Food and Agricultural Immunology

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/cfai20

Effects of long-term dietary supplementation of

fermented wheat bran on immune performance
and inflammatory response in laying hens

Yu Wang, Beibei He, Kuanbo Liu, Jingjing Shi, Aike Li, Junlin Cheng, Yuanyuan
Wei, Shuangshuang Guo, Yongwei Wang & Binying Ding

To cite this article: Yu Wang, Beibei He, Kuanbo Liu, Jingjing Shi, Aike Li, Junlin Cheng,
Yuanyuan Wei, Shuangshuang Guo, Yongwei Wang & Binying Ding (2022) Effects of
long-term dietary supplementation of fermented wheat bran on immune performance and
inflammatory response in laying hens, Food and Agricultural Immunology, 33:1, 150-166, DOI:
10.1080/09540105.2021.2025346

To link to this article: https://doi.org/10.1080/09540105.2021.2025346

© 2022 The Author(s). Published with

license by Taylor & Francis Group, LLC.

Published online: 21 Feb 2022.

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FOOD AND AGRICULTURAL IMMUNOLOGY
2022, VOL. 33, NO. 1, 150–166
https://doi.org/10.1080/09540105.2021.2025346

Effects of long-term dietary supplementation of fermented

wheat bran on immune performance and inflammatory
response in laying hens
Yu Wanga,b, Beibei Heb, Kuanbo Liub, Jingjing Shib, Aike Lib, Junlin Chengb,
Yuanyuan Weia, Shuangshuang Guoa, Yongwei Wangb and Binying Dinga
a
Hubei Key Laboratory of Animal Nutrition and Feed Science, Wuhan Polytechnic University, Wuhan,
People’s Republic of China; bAcademy of National Food and Strategic Reserves Administration, Beijing,
People’s Republic of China

ABSTRACT ARTICLE HISTORY

This study was conducted to investigate the effects of solid-state Received 2 November 2021
fermented wheat bran on immune performance and inflammatory Accepted 30 December 2021
response in laying hens. A total of 225 18-week-old Hy-Line
KEYWORDS
brown-egg laying hens were randomly divided into 3 groups with Fermented wheat bran;
5 replicates per group and 15 hens per replicate. Laying hens were immune performance;
fed a basal diet (corn-soybean meal diet) supplemented with 0 inflammatory response;
(control group), 10% wheat bran and 10% fermented wheat bran, laying performance;
respectively. The results showed: (1) Compared to wheat bran reproductive hormones;
group, the contents of crude protein, trichloroacetic acid-soluble laying hens
protein, dietary fibre (DF), mannan and total polyphenols in wheat
bran were increased by solid state fermentation. (2) Compared to
the control group, fermented wheat bran increased the levels of
serum biochemical parameters, reproductive hormones,
immunoglobulins and anti-inflammatory factors. Therefore, long-
term dietary supplementation of 10% fermented wheat bran plays
an important role in improving the immune performance of laying
hens.

Introduction
With the growth of global population, competition between grain and animal feed has
become increasingly serious, which will threaten grain security (Kim et al., 2019). Along
with the intensive feeding mode and the prohibition of antibiotics, some physiological
health problems may occur during laying stage, such as decreased immune performance,
which further reduces the resistance of laying hens to diseases, and ultimately leads to
decreased laying performance and egg quality. Soybean meal is the main protein feedstuff
in animal feed because of its high feeding value (Banaszkiewicz, 2011). However, the con-
tradiction between supply and demand of soybeans is greatly severe in China, so it is critical

CONTACT Yongwei Wang wyw@ags.ac.cn Academy of National Food and Strategic Reserves Administration,
Beijing 100037, People’s Republic of China; Binying Ding dbying7471@126.com Hubei Key Laboratory of Animal
Nutrition and Feed Science, Wuhan Polytechnic University, Wuhan 430023, People’s Republic of China
© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License
(http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any
medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
 FOOD AND AGRICULTURAL IMMUNOLOGY 151

to expand the feed resources to substitute soybean meal. Wheat bran, a by-product of wheat
starch process, is a rich feedstuff. The global annual output is 112 million tons, and China
accounts for 20 million tons (Hell et al., 2014; Prückler et al., 2014). However, wheat bran
contains 10%∼26% arabinoxylan, 11% cellulose, and 5.6% lignin (Apprich et al., 2014).
These non-starch polysaccharides (NSP) limit the application proportion of wheat bran
in animal dites (Balandrán-Quintana et al., 2015; Onipe et al., 2015).
Biological fermentation can improve the nutritional properties of wheat bran.
Microbial species play a key role in fermentation process. Zhao et al. (2017) discovered
that when wheat bran was fermented using commercial baker’s yeast and lactic acid bac-
teria, the levels of phytic acid and arabinoxylan were reduced, cell wall components were
degraded, and more taste compounds were produced. The total phenols content of wheat
bran increased during yeast fermentation, and the taste compounds were primarily 4-
ethyl-2-methoxyphenol (Tu et al., 2020). Furthermore, wheat bran fermented with Tri-
choderma might boost xylanase and cellulase activity. Lin and Lee (2020a) and Chu et al.
(2016) reported that dietary supplementation with 5% fermented wheat bran could effec-
tively improve the growth performance and intestinal health of broilers. Teng et al.
(2017) also reported that wheat bran fermented with Bacillus amylolytic increased the
content of lactobacillus and lactic acid in the ileum of broilers. Although the fermenta-
tion process increased the level of bioactive substances in wheat bran, the effect of fer-
mented wheat bran on laying hens needs to be further studied. Therefore, this study
was conducted to investigate the effects of long-term dietary supplementation of fermen-
ted wheat bran on laying performance, egg quality, serum biochemical indexes, repro-
ductive hormones, immune performance and Inflammatory response in laying hens.

Materials and methods

Preparation of solid-state fermented wheat bran
The basal solid-state fermentation substrates containing 98% wheat bran and 2% glucose
were mixed with water at a ratio of 1:0.8 (w:w), Lactobacillus plantarum, Bacillus subtilis,
Saccharomyces cerevisiae and Aspergillus fragrans were mixed at a ratio of 3:2:4:1 with an
inoculation concentration of 0.5% (v/v). Saccharomyces cerevisiae was purchased from
the Angel Yeast Co., Ltd (Yichang, China), and the other strains were preserved by
our laboratory. The mixture was fermented in stainless steel fermentation apparatus
(Figure 1, diameter 1360 mm, height 1360 mm) at 30°C for 3 days and stirred daily.
The contents of Lactobacillus, Bacillus subtilis, yeast and Aspergillus in fermented wet
wheat bran were 7.78, 5.62, 5.24 and 4.93 log CFU/g, respectively, which were determined
according to ISO (2013). Fresh samples of wet wheat bran were dried in a fluidized bed.

Determination of nutrient composition

The moisture (AOAC, 934.01), dietary fibre (AOAC, 993.21), crude fibre (AOAC, 962.09),
crude ash (AOAC, 942.05), crude fat (AOAC, 920.39), and crude protein (AOAC, 990.03)
were determined according to the AOAC method (AOAC, 2007). The total phosphorus
was determined by an ultraviolet spectrophotometer (MAPADA P9, Shanghai, China),
and gross energy was measured by the oxygen bomb calorimeter (IKA C6000, Germany),
152 Y. WANG ET AL.

Figure 1. Schematic of fermenter. Generator (a), thermometer (b), stirrer (c), Material outlet (d),feed
inlet (e).

according to the AOCS method (AOCS, 2009). The total polyphenols were measured by
Folin-phenol method using gallic acid as a calibration curve (Emmons et al., 1999). The tri-
chloroacetic acid-soluble protein was measured according to Zhang and Shen (2013). Crude
polysaccharides were measured by phenol-sulfuric acid method with glucose as the reference
(Dubois et al., 1956). The mannan was determined by high performance liquid chromato-
graphy with a standard curve derived from gradient concentrations of mannose solution
(Waters e2695, USA). The specific determination conditions were as follows: 250 nm UV
wavelength, mobile phase consisted of acetonitrile and ammonium formate at a ratio of
2:8, the column size was C18 (250 mm × 4.6 mm × 5μm), and the sample volume was 10
μL. The three mycotoxins were determined by high performance liquid chromatography
(Waters e2695, USA), using methanol, acetonitrile and water as mobile phases, the chroma-
tographic column was C18, in which C18 (150 mm × 4.6 mm × 5 μm) was used for zearale-
none (ZEN) and deoxynivalenol (DON), and C18 (250 mm × 4.6 mm × 5 μm) was used for
aflatoxin B1 (AFB1) (Herzallah, 2009; Rahmani et al., 2010).

Birds and experimental management

Laying hens were raised and handled in accordance with the guidelines of the Animal
Ethics Committee of the Academy of National Food and Strategic Reserves Administration
(Beijing, China). The animal experiment was performed at the Wuqing base of the
National Food and Strategic Reserves Administration (Tianjing, China).
 FOOD AND AGRICULTURAL IMMUNOLOGY 153

Table 1. Ingredients and nutrient level of experimental diets for laying hens.
Experimental diets
Item Control Wheat bran Fermented wheat bran
Ingredients (%, as-fed basis)
Corn 62.58 53.60 53.47
Soybean meal 24.53 17.80 19.60
Corn gluten meal 3.46 1.94
Wheat bran 10.00
Fermented wheat bran 10.00
Dicalcium phosphate 1.05 1.00 0.98
Limestone 10.00 10.00 10.00
Soybean oil 0.66 2.68 2.65
Vitamin premix1 0.30 0.30 0.30
Mineral premix2 0.20 0.20 0.20
Choline Chloride (50%) 0.10 0.10 0.10
Salt 0.30 0.30 0.30
DL-methionine (98%) 0.153 0.14 0.147
L-Lysine• HCl (98%) 0.084 0.32 0.242
Threonine (98%) 0.06 0.03
Complex enzyme3 0.02 0.02 0.02
Phytase 0.02 0.02 0.02
Total 100 100 100
Calculated nutrient value
Crude protein 16.65 16.66 16.65
Metabolizable energy (MJ/kg) 11.30 11.30 11.30
Total phosphorus 0.59 0.61 0.62
Available phosphorus 0.39 0.39 0.39
Calcium 4.00 4.00 4.00
Lysine 0.88 0.88 0.88
Methionine 0.40 0.40 0.40
Note: 1Vitamin premix provided the following per kilogram of diet: vitamin A, 12,500 IU; vitamin D3, 2,500 IU; vitamin E,
15 IU; vitamin K3,2.65 mg; vitamin B1, 2 mg; vitamin B2, 6 mg; vitamin B12, 0.025 mg;nicotinic acid, 50 mg; calcium
pantothenate, 12 mg; biotin, 0.0325 mg;folic acid, 1.25 mg
2
The mineral premix provided the following per kg of diet: iron, 80 mg;copper, 8 mg; manganese, 100 mg; zinc, 75 mg;
iodine, 0.35 mg; selenium,0.15 mg.
3
Complex enzymemainly includes: hydrolyase, Pectinase, β-mannanase, cellulase, β-glucanase, protease.

A total of 225 18-week-old Hy-Line brown-egg laying hens were selected and ran-
domly divided into 3 treatments with 5 replicates per treatment and 15 laying hens
per treatment. Hens in three groups were fed a corn-soybean meal basal diet and two
experimental diets, in which the basal diet was supplemented with 10% wheat bran or
10% fermented wheat bran to partly replace soybean meal with iso-nitrogen and iso-
energy principle. The composition and nutrient levels of three diets (Table 1) met or
exceeded the nutrient requirements of Chinese feeding standard of chicken (NY/T,
2004). The experiment lasted for 27 weeks and was divided into four stages (18∼27
weeks, 28∼34 weeks, 35∼39 weeks, and 40∼44 weeks).
Laying hens were kept in battery cages with 16 h of light, and the room temperature
was controlled at 20°C∼25°C, humidity 30%∼60%. According to the management pro-
cedures, regular ventilation, disinfection, and vaccination were carried out.

Laying performance
Eggs were collected every afternoon. The egg number and weight, laying rate, broken egg
rate, feed intake and feed to egg ratio were all recorded and calculated at 18∼27 weeks,
28∼34 weeks, 35∼39 weeks and 40∼44 weeks.
154 Y. WANG ET AL.

Egg quality
Three eggs were randomly selected from each replicate at the 27th, 34th and 39th weeks
to determine egg quality. Each egg was weighed individually. The egg shell thickness
Gauge and Egg Force Reader (ORKA Food Technology Ltd, Ramat Hasharon, Israel)
were used to measure the eggshell strength and thickness, respectively. The egg shape
index (ratio of longitudinal diameter to transverse diameter) was measured by Vernier
Callipers (Syntek, China). The Egg Analyzer (ORKA Food Technology Ltd, Ramat
Hasharon, Israel) was used to measure yolk colour, albumen height and Haugh unit.

Serum biochemical parameters

Blood was collected from the wing veins of randomly selected laying hens before slaugh-
ter. The blood was centrifuged to obtain the serum, which was stored in the refrigerator
at −20°C. Analyses were performed using an automatic biochemical analyzer (Mairui BS-
420 autoanalyzer, Shenzhen, China). Serum triglyceride (TG), high density lipoprotein
(HDL), trimethyl aminoxide (TMAO), total cholesterol (TC), uric acid (UA), albumin
(ALB), globulin (GLB), total protein (TP), calcium (Ca) and phosphorus (P) were deter-
mined by the colorimetric method. The kits were purchased from a commercial company
(BioSino Bio-Technology and Science Inc, Beijing, China).

Serum reproductive hormone

The serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH),
estradiol (E2), progesterone (P4) and gonadotropin-releasing hormone (GnRH) in
laying hens were determined by ELISA diagnostic kits from the Beijing Sino-UK Institute
of Biological Technology (Beijing, China).

Serum immunology and inflammatory factors

The serum immunoglobulin and lipopolysaccharide (LPS) levels were determined by col-
orimetry using kits (BioSino Bio-Technology and Science Inc, Beijing, China), and the
serum levels of interleukin-2 (IL-2), interleukin-10 (IL-10) and interferon-γ (IFN-γ)
were analyzed by kits from Beijing Sino-UK Institute of Biological Technology
(Beijing, China).

Immune indexes of jejunal mucosa

The jejunum was collected and the content was removed. Mucosa was scraped with glass
slides, put in a 1.5 mL centrifuge tube, and stored in a refrigerator at −20°C. After
thawing, normal saline was added at a proportion of 1:9, and then homogenized with
a homogenizer (Wheaton, USA), and centrifuged for 10 min at 4000 rpm, 4°C. The
supernatant was collected for assay (Chen et al., 2014). The secretory immunoglobulin
A (sIgA, CSB-E10097Ch) and interleukin-2 (IL-2, CSB-E06755Ch) were determined
by ELISA kits (CUSABIO, Wuhan, China).
 FOOD AND AGRICULTURAL IMMUNOLOGY 155

Statistical analysis
The statistical analysis was performed with SPSS 20.0 software. The indexes of wheat
bran before and after fermentation were analyzed by an independent T-test. Laying per-
formance and egg quality were analyzed by two-way ANOVA, and multiple comparisons
were performed by the Duncan method. There was statistical significance at P≤ 0.05.

Results
Nutrient composition of wheat bran and fermented wheat bran
The changes in the nutrient composition of wheat bran by fermentation are shown in Table 2.
After fermentation, the contents of crude protein, trichloroacetic acid-soluble protein, crude
ash, gross energy, soluble dietary fibre, insoluble dietary fibre, total dietary fibre, mannan and
total polyphenols in wheat bran were significantly increased (P < 0.05), while the levels of
crude fat, crude fibre, aflatoxin B1 and zearalenone levels were effectively reduced (P < 0.05).

Laying performance
The effects of different dietary treatments and different production periods on the laying
performance of laying hens were displayed in Table 3. There were no significant effects
on the laying performance parameters among all the groups (P > 0.05). With the laying
time increasing, the egg production, feed conversion ratio and broken egg rate of laying
hens decreased, whereas the average daily feed intake, average egg weight and laying rate
were increased (P < 0.05).

Egg quality
The egg quality indexes of 3 experimental treatments and 3 experimental periods were
summarized in Table 4. Different dietary treatments and experiment periods had no

Table 2. Composition changes of wheat bran before and after fermentation (%, air-dry basis).
Component Wheat bran Fermented wheat bran P-value
Dry matter 90.03 94.45
Crude protein 14.94 ± 0.06a 18.02 ± 1.23b 0.005
Trichloroacetic acid-soluble protein 1.48 ± 0.01a 5.08 ± 1.84b 0.005
Crude fat 3.78 ± 0.06b 2.40 ± 0.39a 0.001
Crude fibre 16.01 ± 0.01b 14.65 ± 0.68a 0.044
Crude ash 5.34 ± 0.06a 7.83 ± 0.34b < 0.001
Total phosphorus 1.17 ± 0.01 1.21 ± 0.09 0.061
Gross energy (MJ/kg) 8.15 ± 0.01a 8.89 ± 0.41b 0.019
Nitrogen-free extract 51.04 ± 0.34 48.21 ± 3.63 0.072
Soluble dietary fibre 1.11 ± 0.08a 2.57 ± 0.87b 0.032
Insoluble dietary fibre 30.12 ± 0.08a 46.08 ± 1.66b < 0.001
Total dietary fibre 31.24 ± 0.17a 49.42 ± 2.33b < 0.001
Mannan 0.23 ± 0.01a 0.49 ± 0.13b 0.024
Total polyphenol (mg/g) 3.27 ± 0.01a 10.07 ± 2.57b < 0.001
Crude polysaccharide (mg/g) 9.02 ± 0.10 16.24 ± 12.24 0.189
Aflatoxin B1 (μg/kg) 0.22 ± 0.01b 0.13 ± 0.02a < 0.001
Zearalenone (μg/kg) 6.36 ± 0.49b 1.26 ± 0.25a < 0.001
Deoxynivalenol (μg/kg) 924.20 ± 20.99 889.18 ± 15.73 0.081
ab
Note: Means within a row with different letters differ significantly (P < 0.05).
 156
Table 3. Effects of fermented wheat bran on laying performance ofhens/

Y. WANG ET AL.
Item Treatment Time Week P-value
Week 18∼27 Week 28∼34 Week 35∼39 Week 40∼44 18∼27 28∼34 35∼39 40∼44 Treatment Time Treatment×Time
c b a a
Egg production Control 37.92 ± 2.08 33.94 ± 0.47 24.90 ± 0.37 24.06 ± 0.34 39.34 33.84 24.74 23.72 0.296 <0.001 0.149
Wheat bran 37.60 ± 2.34 33.68 ± 0.75 24.84 ± 0.88 23.56 ± 0.50
Fermented 42.50 ± 1.04 33.90 ± 0.88 24.48 ± 0.82 23.54 ± 0.54
wheat bran
Average daily feed Control 99.98 ± 1.37 116.70 ± 4.04 115.12 ± 2.01 124.18 ± 2.77 101.55a 118.89b 116.37b 115.11b 0.733 <0.001 0.085
intake (g)
Wheat bran 100.44 ± 3.40 119.00 ± 2.12 116.94 ± 2.13 111.98 ± 6.70
Fermented 104.22 ± 2.36 120.52 ± 2.85 117.06 ± 2.13 109.18 ± 5.99
wheat bran
feed conversion Control 2.67 ± 0.10 1.99 ± 0.05 2.08 ± 0.05 2.19 ± 0.06 2.64b 2.04a 2.12a 2.04a 0.636 <0.001 0.402
ratio
Wheat bran 2.75 ± 0.24 2.06 ± 0.04 2.10 ± 0.06 1.99 ± 0.09
Fermented 2.51 ± 0.08 2.11 ± 0.04 2.11 ± 0.09 1.96 ± 0.08
wheat bran
Average egg Control 57.66 ± 0.37 61.56 ± 0.72 62.32 ± 0.71 63.76 ± 1.14 57.30a 61.39b 62.43c 64.03d 0.078 <0.001 0.951
weight (g)
Wheat bran 57.48 ± 0.46 61.80 ± 0.44 63.08 ± 0.36 64.48 ± 0.40
Fermented 56.76 ± 0.20 60.80 ± 0.40 61.88 ± 0.37 63.86 ± 0.18
wheat bran
Laying rate (%) Control 65.40 ± 3.58 94.26 ± 1.30 88.90 ± 1.33 89.12 ± 1.29 67.85a 93.99c 88.36b 87.84b 0.556 <0.001 0.368
Wheat bran 64.86 ± 4.04 93.56 ± 2.06 88.76 ± 3.16 87.22 ± 1.88
Fermented 73.28 ± 1.82 94.14 ± 2.42 87.42 ± 2.91 87.18 ± 2.04
wheat bran
Broken egg rate Control 1.31 ± 0.24 0.55 ± 0.24 1.28 ± 0.19 1.04 ± 0.41 1.47b 0.59a 1.05ab 1.15ab 0.114 0.045 0.457
(%)
Wheat bran 1.60 ± 0.14 0.83 ± 0.20 1.17 ± 0.39 1.81 ± 0.91
Fermented 1.50 ± 0.23 0.40 ± 0.06 0.69 ± 0.37 0.61 ± 0.15
wheat bran
Note: abcd Means within a row with different letters differ significantly (P < 0.05).
 FOOD AND AGRICULTURAL IMMUNOLOGY 157

effect on eggshell strength or Haugh unit of laying hens (P > 0.05). The egg weight, egg-
shell thickness, yolk colour and albumen height at 34 and 39 weeks were significantly
higher than those at 27 weeks (P < 0.05). The egg shape index at 34 weeks was signifi-
cantly lower than that at 27 weeks, while the egg shape index at 39 weeks was significantly
higher (P < 0.05). The albumen height of laying hens at 39 weeks was significantly higher
than that at 27 and 34 weeks (P < 0.05). Compared to the control group, supplementation
of wheat bran or fermented wheat bran significantly increased egg yolk colour (P < 0.05).

Serum biochemical parameters

The effects of fermented wheat bran on serum biochemical parameters of laying hens
were presented in Table 5. Supplementation of wheat bran or fermented wheat bran sig-
nificantly affected the levels of TMAO, ALB, TP and P in the serum of laying hens (P <
0.05). Compared to the control group, ALB, TP and P levels in the serum of laying hens
supplemented with fermented wheat bran were significantly increased (P < 0.05). Com-
pared to the control group, serum TMAO concentrations in the wheat bran group were
significantly increased, but no significant effect was found on TMAO in the fermented
wheat bran group (P < 0.05).

Serum reproductive hormones

The effect of fermented wheat bran on serum reproductive hormone levels was displayed
in Table 6. Compared to the control group, supplementation of wheat bran significantly
increased the levels of E2 and GnRH in the serum of laying hens (P < 0.05), while the sup-
plementation of fermented wheat bran had no significant effects on the levels of repro-
ductive hormones (P > 0.05).

Serum immunoglobulin and inflammatory factors

The levels of immunoglobulin and inflammatory factors were shown in Table 7. Sup-
plementation of wheat bran or fermented wheat bran significantly affected serum
levels of IgA, IgM, IL-2 and IFN-γ(P < 0.05). Compared to the control group, supplemen-
tation of wheat bran significantly increased serum IgM levels (P < 0.05), but significantly
decreased serum IL-2 and IFN-γ levels of laying hens (P < 0.05). The supplementation of
wheat bran increased the serum IgA content of laying hens, but the difference was not
significant compared to the control group (P > 0.05).

Immune indexes of jejunal mucosa

The effects of fermented wheat bran on IL-2, sIgA and IFN-γ in the jejunal mucosa of
laying hens were presented in Figure 2. Supplementation of wheat bran significantly
increased the level of IL-2 in the jejunum mucosa (P < 0.05), but significantly decreased
the level of sIgA in the jejunum mucosa (P < 0.05). The addition of fermented wheat bran
significantly decreased the level of IL-2 in jejunum mucosa (P < 0.05), but significantly
increased the level of sIgA in jejunum mucosa (P < 0.05).
 158
Table 4. Effect of fermented wheat bran on egg quality of laying hens.
Item Treatment Time Treatment Week P-value
Wheat Fermented

Y. WANG ET AL.
Week 27 Week 34 Week 39 Control bran wheat bran 27 34 39 Treatment Time Treatment×Time
a b c
Egg weight (g) Control 58.29 ± 0.73 61.57 ± 1.00 65.62 ± 1.13 61.82 62.42 62.84 58.09 60.83 67.71 0.594 <0.001 0.003
Wheat bran 56.64 ± 0.73 60.95 ± 0.83 69.66 ± 0.64
Fermented 59.66 ± 0.78 59.77 ± 1.08 67.85 ± 0.35
wheat bran
Eggshell Control 41.48 ± 2.64 45.72 ± 1.39 44.28 ± 2.22 43.83 41.23 44.69 41.73 42.57 45.28 0.123 0.142 0.789
strength (N)
Wheat bran 39.19 ± 2.69 43.79 ± 1.98 40.72 ± 2.53
Fermented 45.23 ± 1.50 46.61 ± 1.79 42.72 ± 1.75
wheat bran
Eggshell Control 0.45 ± 0.01 0.47 ± 0.01 0.47 ± 0.01 0.47 0.47 0.48 0.46 0.49 0.47 0.140 0.076 0.818
thickness
(mm)
Wheat bran 0.46 ± 0.01 0.49 ± 0.01 0.46 ± 0.02
Fermented 0.48 ± 0.01 0.49 ± 0.01 0.48 ± 0.01
wheat bran
Egg shape Control 1.27 ± 0.01 1.25 ± 0.01 1.32 ± 0.01 1.28 1.28 1.28 1.27b 1.25a 1.32c 0.979 <0.001 0.869
index
Wheat bran 1.26 ± 0.01 1.26 ± 0.01 1.32 ± 0.01
Fermented 1.26 ± 0.01 1.25 ± 0.01 1.32 ± 0.01
wheat bran
Yolk colour Control 3.07 ± 0.29 4.67 ± 0.15 6.07 ± 0.24 4.60a 6.62b 6.33b 4.00a 6.00b 7.38c <0.001 <0.001 0.675
Wheat bran 4.80 ± 0.27 6.80 ± 0.36 8.27 ± 0.22
Fermented 4.17 ± 0.32 6.66 ± 0.58 7.80 ± 0.25
wheat bran
Albumen Control 6.92 ± 0.28 7.08 ± 0.27 7.35 ± 0.15 7.12 7.28 7.49 7.03a 7.06a 7.73b 0.520 0.035 0.842
height (mm)
Wheat bran 6.89 ± 0.52 7.01 ± 0.57 7.93 ± 0.25
Fermented 7.33 ± 0.50 7.11 ± 0.18 7.93 ± 0.08
wheat bran
Haugh unit Control 82.43 ± 1.91 83.26 ± 1.43 83.69 ± 0.92 83.13 83.51 85.20 82.63 83.20 85.70 0.607 0.284 0.929
Wheat bran 81.52 ± 4.05 82.54 ± 3.96 86.47 ± 1.52
Fermented 84.28 ± 3.02 83.96 ± 1.11 86.93 ± 0.41
wheat bran
Note: abc Means within a row with different letters differ significantly (P < 0.05).
 FOOD AND AGRICULTURAL IMMUNOLOGY 159

Table 5. Effects of fermented wheat bran on serum metabolites of laying hens.

Item Control Wheat bran Fermented wheat bran P-value
TG (mmol/L) 1.52 ± 0.37 1.10 ± 0.33 1.15 ± 0.06 0.443
HDL (mmol/L) 1.44 ± 0.21 1.61 ± 0.22 1.80 ± 0.08 0.422
TMAO (mg/L) 0.08 ± 0.01a 0.11 ± 0.01b 0.08 ± 0.01a <0.001
TC (mmol/L) 2.28 ± 0.50 2.14 ± 0.17 3.29 ± 0.25 0.095
UA (μmol/L) 242.08 ± 22.22 184.58 ± 12.22 228.07 ± 17.99 0.096
ALB (g/L) 11.61 ± 0.89a 12.79 ± 0.71ab 14.61 ± 0.16b 0.045
GLB (g/L) 9.70 ± 0.51 9.16 ± 1.20 9.88 ± 1.14 0.869
TP (g/L) 21.31 ± 0.51a 21.95 ± 0.56a 24.48 ± 1.00b 0.020
Ca (mmol/L) 2.72 ± 0.24 2.49 ± 0.06 3.03 ± 0.03 0.099
P (mmol/L) 1.29 ± 0.28a 1.20 ± 0.10a 2.05 ± 0.08b 0.021
Note: TG, triglyceride; HDL, high density lipoprotein; LDL, low density lipoprotein; TMAO, trimethyl aminoxide; TC, total
cholesterol; UA, uric acid; ALB, albumin; GLB, globulin; TP,total protein; Ca, calcium; P, phosphorus.
ab
Means within a row with different letters differ significantly (P < 0.05).

Table 6. Effects of fermented wheat bran on serum hormone levels of laying hens.
Item Control Wheat bran Fermented wheat bran P-value
FSH (mIU/mL) 14.10 ± 0.60 18.75 ± 2.77 12.74 ± 0.70 0.087
LH (mIU/mL) 20.71 ± 1.25ab 26.56 ± 2.96b 18.11 ± 1.30a 0.044
a b a
E2(pg/mL) 148.90 ± 6.67 188.91 ± 18.99 141.47 ± 1.91 0.045
P4 (ng/mL) 7.39 ± 1.53 15.58 ± 6.29 5.79 ± 0.71 0.228
GnRH (pg/mL) 38.43 ± 1.47a 49.78 ± 2.93b 33.18 ± 1.30a 0.001
Note: FSH,follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; P4, progesterone; GnRH, gonadotropin-
releasing hormone
ab
Means within a row with different letters differ significantly (P < 0.05).

Table 7. Effects of fermented wheat bran on serum immunology and inflammatory factors of laying
hens.
Item Control Wheat bran Fermented wheat bran P-value
IgA (g/L) 2.03 ± 0.10ab 2.60 ± 0.30b 1.80 ± 0.09a 0.049
IgG (g/L) 3.75 ± 0.22 4.38 ± 0.40 3.27 ± 0.09 0.064
IgM (g/L) 1.07 ± 0.09a 1.30 ± 0.04b 0.99 ± 0.04a 0.022
LPS (EU/mL) 0.57 ± 0.03 0.50 ± 0.03 0.61 ± 0.08 0.318
IL-2 (pg/mL) 390.73 ± 19.56b 280.72 ± 13.63a 440.08 ± 19.20b <0.001
IL-10 (pg/mL) 14.82 ± 0.28 16.72 ± 1.88 13.95 ± 0.45 0.298
IFN-γ (pg/mL) 65.23 ± 0.41b 50.68 ± 1.55a 67.49 ± 1.77b <0.001
Note: IgA,immunoglobulin A;IgG, immunoglobulin G; IgM, immunoglobulin M; LPS,lipopolysaccharide;IL-2, interleukin-2;
IL-10, interleukin-10; IFN-γ, interferon-γ.
ab
Means within a row with different letters differ significantly (P < 0.05).

Discussion
Wheat bran contains 15% crude protein, which can be used as an alternative protein or
energy feedstuff in animal diets (Balandrán-Quintana et al., 2015). However, the high
level of dietary fibre, arabinoxylan and phytic acid in wheat bran may reduce nutrient
digestion and absorption (Noblet & Goff, 2001; Stevenson et al., 2012). Generally, the
proportion of wheat bran added to the diet is not more than 8% in practice. After
solid-state fermentation of wheat bran, the contents of aroma substances and phenols
were increased, while arabinoxylan, phytic acid and acidity decreased, which may
increase the nutritional value of wheat bran and application proportion in poultry
diets (Spaggiari et al., 2020). There were two main reasons for the relative increase of
crude protein level of wheat bran after solid-state fermentation: 1) Fermented material
160 Y. WANG ET AL.

Figure 2. Effects of wheat bran and fermented wheat bran on IL-2(A) and sIgA(B) contents of jejunal
mucosa of laying hens. abc Means among without the same letter are significantly different(P < 0.05).
Values are expressed as the mean ± SE (n = 5). IL-2,interleukin-2; sIgA,secretory immunoglobulin A.

concentration effect at the expense of carbohydrates loss in the process of microbial fer-
mentation. This is probably the main reason and can be assessed by dry matter recovery
rate; 2) The bacterial protein synthesis by microorganisms using non-protein nitrogen,
but this cause is hard to accurately analyze (Rozan et al., 2018). The reduction of
crude fibre can be attributed to cellulase degradation by specific microorganisms
during fermentation (Oladapo Olukomaiya et al., 2019). The combined action of fermen-
tation can break down the lignocellulose in the cell wall and release polyphenols, thus the
content of total polyphenols in fermented wheat bran were increased (Yin et al., 2018).
In the present study, the diet supplemented with 10% wheat bran or fermented wheat
bran maintained the same nutritional level by adjusting diet composition, which had no
effect on egg weight and laying rate. Wanzenbck et al. (2020) and Huang et al. (2021) also
reported that dietary supplementation of 10% wheat bran or fermented wheat bran had
no negative effect on the laying performance and egg quality of laying hens. Laying per-
formance is affected by multiple factors, such as breed, laying cycle, light duration, temp-
erature, nutrient level, environmental conditions and age. Consistent with the results of
this study, the feed conversion rate of laying hens was decreased, while the laying rate was
 FOOD AND AGRICULTURAL IMMUNOLOGY 161

increased after 28 weeks of age (Bovera et al., 2014), which originated from the high yield
period of laying hens at about 28 weeks of age.
Egg quality is a parameter to evaluate laying performance and economic benefits
(Iskender et al., 2017). This study indicated yolk colour was more yellow in the wheat
bran group or fermented wheat bran group than that in the control group, which
might be owing to the presence of corn gluten meal in the feed formula, which is in
favour of pigment deposition (Galobart et al., 2004). Compared with 28-week-old
eggs, 40-week-old eggs had higher egg weight and yolk colour (Padhi et al., 2013),
which was consistent with the egg quality characteristics of 34-week and 39-week-old
laying hens compared with 27-week-old eggs in this experiment.
TMAO, as an intermediate product of lipid metabolism, is also one of the parameters
to indicate lipid metabolism. It was reported that TMAO is related to cholesterol metab-
olism and vascular sclerosis, and its content is positively correlated with cardiovascular
disease (Ufnal et al., 2015; Yang et al., 2019). Compared to wheat bran, long-term sup-
plementation of fermented wheat bran could significantly reduce the level of TMAO,
which may be related to the increase in active components of wheat bran and the
changes of intestinal microorganisms during fermentation (Coutinho-Wolino et al.,
2021). As an important index of protein metabolism, the level of total protein in
serum is positively correlated with protein deposition, which can promote animal
growth, and albumin also plays an important role in maintaining animal nutrition. In
this study, it was found that supplementation of fermented wheat bran could effectively
increase the levels of total protein and albumin in the serum of laying hens. This might be
related to the increase of trichloroacetic acid-soluble protein levels and the decrease of
phytic acid content in the fermentation process. However, Lin and Lee (2020a) reported
that diet supplemented with of 5% or 10% fermented wheat bran had no significant effect
on TP and ALB in the serum of broilers, which might be attributed to the different fer-
mentation process of wheat bran. Calcium and phosphorus, as the constituent elements
of eggshells, are mainly derived from blood and bones (De Vries et al., 2010). Supplemen-
tation of fermented wheat bran increased the content of serum phosphorus in laying
hens, which was beneficial to the formation of the eggshell.
There is a close correlation between reproductive hormone level and the laying per-
formance of laying hens (Cui et al., 2021; Onagbesan et al., 2006). Genetic diversity
and endocrine hormone levels mostly limit egg production behaviours (Du et al.,
2020). GnRH mainly stimulates egg-laying behaviour, whereas FSH and LH are
related to the growth and development of follicles, and E2 and P4 act on the hypothala-
mus and pituitary to promote ovulation by promoting the release of LH (Hernandez & Ba
Hr, 2003; Wang et al., 2013). Supplementation of wheat bran increased the serum E2 and
GnRH levels of laying hens, while supplementation of fermented wheat bran had no
effect on these hormones. From the level of reproductive hormones, supplementation
of wheat bran or fermented wheat bran had no adverse effect on laying performance,
which was consistent with the results of this study on laying performance.
Immunoglobulin levels are closely related to animal immune function, especially
humoral immunity (Dalakas, 1997). In this study, supplementation of wheat bran
increased the levels of IgA and IgM in the serum of laying hens, while supplementation
of fermented wheat bran had no significant effect on the level of immunoglobulins in
laying hens. Lin and Lee (2020b) found that supplementation of wheat bran or
162 Y. WANG ET AL.

fermented wheat bran had no effect on serum IgG levels. As the main component of the
cell wall of gram-negative bacteria, LPS induces the release of inflammatory mediators
from a variety of immune cells (Glauser, 1996). Cytokines play a regulatory role in
immunity and inflammation by transmitting information. According to their role in
the process of inflammation, cytokines can be classified into pro-inflammatory
factors that induce deterioration and anti-inflammatory factors that promote healing
(Dinarello, 2000). Th1-cells release pro-inflammatory compounds such as IL-2 and
IFN-γ, whereas Th2-cells secrete anti-inflammatory substances such as IL-10 (Tayal
& Kalra, 2008). In this study, the level of IL-2 and IFN-γ was decreased. So the sup-
plementation of wheat branenhanced the anti-inflammatory ability of laying hens,
which is related to the effect of arabinoxylan contained in wheat bran (Fadel et al.,
2018; Li et al., 2015). Supplementation of fermented wheat bran had no effect on
pro-inflammatory factors in the serum of broilers (Chuang et al., 2021), which was con-
sistent with the results of this study.
Secreted IgA is produced on the surface of the mucosa that can remove viruses and
pathogenic microorganisms. It has a protective effect by aggregating pathogens by
adsorption on the intestinal tract of animals (Corthésy, 2010; Williams & Gibbons,
1972). Shang et al. (2020) found that dietary supplementing with 3% fermented
wheat bran could increase the level of sIgA in the jejunal mucosa of broilers, while
decreasing the level of several pro-inflammatory factors, which was consistent with
the current results. In this study, it was found that the jejunal mucosa sIgA level
increased and the IL-2 level decreased after the supplementation of fermented wheat
bran. The enhancement of jejunal immunity may be related to the increase of small
peptides and the changes of intestinal microorganisms during fermentation (Sugiharto
& Ranjitkar, 2019).

Conclusions
In summary, long-term dietary supplementation of 10% fermented wheat bran could
improve the immune performance and laying performance of laying hens by influencing
serum biochemical, reproductive hormone and inflammatory response. Moreover, fer-
mented wheat bran could effectively promote protein and lipid metabolism of laying
hens. More studies are needed to clarify the underlying mechanisms of fermented
wheat bran in modulating the immune performance of laying hens.

Abbreviations
DF, dietary fibre; NSP, non-starch polysaccharides; ZEN, zearalenone; DON, deoxyniva-
lenol; AFB1, aflatoxin B1; TG, Triglyceride; HDL, high density lipoprotein; TMAO, tri-
methyl aminoxide; TC, total cholesterol; UA, uric acid; ALB, albumin; GLB, globulin; TP,
total protein; Ca, calcium; P, phosphorus; FSH, follicle-stimulating hormone; LH, lutei-
nizing hormone; E2, estradiol; P4, progesterone; GnRH, gonadotropin-releasing
hormone; LPS, lipopolysaccharide; IL-2, interleukin-2; IL-10, interleukin-10; IFN-γ,
interferon-γ; sIgA, secretory immunoglobulin A; IgA, immunoglobulin A; IgG, immuno-
globulin G; IgM, immunoglobulin M.
 FOOD AND AGRICULTURAL IMMUNOLOGY 163

Acknowledgments
We thank Jishan An for the preparation of solid-state fermented wheat bran, and associate pro-
fessor Shuangshuang Guo is also appreciated for critical reading of the manuscript.

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

Funding
This work was supported by National Key R&D Program of China: [Grant Number
2021YFD1300301]; Academy of National Food and Strategic Reserves Administration, P.R.
China: [Grant Number ZX1907].

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