Effects of moldy corn on the performance, antioxidant capacity, immune
function, metabolism and residues of mycotoxins in eggs, muscle, and edible
viscera of laying hens
Fenghua Zhu,* Lianqin Zhu,y Jindong Xu,z Yuchang Wang,y and Yang Wang*,1
College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, P.R. China; yCollege
*
of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, P.R. China; and zCollege of Science and
Information, Qingdao Agricultural University, Qingdao 266109, P.R. China
ABSTRACT Mycotoxins, including aflatoxin B1
(AFB1), zearalenone (ZEN) and deoxynivalenol
(DON), are common contaminants of moldy feeds.
Mycotoxins can cause deleterious effects on the health of
chickens and can be carried over in poultry food prod-
ucts. This study was conducted to investigate the effects
of moldy corn (containing AFB1, ZEN, and DON) on
the performance, health, and mycotoxin residues of lay-
ing hens. One hundred and eighty 400-day-old laying
hens were divided into 4 treatments: basal diet (Con-
trol), basal diet containing 20% moldy corn (MC20),
40% moldy corn (MC40) and 60% moldy corn (MC60).
At d 20, 40, and 60, the performance, oxidative stress,
immune function, metabolism, and mycotoxin residues
in eggs were determined. At d 60, mycotoxin residues in
muscle and edible viscera were measured. Results
showed the average daily feed intake (ADFI) and laying
performance of laying hens were decreased with moldy
corn treatments. All the moldy corn treatments also
induced significant oxidative stress and immunosuppres-
sion, reflected by decreased antioxidase activities, con-
Key words: moldy corn, aflatoxin B1, zearalenone, deoxynivalenol, laying hen
INTRODUCTION
Mycotoxins are natural small-molecule contaminants
produced as secondary metabolites by fungi (Steyn,
1995). Contamination by mycotoxins occurs frequently
in chicken feed, including maize and other cereals (Iqbal
et al., 2014). Economic losses due to mycotoxin contami-
nation occur at all levels of food and feed production
including crop and animal production, processing and
distribution (Galvano et al., 2001). According to a 10-
Ó 2023 The Authors. Published by Elsevier Inc. on behalf of Poultry
Science Association Inc. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
Received November 19, 2022.
Accepted January 10, 2023.
1
Corresponding author: yangwang@qau.edu.cn
1
tents of cytokines, immunoglobulins, and increased
malonaldehyde level. Moreover, the activities of aspar-
tate aminotransferase and alanine transaminase were
increased by moldy corn treatments. The lipid metabo-
lism was influenced in laying hens receiving moldy corn,
reflected by lowered levels of total protein, high density
lipoprotein cholesterol, low density lipoprotein choles-
terol, total cholesterol, and increased total triglyceride
as well as uric acid. The above impairments were aggra-
vated with the increase of mycotoxin levels. Further-
more, AFB1 and ZEN residues were found in eggs,
muscle, and edible viscera with moldy corn treatments,
but the residues were below the maximum residue limits.
In conclusion, moldy corn impaired the performance,
antioxidant capacity, immune function, liver function,
and metabolism of laying hens at d 20, 40, and 60. Moldy
corn also led to AFB1 residue in eggs at d 20, 40, and 60,
and led to both AFB1 and ZEN residues in eggs at days
40 and 60, and in muscle and edible viscera at d 60. The
toxic effects and mycotoxin residues were elevated with
the increase of moldy corn levels in feed.
2023 Poultry Science 102:102502
https://doi.org/10.1016/j.psj.2023.102502
year survey, in 41.1, 38.5, and 20.9% of feed and feed
raw materials (e.g., maize, wheat, soybean) samples
from South Asia, Sub-Saharan Africa, and Southeast
Asia, respectively, the AFB1 contents exceeded the max-
imum level (20 mg/kg). DON and ZEN concentrations
were also correlated in maize (Gruber-Dorninger et al.,
2019). In addition, from yr 2016 to 2017, the positive
detection rates for AFB1, ZEN, and DON in maize sam-
ples collected from 21 provinces in China were 96.1,
92.05, and 98.15%, respectively, and these 4 toxins were
detected at the same time in 92.25% of the samples (Ma
et al., 2018). Aflatoxins (AF), produced by Aspergillus
spp., are known to have strong hepatotoxic and carcino-
genic effects and are regulated by feed/food law in at
least 100 countries (van Egmond and Jonker, 2004).
2 ZHU ET AL.

Among the various types of AF, aflatoxin B1 (AFB1) is Table 1. Composition and calculated nutrient content of basal
most commonly encountered and it is also considered to diets (air dry basis).
have higher toxicity than other aflatoxins (Yunus et al., Ingredients Content Nutrients2 Content
2011). To date, the most frequently found mycotoxins
Corn, % 62.50 ME, MJ/kg 10.86
are deoxynivalenol (DON) and its acetylated deriva- Soybean meal, % 24.00 CP, % 15.70
tives, which can affect animal and human health causing Limestone, % 8.50 AP, % 0.48
acute temporary nausea, vomiting, diarrhea, abdominal Premix1, % 5.00 Ca, % 3.70
Total, % 100.00 Cl, % 0.19
pain, headache, dizziness, and fever (Sobrova et al., Na, % 0.30
2010). Moreover, zearalenone (ZEN) resembles the Lys, % 0.80
structure of naturally occurring estrogens. This property Met, % 0.32
Cys, % 0.33
of ZEN determines its ability to bind to estrogen recep-
1
tors, exerting estrogenic and anabolic effects on animals The premix provided the following per kg of diets: VA 11,000 IU, VD3
600 IU, VE 20 IU, VK3 2 mg, VB1 1.6 mg, VB2 4.5 mg, VB6 5 mg, VB12
(Rogowska et al., 2019). Besides, reports have indicated 0.03 mg, folic acid 2 mg, pantothenic acid 10 mg, biotin 0.1 mg, choline
that AFB1, DON, and ZEN can also impair the immune 50 mg, Cu 10 mg, Fe 35 mg, Zn 50 mg, Mn 60 mg, Se 0.30 mg, I 0.5 mg,
function, antioxidant ability and biochemical metabo- limestone 0.175 g, calcium dihydrogen phosphate 0.6 g, Lys 25 mg, Met
40 mg, sodium bicarbonate 0.15 g.
lism. For example, Rajput et al. (2017) demonstrated 2
All nutrient contents were calculated.
that AFB1 significantly influenced the serum biochemi-
cal parameters, decreased immunoglobulin (Ig) levels,
and reduced antioxidase activities of chicken. Azizi et al.
(2021) revealed that dietary DON feeding to chickens mycotoxin residues were investigated. Overall, the
caused an enhancement in blood aspartate transami- results of our study are helpful to raise awareness of the
nases (AST) content and a decrease in blood total pro- health risks associated with these toxins.
tein (TP). The antioxidase activities and antioxidant
gene expressions of chickens were also reduced by DON
treatment (Yang et al., 2017). Additionally, both of the MATERIALS AND METHODS
humoral and cellular immunity can be impaired by
DON (Li et al., 2014). As for the ZEN, Xu et al. (2021)
Diets
found that ZEN treatment increased activities of AST For the preparation of moldy corn, plastic film and
and alanine transaminase (ALT), decreased antioxidase insulation pad were used to cover the corn. Water was
capacity, and elevated inflammatory cytokine gene sprayed once a week and the corn was stirred evenly.
expressions of chickens. The corn was mildewed under natural conditions at 14
Human can also easily be exposed to mycotoxins to 20°C and 65 to 70% humidity for 30 d. After 30 days
through foods of plant origin (cereal grains) or foods of mildew, the contents of AFB1, DON, ZEN, fumonisin
animal origin (muscle meat, milk, egg) (Wang et al., (FB) and T-2 were measured to be 125 mg/kg, 1,220
2018). Mycotoxin residue has been found in eggs, mus- mg/kg, 4.9 mg/kg, 2.37 mg/kg, and 45 mg/kg, respec-
cle, and organs and its concentration is high in liver, tively, by using UPLC-MS/MS. Then, 0.3% calcium
egg, kidney, and thigh region (Herzallah, 2013). Fern
an- propionate and sweetener were added to inhibit mildew
dez et al. (1994) found 0.05 mg/kg residue of AFB1 in and mask odor. The prepared moldy corn was used to
muscle of laying hen exposed to 5 mg/kg AF-contami- replace normal corn in the basal diet for Fusarium myco-
nated feed. Feeding birds diet contaminated with AF toxin-contaminated diet preparation. The composition
(123.0 mg/kg), or AF and ZEN (123.0 and 260.2 mg/kg, and nutrient levels of the basal diet was listed in Table 1.
respectively) resulted in the AF residues in eggs between
0.01 and 0.21 mg/kg (Jia et al., 2016). Although in some
cases low-levels of mycotoxin contamination may not Experimental Design
affect animal growth or performance, mycotoxins may
be carried over into animal liquid and tissue products One hundred and eighty 400-day-old Hy-Line Brown
(Fowler et al., 2015). Aly and Anwer (2009) showed laying hens were reared in three-layer stepped cage and
that the egg production and weight were not affected in equally divided into 4 groups (Control, MC20, MC40,
laying birds fed 25 to 100 mg AF/kg feed, yet there was and MC60) with 5 replications of 9 birds each for a 7-d
residue in eggs. Mycotoxin residues poses a risk to food pre-feeding period and 60-d formal period. Three birds
security and safety because increased residues in animal were reared separately in a single cage (0.47 m £ 0.36
products cause economic losses through border rejection m £ 0.38 m). Laying hens in the Control, MC20, MC40,
in the global and local market ultimately aggravating and MC60 groups were fed with the basal diet, basal
the macro- and micronutrient insecurity especially in diet containing 20% moldy corn, 40% moldy corn, and
developing countries (Adegbeye et al., 2020). 60% moldy corn, respectively (Figure 1). Fresh water
In order to provide more in vivo evidence for the toxic- and feed were provided ad libitum. The temperature of
ity and residue of mycotoxins in laying hens, different the room was set at 21°C. The animal experiment was
percentage of moldy corn were fed to chickens for 20, 40, approved and performed in accordance with the guide-
and 60 d. At the endpoints, the performance, antioxi- lines of Ethics and Animal Welfare Committee of Qing-
dant ability, immune function, metabolism, and dao Agricultural University.
 ADVERSE EFFECTS OF MOLDY CORN ON LAYING HENS
Figure 1. Experimental design.
Measurement of Performance
On a replicate basis, the feed intake, number of
eggs, egg weight, number of broken eggs, and number
of dead hens were recorded daily. And the average
daily feed intake (ADFI), average egg weight, laying
rate, feed/egg ratio, broken egg rate, and death/cull-
ing rate were calculated. ADFI = total daily feed
consumption/number of hens; average egg weight =
average daily total egg weight/total number of eggs;
laying rate = (total number of eggs/laying hens num-
ber/days) £ 100%; broken egg rate = (number of
broken eggs//total number of eggs) £ 100%; feed/
egg = ADFI/(average daily total egg weight/number
of hens); death/culling rate = number of dead hens/
total number of hens.
Sample Collection
At d 20, 40, and 60, one hen of each cage (3 hens
of each replicate) were randomly selected and eutha-
nized and blood samples were collected from the wing
vein into vacuum tubes containing an anticoagulant
and centrifuged for 10 min (3,000 g) at 4°C. Pure
plasma samples were collected and stored in 1.5 mL
Eppendorf tubes at 20°C. At d 20, 40, and 60, three
eggs from each replicate were collected, mixed into
one sample and dried in the oven at 60°C. Thereafter,
dried eggs were smashed and passed through 20 mesh
sieves. Egg samples were then collected and stored at
20°C for further detection of mycotoxin residues. At
d 60, the liver, breast muscle, leg muscle, and heart
from 1 hen of each cage were collected for the detec-
tion of mycotoxin residues (Figure 1).
3
Analysis of Biochemical Indices in Plasma
Commercial kits (Nanjing Jiancheng Bioengineering
Institute, Nanjing, China) were used to measure the
activities of superoxidase (SOD; cat. no. A001-3-2), glu-
tathione peroxidase (GSH-Px; cat. no. A005-1-2), AST
(cat. no. C010-2-1), and ALT (cat. no. C009-2), and the
levels of total antioxidant capacity (T-AOC; cat. no.
A015-1-2), malonaldehyde (MDA; cat. no. A003-1),
IgA (cat. no. H108-1-1), IgG (cat. no. H106-1-1), IgM
(cat. no. H109-1-1), interleukin 1b (IL-1b; cat. no.
H002), IL-6 (cat. no. H007-1-1), tumor necrosis factor a
(TNF-a; cat. no. H052-1), TP (cat. no. A045-1-1), total
cholesterol (T-CHO; cat. no. A111-1-1), total triglycer-
ides (TG; cat. no. A110-1-1), high density lipoprotein
cholesterol (HDL-C; cat. no. A112-1-1), low density
lipoprotein cholesterol (LDL-C; cat. no. A113-1-1), and
uric acid (UA; cat. no. C012-2-1) were detected spectro-
photometrically according to manufacturer’s protocol.
Measurement of Mycotoxins
The contents of mycotoxins in the feed, muscle, edible
viscera, and eggs were measured using UPLC-MS/MS
(Agilent Technologies). Briefly, 10 mL of 0.05 mol/L
sodium acetate (pH = 4.8) and 0.025 mL glucuronidase/
sulfate complex enzyme were added in 3 g samples, vor-
tex-mixed and vibrated in gas bath at 37°C for 12 h.
Then, 10 mL acetonitrile (containing 2% formic acid),
4 g MgSO4 and 1.5 g NaCl were added, and the sample
were extracted by vibration for 2 h. After centrifuge at
5,000 r/min for 5 min, the upper acetonitrile phase
(8 mL) was pipetted into a 10 mL tube and diluted with
2 mL water and vortex-mixed. The extract (2.5 mL) was
loaded into a 3 mL Captiva EMR-Lipid purification
tube (Agilent Technologies, Santa Clara, California)
4 ZHU ET AL.
and allowed to flow through the purification tube by
gravity. After the extract was completely eluted, vac-
uum was applied to evacuate the purification tube.
Transfer 0.5 mL eluate to an autosampler tube and add
0.3 mL of 0.1% formic acid in 5 mmol/L ammonium for-
mate. The mixed solution was filtered through a 0.22-
mm membrane filter and injected into the UPLC-MS/
MS system for analysis.
Chromatographic separation was carried out using a 50
mm £ 2.1 mm i.d., 2.7 mm, C18 column (Waters, Mil-
ford, MA). A 5 mmol/L ammonium formate containing
0.1% formic acid was used as the mobile phase at a flow
rate of 0.3 mL min1. The column temperature was main-
tained at 40°C and the injection volume was 5 mL. The
UPLC system was coupled to a triple quadrupole tandem
mass spectrometer (TQ Detector; Waters) with an elec-
trospray ion source (ESI). The MS detector was operated
in positive-ion mode with the following settings: capillary
voltage, 3.5 kV; source temperature, 120°C; desolvation
temperature, 350°C; desolvation gas flow, 650 l h1; and
cone gas flow, 50 l h1. High purity nitrogen was used as
the collision gas, and the collision gas flow was 0.20 mL
min1. Cone voltage and collision energy were optimized
by infusion of each individual analyte. Detection was car-
ried out in multiple reaction monitoring (MRM) mode.
MRM parameters for MS detection of the 3 mycotoxins
are summarized in Table S1.
Method Validation
The validation parameters considered were linearity
(R2), recovery rate, precision (RSD), and limit of detec-
tion (LOD) and quantification (LOQ). Recovery rate
and RSD were evaluated via blank samples spiked at dif-
ferent concentrations. Calculations were based on rela-
tive peak areas, that is, the peak area of the compound
of interest divided by the peak area of the corresponding
IS added to the same sample (Emmanuel et al., 2020).
As for the validation results, the upper limit of recov-
ery rate varied between 92.2% and 112.7% for egg, liver,
heart, leg muscle, and breast muscle. The lower limit of
recovery rate varied between 80.5% and 99% for egg,
heart, leg muscle, and breast muscle. RSD were found to
be adequate for the different mycotoxins (Table S2).
The LOD values for AFB1, ZEN and DON were 0.0015
mg/kg, 0.06 mg/kg, and 2.7 mg/kg, respectively. The
LOQ values for AFB1, ZEN, and DON were 0.0045 mg/
kg, 0.20 mg/kg, and 9.13 mg/kg, respectively (Table S3).
Table 2. Mycotoxins contents in moldy feed.
Control MC20
AFB1 (mg/kg) 0.10 25.36
ZEN (mg/kg) 51.70 245.05
DON (mg/kg) 0.20 0.99
FB (mg/kg) 1.70 0.49
T-2 (mg/kg) Not detected 9.00
Abbreviations: AFB1, aflatoxin B1; ZEN, zearalenone; DON, deoxynivalenol; FB, fumonisin; MC20, basal diet containing 20% moldy corn; MC40,
basal diet containing 40% moldy corn; MC60, basal diet containing 60% moldy corn.
The criteria of linearity (R2 ≥ 0.997) were fulfilled for all
the mycotoxins studies.
Statistical Data Analysis
All data were subjected to analysis of variance (moldy
corn percentage) as an entirelyrandomized design using
the general linear model (GLM) of SAS (SAS Institute
Inc, version 9.3, Cary, North Carolina), followed by
Duncan’s multiple comparisons test. Results were pre-
sented as the means § standard deviation (SD). The
differences were considered significant at P < 0.05.
RESULTS
Contents of AFB1, ZEN, and DON in the
Experimental Diets
The AFB1, ZEN, DON and FB levels in the diets of
Control group were 0.10 mg/kg, 51.7 mg/kg, 0.20 mg/kg,
and 1.70 mg/kg, respectively, which were all below the
mycotoxin limit standard of China. The AFB1, ZEN,
DON, FB, and T-2 levels in the diets of MC20 group
were 25.36 mg/kg, 245.05 mg/kg, 0.99 mg/kg,
0.49 mg/kg, and 9 mg/kg, respectively, and the AFB1
level was higher than the mycotoxin limit standard of
China. The AFB1, ZEN, DON, FB, and T-2 levels in the
diets of MC40 group were 51.62 mg/kg, 505.73 mg/kg,
2.12 mg/kg, 0.96 mg/kg, and 18 mg/kg, respectively. The
AFB1 and ZEN levels were higher than the mycotoxin
limit standard of China. Moreover, the AFB1, ZEN,
DON, FB and T-2 levels in the diets of MC60 group were
76.50 mg/kg, 750.30 mg/kg, 3.34 mg/kg, 1.43 mg/kg, and
27 mg/kg, and levels of AFB1, ZEN, and DON were all
higher than the mycotoxin limit standard of China
(Table 2). Thus, by using these experimental diets, the
toxic effects of moldy corn can be investigated.
Effects of Moldy Corn on the Performance of
Laying Hens
From d 1 to d 20, the ADFI was significantly
decreased in all the moldy corn treatments (P < 0.05)
and the value in MC60 group was lower than that of the
MC20 and MC40 groups (P < 0.05). The laying rate was
significantly decreased by all the moldy corn treatment
groups (P < 0.01) in a dose-dependent manner. The
average egg weight was also lowered in the MC60 group
Mycotoxin limit standard
MC40 MC60 (GB 13078-2017, China)
51.62 76.5 20
505.73 750.3 500
2.12 3.34 3
0.96 1.43 20
18.00 27.00 500
 ADVERSE EFFECTS OF MOLDY CORN ON LAYING HENS
compared to other groups (P < 0.05). The broken egg
rate was significantly elevated in the MC60 group com-
pared to the Control and MC20 groups (P < 0.01).
From d 1 to d 40, the ADFI was significantly
decreased all the moldy corn treatments (P < 0.05), and
the value in MC60 group was lower than that of the
MC20 and MC40 groups (P < 0.01). The laying rate was
also significantly decreased in all the moldy corn treat-
ments (P < 0.05) in a dose-dependent manner. The aver-
age egg weight and feed/egg ratio were significantly
reduced in the MC40 and MC60 groups (P < 0.05). The
MC60 treatment led to elevated broken egg rate (P <
0.01), which was higher than that of the MC20 (P <
0.01) and MC40 (P < 0.05) groups.
Moreover, from d 1 to d 60, the ADFI, laying rate, and
average egg weight were significantly decreased in all the
moldy corn treatments (P < 0.05) in a dose-dependent
manner. The feed/egg ratio was significantly increased
in the MC40 and MC60 groups (P < 0.01), and the
MC60 treatment led to a higher feed/egg ratio than that
of the MC20 treatment (P < 0.05). The broken egg rate
was significantly elevated in all the moldy corn treat-
ments (P < 0.05), and was higher in MC60 treatment
than in that of the MC20 and MC40 groups (P < 0.01).
The death/culling rate was not significantly altered
among groups (P > 0.05; Table 3).
Effects of Moldy Corn on the Antioxidant
Capacity of Laying Hens
At d 20, the T-AOC level was significantly decreased
by the MC40 and MC60 treatments (P < 0.05). The
SOD activity was significantly decreased by all the
moldy corn treatments (P < 0.01), and was lower in the
MC60 group than that in the MC20 and MC40 groups
5
(P < 0.01). The GSH-Px activity was significantly
decreased in all the moldy corn treatments (P < 0.05),
and was lower in the MC60 group than that in the
MC20 group (P < 0.05). The MDA content was signifi-
cantly increased in MC40 and MC60 groups (P < 0.01)
in a dose-dependent manner.
At d 40, the T-AOC level and SOD activity were sig-
nificantly decreased in the MC40 and MC60 groups (P <
0.05). The GSH-Px activity was significantly lowered (P
< 0.01), while the MDA content was significantly
increased in all the moldy corn treatment groups (P <
0.01) in a dose-dependent manner.
At d 60, the T-AOC level and SOD activity were sig-
nificantly decreased in all the moldy corn treatments in
a dose-dependent manner. The GSH-Px activity was
also significantly decreased by all the moldy corn treat-
ments (P < 0.01), and was lower in the MC60 group
than in the MC20 (P < 0.01) and MC40 (P < 0.05)
groups. Conversely, all the moldy corn treatments led to
significantly increased MDA content (P < 0.01), which
reached the highest level in MC60 group (Table 4).
Effects of Moldy Corn on the Immune
Function of Laying Hens
At d 20, the IFN-a and IL-1b concentrations were sig-
nificantly decreased by all the moldy corn treatments (P
< 0.01), and were lower in the MC60 group than that in
the MC20 and MC40 groups (P < 0.05). The IL-6 and
IgA concentrations were significantly reduced in the
MC40 and M60 groups (P < 0.01) in a dose-dependent
manner. The IgG concentration was significantly
reduced by all the moldy corn treatments (P < 0.01) in a
dose-dependent manner. Moreover, the IgM

Table 3. Effects of moldy corn on the laying performance of laying hens.

Treatments
days Items Control MC20 MC40 MC60 P value
20 ADFI, g 112.56 § 4.61 A,a
104.27 § 4.36AB,b
97.73 § 7.19BC,b
87.39 § 6.26C,c
< 0.0001
Laying rate, % 88.22 § 2.68A 82.22 § 2.22B,a 76.44 § 3.68BC,b 71.33 § 3.72C,c < 0.0001
Average egg weight, g 62.68 § 0.68A 61.77 § 0.65A 61.29 § 0.65AB,a 58.65 § 2.76B,b 0.004
Feed/egg 2.03 § 0.09 2.05 § 0.07 2.09 § 0.19 2.09 § 0.13 0.866
Broken egg rate, % 0.00 § 0.00B 0.28 § 0.38B 0.59 § 0.63AB 1.23 § 0.64A 0.007
Death/culling rate, % 0.00 § 0.00 0.00 § 0.00 0.00 § 0.00 0.00 § 0.00 -
40 ADFI, g 110.67 § 5.47A,a 103.09 § 4.41AB,b 97.79 § 3.84B 87.66 § 4.62C < 0.0001
Laying rate, % 84.33 § 1.59A,a 80.22 § 3.46AB,b 77.89 § 1.98BC,b 73.78 § 1.27C,c < 0.0001
Average egg weight, g 63.10 § 0.82A 59.94 § 2.74AB 57.89 § 2.61BC,a 53.39 § 3.64C,b 0.000
Feed/egg 2.08 § 0.10b 2.15 § 0.04ab 2.17 § 0.04a 2.23 § 0.06a 0.015
Broken egg rate, % 0.13 § 0.29B 0.29 § 0.39B 0.71 § 0.49AB,b 1.67 § 1.00A,a 0.004
Death/culling rate, % 0.00 § 0.00 0.00 § 0.00 0.00 § 0.00 0.00 § 0.00 -
60 ADFI, g 107.80 § 1.61A,a 101.26 § 3.53AB,b 95.02 § 2.08BC,c 87.54 § 7.44C,d 0.0001
Laying rate, % 83.22 § 2.40A,a 79.22 § 2.88AB,b 75.33 § 2.06BC,c 70.78 § 2.50C,d 0.0001
Average egg weight, g 59.81 § 1.86A 54.39 § 2.14B 50.79 § 1.97C 47.05 § 1.41D 0.0001
Feed/egg 2.17 § 0.11B 2.36 § 0.17AB,b 2.49 § 0.14A,ab 2.63 § 0.19A,a 0.002
Broken egg rate, % 0.13 § 0.30C,b 0.98 § 0.39BC,a 1.18 § 0.65B 2.35 § 0.53A 0.0001
Death/culling rate, % 0.00 § 0.00 0.00 § 0.00 2.22 § 4.97 4.44 § 6.09 0.261
Abbreviations: ADFI, average daily feed intake; MC20, basal diet containing 20% moldy corn; MC40, basal diet containing 40% moldy corn; MC60,
basal diet containing 60% moldy corn.
Results are represented as mean values § SD (n = 5 per group).
a,b,c,d
Mean value within a role with no common superscript differ significantly (P < 0.05).
A,B,C,D
Mean value within a role with no common superscript differ very significantly (P < 0.01).
6 ZHU ET AL.

Table 4. Effects of moldy corn on the antioxidant capacity of laying hens.

Treatments
days Items Control MC20 MC40 MC60 P value
20 T-AOC (mmol/L) 6.29 § 0.33 a
6.25 § 0.10ab
6.02 § 0.07bc
5.91 § 0.15c
0.017
SOD (U/mL) 241.13 § 6.63A 224.27 § 6.60B 219.05 § 5.18B 206.63 § 6.49C < 0.0001
GSH-Px (U/mL) 81.77 § 5.71A,a 74.80 § 5.06AB,b 70.47 § 4.89B,bc 68.10 § 2.47B,c 0.002
MDA (nmol/mL) 5.32 § 0.07C 5.42 § 0.05C 5.61 § 0.07B 6.01 § 0.09A < 0.0001
40 T-AOC (mmol/L) 6.22 § 0.08A 6.11 § 0.24AB,a 5.87 § 0.10BC,b 5.76 § 0.10C 0.000
SOD (U/mL) 253.80 § 7.33A 245.30 § 7.51AB,a 235.90 § 7.84BC,b 225.59 § 3.97C,c < 0.0001
GSH-Px (U/mL) 80.11 § 2.22A 75.04 § 1.73B 73.50 § 3.16BC,a 69.08 § 3.09C,b < 0.0001
MDA (nmol/mL) 5.82 § 0.12C 6.21 § 0.11B,b 6.45 § 0.17B,a 6.94 § 0.17A < 0.0001
60 T-AOC (mmol/L) 6.18 § 0.10A,a 6.02 § 0.08Ab 5.72 § 0.08B,c 5.58 § 0.11B,d < 0.0001
SOD (U/mL) 270.28 § 2.53A,a 263.18 § 6.74A,b 252.45 § 6.19B 240.34 § 3.93C < 0.0001
GSH-Px (U/mL) 81.07 § 1.98A 75.62 § 2.01B 73.48 § 1.94BC,a 70.51 § 1.24C,b < 0.0001
MDA (nmol/mL) 6.30 § 0.08C 6.87 § 0.09B 7.08 § 0.08Ab 7.21 § 0.11A,a < 0.0001
Abbreviations: MDA, malondialdehyde; MC20, basal diet containing 20% moldy corn; MC40, basal diet containing 40% moldy corn; MC60, basal diet
containing 60% moldy corn; GSH-Px, glutathione peroxidase; SOD, superoxide dismutase; T-AOC, total antioxidant capacity.
Results are represented as mean values § SD (n = 15 per group).
a,b,c
Mean value within a role with no common superscript differ significantly (P < 0.05).
A,B,C
Mean value within a role with no common superscript differ very significantly (P < 0.01).

concentration in the MC60 group was much lower than
that of other groups (P < 0.05).
At d 40, the IFN-a and IL-1b concentrations were sig-
nificantly decreased by all the moldy corn treatments (P
< 0.05), and were lower in the MC40 and MC60 groups
than in MC20 group (P < 0.05). The concentrations of
IL-1b, IL-6, IgA, and IgG were also decreased by all the
moldy corn treatments (P < 0.01). The IL-1b concentra-
tion in the MC40 and MC60 groups were lower than
that of the MC20 group (P < 0.01). The IL-6 and IgA
concentrations were decreased in a dose-dependent man-
ner. The IgG concentration in the MC60 group was
lower than that of MC20 (P < 0.01) and MC40 (P <
0.05) groups. Besides, the IgM concentration was
reduced by MC40 (P < 0.05) and MC60 (P < 0.01) treat-
ments.
At d 60, the concentrations of IFN-a, IL-1b, IL-6, IgA,
and IgG were significantly reduced in all the moldy feed
treatment groups (P < 0.01). Moreover, the IFN-a con-
centration of MC60 group was lower than that of the
MC20 (P < 0.01) and MC40 (P < 0.05) groups. The IL-
1b concentration in the MC40 and MC60 groups was
lower than that of the MC20 group (P < 0.01). More-
over, all the moldy corn treatments decreased IL-6 (P <
0.01) and IgA (P < 0.05) concentrations in a dose-depen-
dent manner. The IgG concentration was decreased in
the MC60 group compared to that of the MC20 group
(P < 0.01). Additionally, the IgM concentration was
reduced in the MC40 and MC60 groups (P < 0.01) in a
dose-dependent manner (P < 0.05; Table 5).
Effects of Moldy Corn on the Metabolism of
Laying Hens
At d 20, the MC40 and MC60 contributed to the
increase of AST activity (P < 0.01). The ALT activity
and TG content were only increased in the MC60 group
(P < 0.05). The contents of TP and HDL-C were
decreased in the MC40 and MC60 groups (P < 0.05).
The LDL-C content was reduced in the MC60 group
compared to the Control and MC20 groups (P < 0.01).
Moreover, all the moldy corn treatments decreased the
T-CHO content (P < 0.01) and increased the UA con-
tent (P < 0.05).
At d 40, the AST activity and TP content were
increased, while HDL-C and TG contents were
decreased in the MC40 and MC60 groups (P < 0.05).
The ALT activity and T-CHO content were increased
by all the moldy corn treatments (P < 0.05). Besides, all
the moldy corn treatments elevated the UA content (P
< 0.01), which reached the highest in the MC60 group.
At d 60, the AST activity was increased by all the
moldy corn treatments (P < 0.01). The ALT activity,
TG content and UA concentration were increased, while
LDL-C content was decreased in the MC40 and MC60
treatments (P < 0.05). On the contrary, the TP, HDL-
C, and T-CHO contents were decreased by all the moldy
corn treatments (P < 0.05; Table 6).
Effects of Moldy Corn on the Residues of
Mycotoxins in Eggs
At d 20, the AFB1 residue in eggs were increased in
the MC40 and MC60 groups (P < 0.01) in a dose-depen-
dent manner. Moreover, there were no ZEN and DON
residues in eggs of all the moldy corn treatment groups.
At d 40, the AFB1 residue in eggs were found in the
MC40 and MC60 groups. The ZEN residue was only
found in the eggs of the MC60 group. There were no
detectable DON residues in eggs of all the moldy corn
treatment groups.
At d 60, no AFB1 and ZEN were found in the Control
and MC20 groups, but the AFB1 and ZEN residues in
eggs were increased in the MC40 and MC60 groups, and
the ZEN content in the MC60 group was higher than
that of the MC40 group (P < 0.01). There were no DON
residues in eggs of all the moldy corn treatment groups
(Figure 2).
 ADVERSE EFFECTS OF MOLDY CORN ON LAYING HENS 7
Table 5. Effects of moldy corn on the immune function of laying hens.

Treatments
days Items Control MC20 MC40 MC60 P value
20 IFN-a (pg/mL) 189.36 § 12.15A
163.15 § 14.12 B,a
157.83 § 15.32B,a
140.12 § 10.69B,b
0.000
IL-1b (ng/L) 22.32 § 1.12A 18.56 § 1.22B 17.39 § 1.53BC,a 15.22 § 1.63C,b < 0.0001
IL-6 (ng/L) 25.12 § 0.50A 24.22 § 1.29A 20.27 § 0.32B 15.87 § 0.30C < 0.0001
IgA (mg/mL) 10.73 § 0.92A 9.55 § 0.89A 7.63 § 0.77B,a 6.44 § 0.93B,b < 0.0001
IgG (mg/mL) 98.24 § 0.98A 92.15 § 1.35B 82.32 § 1.67C,a 79.88 § 1.22C,b < 0.0001
IgM (ng/mL) 5.82 § 0.32A 5.67 § 0.28A 5.55 § 0.26AB,a 4.96 § 0.45B,b 0.004
40 IFN-a (pg/mL) 181.42 § 14.25A,a 160.25 § 13.23AB,b 146.52 § 12.32Bb,c 139.26 § 14.25B,c 0.001
IL-1b (ng/L) 21.25 § 1.17A 17.53 § 1.06B 14.22 § 1.78C 12.33 § 1.84C < 0.0001
IL-6 (ng/L) 23.15 § 0.41A 17.66 § 0.45B 16.35 § 0.63C 14.22 § 0.62D < 0.0001
IgA (mg/mL) 9.86 § 0.53A 8.21 § 0.42B 6.54 § 0.91C,a 5.32 § 0.94C,b < 0.0001
IgG (mg/mL) 94.55 § 4.19A 84.35 § 4.48B 79.87 § 4.70BC,a 73.27 § 5.42C,b < 0.0001
IgM (ng/mL) 5.76 § 0.32A,a 5.52 § 0.36A,ab 5.21 § 0.27AB,b 4.62 § 0.38B,c 0.000
60 IFN-a (pg/mL) 175.89 § 10.12A 150.32 § 9.72B 142.17 § 11.35BC,a 125.82 § 12.37C,b < 0.0001
IL-1b (ng/L) 20.24 § 1.23A 16.54 § 2.24B 12.33 § 1.42C 11.24 § 1.55C < 0.0001
IL-6 (ng/L) 21.55 § 0.56A 16.83 § 0.43B 15.69 § 0.47C 13.22 § 0.41D < 0.0001
IgA (mg/mL) 9.22 § 0.43A 6.47 § 0.25B 5.32 § 0.38C,a 4.81 § 0.36C,b < 0.0001
IgG (mg/mL) 93.18 § 4.56A 82.15 § 4.65B 76.55 § 5.16BC 72.32 § 5.26C < 0.0001
IgM (ng/mL) 5.55 § 0.42A 5.21 § 0.32AB 4.82 § 0.25BC,a 4.27 § 0.32C,b 0.000
Abbreviations: IFN-a, interferon-a; IL-1b, interleukin-1b; IL-6, interleukin-6; IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin
M; MC20, basal diet containing 20% moldy corn; MC40, basal diet containing 40% moldy corn; MC60, basal diet containing 60% moldy corn.
Results are represented as mean values § SD (n = 15 per group).
a,b,c
Mean value within a role with no common superscript differ significantly (P < 0.05).
A,B,C,D
Mean value within a role with no common superscript differ very significantly (P < 0.01).

Effects of Moldy Corn on the Residues of
Mycotoxins in Muscle and Edible Viscera
According to Figure 3, AFB1 residues in liver were
increased in all the moldy corn treatments in a dose-depen-
dent manner. In heart, the AFB1 residue was increased in
the MC40 and MC60 groups (P < 0.01) in a dose-
dependent manner. Besides, the AFB1 was only found in
the leg muscle of MC60 group. Moreover, in the moldy
corn treatments, the ZEN residues were only detected in
liver and leg muscle. The MC40 and MC60 treatments
increased ZEN residues in the liver in a dose-dependent
manner (P < 0.01). The MC60 treatment also led to
increased ZEN residues in leg muscle (Figure 4).

Table 6. Effects of moldy corn on the metabolism of laying hens.

Treatments
days Items Control MC20 MC40 MC60 P value
20 AST (U/L) 28.32 § 1.25 C
29.89 § 1.38BC
31.10 § 1.35AB
32.45 § 1.05A 0.001
ALT (U/L) 3.58 § 0.25b 3.54 § 0.35b 3.97 § 0.49ab 4.15 § 0.21a 0.031
TP (mg/mL) 52.89 § 0.80a 51.96 § 1.41ab 51.01 § 1.35bc 50.11 § 1.25c 0.015
HDL-C (mmol/L) 1.60 § 0.14a 1.55 § 0.12ab 1.42 § 0.14bc 1.35 § 0.11c 0.024
LDL-C (mmol/L) 0.64 § 0.06A 0.64 § 0.03A 0.56 § 0.09AB 0.50 § 0.05B 0.006
T-CHO (mmol/L) 2.98 § 0.07A 2.66 § 0.08B,a 2.67 § 0.12B,a 2.52 § 0.09B,b < 0.0001
TG (mmol/L) 18.25 § 1.13b 18.31 § 2.11b 18.88 § 1.24b 20.91 § 0.97a 0.031
UA (mmol/L) 280.26 § 8.54B,c 296.89 § 13.55AB,b 310.22 § 6.54A,ab 316.04 § 9.82A,a 0.000
40 AST (U/L) 31.24 § 1.43b 33.64 § 1.75ab 34.10 § 1.69a 35.20 § 2.72a 0.036
ALT (U/L) 4.11 § 0.12B 4.41 § 0.11A,b 4.54 § 0.08A,ab 4.67 § 0.21A,a < 0.0001
TP (mg/mL) 54.01 § 1.34A 52.33 § 1.26A 48.01 § 2.24B 47.06 § 1.85B < 0.0001
HDL-C (mmol/L) 2.11 § 0.28a 2.02 § 0.28ab 1.78 § 0.13bc 1.69 § 0.10c 0.022
LDL-C (mmol/L) 0.68 § 0.05a 0.65 § 0.04a 0.64 § 0.10a 0.54 § 0.03b 0.014
T-CHO (mmol/L) 4.21 § 0.18A,a 3.92 § 0.08A,b 3.94 § 0.22A,b 2.84 § 0.18B < 0.0001
TG (mmol/L) 19.66 § 1.18b 20.02 § 1.24b 22.17 § 2.12a 22.69 § 1.47a 0.014
UA (mmol/L) 288.12 § 3.28C,b 300.97 § 5.32BC,a 302.15 § 9.24B 316.89 § 8.53A < 0.0001
60 AST (U/L) 35.15 § 1.66B 38.32 § 1.76A 38.45 § 1.05A 38.69 § 1.55A 0.006
ALT (U/L) 5.08 § 0.34B,c 5.32 § 0.11B,bc 5.48 § 0.11AB,b 5.86 § 0.38A,a 0.002
TP (mg/mL) 53.64 § 0.83A,a 51.77 § 1.54A,b 49.13 § 1.12B 48.06 § 1.23B < 0.0001
HDL-C (mmol/L) 2.34 § 0.08A 2.11 § 0.07B,a 2.04 § 0.06B,ab 1.97 § 0.08B,b < 0.0001
LDL-C (mmol/L) 1.05 § 0.06a 0.96 § 0.08ab 0.85 § 0.22bc 0.74 § 0.11c 0.011
T-CHO (mmol/L) 3.82 § 0.12A 3.41 § 0.15B,a 3.41 § 0.18B,a 3.11 § 0.21B,b < 0.0001
TG (mmol/L) 21.52 § 0.55b 22.65 § 0.93ab 23.48 § 1.42a 23.56 § 0.83a 0.016
UA (mmol/L) 305.79 § 4.05C 315.56 § 8.69BC 320.24 § 8.40B 335.61 § 7.62A < 0.0001
Abbreviations: AST, aspartate transaminase; ALT, alanine transaminase; TP, total protein; HDL-C, high density lipoprotein cholesterol; LDL-C, low
density lipoprotein cholesterol; MC20, basal diet containing 20% moldy corn; MC40, basal diet containing 40% moldy corn; MC60, basal diet containing
60% moldy corn; T-CHO, total cholesterol; TG, total triglycerides; UA, uric acid.
Results are represented as mean values § SD (n = 15 per group).
a,b,c,d
Mean value within a role with no common superscript differ significantly (P < 0.05).
A,B,C
Mean value within a role with no common superscript differ very significantly (P < 0.01).
8 ZHU ET AL.

Figure 2. Effects of moldy corn on the mycotoxin contents in eggs. Values are means with their SD represented by vertical bars (n = 5 per
group). End-point means without a common letter are significantly different (a or b, P < 0.05; A or B, P < 0.01).

Figure 3. Effects of moldy corn on the AFB1 contents in muscle and edible viscera of laying hens. Values are means with their SD represented by
vertical bars (n = 15 per group). End-point means without a common letter are significantly different (A, B or C, P < 0.01).

Furthermore, the DON was not found in all the tested
muscle and edible viscera with moldy corn treatments.
DISCUSSION
Dietary mycotoxins in laying hens can precipitate egg
production defects, leading to heavy economic losses. In
China, co-contamination with AFB1, ZEN, and DON
was commonly found in feed and feed ingredients (Ma
et al., 2018). The knowledge of single toxin mechanisms
of action may help predicting the potential interactive
effects of mixtures, but the relative concentration of
each toxin in the mixture may influence the combined
final effect (Tavares et al., 2013). Considering that natu-
rally contaminated feed could contain multiple toxins, it
is important to research the toxicity and clinical signs
when more than one mycotoxin is present (Christofidou
et al., 2015; Jia et al., 2016).
In the present study, the AFB1 content in the MC20
group, the AFB1 and ZEN contents in the MC40 groups,
and the AFB1, ZEN, DON contents in the MC60 group

Figure 4. Effect of moldy corn on ZEN contents in muscle and edible viscera of laying hens. Values are means with their SD represented by ver-
tical bars (n = 15 per group). End-point means without a common letter are significantly different (A, B or C, P < 0.01).
 ADVERSE EFFECTS OF MOLDY CORN ON LAYING HENS
were higher than the limit standard. Although relatively
high percentages of moldy corn were used in the present
study, the levels of amino acids and vitamins in the 5%
premix fully meet the requirements of laying hens. In
addition, previous studies implied that mycotoxins, such
as DON, had no significant influence on the nutrient com-
position of feed materials (Pietsch et al., 2014; Holanda
and Kim, 2020). Bartov et al. (1982) also reported that
the fat concentration of wetted whole corn did not change
during storage, and no significant changes in fatty acid
composition or in vitamin E, carotenoids, or protein con-
centration were observed. Thus, the toxic effect of moldy
corn on laying hens can be considered as the consequences
of toxic effects of mycotoxins.
The effects of moldy corn on the performance and
health of laying hens were first investigated. Results
showed that the moldy corn treatments decreased the
ADFI, average egg weight, laying rate, and increased
broken egg rate. Moreover, low percentage of moldy
corn (MC20) could cause significant changes of ADFI
and laying rate. Higher level of moldy corn (MC40 or
MC60) could alter the average egg weight and broken
egg rate significantly at d 20 and d 40. Moldy corn treat-
ments also led to higher feed/egg ratio at d 40 and d 60.
No death/culling occurred at d 20 and d 40, but
occurred in the MC40 and MC60 groups at d 60, imply-
ing there was an additive effect of higher levels of AFB1,
ZEN and DON on the reduction of performance. Using
naturally contaminated diets containing similar AF and
ZEN levels (AF: 123.0 mg/kg, ZEN: 260.2 mg/kg), Jia
et al. (2016) found that naturally contaminated diets
also led to lower egg production, feed intake and shell
thickness of laying hens. Zhao et al. (2021) reported that
dietary supplementation of 0.15 mg/kg AFB1,
1.5 mg/kg DON, and 0.12 mg/kg OTA decreased total
feed intake, total egg weight, and egg laying rate, but
increased feed/egg ratio and mortality, and the
increased mortality may be due to the toxicity of OTA
and higher AFB1 concentration than that of our study.
Mycotoxins can evoke oxidative stress by generating
reactive oxygen species (ROS). The main consequences
of ROS induced by mycotoxins, such as AFB1, DON,
and ZEN, are damage to DNA and mitochondrial lesions
(Liu and Wang, 2016; Da Silva et al., 2018). Mycotoxins
can change the intracellular antioxidant mechanisms
such as Nrf2, SOD, GSH-PX, and CAT expressions,
inhibiting antioxidase activity and lead to increase in
lipid peroxidation, which is evaluated through the
amount of MDA (Da Silva et al., 2018). It has been
reported that chickens fed with 100 mg/kg of AFB1 had
significantly decreased antioxidant enzyme activities of
total SOD, CAT, GSH-Px, and T-AOC but increased
MDA content (Guo et al., 2021). Naturally contami-
nated diet also caused lower SOD activity and higher
MDA level in the chicken serum (Christofidou et al.,
2015). In vitro studies also displayed that various myco-
toxins increased lipid peroxidation, and decreased levels
of antioxidase (Lautert et al., 2014; Azizi et al., 2021).
Similarly, here we also found that moldy corn treat-
ments induced obvious oxidative stress by decreasing T-
9
AOC, SOD, GSH-Px, and increasing MDA level at d 20,
40, and 60. Moreover, the activities of GSH-Px and SOD
were sensitive to low level of moldy corn as they were sig-
nificantly altered by MC20 treatment. Moreover, the
MDA level was increased in the moldy corn treatments
in a dose-dependent manner. Our results were consistent
with previous study indicating that ZEN or DON dis-
played synergistic effects with AFB1 on oxidative dam-
age (Lei et al., 2013).
Studies have demonstrated that immunosuppressive
level elevated under mycotoxin contaminations (Berek
et al., 2001; Awad et al., 2013). Mycotoxin-induced
immunosuppression may be established as depressed T
or B lymphocyte activity, suppressed immunoglobulin
and antibody production, reduced complement or inter-
feron activity, and impaired macrophage/neutrophil-
effector activities. Besides, the inhibition of DNA, RNA,
and protein synthesis via a variety of different mecha-
nisms appears to be directly or indirectly responsible for
the immunosuppressive action of many mycotoxins
(Corrier, 1991). The immunosuppressive potency of var-
ious mycotoxins differs substantially. In the study by
Berek et al. (2001), among the mycotoxins tested, T-2
toxin, fusarenon X, nivalenol, and DON showed the
highest immunosuppressing effect in vitro. Studies in
chickens also suggested that DON in feed suppressed the
antibody to infectious bronchitis vaccine and Newcastle
disease virus, and decreased plasma TNF-a (Awad
et al., 2013); ZEN also decreased IL-6 levels in splenic
lymphocytes (Wang et al., 2012). In the current study,
laying hens fed with moldy corn showed decreased IFN-
a, IL-1b, IL-6, IgA, IgG, and IgM concentrations. More-
over, IFN-a, IL-1b, IgG were significantly altered by
MC20 treatment at d 20, but IgM was only significantly
altered by MC60 at d 20, and by MC40 and MC60 for
longer days, implying IgM was less sensitive to moldy
corn than other parameters. Additionally, when more
mycotoxins in the moldy corn were above the limit stan-
dard, the immunosuppressive effect was additive. Previ-
ous study also suggested that combined exposures to
alternariol, ZEN, and DON affected the immune func-
tion of THP-1 cells additively (Solhaug et al., 2016),
unfortunately, the combined toxicological effects of
AFB1, ZEN and DON on immunity were not investi-
gated in chickens in other studies.
Mycotoxins are hepatotoxicity, the levels of serum
ALT and AST were increased in chickens fed with myco-
toxin contaminated diet ( Faixov
a et al., 2010; Gholami-
Ahangaran et al., 2016). Similarly, the moldy corn treat-
ments in the present study also elevated the activities of
ALT and AST. The liver plays a key role in lipid metab-
olism. Therefore, we further investigated the lipid
metabolism. In the present study, the T-CHO content
was decreased, while TG content was elevated by moldy
corn treatments at day 20, 40 and 60. These findings
were in line with the results by Zhang, in which 25 mg/
kg AFB1 in feed increased TG (+12.5%) and decreased
T-CHO (14.17%) in the chicken blood compared to
the control group (Zhang, 2018). Besides, the present
data demonstrated that the TP level was decreased by
10 ZHU ET AL.
moldy corn treatments. Decreased TP level was also
observed in chickens fed mycotoxin-contaminated diet
(Klap
acov
a et al., 2011; Gholami-Ahangaran et al.,
2016). Moreover, the HDL-C and LDL-C contents were
lowered by moldy corn treatment at days 20, 40 and 60.
Similar to our results, Raei et al. (2022) reported that
AFB1 contamination in diet also significantly reduced
the HDL-C in chickens. UA is a modulator of lipid
metabolism, which can affect fat accumulation through
the stimulation of oxidative stress (Lima et al., 2015).
Contaminated grains in broilers caused significant linear
increases in serum UA concentration (Swamy et al.,
2002). In the current study, moldy corn treatments ele-
vated UA concentration at days 20, 40 and 60. It is
reported that mixtures of ZEN or FB1 and DON display
synergistic effects in lipid metabolism (Kouadio et al.,
2007). Here, the lipid metabolism parameters, such as
TG and TP were lower in MC40 group than that in
MC20 group, suggesting that higher level of ZEN and
AFB1 may has synergistic effects.
Mycotoxins can diffuse into foods and accumulate in the
fat parts (Herzallah, 2009). In eggs, milk and meat prod-
ucts, different kinds of mycotoxins have been found (Her-
zallah, 2009; Qu et al., 2019). Low transmission rates of
mycotoxin (below 1%) from feed to eggs are often reported
(Sypecka et al., 2004; Meerpoel et al., 2020). Oliveira et al.
(2000) investigated effects of 100, 300, or 500 mg/kg AFB1
in feed on the AFB1 residue in eggs, and found that AFB1
residues were detected only in eggs of hens given 500 mg/
kg AFB1, at levels that ranged from 0.05 to 0.16 mg/kg.
Previous study also reported relative lower level of AFB1
in the examined egg samples (Fern
andez et al., 1994). Sim-
ilar results were obtained in the present study, demonstrat-
ing that 25.36 to 76.5 mg/kg AFB1 in diets led to 0.0049
to 0.0077 mg/kg AFB1 residue in eggs. To our knowledge,
there is no fixed maximum limits for AFB1 residues in
chicken eggs. However, Pourelmi et al. (2013) stipulated a
limit of 12 ng/mL AFB1 for table eggs. The European
Union (EU) also has set maximum levels for AFB1 and
total AFs in different foods for direct human consumption,
ranging from 2 to 15 mg/kg for AFB1 and total AFs,
respectively (Ebrahem et al., 2014). The AFB1 contamina-
tions in our study were lower than the aforementioned egg
residuals, and none of the samples exceeded the limits.
Moreover, using diets containing 5, 7.5, and 10 mg/kg
DON together with 137.5, 206, and 275 mg/kg ZEN,
Sypecka et al. (2004) found no ZEN residue in eggs. Incon-
sistently, in the present study, the ZEN was detected in
eggs in the MC40 and MC60 groups. This result may be
caused by the higher ZEN contents in the MC40 (505.73
mg/kg) and MC60 (750.3 mg/kg) diets. The European
committee has regulated the maximum levels of ZEN rang-
ing between 20 and 100 ppb in various food commodities
(Richard, 2007), which was higher than the ZEN level in
eggs of the present study. Moreover, the DON residues
were not detectable in all groups at different days in this
study. Similarly, previous studies also showed that DON
was not detectable in eggs after chickens were fed a diet
contaminated with DON (El-Banna et al., 1983; Valenta
and D€ anicke, 2005). The above findings indicate that the
transmission rate of AFB1 from moldy feed to egg is low,
and ZEN residue can not be detected in eggs when the
ZEN content is lower than that in the MC20 group
(245.05 mg/kg). Moreover, levels of DON lower than that
in the MC60 diet (3.34 mg/kg) may be too low for it to be
detected in eggs.
Mycotoxins in muscle and edible viscera may also
damage human health. Previous study showed that the
AFB1 residue levels in liver of chickens fed 1,600 mg/kg
AFB1 ranged from 0.83 to 3.51 ng/g (Hussain et al.,
2010). In the present study, 25.36, 51.62, and 76.5 mg/
kg AFB1 in the MC20, MC40, and MC60 diets led to
0.015, 0.0341, and 0.053 mg/kg liver AFB1 residue, and
the transmission rate was consistent with the study by
Hussain et al. (2010). The AFB1 residue levels in muscles
of young chicks fed 6,400 mg/kg AFB1 was 3.27 ng/g,
and the transmission rate of AFB1 from feed to liver was
higher than our result. This may be due to the combina-
tion of multiple mycotoxins, because the combined
effects of mycotoxins in feed resulted in increased resi-
dues and in broiler chickens’ organs (Chang et al.,
2020). Moreover, the Food and Drug Administration
(FDA) tolerance level of AF in human food is 20 mg/kg
(El-Nabarawy et al., 2020), which was higher than the
detected AFB1 levels in muscle and edible viscera of the
present study. Due to the global occurrence of ZEN, the
European Food Safety Authority (ESFA) has set guide-
lines for the maximum levels of ZEN allowable in various
foodstuffs (Thapa et al., 2021), which were also higher
than the detected ZEN levels in liver and leg muscle in
the present study. Moreover, report showed that
2.5 mg/kg ZEN in feed led to around 1,100 ng/kg ZEN
residue in liver (Jia et al., 2022), and the transmission
rate of ZEN from feed to liver was a little higher than
that in our study. This may be due to the high ZEN
metabolism in chickens (Tardieu et al., 2021), as
D€anicke et al. (2002) reported that a-ZEL was detect-
able in the liver, whereas ZEN was not detected in either
breast meat or the liver of laying hens. However, the
ZEN metabolism should be further investigated.
In conclusion, the moldy corn with excessive AFB1,
ZEN, DON above the limits can impair the ADFI and
laying performance of laying hens. Moreover, the moldy
corn also decreased antioxidant capacity, increased
immunosuppression, impaired liver function and metab-
olism. Although AFB1 and ZEN residues were found in
eggs, muscle and edible viscera, the residue levels were
relatively low and may not have adverse impact on
human health. In addition, the toxic effects and myco-
toxin residues (AFB1 and ZEN) were increased with the
increase of moldy corn levels in feed. However, the com-
bination effects of multiple mycotoxins including AFB1,
ZEN and DON, should be further explored by using
binary and ternary mixtures.
ACKNOWLEDGMENTS
This work was supported by the Public Sector Science
and Technology Support Program of Qingdao (Grant
 ADVERSE EFFECTS OF MOLDY CORN ON LAYING HENS
No. 12-1-3-32-nsh), the National Modern Agricultural
Industry Technology System Project of Shandong Prov-
ince (SDAIT-11-07), National Natural Science Founda-
tion of China (Grant No. 32102586) and the Shandong
Provincial Natural Science Foundation (Grant No.
ZR2020QC183) .
Author contributions: Fenghua Zhu: conceptualiza-
tion, visualization, funding acquisition. Lianqin Zhu:
data circulation. Jindong Xu: visualization. Yuchang
Wang: investigation. Yang Wang: data circulation, writ-
ing-original draft.
DISCLOSURES
The authors declare that the research was conducted
in the absence of any commercial or financial relation-
ships that could be construed as a potential conflict of
interest.
SUPPLEMENTARY MATERIALS
Supplementary material associated with this article
can be found in the online version at doi:10.1016/j.
psj.2023.102502.
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