Bacterial xylanase supplementation improves nutrient utilization, gut integrity, and microbial metabolism in broilers fed energy-reduced diets

Abstract

This study evaluated the effects of bacterial xylanase supplementation on growth performance, nutrient digestibility, intestinal integrity, and microbial metabolic function in broilers fed energy-reduced diets. A total of 1,050 one-day-old male Ross 308 broiler chicks were randomly assigned to three dietary treatments, each comprising 14 replicates of 25 birds: a positive control (CON; standard corn–soybean meal diet), a negative control with reduced energy (NC; −85 kcal/kg), and an energy-reduced diet supplemented with bacterial xylanase (NCX; 100 g/ton Belfeed Xylanase™). During the starter phase, broilers fed the NC diet exhibited higher feed intake and FCR compared with those fed the CON and NCX diets (P < 0.05), with no significant difference between the CON and NCX diets. Apparent digestibility of dry matter, crude protein, and fat did not differ among dietary treatments (P > 0.05). However, broilers fed the NCX diet showed higher (P < 0.05) digestibility of crude fiber, NDF, and ADF than those fed the CON or NC diets. Apparent metabolizable energy was higher in broilers fed the CON and NCX diets compared with the NC diet. Furthermore, broilers receiving the CON and NCX diets exhibited significantly lower serum fluorescein isothiocyanate–dextran concentrations than those fed the NC diet, indicating improved intestinal barrier integrity. Bacterial xylanase supplementation increased microbial alpha diversity and altered beta diversity clustering, with enrichment of beneficial taxa such as Bifidobacteriaceae and Lactobacillaceae. Functional metagenomic prediction suggested greater representation of carbohydrate metabolism and energy production pathways in the NCX diet, whereas the NC diet was associated with enrichment of stress-related and xenobiotic degradation pathways. Overall, bacterial xylanase supplementation mitigated the adverse effects of dietary energy reduction by improving fiber utilization, maintaining gut integrity, and modulating the cecal microbiota toward a more favorable metabolic profile.

Introduction

Feed cost represents the largest expense in broiler production, with dietary energy accounting for the greatest proportion of formulation costs. Corn and soybean meal serve as the primary energy and protein sources in poultry diets; however, volatility in their availability and price has intensified interest in nutritional strategies that enhance nutrient utilization while permitting reductions in dietary energy without compromising bird performance (Morgan et al., 2021; Moita and Kim, 2022).

Exogenous carbohydrases, particularly xylanase, are widely applied in poultry nutrition to mitigate the anti-nutritive effects of non-starch polysaccharides (NSP) in cereal-based diets (Bedford and Apajalahti, 2018). In corn–soybean meal diets, arabinoxylans (AX) constitute the predominant NSP fraction and occur mainly in an insoluble form, which restricts access of endogenous enzymes to encapsulated starch and protein, thereby reducing energy utilization (Kim et al., 2022; Davies et al., 2024). Although limited fermentation of NSP in the hindgut may yield short-chain fatty acids (SCFAs) that support gut health, the net effect of insoluble AX in corn-based diets remains largely anti-nutritive (Kouzounis et al., 2022).

Xylanase hydrolyzes the xylan backbone of AX, disrupting the cell wall matrix and releasing encapsulated nutrients, which can improve nutrient digestibility and energy recovery. In addition, the resulting arabinoxylo-oligosaccharides (AXOS) and xylo-oligosaccharides (XOS) may act as fermentable substrates for beneficial gut microbiota, supporting intestinal function through enhanced short-chain fatty acid production (González-Ortiz et al., 2019; Bautil et al., 2020; Šimić et al., 2023). Bacterial xylanases, in particular, are considered effective in corn-based diets due to their preference for insoluble AX fractions, which dominate these feed ingredients (Moers et al., 2003; Stefanello et al., 2016).

Despite extensive use of xylanase, broiler responses remain variable and are influenced by factors such as cereal type, dietary energy level, enzyme dose, bird age, and baseline gut microbiota composition (Nusairat and Wang, 2021; Van Hoeck et al., 2021; Saleh et al., 2024). In energy-reduced diets, growth performance and gut health are often compromised; however, xylanase supplementation may partially offset these negative effects by improving nutrient release and energy utilization (Aftab and Bedford, 2018; Wang et al., 2021; Stefanello et al., 2025). Notably, limited information is available on the ability of bacterial xylanase to counteract performance depression while concurrently modulating nutrient utilization and cecal microbiome composition in energy-reduced, corn–soybean meal diets dominated by insoluble arabinoxylans.

The reduction of dietary metabolizable energy by 85 kcal/kg was selected based on previous studies demonstrating that energy reductions within the range of 80–100 kcal/kg are sufficient to induce measurable performance depression while remaining within practical limits for commercial broiler production and enzyme efficacy evaluation (Cruz et al., 2024: Stefanello et al., 2025).

Therefore, the objective of this study was to evaluate the effects of bacterial xylanase supplementation on growth performance, nutrient utilization, and cecal microbiome composition in broiler chickens fed corn–soybean meal diets formulated with reduced metabolizable energy.

Materials and methods

Ethics statement

The experimental protocol was approved by the Institutional Animal Care and Use Committee of Kasetsart University (Approval No. ACKU66-AGK-038), and conducted in accordance with the university’s regulations on animal experiments.

Birds, management, and experimental treatments

A total of 1,242 one-day-old male Ross 308 broiler chicks (45.5 ± 0.5 g initial body weight) were obtained from a commercial hatchery. Of these, 1,050 birds were used for growth performance, intestinal permeability, and gut microbiome assessments, while the remaining 192 birds were allocated for nutrient digestibility evaluation. The 1,050 chicks were randomly distributed into 42 replicate pens (1.20 × 2.35 m) with 25 birds per pen and assigned to three dietary treatments for 35 days. Each dietary treatment had 14 replicates. The dietary treatments consisted of: Positive control (CON): standard corn–soybean meal-based diet; Negative control (NC): energy-reduced diet (−85 kcal/kg); and NC + xylanase (NCX): energy-reduced diet supplemented with bacterial xylanase (Belfeed Xylanase™, 100 g/ton; Jefo Nutrition Inc., St-Hyacinthe, QC, Canada).

All diets were formulated to meet the nutrient requirements of Ross 308 broilers (Aviagen, 2018), with corn and soybean meal serving as the primary ingredients. Diets were pelleted, and xylanase was incorporated into the basal mash before pelleting. Representative samples from each dietary phase were analyzed for proximate composition, calcium, and phosphorus following AOAC (2005) methods, while crude fiber was determined according to the Weende method. The ingredient composition, calculated and analyzed nutrient profiles of the experimental diets are presented in Table 1, Table 2, Table 3, respectively. Rice husk was used as litter at a depth of approximately 5 cm. Feed was provided in hanging tube feeders, and fresh water was available ad libitum via an automatic bell drinker system. Birds were housed in a closed, temperature-controlled facility with appropriate ventilation and lighting (23 h light:1 h dark from days 1–7 and 20 h light:4 h dark from days 8–35). Vaccinations were administered as follows: Newcastle disease (ND; B1 strain) and infectious bronchitis (IB) at 7 days via nasal drops, infectious bursal disease (IBD) at 14 days via oral drops, and ND (LaSota strain) and IB at 21 days via nasal drops. A three-phase feeding program was implemented: starter (days 1–10), grower (days 11–28), and finisher (days 29–35).

Table 1.

Ingredient composition of experimental diets.

Ingredient (%)	Starter (day 0-10)			Grower (day 11-28)			Finisher (day 29-35)
	CON	NC	NCX	CON	NC	NCX	CON	NC	NCX
Corn	41.89	43.69	43.69	36.23	38.13	38.13	32.43	34.33	34.33
Soybean meal (48 %CP)	31.64	31.30	31.30	27.13	26.80	26.80	20.89	20.56	20.56
Wheat	10.00	10.00	10.00	15.00	15.00	15.00	20.00	20.00	20.00
Full fat soybean	6.00	6.00	6.00	8.00	8.00	8.00	10.00	10.00	10.00
Cassava chip	5.00	5.00	5.00	7.50	7.50	7.50	10.00	10.00	10.00
Soybean oil	1.55	-	-	2.84	1.26	1.26	3.85	2.27	2.27
Mono Dicalcium Phosphate (21/16)	1.29	1.29	1.29	1.01	1.01	1.01	0.75	0.75	0.75
Limestone	1.13	1.14	1.14	1.03	1.03	1.03	0.93	0.93	0.93
Salt	0.19	0.19	0.19	0.22	0.21	0.21	0.22	0.23	0.22
Broiler premix	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20
DL-Methionine	0.32	0.32	0.32	0.28	0.27	0.27	0.25	0.25	0.25
L-Lysine	0.27	0.28	0.28	0.21	0.22	0.22	0.20	0.21	0.21
L-Threonine	0.14	0.14	0.14	0.10	0.10	0.10	0.08	0.08	0.08
L-Valine	0.05	0.05	0.05	0.02	0.02	0.02	0.02	0.02	0.02
L-Isoleucine	0.01	0.02	0.02	-	-	-	-	-	-
L-Arginine	0.04	0.04	0.04	-	-	-	-	-	-
Sodium bicarbonate	0.14	0.20	0.20	0.10	0.10	0.10	0.09	0.10	0.10
Choline chloride 60 %	0.10	0.10	0.10	0.09	0.09	0.09	0.09	0.09	0.09
Coccidiostat	0.05	0.05	0.05	0.05	0.05	0.05	-	-	-
Phytase	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01
Bacterial xylanase1	-	-	0.01	-	-	0.01	-	-	0.01
Total	100	100	100	100	100	100	100	100	100

CON = control diet; NC = negative control diet (reduced energy by 85 kcal/kg); NCX = negative control diet supplemented with 100 g/ton bacterial xylanase.

¹Derived from Bacillus subtilis.

Table 2.

Calculated nutrient composition of experimental diets.

	Starter (0-10 d)			Grower (11-28 d)			Finisher (29-35 d)
Nutrient (%)	PC	NC	NCX	PC	NC	NCX	PC	NC	NCX
Dry matter	88.52	88.35	88.35	88.56	88.37	88.37	88.58	88.39	88.39
Crude protein	23.00	23.00	23.00	21.50	21.50	21.50	19.50	19.50	19.50
Crude fat	4.83	3.35	3.35	6.32	4.81	4.81	7.57	6.06	6.06
Linoleic acid	2.40	1.64	1.64	3.16	2.38	2.38	3.79	3.01	3.01
Crude fiber	3.51	3.53	3.53	3.46	3.47	3.47	3.33	3.35	3.35
Ash	5.56	5.60	5.60	5.15	5.15	5.15	4.67	4.68	4.68
ME (kcal/kg)	3000	2915	2915	3100	3015	3015	3200	3115	3115
Dig. Lysine	1.28	1.28	1.28	1.15	1.15	1.15	1.02	1.02	1.02
Dig. Methionine	0.62	0.62	0.62	0.56	0.56	0.56	0.51	0.51	0.51
Dig. Cystine	0.33	0.33	0.33	0.31	0.31	0.31	0.29	0.29	0.29
Dig. Methionine + Cystine	0.95	0.95	0.95	0.87	0.87	0.87	0.80	0.80	0.80
Dig. Threonine	0.86	0.86	0.86	0.77	0.77	0.77	0.68	0.68	0.68
Dig. Tryptophan	0.26	0.26	0.26	0.24	0.24	0.24	0.22	0.22	0.22
Dig. Arginine	1.37	1.37	1.37	1.24	1.24	1.24	1.09	1.09	1.09
Dig. Valine	0.96	0.96	0.96	0.87	0.87	0.87	0.78	0.78	0.78
Dig. Isoleucine	0.86	0.86	0.86	0.79	0.79	0.79	0.70	0.70	0.70
Dig. Leucine	1.58	1.59	1.59	1.46	1.47	1.47	1.30	1.31	1.31
Lysine	1.43	1.43	1.43	1.30	1.30	1.30	1.16	1.16	1.16
Arginine	1.53	1.53	1.53	1.39	1.39	1.39	1.24	1.24	1.24
Methionine	0.66	0.66	0.66	0.59	0.59	0.59	0.54	0.54	0.54
Met + Cys	1.04	1.04	1.04	0.96	0.96	0.96	0.88	0.88	0.88
Cystine	0.38	0.38	0.38	0.36	0.37	0.37	0.34	0.34	0.34
Threonine	0.97	0.97	0.97	0.88	0.88	0.88	0.78	0.78	0.78
Tryptophan	0.28	0.28	0.28	0.26	0.26	0.26	0.23	0.23	0.23
Gly + Ser	1.90	1.90	1.90	1.79	1.79	1.79	1.63	1.63	1.63
Histidine	0.60	0.60	0.60	0.56	0.56	0.56	0.50	0.50	0.50
Isoleucine	0.97	0.97	0.97	0.89	0.89	0.89	0.80	0.80	0.80
Leucine	1.81	1.81	1.81	1.68	1.69	1.69	1.51	1.52	1.52
Valine	1.10	1.10	1.10	1.00	1.00	1.00	0.91	0.91	0.91
Phenylalanine	1.06	1.06	1.06	1.00	1.00	1.00	0.90	0.90	0.90
Calcium	0.96	0.96	0.96	0.87	0.87	0.87	0.78	0.78	0.78
Phosphorus-total	0.64	0.64	0.64	0.56	0.57	0.57	0.49	0.49	0.49
Phosphorus-avail	0.48	0.48	0.48	0.44	0.44	0.44	0.39	0.39	0.39
Potassium	0.98	0.98	0.98	0.95	0.95	0.95	0.88	0.88	0.88
Choline (mg/kg)	1700	1700	1700	1600	1600	1600	1500	1500	1500
Sodium	0.16	0.18	0.18	0.16	0.16	0.16	0.16	0.16	0.16
Chloride	0.23	0.23	0.23	0.23	0.23	0.23	0.23	0.23	0.23
DEB2 (mg/kg)	256	263	263	248	247	247	231	231	231

¹CON = basal diet; NC = reduced energy diet (−85 kcal/kg); NCX = NC + 100 g/ton bacterial xylanase. ²DEB, dietary electrolyte balance.

Table 3.

Analyzed nutrient composition of experimental diets.

Nutrient	Experimental diet1
	CON	NC	NCX
Starter diet (0-10 d)
Dry matter (%)	87.40	87.60	87.80
Protein (%)	22.80	22.50	22.60
Fat (%)	4.57	3.14	3.50
Fibre (%)	2.23	2.16	2.43
Ash (%)	5.23	5.24	5.28
Calcium (%)	0.79	0.80	0.81
Total phosphorus (%)	0.62	0.63	0.60
Gross energy (kcal/kg)	3,988	3,888	3,888
Grower diet (11-28 d)
Dry matter (%)	88.30	88.20	88.50
Protein (%)	21.40	21.40	21.60
Fat (%)	6.27	4.60	4.56
Fibre (%)	2.39	2.40	2.63
Ash (%)	4.92	4.79	4.92
NDF (%)	6.77	6.51	6.47
ADF (%)	3.40	3.70	3.71
Calcium (%)	0.68	0.69	0.69
Total phosphorus (%)	0.55	0.55	0.56
Gross energy (kcal/kg)	4,107	4,007	4,007
Finisher diet (29-35 d)
Dry matter (%)	88.70	88.50	88.30
Protein (%)	20.00	19.80	20.00
Fat (%)	7.85	6.22	6.25
Fibre (%)	2.27	2.12	2.36
Ash (%)	4.69	4.74	4.68
Calcium (%)	0.60	0.62	0.61
Total phosphorus (%)	0.47	0.49	0.49
Gross energy (kcal/kg)	4,187	4,088	4,087

¹CON = basal diet; NC = reduced energy diet (−85 kcal/kg); NCX = NC + 100 g/ton bacterial xylanase.

Growth performance

Body weights of all birds in each pen were recorded on days 1, 10, 28, and 35 to determine body weight gain (BWG). Feed intake was measured for each period by recording the amount of feed offered and refused for each replicate pen. Average feed intake (FI) was calculated for each feeding phase, and the feed conversion ratio (FCR) was determined as the ratio of feed consumed to BWG, with adjustments made for mortality. Mortality was recorded daily, and percentage mortality was calculated as the proportion of birds that died relative to the initial number of birds in each treatment group.

Nutrient digestibility

A total of 192 one-day-old male Ross 308 broiler chicks were used in the study. The chicks were randomly allocated to 24 pens, with 8 birds per pen. The pens were randomly assigned to three dietary treatments (CON, NC and NCX), resulting in eight replicate pens per treatment. Each pen served as the experimental unit for digestibility measurements. The birds were maintained under the previously described management conditions until day 18, when they were transferred to stainless-steel metabolic cages for the digestibility trial. From days 19 to 28, the birds were fed their respective experimental diets containing 0.30 % titanium dioxide (TiO₂) as an indigestible marker. Feed and water were provided ad libitum. On day 28, all birds were humanely euthanized by cervical dislocation following a 12-hour feed withdrawal. Ileal digesta were collected from the segment extending from Meckel’s diverticulum to 2 cm anterior to the ileo–ceco–colonic junction. Digesta from birds within each replicate were pooled, immediately frozen, freeze-dried, ground, and stored at −20°C until analysis. Feed and digesta samples were analyzed for dry matter (DM), crude protein (CP), ether extract (EE), crude fiber (CF), ash, neutral detergent fiber (NDF), acid detergent fiber (ADF), and gross energy (GE) according to AOAC (2005) procedures. Titanium dioxide concentration was determined spectrophotometrically after acid digestion as described by Short et al. (1996). Apparent ileal digestibility coefficients of nutrients were calculated using the marker ratio method, and apparent metabolizable energy (AME) was estimated according to Pirgozliev and Bedford (2013).

Intestinal permeability (FITC-d assay)

Intestinal barrier function was assessed using fluorescein isothiocyanate–dextran (FITC-d; 3–5 kDa; Sigma-Aldrich, Sweden) following established procedures with modifications (Baxter et al., 2017). On day 35, one bird per pen (n = 14 per treatment), representing the average body weight of the pen was randomly selected, fasted for two hours and orally gavaged with FITC-d at a dose of 4.16 mg/kg body weight dissolved in 1 mL Milli-Q water. Blood samples were collected from the wing vein 1 h post-gavage, allowed to clot at room temperature for 3 h, and centrifuged at 3,500 × g for 15 min to obtain serum. FITC-d concentrations were determined using a microplate reader (Synergy HT, BioTek Instruments, USA) with excitation and emission wavelengths set at 485 and 528 nm, respectively. A standard curve was generated by serial dilution of FITC-d in Milli-Q water, and serum FITC-d concentrations (µg/mL) were interpolated from the curve, with higher values indicative of increased intestinal permeability.

Cecal sample collection

At 35 days of age, two birds per replicate were humanely euthanized, and cecal contents were aseptically collected into sterile tubes. Samples were immediately preserved in RNAlater (Qiagen, Maryland, USA) to stabilize nucleic acids and were stored at 4°C for 24 h before being transferred to −80°C for long-term storage until DNA extraction.

DNA extraction

Genomic DNA was extracted from cecal content samples collected at day 35 using the DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. To minimize bias arising from nucleic acid degradation, samples were preserved in DNA/RNA Shield™ (Cat. No. R1100; Zymo Research, California, USA) during collection. The concentration and purity of extracted DNA were assessed using the QIAxcel Advanced system (Qiagen, Hamburg, Germany). High-quality DNA samples were retained for downstream 16S rRNA sequencing. Amplification and sequencing of the V3–V4 hypervariable region of the bacterial 16S rRNA gene were performed using the Illumina NextSeq platform, as described below.

Amplification of the 16S rRNA V3–V4 region and illumina sequencing

The V3–V4 region of the bacterial 16S rRNA gene was amplified by PCR using primers 341F (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGAGGCAGCAG-3′) and 806R (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATTACCGCGGCTGCTGG-3′).

Each 25 μL reaction contained 1 × Phusion Hot Start II High-Fidelity PCR Master Mix (Cat. No. F-565S; Thermo Fisher Scientific, Massachusetts, USA), 0.2 μM of each primer, and ∼10 ng of template DNA. The PCR program included an initial denaturation at 95°C for 3 min, followed by 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 5 min in a thermal cycler (Blue-Ray Biotech, Taipei, Taiwan). Amplicon size (∼550 bp) was verified by electrophoresis on a 1.5 % agarose gel. DNA quality and concentration were assessed using a QFX Fluorometer (DeNovix, Delaware, USA) and a QIAxcel Advanced system (Qiagen, Hamburg, Germany).

Amplicons were purified with AMPure XP beads (Cat. No. A63881; Beckman Coulter, Indiana, USA) to remove free primers and primer-dimers. Dual indices and Illumina sequencing adapters were then attached using the Nextera XT Index Kit (Illumina, San Diego, CA, USA) following the manufacturer’s protocol. A second AMPure XP bead purification was performed before quantification of the final library. After normalization, 5 μL of each purified amplicon was pooled to construct the sequencing library. The pooled library was quantified using a dsDNA fluorescent dye method on a QFX Fluorometer, and fragment length distribution was evaluated using a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). Prior to sequencing, the library was diluted to 2 nM with resuspension buffer, spiked with 2 % PhiX Control (Illumina), and further diluted to a final concentration of 1.5 pM. Indexed primers and sequencing primers were added, and the prepared library was loaded into an Illumina reagent cartridge. Sequencing was performed on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) with a paired-end run of 2 × 151 bp.

Microbial diversity and functional prediction using PICRUSt

Paired-end reads generated from the Illumina MiSeq platform were processed using the Quantitative Insights into Microbial Ecology (QIIME, version 1.9.1). Sequences were clustered into operational taxonomic units (OTUs) using the UCLUST-based open-reference protocol at 97 % similarity against the PKSS 4.0 reference database. Alpha diversity indices, including Chao1 and Shannon, were calculated and compared among treatments using the Kruskal–Wallis test. Beta diversity was assessed with Bray–Curtis dissimilarity based on 46,146 randomly subsampled sequences per sample. Principal coordinate analysis (PCoA) plots were generated using both Bray–Curtis and Jaccard distance matrices, and statistical significance of community clustering was evaluated using PERMANOVA and the Adonis test. Differentially abundant OTUs among treatment groups were identified with the DESeq2 method (negative binomial model). The functional potential of the cecal microbiota was inferred using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 (PICRUSt2) within QIIME2 (version 2019.10). The BIOM-format OTU table was processed with the q2-picrust2 plugin, and functional gene prediction was performed against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. KEGG Ortholog (KO) assignments were further annotated with Enzyme Commission (EC) numbers, and metabolic pathways were reconstructed using the MetaCyc database (Khongthong et al., 2023). Functional profiles were normalized and summarized per sample, and downstream statistical analyses were conducted in RStudio (R Foundation for Statistical Computing, Vienna, Austria) using the vegan and ggplot2 packages. Functional comparisons across treatments were visualized with STAMP (version 2.1.3).

Statistical analysis

Data for growth performance, nutrient digestibility, and intestinal permeability (FITC-d assay) were analyzed using a one-way analysis of variance (ANOVA) under the General Linear Model (GLM) procedure of SAS® OnDemand for Academics (SAS Institute, Cary, NC, USA). Pen was considered the experimental unit. Treatment means were separated using Duncan’s multiple range test, and significance was declared at P < 0.05. Mortality data were expressed as percentages and subjected to arcsine square root transformation prior to analysis.

For cecal microbiota analysis, differences in alpha-diversity indices among treatments were evaluated using the Kruskal–Wallis test. Beta-diversity was assessed using Bray–Curtis and Jaccard distance matrices and visualized by principal coordinate analysis (PCoA). Group differences in Beta-diversity were tested by permutational multivariate analysis of variance (PERMANOVA, 999 permutations) and the Adonis method. Differentially abundant taxa at the OTU level were identified using DESeq2. Predicted microbial functional pathways from PICRUSt2 were compared across treatments using the Statistical Analysis of Metagenomic Profiles (STAMP, version 2.1.3). Welch’s two-sided t-test was applied for pairwise comparisons.

Results

Growth performance

During the starter phase (d 1–10), birds fed the NC diet had higher feed intake and FCR compared with the CON and NCX groups (P < 0.05), whereas no difference was observed between the CON and NCX birds (Table 4). No significant differences were observed in BW, BWG, or mortality among treatments during this period. Dietary treatments did not significantly affect BW, BWG, FI, FCR, mortality, or European Production Efficiency Factor (EPEF) (P > 0.05) during d 11–28, d 29–35, or over the entire experimental period.

Table 4.

Effects of bacterial xylanase supplementation on growth performance of broilers from 0 to 35 days of age.

Items	Dietary treatment			Pooled SE	P-value
	CON	NC	NCX
Initial BW (g)	43.96	43.82	44.12	0.12	0.568
Day 0-10
BW (g)	338	337	335	1.17	0.514
Uniformity (%)	75.38	72.86	71.71	1.57	0.637
Weight gain (g/bird)	294	293	291	1.18	0.481
Feed intake (g/bird/d)	322b	329 a	323b	1.15	0.019
FCR	1.097b	1.124a	1.110b	0.01	0.014
Mortality, (%)	0.29	0.00	0.00	0.09	0.377
Day 11-28
BW (g)	1,964	1,958	1,959	4.92	0.853
Uniformity (%)	82.79	81.80	79.04	1.17	0.404
Weight gain (g/bird)	1,626	1,621	1,624	4.67	0.908
Feed intake (g/bird/d)	2,058	2,070	2,046	12.59	0.756
FCR	1.266	1.277	1.260	0.01	0.593
Mortality, (%)	1.43	1.14	0.57	0.31	0.295
Day 29-35
BW (g)	2,747	2,736	2,740	12.94	0.950
Uniformity (%)	61.20	58.33	58.90	1.58	0.745
Weight gain (g/bird)	783	779	782	9.63	0.986
Feed intake (g/bird/d)	1,335	1,342	1,320	11.14	0.725
FCR	1.710	1.730	1.697	0.02	0.746
Mortality, (%)	0.57	0.58	0.29	0.20	0.807
Day 0-35
BW (g)	2,747	2,736	2,740	12.94	0.950
Uniformity (%)	61.20	58.33	58.90	1.58	0.745
Weight gain (g/bird)	2,703	2,692	2,696	12.92	0.951
Feed intake (g/bird/d)	3,715	3,741	3,689	20.37	0.590
FCR	1.375	1.390	1.369	0.01	0.330
Mortality, (%)	2.29	1.71	0.86	0.36	0.116
EPEF	547.32	544.67	558.43	4.27	0.382

^a,b Means within the same row with different superscripts differ significantly (P < 0.05).

CON = basal diet; NC = reduced energy diet (−85 kcal/kg); NCX = NC + 100 g/ton bacterial xylanase.

FCR = feed conversion ratio; BW = body weight; EPEF = European production efficiency factor; SE = standard error.

Nutrient digestibility

Apparent ileal digestibility of dry matter, crude protein, and ether extract was not affected by the dietary treatments (P > 0.05) (Table 5). However, birds fed the NCX diet had higher (P < 0.05) crude fiber, NDF, and ADF digestibility compared with those fed the CON and NC diets. In addition, birds in the NCX group exhibited greater (P < 0.05) AME than those in the NC group, while the values for the CON group were similar to those of NCX birds.

Table 5.

Effect of bacterial xylanase on ileal nutrient digestibility of broiler chickens.

Items	Dietary treatment1			Pooled SE	P-value
	CON	NC	NC+ Xylanase
Dry matter (%)	91.3	91.4	94.4	0.72	0.129
Crude Protein (%)	79.8	79.6	81.0	0.33	0.199
Crude Fat (%)	93.9	94.9	94.5	0.35	0.497
Crude Fiber (%)	28.2b	30.5b	39.0a	1.91	0.044
NDF (%)	24.4b	21.3b	34.5a	2.08	0.017
ADF (%)	17.7b	20.7b	31.6a	1.68	<0.001
AME (kcal/kg)	3,004a	2,928b	2,996a	12.25	0.015

^a,b Means within the same row with different superscripts differ significantly (P < 0.05).

¹CON = basal diet; NC = reduced energy diet (−85 kcal/kg); NCX = NC + 100 g/ton bacterial xylanase.

NDF = neutral detergent fiber; ADF = acid detergent fiber; AME = apparent metabolizable energy; SE = standard error.

Intestinal permeability (FITC-d assay)

The NC birds exhibited higher FITC-d levels compared to the CON and NCX groups (P < 0.01), whereas no difference was observed between the CON and NCX birds (Fig. 1).

Fig. 1.

Effects of bacterial xylanase supplementation on serum fluorescein isothiocyanate-dextran (FITC-d) levels in broilers at 35 days of age. CON = basal diet; NC = reduced energy diet (−85 kcal/kg); NCX = NC + 100 g/ton bacterial xylanase.

Ceca microbial diversity

Alpha-diversity was evaluated based on estimators of species richness, the Chao1 index (Fig. 2A), and total observed species (Fig. 2C), which were significantly higher (P < 0.05) in the NCX group compared with both CON and NC, indicating greater species richness. Similarly, the Shannon index (Fig. 2B) was significantly higher (P < 0.05) in NCX compared with NC, with CON showing intermediate values. Beta-diversity analysis, assessed using principal coordinate analysis (PCoA) based on Bray–Curtis distances (Fig. 3A), revealed distinct clustering of microbial communities among dietary treatments, with NCX samples separating from NC and partially overlapping with CON. Likewise, Jaccard analysis (Fig. 3B) showed a clear separation between NCX and NC groups, whereas CON samples overlapped with both.

Fig. 2.

Alpha diversity indices of cecal microbiota in broilers at 35 days of age. (A) Chao1 richness index, (B) Shannon diversity index, and (C) total observed OTUs. CON = basal diet; NC = reduced energy diet (−85 kcal/kg); NCX = NC + 100 g/ton bacterial xylanase.

Fig. 3.

Beta-diversity analysis of the cecal microbiome in 35-day-old broilers, assessed using Bray–Curtis (A) and Jaccard (B) distance metrics. CON = basal diet; NC = reduced energy diet (−85 kcal/kg); NCX = NC + 100 g/ton bacterial xylanase.

The taxonomic distributions of the cecal microbiota at the phylum, family, genus, and species levels of 35-day-old broilers are shown in Fig. 4. At the phylum level (Fig. 4A), Firmicutes and Bacteroidetes were dominant across all treatments, followed by Actinobacteria and Proteobacteria. Firmicutes were significantly more abundant in the NCX group compared with CON and NC, whereas Bacteroidetes were reduced in NCX (P < 0.05). Actinobacteria and Proteobacteria were present in lower proportions and showed no significant differences among treatments. At the family level (Fig. 4B), the predominant taxa included Ruminococcaceae, Lachnospiraceae, Bacteroidaceae, and Lactobacillaceae. The abundance of Ruminococcaceae and Lachnospiraceae was significantly higher in NCX compared with CON and NC groups (P < 0.05), whereas Bacteroidaceae was enriched in NC relative to CON and NCX. Lactobacillaceae showed a higher relative abundance in CON birds compared with NC and NCX. At the genus level (Fig. 4C), Bacteroides, Faecalibacterium, Lactobacillus, and Ruminococcus were the dominant genera. The NCX group exhibited significantly increased proportions of Faecalibacterium and Ruminococcus (P < 0.05), while Bacteroides was more abundant in NC birds. Lactobacillus abundance was greater in CON birds compared with NC and NCX. At the species level (Fig. 4D), Bacteroides vulgatus, Faecalibacterium prausnitzii, Lactobacillus reuteri, and Ruminococcus bromii were the major species detected. NCX supplementation significantly enriched F. prausnitzii and R. bromii, while B. vulgatus was more prominent in NC birds (P < 0.05). L. reuteri was relatively higher in the CON group compared with the NC and NCX.

Fig. 4.

Relative abundance (%) of cecal microbiota at (A) phylum, (B) family, (C) genus, and (D) species levels in broilers at day 35. CON = basal diet; NC = reduced energy diet (−85 kcal/kg); NCX = NC + 100 g/ton bacterial xylanase.

The functional profiles of the cecal microbiota of 35-day-old broilers revealed several significantly altered metabolic pathways among the dietary treatments (Fig. 5). Compared with the NC group, the CON group showed significant changes in nine metabolic pathways, including ferulate and sinapate biosynthesis, chlorpyrifos degradation, phosphatidylcholine biosynthesis III–V, phenol degradation I (aerobic), chlorinated phenols degradation, and fluoroacetate degradation (P < 0.05) (Fig. 5A). In contrast, the visual cycle I (vertebrates) pathway was higher in NC than in CON. Eleven pathways differed significantly between CON and NCX (P < 0.05) (Fig. 5B). The NCX group was enriched in the visual cycle I (vertebrates) and diacylglycerol and triacylglycerol biosynthesis compared with CON. Conversely, CON exhibited higher predicted abundance of multiple pathways, including aliphatic glucosinolate biosynthesis (side-chain elongation), salicylate biosynthesis I, 3-methylbutanol biosynthesis (engineered), camalexin biosynthesis, 2-methylcitrate cycle I, L-histidine biosynthesis, pyruvate fermentation to isobutanol, and DIBOA-glucoside biosynthesis. Five pathways were significantly different between NC and NCX (P < 0.05) (Fig. 5C). The NCX group had higher predicted representation of L-histidine degradation (III and VI), neurosporaxanthin biosynthesis, and 4-hydroxymandelate degradation. whereas the NC group was enriched for aerobic respiration III (alternative oxidase pathway).

Fig. 5.

Predicted functional pathways of the cecal microbiome in broilers fed different dietary treatments at day 35. (A) CON vs NC, (B) CON vs NCX, and (C) NC vs NCX. CON = basal diet; NC = reduced energy diet (−85 kcal/kg); NCX = NC + 100 g/ton bacterial xylanase.

Discussion

During the starter phase, broilers fed the energy-reduced diet exhibited higher feed intake and poorer feed conversion, reflecting the well-established tendency of birds to increase feed consumption to compensate for reduced dietary energy density. However, this compensatory response is often inefficient during early growth due to limitations in gut capacity and nutrient utilization. Bacterial xylanase supplementation mitigated these effects by improving feed efficiency during the starter phase, indicating enhanced nutrient release from the corn-soybean meal matrix possibly due to enhanced degradation of arabinoxylans and reduced digesta viscosity (Bedford and Apajalahti, 2022; Moita et al., 2022). These findings agree with previous reports that xylanase supplementation enhances feed efficiency in broilers fed corn–soybean–based diets (Chen et al., 2023; Cruz et al., 2024; Stefanello et al., 2025).

The absence of significant effects on growth performance during the grower, finisher, and overall phases indicates that moderate dietary energy reduction did not impair broiler performance, irrespective of enzyme supplementation. This response likely reflects compensatory growth, maturation of the digestive system, and the strong adaptive capacity of modern broiler genotypes. As birds age, improvements in endogenous enzyme secretion, nutrient utilization, and gut function reduce sensitivity to dietary energy dilution and exogenous enzymes. Consequently, the performance response to bacterial xylanase becomes less pronounced beyond the starter phase. Similar observations have been reported, showing diminished responses to enzyme supplementation and maintained growth performance in older broilers fed energy-reduced diets due to enhanced endogenous digestive capacity and gut adaptation (Kouzounis et al., 2021; Morgan et al., 2022). Nonetheless, the consistent early improvement in feed conversion highlights the importance of xylanase during critical periods of intestinal development.

Digestibility was assessed at 28 d of age, during the grower phase, when digestive capacity is considered physiologically stable and ileal digestibility estimates are more reliable and less influenced by early post-hatch gut development. Furthermore, ileal digestibility measurements at this age provide nutritionally relevant estimates of nutrient utilization while avoiding the confounding effects associated with total tract measurements (Ravindran, 2013). Although pooling digesta within pens limits assessment of within-pen variability, this approach is appropriate when the pen is the experimental unit and has been widely used in poultry ileal digestibility studies to obtain representative pen-level estimates of nutrient utilization (de Vries et al., 2023; Yun et al., 2023; Venter et al., 2024).The improvement in apparent digestibility of dietary fiber fractions, and apparent metabolizable energy confirms the targeted action of bacterial xylanase against insoluble arabinoxylans, the dominant non starch polysaccharide fraction in corn-based diets. By disrupting the structural polysaccharide network, xylanase increases access of endogenous enzymes to encapsulated starch and protein, thereby enhancing energy utilization. Similar trends have been reported by Chen et al. (2023) and Cruz et al. (2024), who observed enhanced fiber degradation and energy yield in broilers fed low-energy diets supplemented with xylanase.

Energy reduction negatively affected gut barrier integrity, as evidenced by increased serum FITC-d concentrations, indicating increased paracellular leakiness (Wiersema et al., 2023). Energy deficiency may impair epithelial maintenance and tight junction protein synthesis, weakening the barrier and allowing greater translocation of FITC-d across the mucosa. Bacterial xylanase supplementation restored barrier function to levels comparable with the energy sufficient control. This improvement likely reflects a combination of reduced digesta viscosity and shorter digesta retention time (Bedford and Apajalahti, 2022; Moita et al., 2022), improved substrate fermentation, and enhanced production of SCFAs, particularly butyrate, which supports epithelial energy supply and tight junction integrity (Vuong et al., 2021; Wang et al., 2024). These findings demonstrate that xylanase not only improves nutrient utilization but also contributes to intestinal resilience under nutritional stress.

Microbiome analyses demonstrated that bacterial xylanase supplementation reshaped microbial diversity and community structure under energy reduced conditions, indicating a coordinated ecological response rather than isolated taxonomic shifts. The elevation of alpha diversity indices, including Chao1, Shannon, and observed OTUs, in the NCX group reflects increased microbial richness and evenness, supporting the concept that xylanase derived xylo oligosaccharides act as stimbiotic substrates that selectively promote beneficial bacterial populations (Amir et al., 2023). Increased microbial diversity is widely associated with greater ecosystem stability and resilience against nutritional and enteric challenges in broilers (Kouzounis et al., 2022; Morgan et al., 2022).

Beta diversity analyses further reinforced this interpretation, as microbial communities in NCX clustered closer to the energy sufficient control than to the unsupplemented energy reduced group. This partial convergence toward a control like microbiome suggests that bacterial xylanase can mitigate diet induced microbial disruption even under reduced dietary energy. Similar shifts in community structure following enzyme supplementation have been reported in broilers fed nutritionally challenged diets, supporting the role of carbohydrases in steering microbial assembly toward more functionally efficient configurations (Cruz et al., 2024; Vergas et al., 2024).

At broader taxonomic levels, Firmicutes and Bacteroidetes remained dominant across treatments, consistent with established poultry microbiome profiles (Rassmidatta et al., 2024; Insawake et al., 2024, 2025). However, the increased relative abundance of Firmicutes and reduced proportion of Bacteroidetes in NCX compared with NC suggests enhanced butyrate producing capacity, as Firmicutes include many key fiber fermenting and butyrate producing taxa, whereas Bacteroidetes are more strongly associated with acetate and propionate production (Onrust et al., 2015; Riviere et al., 2016; Singh et al., 2021). This phylum level redistribution aligns with improved intestinal health and energy utilization through more efficient fiber fermentation (Kouzounis et al., 2021). Wang et al. (2021) reported modulation of phylum-level ratios by xylanase in corn-based supplemented diets, supporting this directional shift.

At finer taxonomic resolution, bacterial xylanase supplementation selectively enriched Bifidobacteriaceae and Brevibacteriaceae at the family level, with corresponding increases in Bifidobacterium and Brevibacterium compared with NC and CON. These taxa are closely associated with gut health due to their capacity to ferment arabinoxylan derived oligosaccharides and produce SCFAs, particularly butyrate (Moita et al., 2022; Wang et al., 2024). The enrichment of Bifidobacterium is of particular relevance, as this genus participates in cross feeding interactions with butyrate producing commensals such as Faecalibacterium prausnitzii, whereby acetate and lactate generated during oligosaccharide fermentation are further converted to butyrate, supporting intestinal barrier integrity and epithelial energy supply (Riviere et al., 2016).

At the species level, NCX birds showed increased abundance of Bifidobacterium pseudolongum and Corynebacterium stationis, both regarded as beneficial core commensals involved in complex polysaccharide degradation and SCFA synthesis. The enrichment of Bifidobacterium pseudolongum further indicates enhanced fermentative activity and a microbial profile associated with improved intestinal health. In contrast, NC birds exhibited a higher relative abundance of Corynebacterium ammoniagenes, which may reflect a microbiota operating under nutritional stress with reduced efficiency of beneficial fermentation. These taxonomic shifts are consistent with previous reports showing that xylanase supplementation enriches beneficial bacterial genera and improves gut functionality in broilers fed both wheat- and corn-based diets (Wang et al., 2021, 2024).

PICRUSt2 derived functional predictions provided insight into how bacterial xylanase supplementation may alter the inferred metabolic potential of the cecal microbiome under energy reduced conditions. In comparisons between CON and NC treatments, several predicted pathways associated with phospholipid metabolism, aromatic compound degradation, and xenobiotic related processes such as phenol and chlorinated phenol degradation were enriched in CON, suggesting that the microbiome in energy sufficient diets retained a broader repertoire of predicted metabolic functions. In contrast, NC showed increased representation of pathways such as visual cycle I, which while not directly related to host physiology may reflect shifts in microbial redox balance or stress associated metabolic signatures under energetic restriction.

When comparing CON with NCX, NCX exhibited altered functional predictions including enrichment of visual cycle I and suppression of several pathways highly represented in CON such as glucosinolate and triacylglycerol biosynthesis. These changes suggest that bacterial xylanase supplementation may reorient predicted microbial functional priorities in response to altered substrate availability under energy reduced conditions.

In the direct NC versus NCX comparison, NC showed higher predicted representation of amino acid degradation pathways and alternative respiratory routes, potentially indicating microbiomes experiencing nutrient limitation or metabolic stress. Conversely, NCX favored pathways associated with secondary metabolite and aromatic compound processing, implying that xylanase supplementation may support more targeted and efficient microbial substrate utilization. Collectively, these inferred functional shifts are consistent with the improvements observed in feed efficiency, nutrient digestibility, and intestinal integrity, and support the concept that enzyme supplementation modulates microbial metabolic capacity in a manner that complements host physiology rather than reflecting microbial proliferation alone. These observations are consistent with previous reports. For example, Wang et al. (2021) reported that xylanase supplementation in broilers modulated predicted metabolic pathways, including increases in mineral absorption routes. Also, studies combining xylanase with XOS have demonstrated downstream effects on SCFA and metabolic signaling pathways (Singh et al., 2021).

It should be noted that functional profiles generated using PICRUSt2 represent inferred metabolic potential based on 16S rRNA gene data rather than direct measurements of gene abundance, transcriptional activity, or enzymatic function. Therefore, the presence of certain predicted pathways, including those without obvious relevance to avian physiology, should be interpreted as indicators of microbial metabolic signatures rather than evidence of active biological processes occurring in the host. Future studies employing metagenomic, metatranscriptomic, or metabolomic approaches are required to validate these predictions and confirm their functional relevance.

Conclusions

Feeding broilers an energy-reduced diet impaired feed efficiency at the starter phase, and intestinal barrier integrity compared with birds fed the energy-sufficient diet. However, bacterial xylanase supplementation effectively mitigated these adverse effects, improving feed efficiency, fiber digestibility and gut barrier function. Bacterial xylanase also enhanced microbial diversity and promoted beneficial taxa such as Bifidobacteriaceae, and Lactobacillaceae, supporting a healthier cecal ecosystem. Functional prediction revealed that bacterial xylanase modulated microbial metabolic pathways toward more efficient carbohydrate and aromatic compound metabolism, indicating improved microbial energy utilization. Overall, bacterial xylanase supplementation restored performance and gut health in broilers fed an energy-reduced diet, highlighting its dual role as a nutrient-liberating and gut-modulating enzyme. These findings emphasize the potential of bacterial xylanase as a sustainable nutritional strategy to maintain broiler productivity, microbiota balance and intestinal integrity in reduced-energy feeding programs.

CRediT authorship contribution statement

Benjaphorn Phusathian: Writing – original draft, Resources, Methodology, Investigation, Formal analysis. Koonphol Pongmanee: Resources, Methodology, Investigation, Formal analysis. Yongyuth Theapparat: Resources, Methodology, Investigation, Formal analysis. Nanthawath Saikhwan: Resources, Methodology, Investigation, Formal analysis. Tassanee Trairatapiwan: Resources, Methodology, Investigation, Formal analysis. Chanporn Chaosap: Writing – review & editing, Resources, Methodology, Investigation. Akaradet Seemacharoensri: Resources, Methodology, Investigation. Glenmer Bathan Tactacan: Resources, Methodology, Investigation. Li-Yen Wong: Resources, Methodology, Investigation. Yuwares Ruangpanit: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.