Effects of Phellinus linteus-Fermented Wheat Bran on Growth Performance, Intestinal Morphology, and Modulation of Nrf2-ARE-Associated Gene Expression of Broilers during Summer Conditions

Abstract

This study investigated the effects of Phellinus linteus-fermented wheat bran (FWB) on growth performance, intestinal morphology, and modulation of nuclear factor erythroid 2-related factor 2-antioxidant response element-associated gene expression in broilers during summer conditions. The fiber content and cellulolytic enzyme activity of wheat bran (WB) improved after fermentation. In addition, FWB had a significantly higher secondary metabolite content, such as crude polysaccharides, ergosterol, and crude triterpenoids. Moreover, FWB showed 2,2-diphenyl-1-picrylhydrazyl free-radical scavenging and ferrous ion-chelating abilities. Three hundred 1-day-old broiler chickens (Ross 308) were assigned into five groups fed the control diet or the control diet replaced with 5% WB, 10% WB, 5% FWB or 10% FWB. Over the entire experimental period, broilers in the 5% FWB group had a significantly lower feed conversion ratio than that of broilers in the control group. Assessment of blood characteristics revealed that broilers fed FWB had significantly higher serum superoxide dismutase activity and reduced malondialdehyde production. Measurement of the expression of antioxidant-related genes and proinflammatory cytokines in broiler peripheral blood mononuclear cells showed that partially replacing FWB in broiler diets enhanced the expression of antioxidant-related genes and decreased the expression of proinflammatory cytokines. These results indicated that P. linteus increased the feed value of WB and improved the growth performance and antioxidant capacity of broiler chickens, suggesting its potential for application in broiler diets during summer conditions.

Introduction

Feed cost is the main expenditure of domestic animal production, accounting for approximately 60%–70% of the total production cost. As cereals are the main components of animal feed, the cost of animal production concurrently increases as the prices of global grains rise. To reduce production costs, efforts have been made to use cheaper feed ingredients or develop agricultural byproducts as alternative feed ingredients[1,2,3].

Wheat bran (WB) is a byproduct of wheat processing[4]. Javed et al.[5] have reported that one million tons of wheat may produce up to 0.25 million tons of WB. Owing to the high demand for wheat, the production of WB is high and stable[6], which is favorable for feed application. Accordingly, Hossain et al.[7] have pointed out that approximately 90% of WB is used as animal feed. However, a comparison of commonly used feed ingredients, such as maize and soybean, shows that the high fiber content of WB may adversely affect the digestibility of starch, lipids, and proteins in monogastric animals[8], further influencing growth or production performance[4,9]. The negative effects of excess fiber must be reduced to increase the utilization of agro-by-products in monogastric animals[10].

Several processing methods, classified into physical, chemical, physic-chemical, and biological treatments by Sharma et al.[11], have been used to enhance the utilization of crop residues. Biological methods have gradually replaced physical or chemical treatments owing to cost, safety, and possible negative impacts on the environment[1,12,13]. Using lignocellulolytic fungi or enzymes to process agro-byproducts not only improves the nutritional value of these byproducts, but also reduces the usage of energy and chemicals, further alleviating environmental damage[3,9,14].

To enhance the antioxidant capacity and immunomodulatory functions of broiler diets, Phellinus linteus (PL) was used to ferment WB as a functional feed ingredient to improve growth performance and regulate gut microbial communities in broilers. PL is a perennial medicinal mushroom that parasitizes the trunks of mulberry and other broad-leaved trees[15]. PL exhibits potent antioxidant, anti-inflammatory, and immunomodulatory activities[16,17,18]. In addition, PL exerts protective effects against viral infections by modulating both the innate and adaptive immune responses[19]. However, the slow growth rate of PL fruiting bodies limits its direct application in animal feed. Advances in solid-state fermentation technology have provided an effective strategy for utilizing PL, allowing its bioactive compounds to be incorporated into feed substrates[15]. Notably, PL fermentation enhances the antioxidant properties of cereal-based substrates, including adlay and rice[20], suggesting it has potential as a functional feed additive for broiler nutrition.

In addition to concerns of reducing feed costs, the growth and production performance of domestic animals are affected by many stressors. Most cellular stressors in poultry production are associated with oxidative stress[17]. Therefore, the present study investigated the effects of FWB on growth performance, antioxidant activity, and antioxidant-related gene expression in broiler chickens.

Materials and Methods

Microorganism and WB fermentation

The PL used in this study was kindly provided by a local farm. The fungus was inoculated onto potato dextrose agar (PDA) plates and incubated at 30 °C for activation. After activation, the microorganism was routinely maintained on a PDA plate at 30 °C with regular subcultivation (about 2 weeks). For the starter culture, 10 PL mycelial disks (10 mm diameter) were obtained from the PDA plate and inoculated onto 50 g of sterilized oat with 60% (v/w) moisture content, then incubated at 30 °C for 20 days. FWB was produced by mixing the starter culture with 250 g of sterilized WB with 50% moisture and then incubating it at 30 °C for 18 days.

Fiber content determination and enzyme assay

The neutral detergent fiber (NDF) and acid detergent fiber (ADF) content were determined according to Van Soest et al.[21]. Xylanase, mannanase, and cellulase activities were assayed by measuring reducing sugars using the dinitrosalicylic acid method[22]. Briefly, FWB was extracted in cold water and stirred for 30 min to obtain a crude enzyme extract. The extract was centrifuged at 1,006 × g (Himac CF, Hitachi Koki, Tokyo, Japan) for 10 min and then filtered through Whatman No. 1 filter paper for further enzyme assays. One international unit (IU) of enzyme activity was defined as the quantity of enzyme required to release 1 µm reducing sugar from the substrate per minute under standard assay conditions (55 °C, 1% xylan, pH 5.3 for xylanase; 37 °C, 0.6% locust bean gum, pH 5.5 for mannanase; 50 °C, 1% carboxymethyl cellulose, pH 4.8 for cellulase).

Secondary metabolite measurement

First, FWB was dried in a 50 °C oven for 48 h. Dried samples were ground and passed through an 18-mesh sieve. The ground samples were stored at 4 °C before further use. Extraction conditions were determined based on the measured compounds. In brief, ergosterol was extracted with methanol (MeOH) at 40 °C, adenosine was extracted with 5% ethanol (EtOH) at 80 °C, crude triterpenoids were extracted with 95% EtOH at room temperature (RT), and crude polysaccharides and total phenolic compounds were extracted with distilled water at 95 °C. High performance liquid chromatography (HITACHI, L-6200A Intelligent Pump, Hitachi Co., Ltd., Tokyo, Japan) was used to measure the content of ergosterol and adenosine. Different concentrations of standard products were measured to obtain standard curves, and the ergosterol or adenosine of FWB content were calculated by substituting the area measured using the respective standard curves. Crude triterpenoid content was determined according to the method described by Lu et al.[23]. The amount of crude polysaccharide was measured using the phenol-sulfuric acid assay described by DuBois et al.[24]. The total phenolic compound content was determined using the Folin–Ciocalteu method described by Kujala et al.[25].

In vitro antioxidant capacities

Ground FWB was stirred and extracted with deionized water at 95 °C for 1 h, centrifuged at 3000 rpmfor 10 min, and filtered using Whatman No. 1 filter paper. The filtrates were stored at 4 °C before further use. The free radical-scavenging ability of the FWB was examined according to the method described by Gyamfi et al.[26]. Briefly, 1 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH) in EtOH was prepared and added to the filtrate at different concentrations. After incubation for 30 min, the absorbance at 517 nm was measured against butylated hydroxytoluene (BHT). FWB reducing power was measured using the method described by Oyaizu[27]. In brief, the filtrate was mixed with 0.2 M phosphate buffer (pH 6.6) and 1% potassium ferricyanide. The mixture was incubated at 50 °C for 20 min and the reaction was terminated with 2.5 mL 10% trichloroacetic acid. Approximately 5 mL of this solution was then mixed with the same volume of deionized water and 1 mL 0.1% ferric trichloride and incubated for 10 min. The absorbance was measured at 700 nm using a spectrophotometer with ascorbic acid as the control. The ferrous ion chelating activity of FWB was determined according to the method described by Dinis et al.[28]. Briefly, 0.25 mL filtrate was mixed with 0.025 mL 2 mM ferrous chloride tetrahydrate solution and 0.925 mL MeOH. After 30 s, 0.05 mL 5 mM ferrozine was added to initiate the reaction and the mixture was then left to stand at RT for 10 min. When the reaction was complete, the absorbance of the mixture was measured at 562 nm using a spectrophotometer.

Experimental birds and housing

The feeding trial was conducted during the summer with an average environmental temperature of 30 ± 1.5 °C and humidity of 75 ± 10% at the Experimental Husbandry Farm (National Chung Hsing University (NCHU), Taiwan). All protocols were conducted in accordance with the guidelines by the Animal Care and Use Committee of NCHU (IACUC 107-042). Three hundred (1-day-old male) broiler chickens (Ross 308) were randomly assigned to one of five treatments. For each treatment, there were three replicate pens, with 20 birds per pen (60 birds per treatment). The initial body weight of all birds were similar (approximately 42.1 g/bird). The temperature was maintained at 34 ± 1 °C until the birds were 7 days of age and then was gradually decreased to 27 ± 1 °C until the birds were 21 days of age. After this time, the broilers were maintained at approximately 27 °C.

Feeding schedule and dietary composition

The treatment groups included the control diet, and groups where the control diet was replaced with 5% WB, 10% WB, 5% FWB, or 10% FWB (as shown in Table 1-1 and 1-2). Water and all experimental diets were provided ad libitum. Throughout the experimental period (35 days), diets were formulated to meet the requirements suggested by the Ross Broiler Management Manual (2014) and the NRC (1994).

Table 1-1.  Ingredients and chemical compositions (g/kg as fed) of the diets of broilers (1–21 days)¹.

Ingredients		Control	5% WB	5% FWB	10% WB	10% FWB
	Corn	524.9	458.7	458.4	392.5	391.9
	Wheat bran	0	50.0	0	100.0	0
	Fermented wheat bran	0	0	50.0	0	100.0
	Soybean meal, CP 44%	320.0	167.3	167.3	14.8	14.7
	Fish meal, CP 60%	50.0	50.0	50.0	50.0	50.0
	Full fat soybean meal	41.4	209.8	210.1	377.9	378.7
	Soybean oil	30.0	30.0	30.0	30.0	30.0
	Limestone	11.6	11.6	11.6	11.5	11.5
	Monocalcium phosphate	11.2	11.2	11.2	11.2	11.2
	DL-Methionine	3.4	3.7	3.7	4.1	4.1
	Salt	2.9	2.8	2.8	2.8	2.8
	L-Lysine HCl	1.8	2.1	2.1	2.4	2.3
	Choline-Cl (60%)	0.8	0.8	0.8	0.8	0.8
	Vitamin premix2	1.0	1.0	1.0	1.0	1.0
	Mineral premix3	1.0	1.0	1.0	1.0	1.0
	Total	1000.0	1000.0	1000.0	1000.0	1000.0
Calculated nutrient value
	ME, kcal/kg	3050.0	3050.0	3050.0	3050.0	3050.0
	Dry matter, %	88.28	88.85	89.11	89.42	89.95
	Crude protein, %	23.0	23.0	23.0	23.0	23.0
	Crude fat, %	6.04	8.86	8.64	11.68	11.25
	Calcium, %	1.05	1.05	1.05	1.05	1.05
	Total phosphorus, %	0.73	0.73	0.73	0.72	0.72
	Available phosphorus, %	0.50	0.50	0.50	0.50	0.50
	Lysine, %	1.43	1.43	1.43	1.43	1.43
	Methionine, %	0.73	0.74	0.74	0.76	0.76
	Cysteine, %	0.34	0.32	0.32	0.31	0.31
Analyzed nutrient value
	Dry matter, %	88.22	88.67	89.11	89.05	89.95
	Crude protein, %	23.54	23.48	23.45	23.52	23.40
	Crude fat, %	6.32	8.89	8.63	11.44	11.23

¹Control: basal diet (corn-soybean meal); 5% WB: diet with 5% wheat bran replacement; 10% WB: diet with 10% wheat bran replacement; 5% FWB: diet with 5% fermented wheat bran replacement; 10% FWB: diet with 10% fermented wheat bran replacement.

²Supplied per kg of diet: Vit. A 15000 U; Vit. D3 3000 U; Vit. E 30 mg; Vit. K3 4 mg; Riboflavin 8 mg; Pyridoxine 5 mg; Vit. B12 25 μg; Ca-pantothenate 19 mg; Niacin 50 mg; Folic acid 1.5 mg; Biotin 60 μg.

³Supplied per kg of diet: Co (CoCO₃) 0.255 mg; Cu (CuSO₄･5H₂O) 10.8 mg; Fe (FeSO₄･H₂O) 90 mg; Zn (ZnO) 68.4 mg; Mn (MnSO₄･H₂O) 90 mg; Se (Na₂SeO₃) 0.18 mg. ME, metabolizable energy.

Table 1-2.  Ingredients and chemical compositions (g/kg as fed) of the diets of broilers (22–35 days)¹.

Ingredients		Control	5% WB	5% FWB	10% WB	10% FWB
	Corn	549.5	482.9	482.9	416.3	416.3
	Wheat bran	0	50.0	0	100.0	0
	Fermented wheat bran	0	0	50.0	0	100.0
	Full fat soybean meal	16.6	185.6	185.6	354.4	354.4
	Soybean meal, CP 44%	320.6	167.8	167.8	15.1	15.1
	Fish meal, CP 60%	30.0	30.0	30.0	30.0	30.0
	Limestone	10.6	10.6	10.6	10.6	10.6
	Monocalcium phosphate	12.2	12.2	12.2	12.2	12.2
	Salt	3.4	3.3	3.3	3.2	3.2
	Soybean oil	50.0	50.0	50.0	50.0	50.0
	L-Lysine HCl	1.3	1.5	1.5	1.8	1.8
	DL-Methionine	3.0	3.3	3.3	3.6	3.6
	Choline-Cl (60%)	0.8	0.8	0.8	0.8	0.8
	Vitamin premix2	1.0	1.0	1.0	1.0	1.0
	Mineral premix3	1.0	1.0	1.0	1.0	1.0
	Total	1000.0	1000.0	1000.0	1000.0	1000.0
Calculated nutrient value
	ME, kcal/kg	3175.0	3175.0	3175.0	3175.0	3175.0
	Dry matter, %	88.31	88.88	89.15	89.45	89.98
	Crude protein, %	21.0	21.0	21.0	21.0	21.0
	Crude fat, %	7.56	10.39	10.17	13.22	12.78
	Calcium, %	0.90	0.90	0.90	0.90	0.90
	Total phosphorus, %	0.68	0.67	0.67	0.67	0.67
	Available phosphorus, %	0.45	0.45	0.45	0.45	0.45
	Lysine, %	1.25	1.25	1.25	1.25	1.25
	Methionine, %	0.65	0.66	0.66	0.67	0.67
	Cysteine, %	0.31	0.30	0.30	0.29	0.29
Analyzed nutrient value
	Dry matter, %	88.71	88.79	89.04	89.20	89.48
	Crude protein, %	21.83	21.77	21.67	21.25	21.24
	Crude fat, %	7.23	10.33	10.73	12.49	12.74

¹Control: basal diet (corn-soybean meal); 5% WB: diet with 5% wheat bran replacement; 10% WB: diet with 10% wheat bran replacement; 5% FWB: diet with 5% fermented wheat bran replacement; 10% FWB: diet with 10% fermented wheat bran replacement.

²Supplied per kg of diet: Vit. A 15000 U; Vit. D3 3000 U; Vit. E 30 mg; Vit. K3 4 mg; Riboflavin 8 mg; Pyridoxine 5 mg; Vit. B12 25 μg; Ca-pantothenate 19 mg; Niacin 50 mg; Folic acid 1.5 mg; Biotin 60 μg.

³Supplied per kg of diet: Co (CoCO₃) 0.255 mg; Cu (CuSO₄･5H₂O) 10.8 mg; Fe (FeSO₄･H₂O) 90 mg; Zn (ZnO) 68.4 mg; Mn (MnSO₄･H₂O) 90 mg; Se (Na₂SeO₃) 0.18 mg. ME, metabolizable energy.

Performance and collection of serum and intestinal content

Body weights were recorded at 1, 21, and 35 days of age. Feed consumption was recorded at the end of the starter (21 days) and finisher phases (35 days). Body weight gain and feed conversion ratios (FCR) were calculated using the above data. At the end of the experiment (day 35), six birds (two birds per pen) were randomly selected for sampling. Blood samples were collected from the wing veins and centrifuged at 1006 g for 10 min to obtain serum. The serum was stored at -20 °C until further analysis. After collecting blood samples, the chickens were euthanized by exsanguination, and stunning was performed using an electrical stunning method at a voltage of approximately 110 V. When adjustments were required, the frequency did not exceed 800 Hz. Electrical stunning lasted approximately 3 s and exsanguination was performed within 15 s after confirmation that the broilers were fully unconscious. The abdominal cavity was opened to extract the contents of the ileum and cecum. The intestine and lymphoid organs were removed and weighed to calculate relative organ weights.

Microbial populations of intestinal content

The ileum and cecum digesta were collected and serially diluted in phosphate-buffered saline (PBS) to enumerate the microbial populations. Coliform bacteria were cultured on Chromocult® Coliform agar (Merck KGaA, Darmstadt, Germany) under anaerobic conditions at 37 °C for 24 h. Lactic acid bacteria were cultured on de Man, Rogosa, and Sharpe agar (Becton Dickinson and Company, Franklin Lakes, NJ, USA) under aerobic conditions at 37 °C for 48 h. The number of microflora was calculated and bacterial populations were expressed as log10 colony forming units (CFU) per gram of intestinal content.

Morphometric analysis of the small intestine

At the end of the experiment (35 days), one bird per replicate from each treatment group (three birds per treatment) was randomly selected and sacrificed. The duodenum begins at the junction of the gizzard and extends to the point where the bile and pancreatic ducts converge[29]. The remainder of the small intestine comprises the jejunum and ileum. Based on the definition proposed[30], the jejunum comprises the proximal four-fifths of the jejunoileum, whereas the ileum constitutes the distal one-fifth.

The ileum of each bird was sectioned. Approximately 3 cm of each segment was fixed in 10% formalin for morphometric assays. Formalin-fixed tissues were washed with PBS and embedded in paraffin wax. A sectioned tissue of 3 µm thickness was stained using hematoxylin and eosin. Samples were analyzed by light microscopy and computer software (Motic Image Plus 2.0, Motic Inc., Wetzlar, Germany) was used to measure the villus height and crypt depth in 10 favorably oriented and representative samples per treatment. The ratio of villus height to crypt depth was also calculated.

Evaluation of blood lipid profile

Serum samples were sent to the Health-Medical-Laboratory (Yunlin, Taiwan) to determine the levels of cholesterol, triglycerides, high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C).

Determination of serum antioxidant enzyme activities

Total superoxide dismutase (SOD) and catalase (CAT) activities were assayed using kits purchased from Cayman Chemical Co., Ltd. (Ann Arbor, MI, USA). Serum samples were measured in triplicate at appropriate dilutions to obtain a linear range of standard curves for enzymatic activity. Antioxidant enzyme activity is expressed as units (U) per mL of serum. The amount of malondialdehyde (MDA) was measured using an assay kit from Cayman Chemical Co., Ltd. to evaluate the degree of lipid peroxidation in the serum.

Peripheral blood mononuclear cell (PBMC) isolation

Whole blood was collected from the wing vein using a hypodermic syringe and transferred to tubes containing ethylenediaminetetraacetic acid (EDTA). After centrifugation at 150 × g for 15 min, the supernatant was removed and the same volume of PBS was added. Blood was gently layered onto Ficoll-Paque Plus (GE Healthcare Life Science, Chicago, IL, USA), centrifuged at 200 × g for 30 min, and PBMCs were collected from the gradient interface. Collected PBMCs were washed three times with PBS and centrifuged at 200 × g for 10 min each. After the supernatant was removed, 1 mL of Trizol reagent (Invitrogen, Carlsbad, CA, USA) was added and the mixture was stored at -80 °C before further analysis.

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)

Trizol-treated PBMCs were extracted with chloroform and precipitated with isopropanol to obtain an RNA pellet. The RNA pellet was washed with 75% EtOH and then resuspended in RNase-free water. RNA concentration was determined by spectrophotometry and diluted to 50 ng/μL. Total RNA was reverse transcribed to cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), and stored at -20 °C for further assessments. qPCR was performed using cDNA, Fast SYBR® Green Master Mix (Applied Biosystems), and gene-specific primers using the StepOnePlusTM Real-Time PCR System (Applied Biosystems). Gene-specific primers were designed based on the respective genes of Gallus gallus (chickens). The primer pair features are listed in Table S1.

Statistical analysis

Data have been statistically analyzed using the general linear model procedure of SAS software (1999) following a random arrangement.

The mathematical model is:

  

$Y_{ij}=T_i+{\epsilon }_{ij}$

where, $Y_{ij}$= observed response of a bird in a pen; $T_i$= fixed effect of dietary treatment; and ${\epsilon }_{ij}$= residual error when the pen is regarded as an experimental unit, ${\epsilon }_{ij}~N(0,{\sigma }_{\epsilon }^2)$. The multiple comparison procedure applied after Least-squares means was Tukey’s honestly significant difference test, with a significance level of P < 0.05.

Results

Fiber content and cellulolytic enzyme activity

Fiber content and cellulolytic enzyme activity are listed in Table 2. Cellulolytic enzyme activities were significantly higher in FWB, including xylanase (30.16 U/g dry weight [DW] vs 21.48 U/g DW, P<0.05) and mannanase (20.01 U/g DW vs 16.14 U/g DW, P<0.05). The NDF and ADF content were lower in FWB (32.84% dry matter (DM) and 7.04% DM, respectively).

Table 2.  Analysis of fiber content, cellulolytic enzymes activities, and secondary metabolites of P. linteus fermented wheat bran.

Items			WB	FWB		SEM7
	Fiber content (% DM)
		NDF	36.36a	32.84b		0.49
		ADF	9.64 a	7.04b		0.64
	Cellulolytic enzyme activity (U/g DW)
		Xylanase activity1	21.48b	30.16a		1.06
		Mannanase activity2	16.14b	20.01a		0.31
		Cellulase activity3	17.18	18.00		0.27
	Secondary metabolites
		Crude polysaccharide (mg GCE4/g DW)	78.76b	113.04a		2.85
		Ergosterol (ppm)	13.33b	31.86a		0.65
		Crude triterpenoids (mg OAE5/g DW)	2.99b	4.12a		0.08
		Adenosine (μg/g DW)	72.51b	113.05a		4.13
		Total phenolic compound (mg GAE6/g DW)	3.29b	5.43a		0.04

Values are presented as mean ± SD (n = 6). Means with different superscripts within a row represent significant differences (P < 0.05).

WB: Wheat bran; FWB: P. linteus-fermented wheat bran; DM: Dry matter; DW: Dry weight; NDF: Neutral detergent fiber; ADF: Acid detergent fiber.

¹One unit (U/g) of xylanase activity is defined as the amount of enzyme required to release 1 μmol reducing sugar (xylose equivalent) per minute from 1% xylan at 55 °C.

²One unit (U/g) of mannanase activity is defined as the amount of enzyme required to release 1 μmol reducing sugar (mannose equivalent) per minute from 0.6% locust bean gum at 37 °C.

³One unit (U/g) of cellulase activity is defined as the amount of enzyme required to release 1 μmol reducing sugar (glucose equivalent) per minute from 1% Carboxymethylcellulose sodium salt (CMC) at 50 °C.

⁴GCE: Glucose equivalent

⁵OAE: Oleanolic acid equivalent.

⁶GAE: Gallic acid equivalent.

⁷SEM: standard error of the mean.

Secondary metabolites

The determination of secondary metabolite contents (Table 2) revealed that crude polysaccharide (113.04 mg glucose equivalent [GCE]/g DW), ergosterol (31.86 ppm), crude triterpenoids (4.12 mg oleanolic acid equivalent [OAE]/g DW), adenosine (113.05 μg/g DW) and total phenolic compound (5.43 mg gallic acid equivalent [GAE]/g DW) content were significantly enhanced after PL fermentation.

In vitro antioxidant capacity

First, the DPPH scavenging ability of FWB was measured (Figure 1 (A)). Compared to 0.1 mg/mL BHT, 10 mg/mL FWB scavenged 40% of DPPH radicals and the scavenging effect increased with increasing FWB concentration. Figure 1 (B) shows the reducing power of FWB. Ascorbic acid exhibited maximum reducing power at 0.5 mg/mL and did not increase further with increasing FWB concentration. At 30 mg/mL, FWB showed a reducing power similar to that of ascorbic acid. Finally, ferrous ion chelating ability, (Figure 1 (C)), revealed that compared to 0.1 mg/mL EDTA, FWB exerted 80% chelating capacity at 10 mg/mL.

Fig. 1.

DPPH radical scavenging capacity (A), reducing power (B), and ferrous ion chelating ability (C) of aqueous extracts of P. linteus fermented wheat bran (FWB).

Values are mean ± standard deviation (SD) (n = 4). DPPH, 2,2-diphenyl-1-picrylhydrazyl; BHT, butylated hydroxytoluene; EDTA, ethylenediaminetetraacetic acid.

Growth performance

Table 3 shows the growth performance of 1–35-day-old broiler chickens fed different experimental diets. Broilers fed diets containing 5% and 10% FWB had significantly lower feed intake (FI) than those in the control group from 1–21 days of age, whereas no significant differences were observed in FCR(P<0.05). However, broilers fed the 10% FWB diet exhibited significantly lower body weight gain (BWG) compared to that of the control group at 21 days of age. Broilers fed the 5% and 10% FWB diets also showed significantly lower FI than those in the control group at 22–35 days and 1–35 days of age. In addition, broilers in the 10% FWB group had significantly reduced BWG compared to that of the control group at 35 days of age (P<0.05).

Table 3.  Effects of P. linteus fermented wheat bran on growth performance of 1–35 day-old broilers.

Items		Treatments					SEM3
		Control	5% WB	5% FWB	10% WB	10% FWB
1–21 days
	BW, g/bird1	890.5a	846.8ab	856.2ab	889.3a	807.1b	15.88
	FI, g/bird2	1066.3a	987.1bc	998.8b	1031.5ab	937.4c	19.33
	BWG, g/bird	848.6a	805.1ab	813.8ab	847.3a	764.5b	15.96
	FCR, feed/gain	1.26	1.23	1.23	1.22	1.23	0.020
22–35 days
	BW, g/bird	2385.0a	2240.7bc	2331.5ab	2195.9cd	2070.2d	40.93
	FI, g/bird	2313.8a	2134.4bc	2123.9bc	2227.7ab	2032.1c	35.11
	BWG, g/bird	1494.5a	1393.8ab	1475.3a	1306.6bc	1263.1c	38.31
	FCR, feed/gain	1.55b	1.53bc	1.44c	1.71a	1.61b	0.030
1–35 days
	FI, g/bird	3380.0a	3121.5a	3122.7b	3259.2b	2969.5c	42.99
	BWG, g/bird	2343.1a	2198.9bc	2289.1ab	2153.9c	2027.7d	40.92
	FCR, feed/gain	1.44b	1.42b	1.36c	1.51a	1.46ab	0.016

Control: basal diet (corn-soybean meal); 5% WB: 5% wheat bran; diet with 5% wheat bran; 10% WB: diet with 10% wheat bran; 5% FWB: diet with 5% P. linteus fermented wheat bran; 10% FWB: diet with 10% P. linteus fermented wheat bran; BW: body weight; FI: feed intake; BWG: body weight gain; FCR: feed conversion ratio.

¹Results are provided as the means of three replicates (20 birds/replicate) in each control and treatment group (n = 3).

²Results are provided as the means of 60 birds in each control and treatment group (n = 60).

³SEM: standard error of the mean.

^a-d Means within the same row without the same superscript letter are significantly different (P < 0.05).

Relative organ weight

Table 4 presents the effects of FWB on relative weights of the intestine and lymphoid organs of 35-day-old broilers. The relative jejunal weights of chickens in the 10% WB and 10% FWB groups were significantly higher than that of the control group (P<0.05), whereas there was no significant difference of the 5% WB and 5% FWB relative to the control group.

Table 4.  Effects of P. linteus fermented wheat bran on relative weights of intestine and lymphoid organs of 35-day-old broilers.

Items		Treatments					SEM1
		Control	5% WB	5% FWB	10% WB	10% FWB
─ g/100 g body weight ─
Intestine
	Duodenum	0.40	0.45	0.47	0.53	0.57	0.074
	Jejunum	0.91b	0.98ab	0.94b	1.21a	1.20a	0.098
	Ileum	0.72	0.80	0.77	0.82	0.86	0.053
	Ceca	0.27	0.27	0.25	0.29	0.27	0.017
Lymphoid organs
	Liver	2.30	2.31	2.38	2.33	2.51	0.083
	Spleen	0.09	0.09	0.10	0.08	0.09	0.011
	Bursa	0.20	0.21	0.20	0.21	0.23	0.019

Control: basal diet (corn-soybean meal); 5% WB: 5% wheat bran; diet with 5% wheat bran; 10% WB: diet with 10% wheat bran; 5% FWB: diet with 5% P. linteus fermented wheat bran; 10% FWB: diet with 10% P. linteus fermented wheat bran.

Each value represents the mean of six birds each in the control and treatment groups (n = 6).

¹SEM: standard error of the mean.

^{a, b}Means within the same row without the same superscript letter are significantly different (P < 0.05).

Intestinal microbiota

The effects of FWB on intestinal microbial populations in the broiler ileum and cecum are presented in Table 5. The coliform bacterial population decreased significantly in the ileum of broilers receiving 10% FWB (P<0.05); however, the population was not significantly different in the cecum between treatments. The lactic acid bacteria population in the ileum of chickens receiving 10% FWB was the highest, whereas lactic acid bacteria in the cecum were undectable at the measured concentrations.

Table 5.  Effects of P. linteus fermented wheat bran on intestinal microbial parameter of 35-day-old broilers.

Items		Treatments					SEM1
		Control	5% WB	5% FWB	10% WB	10% FWB
	─ Log CFU/g ─
Coliform bacteria
	Ileum	7.21a	7.28a	6.85ab	7.45a	6.39b	0.26
	Cecum	8.07	8.23	7.95	7.97	8.11	0.14
Lactic acid bacteria
	Ileum	8.35b	8.56ab	8.63ab	8.49ab	8.94a	0.16
	Cecum	ND	ND	ND	ND	ND

Control: basal diet (corn-soybean meal); 5% WB: 5% wheat bran; diet with 5% wheat bran; 10% WB: diet with 10% wheat bran; 5% FWB: diet with 5% P. linteus fermented wheat bran; 10% FWB: diet with 10% P. linteus fermented wheat bran; ND: not detected.

Results are presented as the mean of six birds each in the control and treatment groups (n = 6).

¹SEM: standard error of the mean.

^{a, b}Means within the same row without the same superscript letter are significantly different (P < 0.05).

Intestinal morphology

Table 6 shows the effects of FWB on the ileum morphology of broiler chickens. The ileal villus height of chickens receiving 5% FWB and 10% WB were significantly higher than those of chickens in the 5% WB and 10% FWB groups. The crypt depth of chickens in the 10% WB group was the highest among the treatments, but did not significantly differ from that of chickens in the 10% FWB group. However, the villus:crypt ratio of chickens in the 10% WB and 10% FWB groups was significantly lower than that of the control group (P<0.05).

Table 6.  Effects of P. linteus fermented wheat bran on the ileum morphology of 35-day-old broilers.

Items	Treatments					SEM1
	Control	5% WB	5% FWB	10% WB	10% FWB
Villus height (μm)	1253.2ab	1199.7bc	1301.7a	1275.9ab	1186.2c	20.77
Crypt depth (μm)	149.0c	160.6bc	164.5bc	190.1a	170.3ab	7.01
Villus/ crypt ratio	8.4a	7.7abc	8.1ab	6.9c	7.1bc	0.41

Control: basal diet (corn-soybean meal); 5% WB: 5% wheat bran; diet with 5% wheat bran; 10% WB: diet with 10% wheat bran; 5% FWB: diet with 5% P. linteus fermented wheat bran; 10% FWB: diet with 10% P. linteus fermented wheat bran.

Results are presented as the means of ten spots corresponding to six birds each in the control and treatment groups (n = 6).

¹SEM: standard error of the mean.

^a-cMeans within the same row without the same superscript letter are significantly different (P < 0.05).

Blood lipid profile

The effects of FWB on the blood characteristics of broiler chickens are shown in Table 7. Broilers fed 10% FWB exhibited significantly reduced amounts of LDL-C in serum (P<0.05). However, cholesterol, triglyceride, and HDL-C levels did not significantly differ between treatments.

Table 7.  Effects of P. linteus fermented wheat bran on blood characteristics of 35 day-old broilers.

Items	Treatments					SEM1
	Control	5% WB	5% FWB	10% WB	10% FWB
CHOL (mg/dL)	112.7	116.0	102.7	109.0	109.0	5.94
TG (mg/dL)	21.0	24.0	23.5	24.3	19.7	4.26
HDL-C (mg/dL)	73.0	72.5	75.0	75.0	72.0	3.56
LDL-C (mg/dL)	32.3a	31.0ab	28.3ab	30.0ab	23.0b	2.91

Control: basal diet (corn-soybean meal); 5% WB: 5% wheat bran; diet with 5% wheat bran; 10% WB: diet with 10% wheat bran; 5% FWB: diet with 5% P. linteus fermented wheat bran; 10% FWB: diet with 10% P. linteus fermented wheat bran; CHOL: Cholesterol; TG: Triglyceride; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol.

¹SEM: standard error of the mean.

Each value represents the mean of three replicates (n = 3).

^{a, b}Means within the same row without the same superscript letter are significantly different (P < 0.05).

Serum antioxidant parameter

Table 8 presents levels of serum antioxidant enzymes. The SOD activity of chickens in the 10% WB and both FWB treatments was significantly enhanced compared to that of the control group (P<0.05), and the enhancement effect of FWB increased with increasing FWB concentration. The serum MDA content was measured to investigate the antioxidant effects of FWB. The amount of serum MDA was significantly higher in broilers receiving 10% WB (P<0.05); however, chickens in the 10% FWB group did not exhibit a significant difference compared to that of the control group.

Table 8.  Effects of P. linteus fermented wheat bran on serum MDA and antioxidant enzymes of 35-day-old broilers.

Items	Treatments					SEM1
	Control	5% WB	5% FWB	10% WB	10% FWB
SOD (U/mL)	5.99c	9.85bc	16.04b	15.64b	25.12a	2.50
CAT (U/mL)	18.61	18.35	18.68	22.15	16.33	2.89
MDA (μM)	7.09b	9.49ab	9.12ab	10.97a	7.83b	0.85

Control: basal diet (corn-soybean meal); 5% WB: 5% wheat bran; diet with 5% wheat bran; 10% WB: diet with 10% wheat bran; 5% FWB: diet with 5% P. linteus fermented wheat bran; 10% FWB: diet with 10% P. linteus fermented wheat bran; SOD: Superoxide dismutase; CAT: Catalase; MDA: Malondialdehyde.

¹SEM: standard error of the mean.

Each value represents the mean of six birds each in the control and treatment groups (n = 6).

^a-c Means within the same row without the same superscript letter are significantly different (P < 0.05).

Gene expression in PBMCs

Figure 2 (A)–(G) presents the expression of antioxidant-related genes, and Figure 2 (H)–(N) shows the expression of proinflammatory cytokines. Overall, FWB enhanced the expression of antioxidant genes. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2), which plays an important role in modulating antioxidant capacity, was significantly enhanced in broilers that received FWB. Although there was no significant difference between FWB and WB treatments in heme oxigenase 1 (HO-1) expression, a gene downstream of Nrf2, the HO-1 expression in these treatment groups was higher than that in the control group. The expression levels of glutathione S-transferase (GST), glutamate-cysteine ligase catalytic subunit (GCLC), and SOD, which are downstream genes of Nrf2, are significantly higher in chickens in the FWB group compared to those in the control and WB groups (P<0.05). Reactive oxygen species modulator protein 1 (ROMO1) and NADPH oxygenase 1 (NOX1) promote the generation of reactive oxygen species (ROS). The expression of NOX1 was significantly reduced in chickens receiving FWB and WB (P<0.05), but there was no difference in ROMO1 gene expression.

Fig. 2.

Effects of P. linteus fermented wheat bran (FWB) on mRNA expression levels of antioxidant-related genes and proinflammatory cytokines in chicken peripheral blood mononuclear cells (PBMCs) of 35-day-old broilers.

Values are expressed as the mean ± standard deviation (SD) of six samples (n = 6).

^a-dMeans with different superscript letters are significantly different (P < 0.05).

Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase 1; GST, glutathione S-transferase; GCLC, glutamate-cysteine ligase catalytic subunit; SOD, superoxide dismutase; ROMO1, reactive oxygen species modulator protein 1; NOX-1, NADPH oxygenase 1; NF-κB, nuclear factor-κB; IL-6, interleukin-6; IL-1β, interleukin-1β; NLRP3, Nod-like receptor protein 3; TLR-4, Toll-like receptor 4; iNOS, inducible nitric oxide synthase; COX2, cyclooxygenase 2.

Fig. 2.

Effects of P. linteus fermented wheat bran (FWB) on mRNA expression levels of antioxidant-related genes and proinflammatory cytokines in chicken peripheral blood mononuclear cells (PBMCs) of 35-day-old broilers.

Values are expressed as the mean ± standard deviation (SD) of six samples (n = 6).

^a-dMeans with different superscript letters are significantly different (P < 0.05).

Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase 1; GST, glutathione S-transferase; GCLC, glutamate-cysteine ligase catalytic subunit; SOD, superoxide dismutase; ROMO1, reactive oxygen species modulator protein 1; NOX-1, NADPH oxygenase 1; NF-κB, nuclear factor-κB; IL-6, interleukin-6; IL-1β, interleukin-1β; NLRP3, Nod-like receptor protein 3; TLR-4, Toll-like receptor 4; iNOS, inducible nitric oxide synthase; COX2, cyclooxygenase 2.

Nuclear factor (NF)-κB is an important cytokine that regulates inflammation. These results suggested that addition of WB and FWB promoted the expression of NF-κB, but the effect decreased as the FWB percentage increased. The same expression pattern was observed for Toll-like receptor 4 (TLR-4) (an upstream gene of NF-κB). However, expression of the genes downstream of NF-κB, interleukin (IL)-6, and IL-1β were significantly higher in chickens in the WB groups (P<0.05). There were no effects on IL-6 and IL-1β expression in chickens in FWB groups; in fact, the expression of IL-6 was suppressed. An expression pattern similar to that of IL-1β was noted for Nod-like receptor protein 3 (one of the promoter genes of IL-1β). The activation of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) promotes the production of nitric oxide and prostaglandin E2, which causes further inflammation. Chickens receiving WB significantly suppressed the expression of iNOS, but promoted COX2 expression (P<0.05). However, iNOS expression in chickens in FWB groups was significantly lower than that in chickens in WB groups (P<0.05), whereas COX2 expression did not significantly differ from that of the control group. In summary, FWB exhibited inflammation-suppressive effects.

Discussion

WB has been widely used to feed domestic animals; however, its relatively high fiber content affects the starch, lipid, and protein digestibility of monogastric animals[8], further deteriorating their growth or production performance[8]. This study evaluated the effects of PL–fermented WB supplementation on growth performance, intestinal morphology, gut microbial composition, and associated gene expression in broilers reared during summer conditions.

The WB fiber content significantly decreased after PL fermentation, along with an increase in hemicellulolytic enzyme activities and secondary metabolites. Solid-state fermentation is a commonly used method to produce enzymes and secondary fungal metabolites[1]. PL is a wood-decaying fungus that secretes lignocellulolytic enzymes to degrade fiber content for growth[15], and secondary metabolites accumulate as mushrooms grow. Dashtban et al.[31] have indicated that white-rot fungi selectively degrade lignocellulose, retaining the nutritional component of cellulose. Therefore, these findings suggested that PL fermentation did not increase cellulase activity. These results further revealed that FWB exhibited antioxidant activity. Functional metabolites of PL, such as polysaccharides and phenolic compounds, have antioxidant properties[32]. Liang et al.[20] have shown that the antioxidant activities of adlay and polished rice are enhanced by PL fermentation.

Heat stress is widely recognized as a major environmental challenge in broiler production that leads to impaired growth performance, disrupted gut microbial composition, altered immune status, and intestinal damage[33]. Heat stress significantly reduces the average daily gain (ADG) and body weight in broilers[33], increases the relative abundance of Proteobacteria while decreasing Firmicutes, and elevates the heterophil-to-lymphocyte ratio due to lymphocyte depletion and increased heterophil counts[34]. In addition, heat stress induces intestinal morphological alterations and mucosal injury due to reduced blood flow, compromises nutrient and oxygen supply, and decreases FI[35]. These results revealed that FCR in broilers receiving 5% WB was not adversely affected, but as the FWB amount increased to 10%, the growth performance of broilers deteriorated. Because PL fermentation might effectively reduce the fiber content of WB, broilers receiving 5% FWB exhibited improved FCR compared to that of broilers receiving 5% WB or the control group. Alemawor et al.[36] have shown that substituting broiler diets with 10% Pleurotus ostreatus-fermented cocoa pod husks does not have adverse effects on the growth performance of broilers. However, as the replacement amount increased, the FCR of broilers also increased. Alemawor et al.[36] have indicated that although fungal fermentation decreases the fiber content of substrates, ingestion of excessive fiber still exerts adverse effects on broiler growth performance[37]. Pires-Filho et al.[38] report that broilers fed diets containing graded levels of dried brewer’s grains (0, 20, 40, 60, 80, 100, and 120 g/kg) exhibit a linear reduction in body weight gain from 1–63 days of age, accompanied by a linear increase in the FCR. Similarly, Li et al[39]. have demonstrated that dietary inclusion of 30% wet-fermented brewer’s grains (WFBG) significantly decreases both ADG and average daily feed intake. These negative effects are attributed to the limited capacity of broilers to digest crude fiber, nutritional imbalance, and anti-nutritional factors associated with excessive WFBG inclusion[40]. Furthermore, metabolomic analyses reveal that high-dose WFBG supplementation markedly alters the serum and liver metabolite profiles in broilers, with 504 differentially expressed metabolites (DEMs; 223 upregulated and 281 downregulated) identified in liver tissue and 185 DEMs in serum (159 upregulated and 26 downregulated) compared to that of the control group[40]. These findings suggest that excessive dietary WFBG may disrupt amino acid metabolism and that the identified DEMs may serve as potential biomarkers of high-dose WFBG–induced intestinal dysfunction in broiler chickens. In the present study, dietary supplementation with 10% FWB exerted adverse effects on the growth performance of broilers, which may be attributed to the high fiber content of the diet and its potential interference with amino acid metabolism.

The non-starch polysaccharides in WB increase the viscosity of the chyme in the digestive tract, further affecting the ability of digestive enzymes to exert their effects, resulting in reduced feed efficiency[8]. To improve the digestibility of nutrients, the intestinal microflora proliferate to enhance enzyme secretion, thus increasing intestinal weight[41], which is consistent with the current findings.

This study suggests that FWB favorably improved the gut microflora. Studies have shown that dietary fiber acts as a prebiotic to promote the growth of beneficial bacteria, further reducing the growth or adhesion of harmful bacteria, thereby improving the gut health of the host[42]. Nowak et al.[43] have indicated that fungal polysaccharides promote the growth of Lactobacillus acidophilus and L. rhamnosus, and Reis et al.[44] have identified polysaccharides, glucans, and triterpenoids. Similarly, in the current study, the MeOH and EtOH extracts isolated from the PL fruiting body exhibited inhibitory effects on Staphylococcus aureus, Salmonella typhimurium, and Escherichia coli(data not shown).

The ratio of villus height to crypt depth (V/C ratio) is considered the evaluation index of intestinal digestion and absorption capacity. Montagne et al.[45] report that sticky chymes damage villous cells, leading to villous atrophy. To repair damaged villi, the proliferation rate of crypt cells increases, causing an increase in crypt depth and a reduction in the V/C ratio. The current findings suggest that PL reduced the fiber content of WB, leading to decreased chyme viscosity, improving the V/C ratio.

LDL is one of the carriers of cholesterol, which transports cholesterol from diets or the liver to various tissues for cell utilization[46]. However, it tends to accumulate on the walls of arteries when its concentration is too high, leading to atherosclerosis[46]. Zou et al.[47] report that polysaccharides isolated from PL mycelia reduce triglyceride and LDL-C levels in hyperlipidemic mice. However, the present study only identified an LDL-C-reducing effect in the blood content of broiler chickens.

Antioxidant enzymes, such as SOD and CAT, scavenge ROS or free radicals and prevent oxidative stress or damage[48]. These findings suggest that both WB and FWB diets enhance SOD activity in broiler serum. Surai[48] has indicated that phenolic compounds or flavonoids originating from plants improve the antioxidant capacity of poultry by enhancing SOD activity. Inotilone, isolated from the PL fruiting body, promotes SOD activity in mice with Carr-induced paw edema[49]. Moreover, feruloyl oligosaccharides produced in microbially fermented WB enhance SOD activity in Sprague-Dawley rats[50]. Thus, the present study revealed that SOD activity in chickens in FWB groups was significantly higher than that in chickens in WB groups.

Because WB is a feed ingredient with low energy content[8], the amount of full-fat soybean meal in the present study was increased with WB and F WB content to meet the energy requirements of broilers. Lu and Chiang[23] report that the serum MDA content of mice is proportional to the fat content in their diet, which is also noted in the present study. The MDA level is also an indicator of lipid peroxidation[23]. These findings suggest that increased SOD activity may reduce serum MDA production, an effect that is also reported by Surai[48], who indicates that SOD prevents lipid peroxidation.

To further understand the antioxidant-regulatory effects of FWB, the expression of antioxidant-related genes was determined. The current results showed increased expression of antioxidant-related genes in broilers receiving either WB or FWB, whereas expression levels of Nrf2, GST, GCLC, and SOD were significantly higher in broilers in the FWB groups. Nrf2 is a redox-sensitive transcription factor that plays a key role in regulating the expression of many antioxidant or detoxifying enzymes such as HO-1, GST, and glutamate cysteine ligase (GCL)[51]. HO-1 is an essential cytoprotective component against oxidative tissue injury that metabolizes free heme to iron, CO, and biliverdin[52]. GST catalyzes the conjugation of electrophilic substrates to glutathione (GSH), thus reducing ROS content[53]. GCL is composed of a catalytic subunit (GCLC) and modulatory subunit (GCLM), and is the rate-limiting enzyme in the de novo synthesis of GSH[53]. Kim et al.[54] report that the n-BuOH subfraction of the PL fruiting body increases the expression of Nrf2 and HO-1 in RAW264.7 macrophage cells. In another study, Shon and Nam[55] have shown that butyl alcohol or water extracts of PL enhances GST activity. In addition, the current findings suggest that broilers receiving WB or FWB exhibited reduced NOX-1 expression. Because ROMO1 and NOX-1 have been confirmed as transcription factors involved in the production of intracellular ROS, reducing NOX-1 expression decreases cellular oxidative stress[56].

In addition to cytotoxicity, ROS also cause the proliferation of proinflammatory cytokines, leading to inflammation[56,57]. Hence, in the present study, we investigated the expression of proinflammatory cytokines. These results suggest that the expression of NF-κB, TLR-4, IL-6, IL-1β, and COX-2 were upregulated in broilers that received WB. In contrast, FWB supplementation reduced the expression of proinflammatory cytokines. When TLR-4, a receptor on the cell membrane is stimulated, it promotes NF-κB expression, a widely distributed transcription factor that also regulates the expression of proinflammatory cytokines, such as IL-6, IL-1β, iNOS, and COX-2[59]. Similar to the current results, Shen et al.[58] have shown that polysaccharides isolated from WB promote the expression of TLR-4, NF-κB, and COX-2 in RAW264.7 macrophages. However, the EtOH extracts of PL suppressed the expression of TLR-4 and inhibited the activation of NF-κB in lipopolysaccharide-stimulated RAW264.7 macrophages, further reducing the expression of iNOS and COX-2. Moreover, the expression of IL-6 and IL-1β is inversely proportional to the concentration of EtOH extracts of PL[57]. Wu et al.[59] have also indicated that phenolic compounds derived from PL inhibit the activation of NF-κB, in line with the current findings.

In summary, the fermentation of PL improved the value of WB by increasing the activity of cellulolytic enzymes, secondary metabolite content, and antioxidant capacity. Only one fermentation condition was evaluated in this study. Consequently, further optimization of fermentation parameters, including microbial strains, fermentation time, temperature, and substrate composition, should be conducted to improve the functional properties of FWB. Although supplementation with 5% FWB produced the most consistent effects in the present study, the optimal inclusion level may vary depending on production stage or feeding strategy, and should be further examined.

Ethical Approval

The care and use of all broilers were according to the Regulations of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at Taichung, Taiwan

Funding

This work was supported by National Science and Technology Council (NSTC 112-2313-B-005-043-MY3) and the Ministry of Education (MOE 113-S-0023-A) in Taiwan.

Acknowledgments

The authors would like to thank their colleagues at the National Science and Technology Council (NSTC 112-2313-B-005-043-MY3) and the iEGG and Animal Biotechnology Center from The Feature Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE 114-S-0023-A) in Taiwan for supporting this study. Professor Ching-Hsien Chao assisted with statistical explanations. The funders had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.

Author Contributions

Conceptualization: Tzu Hsiang Wei and Tzu Tai Lee; Data curation: Tzu Hsiang Wei; Formal analysis: Tzu Hsiang Wei and Shen Chang Chang; Funding acquisition: Tzu Tai Lee; Investigation: Min Jung Lin; Methodology: Li Jen Lin; Project administration: Tzu Tai Lee; Resources: Tzu Tai Lee; Software: Tzu Hsiang Wei and Shen Chang Chang; Supervision: Tzu Tai Lee; Validation: Li Jen Lin; Visualization: Tzu Tai Lee; Writing: original draft: Tzu Tai Lee; Writing: review and editing: Tzu Tai Lee.

Conflict of Interest

The authors declare no conflict of interest.

Declaration of AI and AI-assisted Technologies

No AI tools were used in this article.

Supplementary Materials

The online version contains supplementary material available at

https://doi.org/10.2141/jpsa.2026013

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