crude ash (gravimetry; European Comission, 2009), crude protein (Kjeldahl titrimetry; European Comission, 2009), crude fat (gravimetry; (European Comission, 2009)) and crude fibre (gravimetry; (European Comission, 2009)) contents were analysed by Eurofins (Nazareth, Belgium). The measurement of total AX content is described in section 2.2. Calculated nutrient levels were based on the reference values of Foundation CVB (2022). The absence of difference in the macrostructure between the diets was confirmed by measuring the average particle size of the crumble following the dry sieving method described in section 2.2, using a set of sieves with a pore size of 4000, 2000, 1000, 710, 500, 250 and 50 μm.

Table 1.

Composition and nutrient levels of experimental diets (as-fed, g/kg).

Items	Coarse bran (1026 μm)	Medium bran (506 μm)	Fine bran (106 μm)
Ingredients
Wheat	493.55	493.55	493.55
Wheat bran	100.00	100.00	100.00
Soybean meal (48.0% CP)	307.00	307.00	307.00
Soybean oil (37.5 MJ)	58.00	58.00	58.00
Premix1	5.00	5.00	5.00
Limestone fine	15.80	15.80	15.80
Monocalciumphosphate	6.60	6.60	6.60
Salt	1.80	1.80	1.80
Sodium carbonate	3.70	3.70	3.70
L-Lysine HCl	2.80	2.80	2.80
DL-Methionine	3.45	3.45	3.45
L-Threonine	1.65	1.65	1.65
L-Valine 96.5%	0.50	0.50	0.50
L-Isoleucine 90%	0.10	0.10	0.10
Phytase2	0.05	0.05	0.05
Total	1000.00	1000.00	1000.00
Analysed nutrient levels
Dry matter	920	900	908
Crude ash	59	57	58
Crude protein	222	209	219
Crude fat	82	74	77
Crude fibre	42	36	29
Total arabinoxylan	82.5	85.1	77.7
Calculated nutrient levels3
Gross energy, kcal/kg	3850	3740	3790
Carbohydrates	557.0	560.0	554.0
Starch	307.9	307.9	307.9
Sugars	45.2	45.2	45.2
Calcium	8.5	8.5	8.5
Total phosphorus	6.2	6.2	6.2
Available phosphorus	4.5	4.5	4.5
Digestible phosphorus	4.3	4.3	4.3
Magnesium	2.0	2.0	2.0
Potassium	10.2	10.2	10.2
Sodium	1.8	1.8	1.8
Chloride	2.1	2.1	2.1
Digestible lysine, %	1.19	1.19	1.19
Digestible leucine, %	1.38	1.38	1.38
Digestible methionine + cysteine, %	0.91	0.91	0.91
Digestible arginine, %	1.27	1.27	1.27
Digestible threonine, %	0.80	0.80	0.80
Digestible tryptophan, %	0.24	0.24	0.24

¹Premix provided the following per kilogram diets (premix broilers 0.5%, Research Diet Services BV, Wijk bij Duurstede, Utrecht, The Netherlands): vitamin A 10,000 IU; vitamin D₃ 2500 IU; vitamin E 50 mg; vitamin K₃ 2.3 mg; vitamin B₁ 2.0 mg; vitamin B₂ 7.5 mg; vitamin B₆ 3.5 mg; vitamin B₁₂ 20 μg; niacinamide 35 mg; D-pantothenic acid 12 mg; choline chloride 460 mg; folic acid 1.0 mg; biotin 0.2 mg; iron 80 mg; copper 12 mg; manganese 85 mg; zinc 60 mg; iodine 1.2 mg; selenium 0.25 mg; milled maize used as carrier.

²Axtra PHY 500 FTU.

³Nutrient levels calculated using reference values of the Foundation CVB (2022).

2.4. Broiler housing and sampling

A total of 78 one-day-old male broilers (Ross 308) were purchased from a commercial hatchery (Belgabroed NV, Merksplas, Belgium) and randomly divided over 3 floor pens (each 1 m × 1 m) containing wood shavings. Each pen was assigned to one dietary treatment and contained 26 broilers. Conventional heating, ventilation and lighting conditions for broiler housing were applied (Aviagen, 2018). The housing temperature was set to 34 °C at d 1 and gradually decreased to 22.5 °C at d 24 (end of grower phase and experiment). The light schedule consisted of 23 h light and 1 h darkness from d 1 to 7, followed by a schedule of 18 h light and 6 h darkness until d 24. All broilers received water and feed ad libitum. Inspection of the supply of water and feed, housing conditions, health status and mortality was carried out daily. Individual body weights were measured at d 1, 8, 15 and 22 of 26, 9, 9 and 9 broilers, respectively. At d 7 and 24, respectively 14 and 7 broilers per dietary treatment were stunned by electronarcosis followed by decapitation to obtain 7 digesta replicate pools per treatment. On d 7, the digesta was pooled per 2 broilers to collect enough digesta for further analysis. The chyme and digesta of the gizzard, duodenum-jejunum, ileum and caeca were collected by gentle finger stripping the segments. Digesta samples were stored at −20 °C and sub-samples for SCFA analysis at −80 °C until further analysis. The pH of the gizzard digesta was measured immediately after killing using a pH electrode (Liq-Glass, Hamilton, Reno, NV, USA). Aliquots (2.0 g) of ileal digesta were stored on ice for viscosity measurements on the same day. The empty weight of the gizzard and the weight of the full caecal lobes were measured during sampling.

2.5. Determination of ileal digesta viscosity

The viscosity of the ileal digesta was measured on 7 digesta replicate pools per treatment on each sampling day after temporary storage on ice. Aliquots of 2.0 g were centrifuged at 21,000 × g for 15 min using a Himac CT15RE centrifuge (Hitachi, Chiyoda, Tokyo, Japan), after which 500 μL supernatant was transferred to a viscometer (DV II +, Brookfield, Kruibeke, Oost-Vlaanderen, Belgium). Viscosity was measured at 37 °C using a CP40 cone and a shear rate of 90 s⁻¹.

2.6. Determination of fibre particle size distribution in feed and along the precaecal GIT

A 10 g sample of feed, digesta of the gizzard, duodenum-jejunum and ileum was hydrated in 400 mL distilled water for 30 min and divided into 5 size fractions (<38 μm, 38–200 μm, 200–500 μm, 500–710 μm, >1000 μm) by wet sieving using a Vibratory Sieve Shaker (AS 200 control, Retsch GmbH, Aartselaar, Antwerp, Belgium) for 7 digesta replicate pools per treatment and per age. A shaking amplitude of 1.0 mm was applied for 5 min with a water flow of 2 L/min. Another minute of shaking without water flow was applied to discard excess water from the sieves. Each sieved fraction was lyophilised, weighed and ground with a mortar and pestle. Next, the total AX content of each sieved fraction of each GIT segment was determined in duplicate to estimate the distribution of dietary fibre over the different size fractions, following the procedure described in section 2.2. The AX content (%) of each size fraction was multiplied by the mass fraction (%) of each size fraction, resulting in data representing the relative mass distribution of AX over the size fractions, further referred to as the relative AX content per size fraction. This relative AX content was used as an indication for the particle size distribution of fibre in each GIT segment.

2.7. Determination of the caecal volumetric digesta particle size distribution

The volumetric particle size distribution of the caecal digesta was measured with a Laser Diffraction Particle Size Analyzer (LS 13 320, Beckman Coulter Inc., Indianapolis, IN, USA) on 7 digesta replicate pools per treatment and per age, as caecal digesta particles are too small for accurate determination by the wet sieving method described in section 2.6. The laser diffraction particle size analyzer can measure particle sizes ranging from 0.375 to 2000 μm and uses the assumption that all particles are spherical. The 10th percentile (D10), 50th percentile (average particle size, D50) and 90th percentile (D90) were calculated for each measurement, indicating the particle size below which 10%, 50% and 90% of the particles in the sample are classified, respectively.

The contents and particle size of the caecal digesta samples obtained at d 24 were further investigated through brightfield microscopy (Nikon Eclipse 80i microscope, Nikon Inc., Melville, NY, USA). Too little caecal digesta sample of d 7 was available to perform this analysis.

2.8. Determination of ileal and caecal SCFA content

The contents of acetic acid, propionic acid, butyric acid, isobutyric acid and isovaleric acid in the ileal and caecal digesta were measured via GC on 7 digesta replicate pools per treatment and per age, based on the procedure described by Van Craeyveld et al. (2008) with slight adaptations. In short, 20 μL of 2-ethyl butyric acid was added as an internal standard to 500 mg wet digesta, after which 400 μL of sodium-chloride solution (25 g/100 mL) and 200 μL of a 1:1 sulfuric acid solution was added. The samples were vortexed thoroughly and after 2 min, 800 μL of diethyl ether was added. Next, the samples were horizontally shaken at 60 r/min for 2 min at 4 °C, and centrifuged at 3000 × g for 5 min at 6 °C. The ether phase was transferred into vials to measure the SCFA content using a GC (6890 Series A, Agilent Technologies, Wilmington, DE, USA) equipped with a capillary packed column (30 m × 0.53 mm, film thickness 1.0 μm, J&W DB-FFA packed column, Agilent Technologies, Wilmington, DE, USA) using helium (flow rate 20 mL/min) as carrier gas. The total SCFA content was calculated as the sum of acetic acid, propionic acid and butyric acid and expressed as μmol per g wet digesta.

2.9. Statistical analysis

Statistical analyses were performed using JMP Pro 16 Software and R Statistical Software (v4.0.5; R Core Team 2021, R Foundation for Statistical Computing, Vienna, Austria). Significant differences between the means of the different treatments per broiler age were identified by performing a one-way ANOVA using the following model:

$$Y_{ij}=\mu +{\alpha }_i+{ϵ}_{ij}\text{,}$$

where Y_ij is the measured parameter value of the j^th broiler replicate belonging to the i^th dietary treatment (i = coarse bran treatment, medium bran treatment, fine bran treatment); μ is the general mean of the measured parameter; α_i is the main effect of the i^th level of bran particle size (i = coarse, medium, fine); ϵ_ij is the residual error of the model for the j^th broiler replicate belonging to the i^th dietary treatment. Significantly different means were further identified using Tukey's test, after confirming the normality of the data with a quantile-quantile plot. Differences between means were considered significant at P ≤ 0.05 and interpreted as a trend at 0.05 ＜ P ≤ 0.10.

3. Results

3.1. Zootechnical parameters

At d 8, the fine bran treatment resulted in higher individual body weights (P = 0.022) than the coarse bran treatment (Table 2). No effect of dietary treatment on body weight was observed at other ages (P > 0.05). No significant differences between treatments in empty weight of the gizzard, gizzard pH, ileal viscosity or weight of full caecal lobes were detected at either sampling age (Table 3). The mortality rate was 8%, 8% and 4% for the coarse, medium and fine bran treatments, respectively, over d 4 to 24.

Table 2.

Mean individual body weight (g) per treatment at d 1, 8, 15, and 22¹.

Age	Coarse bran (1026 μm)	Medium bran (506 μm)	Fine bran (106 μm)	SEM	P-value
Day 1	44.5	43.4	44.0	0.34	0.412
Day 8	186.8b	209.7ab	211.9a	4.46	0.022
Day 15	563.1	603.4	607.0	9.44	0.107
Day 22	1182.0	1211.2	1181.3	15.07	0.687

Different letters indicate significant differences between treatments within one age group (P < 0.05).

¹Measured on 26, 9, 9 and 9 replicates at d 1, 8 15 and 22, respectively.

Table 3.

The effect of wheat bran particle size on empty gizzard weight, gizzard pH, ileal viscosity and full caecal lobe weight¹.

Items	Coarse bran (1026 μm)	Medium bran (506 μm)	Fine bran (106 μm)	SEM	P-value
d 7
Empty gizzard weight, g	5.90	6.01	6.07	0.088	0.737
Gizzard pH	2.94	3.16	3.26	0.125	0.593
Ileal viscosity, cP	2.75	2.55	3.01	0.165	0.551
Weight full caecal lobes, g	1.95	2.43	2.16	0.148	0.436
d 24
Empty gizzard weight, g	17.27	19.07	18.42	0.604	0.487
Gizzard pH	2.98	3.35	3.11	0.693	0.551
Ileal viscosity, cP	3.34	3.42	3.60	0.176	0.845
Weight full caecal lobes, g	8.65	7.86	8.50	0.116	0.826

¹Measured on 7 biological replicates per dietary treatment and age.

3.2. Fibre particle size distribution in feed and along the GIT

The mass distribution of AX over the different size fractions in the feed and in each GIT segment is shown in Fig. 1 as the relative AX content per size fraction, for each dietary treatment and sampling age (d 7 and 24). An increase in the relative AX content was observed in the size fractions of the feed and digesta that corresponded to the particle size of the bran incorporations. The feed of the coarse bran treatment showed an increased relative AX content in the larger size fractions >1000 (P = 0.034), 500 to 1000 μm (P = 0.005) and 200 to 500 μm (P = 0.002) compared to the fine bran treatment. Accordingly, the feed of the fine bran treatment showed an increase in relative AX content in the smallest size fractions 38 to 200 μm (P < 0.001) and <38 μm (P = 0.002) compared to the coarse bran treatment. A significant increase in the relative AX content in the size fractions corresponding to the included bran sizes was also observed in the digesta at d 7 and 24. On d 7, the coarse bran treatment showed a tendency towards a higher relative AX content in the gizzard's larger size fraction (500–1000 μm) (P = 0.076) and a lower relative AX content in the smaller size fraction (38–200 μm) (P = 0.002) compared to the fine bran treatment. Similar to d 7, the coarse bran treatment also resulted in a higher relative AX content in the gizzard's larger size fractions (200–1000 μm) (P = 0.002) and a lower relative AX content in the smaller size fraction (38–200 μm) (P < 0.001) compared to the fine bran treatment at d 24. Although an increase in relative AX content in the largest size fraction (>1000 μm) in the coarse bran treatment compared to the fine bran treatment was observed in the feed, no significant difference between treatments was observed in this largest fraction in the gizzard at either age (P > 0.05). The digestive tract parts following the gizzard contained too little digesta of this largest size fraction after sieving to measure its relative AX content. In the duodenum-jejunum and ileum, the coarse bran treatment showed a higher relative AX content in the larger size fraction (500–1000 μm) and a lower relative AX content in the smaller size fraction (38–200 μm) compared to the fine bran treatment at both d 7 and 24 (P < 0.05). On d 24, the fine bran treatment additionally resulted in a significant increase in relative AX content in the smallest fraction of the ileum (<38 μm) compared to the coarse bran treatment (P = 0.014).

Fig. 1.

Mass distribution of arabinoxylan over size fractions >1000 to <38 μm in feed and along the gastrointestinal tract at d 7 and 24, presented as the relative arabinoxylan (AX) content (%) per size fraction. Different letters indicate significant differences in relative AX content between treatments within the same size fraction, gastrointestinal tract segment and age (P < 0.05), an asterisk indicates a trend (0.05 ＜ P ≤ 0.10). Measured on 7 replicate pools per dietary treatment and age.

3.3. Caecal digesta particle size distribution

The volumetric particle size distribution of the caecal digesta is represented in Table 4 via the measured average volumetric D10, D50, and D90 values per treatment. The fine bran treatment tended to result in higher values for D10 (P = 0.076), D50 (P = 0.088), and D90 (P = 0.012) compared to the coarse bran treatment on d 7. On d 24, these differences between treatments disappeared (P > 0.05). Overall higher D10 (P = 0.028), D50 (P = 0.029), and D90 (P = 0.034) values were observed on d 24 compared to d 7.

Table 4.

D10, D50 and D90 values (μm) of the caecal digesta particle size distribution measured by laser diffraction¹.

Items	Coarse bran (1026 μm)	Medium bran (506 μm)	Fine bran (106 μm)	SEM	P-value (treatment)	P-value (age)
d 7
D10	0.67∗	0.91	0.98∗	0.058	0.076
D50	2.60∗	7.59	9.01∗	1.220	0.088
D90	17.36b	25.32b	52.33a	6.198	0.012
d 24
D10	4.68	0.89	4.91	1.275	0.370	0.028
D50	27.21	5.61	28.28	6.582	0.298	0.029
D90	81.70	33.55	125.31	20.629	0.172	0.034

D10 = the 10th volumetric percentile; D50 = the 50th volumetric percentile; D90 = the 90th volumetric percentile.

Significant differences in mean volumetric percentile parameters between treatments are indicated by different letters (P < 0.05) and trends by an asterisk (0.05 ＜ P ≤ 0.10) within one age group.

¹Measured on 7 replicate pools per dietary treatment and age.

Microscopic visualisation of caecal digesta samples at d 24 are shown in Fig. 2. In accordance to the laser diffraction measurements, the majority of the visualised particles in the caeca are very small (<50 μm). Occasional larger particles (100–500 μm) are also present for all treatments, and seem to originate from wheat bran based on the visible pericarp and aleurone structures.

Fig. 2.

Microscopic visualisation of caecal digesta samples obtained at d 24 of broilers fed coarse bran (A and B), medium bran (C and D) and fine bran (E and F), with indicated fragments of wheat bran pericarp (p) and aleurone (a) layers.

3.4. Ileal and caecal SCFA content

The total SCFA, acetic acid, propionic acid and butyric acid contents in the caeca on d 7 and 24 are shown in Table 5. On d 7, the coarse bran treatment resulted in higher total SCFA (P < 0.001), acetic acid (P < 0.001) and butyric acid (P = 0.006) concentrations in the caeca compared to the fine bran treatment. The opposite was observed on d 24, where the fine bran treatment resulted in higher total SCFA (P = 0.026), acetic acid (P = 0.018), and propionic acid (P = 0.042) concentrations in the caeca compared to the coarse bran treatment. No significant differences in ileal acetic acid, caecal isobutyric and isovaleric acid concentrations were measured between treatments at both sampling ages (Table S2 and Table S3).

Table 5.

Concentration of total short-chain fatty acids, acetic acid, propionic acid and butyric acid (μmol/g) in caecal digesta¹.

Items	Coarse bran (1026 μm)	Medium bran (506 μm)	Fine bran (106 μm)	SEM	P-value
d 7
Total SCFA	165.02a	114.8b	79.20b	10.175	<0.001
Acetic acid	137.42a	97.23b	61.05b	9.025	<0.001
Propionic acid	8.37	4.93	6.25	3.131	0.113
Butyric acid	19.23a	12.66b	11.90b	5.098	0.006
d 24
Total SCFA	100.05b	120.64ab	147.11a	35.782	0.026
Acetic acid	76.49b	94.79ab	117.14a	5.969	0.018
Propionic acid	8.83b	9.41ab	12.31a	2.962	0.042
Butyric acid	14.74	16.44	17.65	6.568	0.690

SCFA = short-chain fatty acids.

Significant differences between means of treatments within one age group and acid type are indicated by different letters (P < 0.05).

¹Measured on 7 replicate pools per dietary treatment and age.

4. Discussion

In this study, the effect of the particle size of a 10% bran incorporation (coarse bran: 1026 μm, medium bran: 506 μm, fine bran: 106 μm) on body weight, GIT development, fibre particle size along the GIT and caecal fermentation was evaluated. The particle size of the incorporated bran had a limited effect on individual body weight. The increase in body weight observed for the fine bran treatment compared to the coarse bran treatment on d 8, but not on d 22, could be due to the increased exposure of cell contents to digestion of the fine bran. The lower body weights for the coarse bran treatment could also result from an excess of large fibre particles at a young broiler age, possibly hindering digestion and growth. Tejeda and Kim (2021) reported an increase in body weight gain on d 7 and 14 when including fine fibre particles (100 μm) of various origins compared to their coarse counterparts (600 μm) at levels of 4% and 8%, and this effect of particle size also disappeared at a later age. Chewning et al. (2012) further observed increased body weight up to d 21, but not after that, when feeding corn with a particle size of 300 μm compared to 600 μm. Other studies report no effect of fibre particle size on body weight, possibly due to the lower inclusion levels that were applied compared to the current study (Donadelli et al., 2019; Jiménez-Moreno et al., 2010; Pourazadi et al., 2020). Fibre incorporations may modify the macrostructure of the diet (e.g. the pellet or crumble size), consequently affecting feed intake and thus performance (Abdollahi et al., 2018). As the crumble size of the diets in the current trial did not differ, this should not have affected feed intake and performance, despite the lack of measurement of feed intake in this study. The particle size of the bran incorporations did not affect the gizzard weight, pH or ileal viscosity in the current study. The inclusion of coarse fractions in feed has shown to increase gizzard weight and decrease pH in other studies due to the stimulation of the grinding activity of the gizzard (Amerah et al., 2007; Engberg et al., 2002; Jiménez-Moreno et al., 2010; Kheravii et al., 2018, 2017; Svihus, 2011). The reduction in particle size of bran inclusions can further increase the soluble fibre fraction (De Bondt et al., 2020) and, consequently, the ileal viscosity (Bautil et al., 2019). An effect on viscosity was, however, not observed in this trial despite the exclusion of in-feed endoxylanases, which are known to consistently reduce the ileal viscosity in growing broilers (Bautil et al., 2023). It is possible that the high bran inclusion level (10%) by itself already stimulated the gizzard's functioning and increased the ileal viscosity regardless of its particle size, which concealed the different effects of the coarse bran and fine bran treatments. The coarse bran treatment may have increased gizzard fill despite not having increased empty gizzard weight, as the time that is required for sufficient particle size reduction, and thus the retention time in the gizzard, is typically longer for coarse particles (Amerah et al., 2007; Hetland et al., 2004, 2005; Scholey et al., 2020; Svihus, 2011). However, gizzard content weight was not measured in the current study. The effect of the bran incorporations on these parameters might have been more pronounced if a comparison could have been made with a control group without bran incorporation. As the effects of fibre inclusions on broiler performance have already been discussed in multiple previous studies by comparing conventional and fibre-enriched diets, this research study focused on the confined effect of the particle size of the fibre inclusion in broiler feed. This also explains the high bran inclusion level of 10% used in this study since it enabled the detection of the included bran particle size along the entire GIT. Based on the current results, it seems that bran particle size only affects the body weight at a young broiler age and not gizzard weight, pH, caecal lobe weight or ileal viscosity when using a 10% inclusion level.

The measured AX content in the different size fractions of the digesta was used to follow the size distribution of the added dietary fibre along the precaecal GIT, as AX is the main DF component of the incorporated wheat bran. The detected enrichment of AX in the size fractions corresponding to the size of the bran incorporations in both the feed and the precaecal GIT illustrates the feasibility of using AX as an indicator of wheat bran DF along the GIT, despite the presence of AX originating from the basal wheat-based diet. Following AX along the GIT further allowed to study its particle size reduction in the precaecal GIT and the consequences on its fermentation in the hindgut simultaneously. This analysis showed that the fibre particle size distribution in the precaecal GIT is influenced by the addition of fibre with a specific particle size in the feed. The particle size differences between each fraction were maintained throughout the precaecal GIT as illustrated by the increase in relative AX content in the larger size fractions (200–1000 μm) of the digesta of the coarse bran treatment (1026 μm), and by the enrichment in the smaller size fractions (38–200 μm) of the digesta of the fine bran treatment (106 μm). This increase in relative AX content in the size fractions corresponding to the included bran size continued from the gizzard to the ileum at both d 7 and 24. These findings align with the conclusions of Amerah et al. (2007), who observed that the inclusion of coarse particles in the feed will also result in more coarse particles in the small intestine. The absence of digesta in the largest size fraction (>1000 μm) after passage through the gizzard and the marked increase in relative AX content in the smallest size fraction (<38 μm) in the duodenum for all treatments confirm the strong grinding effect of the gizzard at both studied ages regardless of fraction particle size. This indicates that the gizzard acts as a filter to prevent the egress of particles greater than 1000 μm and also cascades larger into smaller particles (Amerah et al., 2007; Svihus, 2011). The faster passage of fine particles through the gizzard, together with the stronger retention of coarser particles, can have further contributed to the relative increase of the finer (<200 μm) compared to the coarser size fractions seen in the duodenum-jejunum for all treatments (Hetland et al., 2004; Svihus, 2011). This faster passage of the finer size fractions is also illustrated by their relative decrease in the gizzard compared to the initial distribution in the feed. The medium bran treatment often showed increased relative AX contents in size fractions similar to the coarse bran treatment, which is mainly due to the selected sieve sizes. The additional increase of relative AX content in the smallest size fraction (<38 μm) in the ileum for the fine bran treatment on d 24, but not on d 7, can imply a more significant enzymatic fibre degradation and solubilisation capacity in the ileum at an older broiler age, related to the maturation of the intestinal microbiome as a function of age (Bautil et al., 2019). These findings show that, despite the clear particle-size-reducing action of the gizzard, the initial particle size of bran inclusions in the feed still affects the fibre particle size distribution in the small intestine.

The direct effect of bran particle size on the particle size distribution along the precaecal GIT did not continue in the caecal lobes. At d 7, the coarse bran treatment surprisingly resulted in the smallest caecal digesta D50, and the fine bran treatment in the largest caecal digesta D50. That the fine bran treatment led to the largest caecal digesta D50 can be the result of an excessive enrichment of particles belonging to the 38 to 200 μm fraction in the ileum compared to the other treatments, caused by the inclusion of the 100 μm fine bran (Fig. 1). Particles within this size fraction can potentially enter the caeca as confirmed by previous measurements of the caecal digesta particle size distribution (Vanderghinste et al., 2025). The enrichment of particles within this size fraction in the precaecal GIT may have caused an overfilling of the caecal lobes of broilers receiving the fine bran treatment. The overlap of the size range of particles found in the caeca (0.98–52.33 μm) with the AX-enriched size fraction in the ileum (38–200 μm) for the fine bran treatment at d 7 supports this hypothesis. This overfilling of the caecal lobes for the fine bran treatment at a young broiler age (d 7) with particles from the 38 to 200 μm size fraction may have led to a suboptimal fermentation, as the immature caecal microbiome at this age could not yet cope with this increased amount of relatively large particles in the caecal lobes (Morgan, 2023). This detrimental effect on caecal fermentation may explain the observed decrease in caecal SCFA production for the fine bran treatment compared to the coarse bran treatment at d 7. At d 24, a broader range of particles is expected to be able to enter the caecal lobes (Bautil et al., 2021; Vanderghinste et al., 2025), and indeed, no significant differences in caecal particle size distribution parameters were observed between treatments. The lowered restriction of the caecal influx of digesta particles at an older age is further illustrated by the overall increase of the caecal D50 with age and the large variation in D90 values measured at d 24 for both the coarse bran and fine bran treatments (Table 4). Due to the broader range of particle sizes that are able to enter the caeca at an older age, no negative effects of possible overfilling of the caeca were associated with a specific particle size of the bran incorporations at this age. The lower values and smaller variations for D10, D50, and D90 of the medium bran compared to the other treatments at d 24 may be explained by the sensitivity of the laser diffraction method to the presence of a few large particles in the samples, together with the different effects of fine and coarse bran in the broiler's GIT. The fine bran is expected to partly pass the size restrictions of the caecal filter at this age, resulting in the occasional caecal influx of relatively large particles (100 μm), while the coarse bran may create a similar increase in particles suitable for caecal entrance by stimulating the grinding activity of the gizzard. If the particle size of the medium bran was too large for this occasional caecal influx, but too small for sufficient gizzard stimulation, the size fraction with particles that could potentially enter the caeca was not enriched in this treatment, resulting in a lower average caecal digesta particle size with less variation between broiler replicates. The absence of observed changes in gizzard weight and pH in this experiment that would support this hypothesis can be due to the in vivo variability of these parameters and to the previously discussed effect of the high bran inclusion level for all treatments, as the gizzard-stimulating effect of coarse fractions has been well documented in previous studies (Jiménez-Moreno et al., 2010; Kheravii et al., 2018, 2017; Svihus, 2011). The microscopic visualisation of caecal digesta at d 24 confirms that the majority of the larger particles (>100 μm) present in the caeca originate from wheat bran, but does not allow a quantitative comparison of the presence of large bran particles in the caecal digesta between treatments. Although most of this bran likely originated from the bran incorporations, given their clear presence in the corresponding size fractions in the precaecal GIT, part of the bran observed in the caecal digesta can also be derived from the general wheat fraction in the diets. Despite the lack of difference in caecal D10, D50, and D90 between the treatments at d 24, the fine bran treatment did result in an increased caecal SCFA concentration compared to the coarse bran treatment. It is possible that including fine bran in the feed still resulted in an increased fraction of fine fibre in the caeca, without being reflected in the caecal D50 due to the large variation in size of the digesta particles that enter the caeca at d 24. Fibre fractions with a reduced particle size have also been previously described to be more prone to microbial fermentation due to the increase in specific surface area and breakage of cell walls (Deroover et al., 2021; Stewart and Slavin, 2009; Vermeulen et al., 2016). Hence, the presence of small (fibre) particles, be it in the caeca (d 7) or in the feed (d 24), resulted in an increased caecal SCFA production. However, the strategy to obtain small fibre particles in the broiler's caeca greatly differs with age and depends on the age-associated caecal influx mechanism. A lower inclusion level than the one in the current study may affect the caecal filling of young birds differently, for example by reducing the observed overfilling effect of the caecal lobes when using fine fibre inclusions and thus decreasing the associated hindrance of the substrate–microbiome interactions. The use of a relatively high bran inclusion level in this study did enable the detection of the preserved bran particle sizes along the precaecal GIT, and showed that the limitations of caecal influx as a function of broiler age play an important role in steering the caecal influx and fermentation of fibre fractions.

5. Conclusion

The replacement of the wheat fraction with 10% wheat bran of 1026, 506, or 106 μm in broiler feed showed that the particle size differences between such fractions are mostly conserved along the precaecal GIT at d 7 and 24. The grinding action of the gizzard was clearly illustrated by the disappearance of the >1000 μm size fraction in the small intestine, but the extent of grinding was not affected by the particle size of the bran incorporations. These results showed that the fibre particle size distribution along the precaecal GIT can be steered by including the desired fibre particle size in the feed. Caecal fermentation was stimulated by the presence of fine fibre particles at both broiler ages, but the caecal influx mechanism at each age was a determining factor for the average caecal digesta particle size and thus fermentation. At a younger age, the coarse bran treatment surprisingly led to the smallest caecal digesta D50 and resulted in the highest caecal SCFA production compared to the fine bran treatment. At an older age, caecal SCFA production was directly stimulated by the inclusion of fine fibre particles in the feed compared to the inclusion of coarse particles. Following the precaecal GIT, the particle size distribution of caecal digesta and consequently the microbial fermentation can be steered by adding fibres of a specific particle size in the feed, but the difference in caecal influx mechanism and microbial fermentation capacity as a function of broiler age should be taken into account.

Credit Author Statement

Paulien Vanderghinste: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Conceptualization. An Bautil: Writing – review & editing, Supervision, Methodology, Conceptualization. Michael R. Bedford: Writing – review & editing, Conceptualization. Gemma González-Ortiz: Writing – review & editing, Conceptualization. Christophe M. Courtin: Writing – review & editing, Supervision, Resources, Methodology, Conceptualization.

Declaration of competing interest

The authors declare no competing financial or personal relationships with other parties that could inappropriately influence this work, and no professional or personal interests in any product, service, or company that might affect the paper’s content. Potential competing interests are as follows: this research was funded by AB Vista; Gemma Gonzalez is currently an employee of AB Vista; Michael Bedford no longer holds a position at AB Vista and has no relevant competing interests.

Appendix A. Supplementary data

The following is the Supplementary data to this article: