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Received: 1 December 2020    Revised: 31 January 2021    Accepted: 19 February 2021

DOI: 10.1111/asj.13540

ORIGINAL ARTICLE

Evaluation of the protective effect of lycopene on growth

performance, intestinal morphology, and digestive enzyme
activities of aflatoxinB1 challenged broilers

Md Touhiduzzaman Sarker  | Zhi Yue Wang | Haiming Yang | Xiaoli Wan |

Asare Emmanuel

College of Animal Science and Technology,

Yangzhou University, Yangzhou City, China
Correspondence
Zhi Yue Wang, Department of Animal
Nutrition and Feed Science, College of
Animal Science and Technology, Yangzhou
University, Wenhui East Road 78# Yangzhou
city, Jiangsu province 225009, China.
Email: dkwzy@263.net
Funding information
Data Center of Management Science,
National Natural Science Foundation of
China -­Peking University, Grant/Award
Number: 31802095
Abstract
The current study was conducted to investigate the protective efficiency of dietary
lycopene (LYC) supplementation on growth performance, intestinal morphology, and
digestive enzyme activities aflatoxinB1 (AFB1) challenged broilers. A total of 240 days
old Arber across male broiler chicks were randomly allocated in five treatments and
six replicates (eight birds per replicate); feed and water were provided ad libitum
during the 42 days experiment. The treatment diets were as follows: (i) Basal diet
(control), (ii) Basal diet + 100 µg/kg AFB1 contaminated diet, (iii) Basal diet + 100 µg/
kg AFB1 + 100 mg/kg LYC1, (iv) Basal diet + 100 µg/kg AFB1 + 200 mg/kg LYC2,
and (v) Basal diet + 100 µg/kg AFB1 + 400 mg/kg LYC3. The results showed that the
addition of LYC to AFB1 contaminated broiler diets significantly increased (p < .05)
average daily gain (ADG) and decreased feed conversion ratio (FCR) compared to the
AFB1 diet. AFB1 diet decreased the intestinal villus height (VH) and crypt depth ratio
(VCR) while increasing the crypt depth (CD). However, dietary LYC supplemented
diets relieved the intestinal morphological alterations. Dietary LYC supplementation
(200 and 400 mg/kg) significantly improved (p  < .05) intestinal digestive enzyme
amylase and lipase activities with AFB1 contaminated diet. These findings suggested
that LYC is a promising feed supplement in the broiler industry, alleviating the harm-
ful effects of AFB1.

KEYWORDS

aflatoxinB1, broiler, growth performance, intestine, lycopene

1 |  I NTRO D U C TI O N increased susceptibility to diseases in several animal species (Peng

Mycotoxins are natural compounds produced by fungi, and afla-
toxinB1 (AFB1) is the most commonly known type of mycotoxins,
which are mainly produced by the Aspergillus flavus and Aspergillus
paraciticus (Diaz et al., 2003). AFB1 is considered the most highly
widespread and toxic type of aflatoxin (Alpsoy & Yalvac, 2011). The
negative effects of AFB1 have also been well documented, includ-
ing reduced performance, decreased immune system function, and
et al., 2016; Shivachandra et al., 2003; Yunus et al., 2011). Previous
researches have shown that poultry was extremely sensitive to the
toxic and carcinogenic action of AFB1, resulting in millions of dollars
in annual losses to farmers due to reduced growth rate, eggs and
meat production, increased susceptibility to disease, and other ad-
verse effects (Guo et al., 2012; Rawal et al., 2010; Shi et al., 2012;
Yunus et al., 2011). Feeding naturally AFB1 contaminated diets in
the starter and grower stage compromised growth performance,

Anim Sci J. 2021;92:e13540. wileyonlinelibrary.com/journal/asj |

© 2021 Japanese Society of Animal Science     1 of 9
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intestinal morphology, changes in digestive physiology, and devel-
opment in ducks (Feng et al., 2017).
Recently, there has been an increasing awareness of the adverse
effects of AFB1 on vulnerable structures in the intestine and the im-
pairment of intestinal integrity (Bouhet & Oswald, 2005). The intes-
tinal tract is the first target organ which is been destructed by the
activities of AFB1 when it enters into the body of the animals, thus,
compared with other organs (liver, lungs, spleen, and kidney). This
toxin exerts greater impacts on the small intestine. Additionally, the
animal's intestinal health status depends on the integrity and inflam-
matory levels of the animals' intestine (Bischoff, 2011). It was shown
that like other mycotoxins, AFB1 altered the intestinal absorbing bar-
rier (Ali Rajput et al., 2017), reduced the activity of digestive enzymes,
and disturbed the energy metabolism of the intestinal cells as well
as the gluconeogenesis, fatty acids synthesis (Ling et al., 2016; Long
et al., 2016; Pinton & Oswald, 2014). Digestive enzymes such as am-
ylase and lipase are secreted by the intestinal tract; they are closely
related to digestion and absorption of nutrients as well as growth and
development. They also play vital tasks such as muscle building, de-
stroying toxins, and breakdown food partials for nutrient absorption.
Lycopene (LYC), a naturally occurring carotenoid found in
tomatoes and tomato products, has drawn considerable atten-
tion as a potential chemopreventive agent (Boeira et al., 2014).
It was one of the most effective antioxidants in the carotenoid
family, and its activity against biological reactive oxygen species
may prevent or ameliorate oxidative damage to cells and tissues
(Boeira et al., 2014; Yonar, 2012). It was known to exert an effec-
tive free radical scavenging activity, and this action could be ben-
eficial to poultry production because free radicals were formed
under stress, fast-­g rowing, high production rates, and intensive
metabolic conditions of poultry (Englmaierová et al., 2011). In
recent years, LYC, as a beneficial carotenoid, has received con-
siderable scientific attention (Sahin et al., 2006, 2014). Dietary
supplementation with LYC has also been associated with lower
risks of cancers, cardiovascular diseases, and other humans and
animals' disorders (Mordente et al., 2011). Moreover, dietary LYC
supplementation is known to protect the animal intestine (struc-
ture and tissue) from damage after coming into contact with
harmful agents like pathogen, toxins, or any other foreign anti-
gen. Mumtaz et al. (2013) demonstrated that supplementation of
LYC with vitamins A, C, and E to radiation exposed rats' intestinal
toxicity could protect against intestinal damage. Previous studies
(Saada et al., 2010; Yilmaz & Kaya, 2019; Yucel et al., 2016) have
demonstrated the beneficial effects of dietary supplementation
of LYC in the AFB1 contaminated diet on the improved intestinal
structure of animals. However, the protective effects of dietary
LYC in the intestine damage on growth performance against
AFB1 challenged broilers have not been reported. Therefore, this
study aimed to evaluate the protective efficiency of different
levels of dietary LYC supplementation on growth performance,
intestinal morphology, and digestive enzyme activities in AFB1
challenged broilers.
2 | M ATE R I A L S A N D M E TH O DS
This experiment was conducted in the Animal Nutrition and Feed
Science Department, College of Animal Science and Technology,
Yangzhou University, China, and all protocols were approved by
Yangzhou University animal care and use committee.
2.1 | Experimental birds, diets, and husbandry
A total of 240 one-­d ay-­o ld male Arbor Acres broilers were ob-
tained from a commercial hatchery (Nantong, Jiangsu, China).
The broiler chicks were randomly assigned to five treatments
with six replicates (eight birds in each pen). Treatments were as
follows: (a) A basal diet containing neither AFB1 nor LYC (Control)
and (b) a basal diet containing 100 µg/kg AFB1 . The other three
treatment groups were supplemented with LYC at 100, 200, and
400 mg/kg on the 100 µg/kg AFB1 . All broilers were raised in
the cage breeding system in the environmentally controlled room
during the whole experimental period. The room temperature
was 32°C–­3 4°C for the first 3 days, then gradually decreased to
22  ± 1°C by 2°C–­3 °C each week and maintained until 42 days
experimental period. All broilers in the experimental period free
access to feed and water with a lighting cycle of 23 hr light and
1 hr dark. Corn-­s oybean-­b ased diets were formulated accord-
ing to NRC (1994) to meet the nutrient requirements from 1 to
21 days (starter) and 22–­42 days (grower) experimental periods
for broilers (Table 1).
AflatoxinB1 (Purity ≥98%, HPLC) and LYC (Purity ≥80%, HPLC)
were purchased from Shanghai Yuanye Biotechnology Co. Ltd.
Previous studies have demonstrated that 100 µg/kg AFB1 in diets
had obvious adverse effects on broilers, and appropriate levels of
LYC supplementation in the diet (200–­4 00 mg/kg) could provide op-
timal protective effects against heat stress broilers (Liu et al., 2016;
Peng et al., 2014; Sahin et al., 2016). Based on this information, an
appropriate toxic concentration and dietary supplementation levels
of LYC were chosen.
2.2 | Samples collection and measurements
The broiler weight and feed intake (FI) were recorded at 21 and
42 days for calculation of average daily gain (ADG) and average daily
feed intake (ADFI) and feed conversion ratio (FCR). At the end of
the experiment, the broilers were euthanized by cervical dislocation
and the duodenum, jejunum, and ileum were removed. The sample
of small intestine tissues (approximately 2 cm from the middle of
duodenum, jejunum, and ileum, respectively) was collected and then
stored in 10% formaldehyde solution for further analysis. Intestinal
digesta (duodenum, jejunum, and ileum) were collected into a plastic
tube to determine digestive enzyme activities, and all samples were
stored at −40°C until analysis.
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TA B L E 1   Composition of feed ingredients (g/kg) and nutrient and stained with hematoxylin and eosin (H&E). The values of villus
level (%) height (VH), crypt depth (CD), and ratio (VCR) on every segment
Items 1–­21 days 22–­42 days were determined using a light microscope (Nikon YS100) attached
to a computer.
Ingredients (g/kg)
Corn 57.10 61.00
Soybean meal 31.00 28.00
2.4.1 | Intestinal enzyme activity assay
Corn gluten meal 4.00 1.60
Soybean oil 3.00 1.30 Trypsin (catalog No. A080-­2), amylase (catalog No. C016-­1), and li-
Dicalcium phosphate 2.00 2.40 pase (catalog No. A054-­1-­1) activities were measured by using cor-
Limestone 1.20 4.00 responding commercial kits purchased from Nanjing Jian Cheng
L-­Lysine 0.20 0.25 Bioengineering institution. The measurements were performed ac-
DL-­Methionine 0.20 0.15 cording to the kit's instructions.
a
Premix   0.31 1.00
Sodium chloride 0.30 0.30
Calculated nutrient levels (%)
2.5 | Statistical analysis
Metabolizable energy (MJ/kg) 12.61 12.96
Statistical analysis of all data was performed using SPSS software
Crude protein 21.36 19.44
(Version 21; SPSS Inc.). One-­way ANOVA with a post hoc test was
Calcium 1.00 0.93
used to elucidate significant differences. Duncan's test was used for
Available phosphorus 0.46 0.39
multiple comparisons when a significant difference was detected.
Lysine 1.09 1.05 Differences were considered to be statistically significant at p < .05,
Methionine 0.56 0.47 and .05 < p <.10 was considered to be a trend towards significant. All
Arginine 1.27 1.16 data were presented as the mean and standard error of the means
Methionine + cysteine 0.91 0.80 (SEM).
a
The premix provided per kilogram of diet: vitamin A (retinyl acetate),
12,000 IU; vitamin D3 (cholecalciferol), 2,500 IU; vitamin E (DL-­
α-­tocopheryl acetate), 20 IU; menadione, 1.3 mg; thiamin, 2.2 mg; 3 | R E S U LT S
riboflavin, 8.0 mg; nicotinamide, 40 mg; choline chloride, 400 mg;
calcium pantothenate, 10 mg; pyridoxine HCl, 4 mg; biotin, 0.04 mg;
folic acid, 1.0 mg; vitamin B12 (cobalamin), 0.013 mg; Fe (from ferrous 3.1 | Growth performance
sulfate), 80 mg; Cu (from copper sulphate), 8.0 mg; Mn (from manganese
sulphate), 110 mg; Zn( from zinc sulfate), 60 mg; I (from calcium iodate), Data from Table 2 represent the effects of dietary treatment on
1.1 mg; Se (from sodium selenite), 0.3 mg.
broiler growth performance. No significant effect (p < .05) was ob-
served in ADFI throughout the experimental period. However, broil-
ers exposed to AFB1 diet had decreased (p < .05) ADG and increased
2.3 | Preparation of intestinal digesta homogenate FCR during the experimental trial compared to the control group.
Broilers fed diet AFB1 + LYC1 improved (p = .007) ADG as compared
Approximately 0.2 g of intestinal digesta samples (duodenum, jeju- to AFB1 at 1–­21 days. Broilers fed diet AFB1 + LYC2 and AFB1 + LYC3
num, and ileum digesta) was homogenized with cold physiological significantly improved (p < .05) the ADG in 22–­42 and 1–­42 days.
saline solution (4°C) at a ratio of 1:9 (wt/vol) using an ultraturrax Additionally, a trend of decreased (p  = .07) in FCR was ob-
homogenizer (Shanghai Jing Xin). The homogenized solution was served in AFB1 + LYC2 group at 22–­42 days while AFB1 + LYC2, and
then centrifuged (cence DL-­5M) at 2,500 rpm for 10 min at 4°C, and AFB1 + LYC3 groups decreased (p < .05) the FCR in 1–­42 days com-
the supernatant was obtained and immediately stored at −20°C for pared to AFB1 group.
further analysis.

2.4 | Intestinal morphology
The fixed small intestine segments (duodenum, jejunum, and
ileum) were soaked through a graded series of ethanol and xylene,
embedded in paraffin, sections at 5 µm with a Leica RM 2016 mi-
crotome (Leica Biosystems Inc.). The sections were deparafinizied
with xylene and rehydrated through graded dilutions of ethanol
3.2 | Intestine morphology
Comparatively, data on duodenal morphology indicated that AFB1
significantly decreased the VH and VCR, while CD was significantly
increased (p < .05) compared to the control. However, dietary LYC
supplementation increased VH and VCR while decreasing (p < .05)
CD as compared to the AFB1 group on 21 and 42 days (Table 3 and
Figure 1). Additionally, on 21 days, broilers fed diet AFB1 jejunal
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TA B L E 2   Effects of LYC on growth performance of broilers fed diets contaminated with AFB1

Dietary treatments

Parameters Control AFB1 AFB1 + LYC1 AFB1 + LYC2 AFB1 + LYC3 SEM p-­value

1–­21  days
ADG (g/day) 32.55a 28.54c 30.76bab 30.47bc 30.35bc 0.364 .007
ADFI (g/day) 48.25 44.09 46.91 45.90 45.77 0.512 .118
FCR (Feed/gain) 1.48b 1.54a 1.52ab 1.50ab 1.50ab 0.006 .039
22–­42  days
ADG (g/day) 70.27a 63.03b 64.48b 68.41a 67.53a 0.648 <.001
ADFI (g/day) 136.07 127.38 128.07 133.70 132.35 1.388 .222
FCR (Feed/gain) 1.93b 2.01a 1.98ab 1.95b 1.96ab 0.010 .074
1–­42  days
ADG (g/day) 51.51a 46.00 c 47.32bc 49.44ab 48.59b 0.492 <.001
ADFI (g/day) 92.37 86.09 86.90 89.64 88.38 0.907 .202
FCR (Feed/gain) 1.78b 1.87a 1.83ab 1.81b 1.81b 0.007 .016

Note: Means with different superscripts (a, b, c) in the same row were significantly different at p < .05, and .05 < p < .10 was considered to be a
tendency towards significant. SEM, pooled standard error of the means (n = 6).
Abbreviations: ADFI, average daily feed intake; ADG, average daily gain; AFB1 +  LYC1-­(100  µg/kg AFB1 + 100 mg/kg LYC); AFB1 + LYC2 (100 µg/kg
AFB1 + 200 mg/kg LYC); AFB1 + LYC3 (100 µg/kg AFB1 + 400 mg/kg LYC); AFB1-­(100  µg/kg AFB1); AFB1, aflatoxinB1; FCR, feed conversion ratio;
LYC, lycopene; SEM, standard error of the mean.

TA B L E 3   Effects of LYC on duodenum morphology of broiler fed with AFB1 contaminated diets at 21 and 42 days

Dietary treatments

Parameters Days Control AFB1 AFB1 + LYC1 AFB1 + LYC2 AFB1 + LYC3 SEM p-­value

a c b ab ab
VH (µm) 21 1,415.75 1,136.01 1,278.17 1,311.32 1,317.35 24.605 .003
42 1801.66a 1529.98c 1645.82b 1673.89b 1,670.21b 20.157 <.001
CD (µm) 21 205.82b 240.67a 217.18b 213.87b 201.85b 3.900 .007
b a b b
42 266.45 320.74 271.00 259.21 254.66b 6.828 .007
VCR (µm/µm) 21 6.91a 4.73c 5.89b 6.17b 6.05ab 0.170 <.001
a b a a
42 6.82 4.81 6.14 6.71 6.69a 0.169 <.001

Note: Means with different superscripts (a, b, c) in the same row were significantly different at p < .05, and .05 < p < .10 was considered to be a
tendency towards significant. SEM, pooled standard error of the means (n = 6).
Abbreviations: AFB1 + LYC1 (100 µg/kg AFB1 + 100 mg/kg LYC); AFB1 +  LYC2-­(100  µg/kg AFB1 + 200 mg/kg LYC); AFB1 +  LYC3-­(100  µg/kg
AFB1 + 400 mg/kg LYC); AFB1-­(100  µg/kg AFB1); AFB1, aflatoxinB1; CD, crypt depth; LYC, lycopene; SEM, standard error of the mean; VCR, villus
height and crypt depth ratio; VH, villus height.

CD was significantly increased while VCR was also significantly de-
creased (p < .05) as compared to the control.
Moreover, on 42 days, fed diet AFB1 recorded a decreased in VH
and VCR compared to the control. On the other hand, irrespective of
the dosage, LYC addition decreased (p = .008) CD while the addition
AFB1 + LYC2 and AFB1 + LYC3 increased (p = .002) the VH and VCR
at 42 days compared to the AFB1 (Table 4). Similarly, data of ileum
morphology on 21 days indicated that broilers fed diet AFB1 signifi-
cantly decreased (p  < .05) VH and VCR while CD was also signifi-
cantly increased (p < .05) as compared to the control. Furthermore,
on 42 days, fed diet AFB1 recorded a decreased in VH and VCR com-
pared to the control. Besides, except CD on 42 days, dietary LYC
supplementation increased VH and VCR on 21 and 42 days while de-
creased (p < .05) CD on 21 days compared to AFB1 group (Table 5).
3.3 | Digestive enzyme activities
Data on duodenum enzyme activities indicated that AFB1 signifi-
cantly decreased (p < .05) the amylase and lipase activities compared
to the control at 21 and 42 days. However, dietary LYC supplementa-
tion AFB1 + LYC2 and AFB1 + LYC3 on 21 days, and AFB1 + LYC2 on
42 days, significantly increased (p < .05) amylase and lipase activities
compared to the AFB1 group (Table 6).
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F I G U R E 1   Histological structure of
Hematoxylin and Eosin (H&E) staining
duodenum at 21 and 42 days of age.
Control, AFB1-­(100  μg/kg AFB1),
AFB1 +  LYC1-­(100  μg/kg AFB1 + 100 μg/
kg LYC), AFB1 +  LYC2-­(100  μg/
kg AFB1 + 200 mg/kg LYC),
AFB1 +  LYC3-­(100  μg/kg AFB1 + 400 mg/
kg LYC). Scale bar–­4 0 µm

TA B L E 4   Effects of LYC on jejunum morphology of broiler fed with AFB1 contaminated diets at 21 and 42 days

Dietary treatments

Parameters Days Control AFB1 AFB1 + LYC1 AFB1 + LYC2 AFB1 + LYC3 SEM p-­value

VH (µm) 21 838.17 769.44 835.42 842.32 829.61 11.767 .133

a b b a a
42 1,229.49 1,067.06 1,090.04 1,222.20 1,213.78 15.390 <.001
CD (µm) 21 153.62b 188.63a 168.72b 157.96b 160.12b 3.606 .008
42 212.26 222.26 223.50 222.40 220.99 3.648 .886
VCR (µm/µm) 21 5.48a 4.07b 5.00a 5.32a 5.20a 0.126 <.001
42 5.82a 4.81c 4.95bc 5.56a 5.44ab 0.101 .002

Note: Means with different superscripts (a, b, c) in the same row were significantly different at p < .05, and .05 < p < .10 was considered to be a
tendency towards significant. SEM, pooled standard error of the means (n = 6).
Abbreviations: AFB1 +  LYC1-­(100  µg/kg AFB1 + 100 mg/kg LYC); AFB1 +  LYC2-­(100  µg/kg AFB1 + 200 mg/kg LYC); AFB1 +  LYC3-­(100  µg/kg
AFB1 + 400 mg/kg LYC); AFB1-­(100  µg/kg AFB1); AFB1, aflatoxinB1; CD, crypt depth; LYC, lycopene; SEM, standard error of the mean; VCR, villus
height and crypt depth ratio; VH, villus height.

TA B L E 5   Effects of LYC on ileum morphology of broiler fed with AFB1 contaminated diets at 21 and 42 days

Dietary treatments

Parameters Days Control AFB1 AFB1 + LYC1 AFB1 + LYC2 AFB1 + LYC3 SEM p-­value

a b a a a
VH (µm) 21 535.11 440.09 513.67 538.10 508.52 10.457 .011
42 1,069.73a 934.83b 1,033.29a 1,047.91a 1,033.83a 11.446 .010
CD (µm) 21 114.52b 153.02a 128.85b 114.63b 107.41b 4.208 <.001
42 177.34 209.51 185.00 181.84 180.31 4.404 .128
a b a a a
VCR (µm/µm) 21 4.72 2.90 4.08 4.73 4.72 0.160 <.001
42 6.09a 4.63b 5.70a 5.91a 5.63a 0.129 <.001

Note: Means with different superscripts (a, b, c) in the same row were significantly different at p < .05, and .05 < p < .10 was considered to be a
tendency towards significant. SEM, pooled standard error of the means (n = 6).
Abbreviations: AFB1 +  LYC1-­(100  µg/kgAFB1 + 100 mg/kg LYC); AFB1 +  LYC2-­(100  µg/kg AFB1 + 200 mg/kg LYC); AFB1 +  LYC3-­(100  µg/kg
AFB1 + 400 mg/kg LYC); AFB1-­(100  µg/kg AFB1); AFB1, aflatoxinB1; CD, crypt depth; LYC, lycopene; SEM, standard error of the mean; VCR, villus
height and crypt depth ratio; VH, villus height.

In addition, broilers fed diet AFB1 jejunum amylase and lipase
activities on 21 and 42 days, trypsin activities on 42 days, signifi-
cantly decreased (p  < .05) compared to the control. On the other
hand, irrespective of the dosage, LYC supplementation AFB1 + LYC2,
and AFB1 + LYC3 increased (p < .05) amylase and lipase activities on
42 days compared to the AFB1 group (Table 7).
Similarly, ileum lipase activities of broilers fed diet AFB1 on
21 days, and amylase and lipase activities on 42 days were signifi-
cantly decreased (p  < .05) compared to the control. Conversely,
dietary LYC supplementation AFB1 + LYC2 and AFB1 + LYC3 signifi-
cantly increased (p < .05) amylase activities on 42 days, compared to
the AFB1 group (Table 8).
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TA B L E 6   Effect of LYC on duodenum digestive enzyme activity of broiler fed with AFB1 contaminated diets at 21 and 42 days

Dietary treatments

Parameters Days Control AFB1 AFB1 + LYC1 AFB1 + LYC2 AFB1 + LYC3 SEM p-­value

Trypsin (U/gprot) 21 2.01 1.49 1.73 1.88 1.78 0.068 .166

42 1.91 1.55 1.81 1.88 1.79 0.063 .437
Amylase (U/gprot) 21 179.51a 135.54c 153.73bc 168.75ab 158.80ab 4.047 .003
a b ab a ab
42 170.91 148.94 155.37 167.52 162.18 2.741 .061
Lipase (U/gprot) 21 3.78a 2.87b 3.26ab 3.66a 3.58a 0.093 .006
a c c ab bc
42 3.59 2.83 2.91b 3.41 3.11 0.074 <.001

Note: Means with different superscripts (a, b, c) in the same row were significantly different at p < .05, and .05 < p < .10 was considered to be a
tendency towards significant. SEM, pooled standard error of the means (n = 6).
Abbreviations: AFB1 +  LYC1-­(100  µg/kgAFB1 + 100 mg/kg LYC); AFB1 +  LYC2-­(100  µg/kg AFB1 + 200 mg/kg LYC); AFB1 +  LYC3-­(100  µg/kg
AFB1 + 400 mg/kg LYC); AFB1-­(100 µg/kg AFB1); AFB1, AflatoxinB1; LYC, Lycopene; SEM, standard error of the mean.

TA B L E 7   Effect of LYC on jejunum digestive enzyme activity of broiler fed with AFB1 contaminated diets at 21 and 42 days

Dietary treatments

Parameters Days Control AFB1 AFB1 + LYC1 AFB1 + LYC2 AFB1 + LYC3 SEM p-­value

Trypsin (U/gprot) 21 1.76 1.46 1.49 1.68 1.65 0.049 .260

a b b ab ab
42 2.23 1.95 1.95 2.17 2.03 0.036 .030
Amylase (U/gprot) 21 177.22a 152.74b 158.96ab 170.99ab 164.92ab 2.966 .063
42 139.00a 113.05c 122.09bc 130.69ab 127.81b 2.072 <.001
a b b ab
Lipase (U/gprot) 21 3.65 2.87 2.98 3.13 3.12ab 0.086 .037
a c c ab ab
42 2.79 1.89 2.07b 2.57 2.44 0.093 .007

Note: Means with different superscripts (a, b, c) in the same row were significantly different at p < .05, and .05 < p < .10 was considered to be a
tendency towards significant. SEM, pooled standard error of the means (n = 6).
Abbreviations: AFB1 +  LYC1-­(100  µg/kg AFB1 + 100 mg/kg LYC); AFB1 +  LYC2-­(100  µg/kg AFB1 + 200 mg/kg LYC); AFB1 +  LYC3-­(100  µg/kg
AFB1 + 400 mg/kg LYC); AFB1-­(100  µg/kg AFB1); AFB1, aflatoxinB1; LYC, lycopene; SEM, standard error of the mean.

4 |  D I S CU S S I O N ADG (Englmaierová et al., 2011), FI (Lira et al., 2010) and could

Poultry is an important international food commodity, and the
broiler is the world's most available and major protein source.
In the recent era, broiler feed is hard to avoid toxic contamina-
tion; it is increasingly recognized that long-­term consumption of
AFB1 contaminated feed has detrimental effects on broiler growth
and performance. In this present study, AFB1 (100 µg/kg) signifi-
cantly depressed the ADG and increased the FCR but did not in-
fluence the ADFI during the whole experimental period. Similar
results had been observed in the previous studies, in which AFB1
(50–­100  µg/kg) could significantly decrease the body weight
gain and FCR of broilers, causing economic losses (Bintvihok &
Kositcharoenkul, 2006; Fan et al., 2013). Another study in ducks
fed diets contaminated with 20 µg/kg of AFB1 had a significantly
reduced ADG and increased FCR (Han et al., 2008). The possible
explanation might be anorexia, listlessness, impairment of general
health and the inhibitory effects of AFB1 on protein synthesis and
lipogeneses.
Several researches conducted by different authors indicated
that dietary supplemented with LYC in chicken diets increased
even alleviate adverse effects of stress conditions on performance
(Sahin et al., 2016). Moreover, LYC supplementation in quail's diets
improved the ADG and FCR even under stress conditions (Sahin
et al., 2006). In the present study, we were observed that supple-
mentation with different levels of LYC (100, 200, and 400 mg/kg)
to the moldy diets ameliorated the growth suppression induced by
AFB1, as evidenced by significantly increased ADG and decreased
FCR. So the toxic effect of AFB1 on growth performance can be
overcome by the addition of LYC in the broiler diet.
The intestine acts as feed digestion, absorption function,
which also acts as an important fighter against all pathogenic
bacteria and toxic substances present in the intestinal lumen. The
intestinal villus and crypt system play an important role in nutri-
ents absorption and animal growth (Hernandez et al., 2006). These
roles provide the conditional environment forming the indicators
of intestinal health status. It has been suggested that shorter villus
height and deeper crypt depth caused poor nutrient absorption,
increased secretion of electrolytes and water in the intestinal
tract, and compromised animals' growth and performance (Li
et al., 2015). In this present study, we found that compared with
SARKER et al. |
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TA B L E 8   Effect of LYC on ileum digestive enzyme activity of broiler fed with AFB1 contaminated diets at 21 and 42 days

Dietary treatments

Parameters Days Control AFB1 AFB1 + LYC1 AFB1 + LYC2 AFB1 + LYC3 SEM p-­value

Trypsin (U/gprot) 21 2.11 1.75 1.93 1.99 1.99 0.077 .721

42 1.89 1.34 1.65 1.86 1.65 0.072 .111
Amylase (U/gprot) 21 154.53 133.74 139.09 150.03 147.94 3.860 .453
a c c ab ab
42 217.33 167.99 188.14b 195.81 193.81 4.510 .070
Lipase (U/gprot) 21 3.75a 2.31b 2.78b 3.03ab 2.93ab 0.141 .017
a b b ab b
42 4.13 3.32 3.39 3.82 3.46 0.097 .028

Note: Means with different superscripts (a, b, c) in the same row were significantly different at p <.05, and .05 < p < .10 was considered to be a
tendency towards significant. SEM, pooled standard error of the means (n = 6).
Abbreviations: AFB1 +  LYC1-­(100  µg/kg AFB1 + 100 mg/kg LYC); AFB1 +  LYC2-­(100  µg/kg AFB1 + 200 mg/kg LYC); AFB1 +  LYC3-­(100  µg/kg
AFB1 + 400 mg/kg LYC); AFB1-­(100  µg/kg AFB1); AFB1, aflatoxinB1; LYC, lycopene; SEM, standard error of the mean.

the control group, the villus height and villus height/crypt depth
in three parts (duodenum, jejunum, and ileum) of the small intes-
tine were noticeably decreased in AFB1 treated group broilers.
These results were in agreement with previous studies observed
by Zhang et al. (2014), who found that 0.3 mg/kg AFB1 decreased
jejunum villus height and the villus height/crypt depth ratio and
disrupted intestinal barrier cells of the broiler chicken at 21 days
of age. Similarly, Wan et al. (2013) found that the intestine villus
height and villus height/crypt depth ratio have been decreased
when the addition of 100 µg/kg AFB1 as their dietary treatment
in ducklings. The possible reasons might be that AFB1 reduced
the nutrient absorption surface area by damaging the broiler in-
testine structure. The above results indicated that dietary AFB1
decreased the broiler intestine function by depressing intestinal
morphological development and induced toxic lesions.
LYC as carotenoid has anticarcinogenic and anticancer proper-
ties, which primarily responsible for its beneficial effects. Recent
studies have found that LYC plays an important role in the preven-
tion of gastrointestinal tract cancer. Yucel et al. (2016) found that
LYC was beneficial in protected intestinal damage against metho-
trexate in rats. A study by Itoh et al. (2004) found that LYC pos-
sessed a significant radio protective effect on villus and crypts in
the small intestine of abdominally radiated (15 Gy) mice. Data from
the current study showed that villus height and villus height/crypt
depth ratio of broiler small intestine was significantly increased
by adding different levels of dietary LYC, thus alleviating AFB1 in-
duced toxicity and improved the histological structure of the small
intestine.
The intestinal enzyme activities have a crucial effect on the an-
imal's physiological processes and life activities (Cho et al., 2012).
It is widely believed that the AFB1 application has malabsorption
syndrome regarding macronutrients and reduced activity of diges-
tive enzymes (Devegowda & Murthy, 2005). It has been demon-
strated in many studies that AFB1 adversely affected production
performance in the poultry industry (Zaghini et al., 2005). The
present study showed a decrease in the intestinal digestive en-
zymes, including trypsin, amylase, and lipase in the AFB1 treated
diet. The possible reason might be ascribed to the damage of the
intestinal structure caused by the AFB1 diet. AFB1 induced distur-
bance of digestive enzyme activity and nutrient transporters may
lead to intestinal disorders, resulting in the alteration of nutrient
digestion, absorption function, and reduced growth performance
of broilers. Matur et al. (2010) found that lipase enzyme activity
decreased in the duodenum after feeding 0.1 mg/kg AFB1 in Ross
female diets.
The current study has shown that the addition of different lev-
els of dietary LYC with AFB1 contaminated diet improved the amy-
lase and lipase activities in the small intestine of broilers. A possible
explanation for the increased enzyme activities might be ascribed
to the improved intestinal morphology and integrity by dietary LYC
supplementation. Also, this result indicated that LYC might have di-
gestive enhancing properties and thus improved the growth perfor-
mance of broilers. Findings of our current study implied that dietary
supplementation of LYC alleviated the detrimental effects of AFB1
on the small intestine, digestive enzyme, and growth performance
of broilers.
5 | CO N C LU S I O N
In conclusion, 100 µg/kg AFB1 in the broiler diet result in some nega-
tive effects manifested by a decrease in ADG, an increase in FCR, a
change in intestinal morphological status, and reduction in digestive
enzyme activities in broilers. Dietary supplementation of different
levels (100, 200, and 400 mg/kg) of LYC exhibited beneficial effects
on growth performance, intestinal morphological status, and diges-
tive enzyme activities of broiler challenged with AFB1. The results of
this study also demonstrated that dietary supplementation of LYC
could be incorporated in broiler diets and exhibit promising effects
to overcome the deleterious effects of AFB1 impact in the broiler
production.
AC K N OW L E D G M E N T S
This study was supported by the National Natural Science
Foundation of China (31802095). The assistance of my teachers and
lab mates in this work is greatly acknowledged.
 |
8 of 9       SARKER et al.
C O N FL I C T O F I N T E R E S T
The authors declare no conflict of interest.
ORCID
Md Touhiduzzaman Sarker  https://orcid.org/0000-0002-0629-7824
REFERENCES
Ali Rajput, S., Sun, L., Zhang, N., Mohamed Khalil, M., Gao, X., Ling, Z.,
Zhu, L., Khan, F., Zhang, J., & Qi, D. (2017). Ameliorative effects of
grape seed proanthocyanidin extract on growth performance, im-
mune function, antioxidant capacity, biochemical constituents, liver
histopathology and aflatoxin residues in broilers exposed to aflatoxin
B1. Toxins, 9, 371. https://doi.org/10.3390/toxin​s9110371
Alpsoy, L., & Yalvac, M. E. (2011). Key roles of vitamins A, C, and E in
aflatoxin B1-­induced oxidative stress. Vitamins and Hormones, 86,
287–­3 05.
Bintvihok, A., & Kositcharoenkul, S. (2006). Effect of dietary cal-
cium propionate on performance, hepatic enzyme activities and
aflatoxin residues in broilers fed a diet containing low levels of
aflatoxin B1. Toxicon, 47, 41–­ 4 6. https://doi.org/10.1016/j.toxic​
on.2005.09.009
Bischoff, S. C. (2011). 'Gut health': A new objective in medicine? BMC
Medicine, 9, 24. https://doi.org/10.1186/1741-­7015-­9-­24
Boeira, S. P., Filho, C. B., Del’Fabbro, L., Roman, S. S., Royes, L. F. F.,
Fighera, M. R., Jessé, C. R., Oliveira, M. S., & Furian, A. F. (2014).
Lycopene treatment prevents hematological, reproductive and histo-
pathological damage induced by acute zearalenone administration in
male Swiss mice. Experimental and Toxicologic Pathology, 66, 179–­185.
https://doi.org/10.1016/j.etp.2014.01.002
Bouhet, S., & Oswald, I. P. (2005). The effects of mycotoxins, fungal food
contaminants, on the intestinal epithelial cell-­derived innate immune
response. Veterinary Immunology and Immunopathology, 108, 199–­
209. https://doi.org/10.1016/j.vetimm.2005.08.010
Cho, I., Yamanishi, S., Cox, L., Methé, B. A., Zavadil, J., Li, K., Gao, Z.,
Mahana, D., Raju, K., Teitler, I., Li, H., Alekseyenko, A. V., & Blaser, M.
J. (2012). Antibiotics in early life alter the murine colonic microbiome
and adiposity. Nature, 488, 621–­626. https://doi.org/10.1038/natur​
e11400
Devegowda, G., & Murthy, T. (2005). Mycotoxins: Their adverse effect in
Poultry and some practical solution in the mycotoxin blue book 1 (pp.
25–­56). Nottingham Univ Press.
Diaz, D. E., Hagler, W. M., Hopkins, B. A., & Whitlow, L. W. (2003).
Aflatoxin binders I: In vitro binding assay for aflatoxin B1 by several
potential sequestering agents. Mycopathologia, 156, 223–­226.
Englmaierová, M., Bubancová, I., Vít, T., & Skrivan, M. (2011). The effect
of lycopene and vitamin E on growth performance, quality and oxida-
tive stability of chicken leg meat. Czech Journal of Animal Science, 56,
536–­543. https://doi.org/10.17221/​4 416-­C JAS
Fan, Y. U., Zhao, L., Ma, Q., Li, X., Shi, H., Zhou, T., Zhang, J., & Ji, C.
(2013). Effects of Bacillus subtilis ANSB060 on growth performance,
meat quality and aflatoxin residues in broilers fed moldy peanut meal
naturally contaminated with aflatoxins. Food and Chemical Toxicology,
59, 748–­753. https://doi.org/10.1016/j.fct.2013.07.010
Feng, G., He, J., Ao, X., & Chen, D. (2017). Effects of maize naturally
contaminated with aflatoxin B1 on growth performance, intestinal
morphology, and digestive physiology in ducks. Poultry Science, 96,
1948–­1955. https://doi.org/10.3382/ps/pew420
Guo, S., Shi, D., Liao, S., Su, R., Lin, Y., Pan, J., & Tang, Z. (2012). Influence
of selenium on body weights and immune organ indexes in ducklings
intoxicated with aflatoxin B 1. Biological Trace Element Research, 146,
167–­170. https://doi.org/10.1007/s1201​1- ­011-­9246-­z
Han, X.-­Y., Huang, Q.-­C ., Li, W.-­F., Jiang, J.-­F., & Xu, Z.-­R . (2008).
Changes in growth performance, digestive enzyme activities and
nutrient digestibility of cherry valley ducks in response to aflatoxin
B1 levels. Livestock Science, 119, 216–­220. https://doi.org/10.1016/j.
livsci.2008.04.006
Hernandez, F., Garcia, V., Madrid, J., Orengo, J., Catalá, P., & Megias, M.
(2006). Effect of formic acid on performance, digestibility, intestinal
histomorphology and plasma metabolite levels of broiler chickens.
British Poultry Science, 47, 50–­ 56. https://doi.org/10.1080/00071​
66050​0 475574
Itoh, Y., Kurabe, T., & Suganuma, H. (2004). The protective effect of ly-
copene against radiation injury to the small intestine of abdominally
radiated mice. The Japan Radiation Research Society Annual Meeting
Abstracts The 47th Annual Meeting of The Japan Radiation Research
Society (p. 59). The Japanese Radiation Research Society.
Li, Y., Zhang, H., Chen, Y. P., Yang, M. X., Zhang, L. L., Lu, Z. X., Zhou, Y. M., &
Wang, T. (2015). Bacillus amyloliquefaciens supplementation alleviates
immunological stress and intestinal damage in lipopolysaccharide-­
challenged broilers. Animal Feed Science and Technology, 208, 119–­
131. https://doi.org/10.1016/j.anife​edsci.2015.07.001
Ling, K.-­H., Wan, M. L. Y., El-­Nezami, H., & Wang, M. (2016). Protective
capacity of resveratrol, a natural polyphenolic compound, against
deoxynivalenol-­induced intestinal barrier dysfunction and bacterial
translocation. Chemical Research in Toxicology, 29, 823–­833. https://
doi.org/10.1021/acs.chemr​estox.6b00001
Lira, R. C., Rabello, C.-­B.-­V., Ludke, M. D. C. M. M., Ferreira, P. V., Lana,
G. R. Q., & Lana, S. R. V. (2010). Productive performance of broiler
chickens fed tomato waste. Revista Brasileira de Zootecnia, 39, 1074–­
1081. https://doi.org/10.1590/S1516​-­35982​01000​0500018
Liu, T., Ma, Q., Zhao, L., Jia, R. U., Zhang, J., Ji, C., & Wang, X. (2016).
Protective effects of sporoderm-­broken spores of ganderma lucidum
on growth performance, antioxidant capacity and immune function
of broiler chickens exposed to low level of aflatoxin B1. Toxins, 8, 278.
https://doi.org/10.3390/toxin​s8100278
Long, M., Yang, S.-­H., Han, J.-­X ., Li, P., Zhang, Y. I., Dong, S., Chen, X.,
Guo, J., Wang, J., & He, J.-­B. (2016). The protective effect of grape-­
seed proanthocyanidin extract on oxidative damage induced by
zearalenone in Kunming mice liver. International Journal of Molecular
Sciences, 17, 808. https://doi.org/10.3390/ijms1​7060808
Matur, E., Ergul, E., Akyazi, I., Eraslan, E., & Cirakli, Z. (2010). The effects
of Saccharomyces cerevisiae extract on the weight of some organs,
liver, and pancreatic digestive enzyme activity in breeder hens fed
diets contaminated with aflatoxins. Poultry Science, 89, 2213–­2220.
https://doi.org/10.3382/ps.2010- ­0 0821
Mordente, A., Guantario, B., Meucci, E., Silvestrini, A., Lombardi, E.,
Martorana, G. E., Giardina, B., & Böhm, V. (2011). Lycopene and
cardiovascular diseases: An update. Current Medicinal Chemistry, 18,
1146–­1163. https://doi.org/10.2174/09298​67117​95029717
Mumtaz, A., Nanda, N., Bhatia, A., Akhtar, R., & Mahmood, S. (2013).
Effect of antioxidant supplementation on digestive enzymes in ra-
diation induced intestinal damage in rats. International Journal of
Radiation Biology, 89, 1061–­ 1070. https://doi.org/10.3109/09553​
002.2013.825062
National Research Council (NRC) (1994). Nutrient requirements of poultry
(9th ed.) National Academy of Sciences Press.
Peng, X. I., Chen, K., Chen, J., Fang, J., Cui, H., Zuo, Z., Deng, J., Chen,
Z., Geng, Y. I., & Lai, W. (2016). Aflatoxin B 1 affects apoptosis and
expression of B ax, B cl-­2, and C aspase-­3 in thymus and bursa of fa-
bricius in broiler chickens. Environmental Toxicology, 31, 1113–­1120.
https://doi.org/10.1002/tox.22120
Peng, X., Zhang, S., Fang, J., Cui, H., Zuo, Z., & Deng, J. (2014). Protective
roles of sodium selenite against aflatoxin B1-­induced apoptosis of jeju-
num in broilers. International Journal of Environmental Research and Public
Health, 11, 13130–­13143. https://doi.org/10.3390/ijerp​h1112​13130
Pinton, P., & Oswald, I. P. (2014). Effect of deoxynivalenol and other Type
B trichothecenes on the intestine: A review. Toxins, 6, 1615–­1643.
https://doi.org/10.3390/toxin​s6051615
SARKER et al.
Rawal, S., Kim, J. E., & Coulombe, R. Jr (2010). Aflatoxin B1 in poul-
try: Toxicology, metabolism and prevention. Research in Veterinary
Science, 89, 325–­331. https://doi.org/10.1016/j.rvsc.2010.04.011
Saada, H. N., Rezk, R. G., & Eltahawy, N. A. (2010). Lycopene protects
the structure of the small intestine against gamma-­radiation-­induced
oxidative stress. Phytotherapy Research, 24, S204–­S208. https://doi.
org/10.1002/ptr.3091
Sahin, K., Onderci, M., Sahin, N., Gursu, M. F., Khachik, F., & Kucuk, O.
(2006). Effects of lycopene supplementation on antioxidant status,
oxidative stress, performance and carcass characteristics in heat-­
stressed Japanese quail. Journal of Thermal Biology, 31, 307–­312.
https://doi.org/10.1016/j.jther​bio.2005.12.006
Sahin, K., Orhan, C., Tuzcu, M., Sahin, N., Hayirli, A., Bilgili, S., & Kucuk,
O. (2016). Lycopene activates antioxidant enzymes and nuclear tran-
scription factor systems in heat-­stressed broilers. Poultry Science, 95,
1088–­1095. https://doi.org/10.3382/ps/pew012
Sahin, K., Yazlak, H., Orhan, C., Tuzcu, M., Akdemir, F., & Sahin, N.
(2014). The effect of lycopene on antioxidant status in rainbow
trout (Oncorhynchus mykiss) reared under high stocking den-
sity. Aquaculture, 418, 132–­ 138. https://doi.org/10.1016/j.aquac​
ulture.2013.10.009
Shi, D., Guo, S., Liao, S., Su, R., Pan, J., Lin, Y., & Tang, Z. (2012). Influence
of selenium on hepatic mitochondrial antioxidant capacity in duck-
lings intoxicated with aflatoxin B 1. Biological Trace Element Research,
145, 325–­329. https://doi.org/10.1007/s1201​1-­011-­9201-­z
Shivachandra, S., Sah, R., Singh, S., Kataria, J., & Manimaran, K. (2003).
Immunosuppression in broiler chicks fed aflatoxin and inoculated
with fowl adenovirus serotype-­4 (FAV-­4) associated with hydroperi-
cardium syndrome. Veterinary Research Communications, 27, 39–­51.
https://doi.org/10.1023/A:10220​58623634
Wan, X., Yang, Z., Yang, W., Jiang, S., Zhang, G., Johnston, S., & Chi, F.
(2013). Toxicity of increasing aflatoxin B1 concentrations from con-
taminated corn with or without clay adsorbent supplementation in
ducklings. Poultry Science, 92, 1244–­1253. https://doi.org/10.3382/
ps.2012-­02748
|
      9 of 9
Yilmaz, S., & Kaya, E. (2019). The protective effect of lycopene on toxicity
of aflatoxin B1 in rats. European International Journal of Science and
Technology, 8(5), 1–­10.
Yonar, M. E. (2012). The effect of lycopene on oxytetracycline-­
induced oxidative stress and immunosuppression in rainbow trout
(Oncorhynchus mykiss, W.). Fish and Shellfish Immunology, 32, 994–­
1001. https://doi.org/10.1016/j.fsi.2012.02.012
Yucel, Y., Tabur, S., Gozeneli, O., Kocarslan, S., Seker, A., Buyukaslan, H., Şavik,
E., Aktumen, A., Ozgonul, A., Uzunkoy, A., & Aksoy, N. (2016). The effects
of lycopene on intestinal injury due to methotrexate in rats. Redox Report,
21, 113–­118. https://doi.org/10.1179/13510​00215Y.00000​00041
Yunus, A. W., Razzazi-­Fazeli, E., & Bohm, J. (2011). Aflatoxin B1 in affect-
ing broiler's performance, immunity, and gastrointestinal tract: A re-
view of history and contemporary issues. Toxins, 3, 566–­590. https://
doi.org/10.3390/toxin​s3060566
Zaghini, A., Martelli, G., Roncada, P., Simioli, M., & Rizzi, L. (2005).
Mannanoligosaccharides and aflatoxin B1 in feed for laying hens:
Effects on egg quality, aflatoxins B1 and M1 residues in eggs, and
aflatoxin B1 levels in liver. Poultry Science, 84, 825–­832. https://doi.
org/10.1093/ps/84.6.825
Zhang, S., Peng, X., Fang, J., Cui, H., Zuo, Z., & Chen, Z. (2014). Effects
of aflatoxin B 1 exposure and sodium selenite supplementation on
the histology, cell proliferation, and cell cycle of jejunum in broil-
ers. Biological Trace Element Research, 160, 32–­ 4 0. https://doi.
org/10.1007/s1201​1-­014-­0 009
How to cite this article: Sarker MT, Wang ZY, Yang H, Wan X,
Emmanuel A. Evaluation of the protective effect of lycopene
on growth performance, intestinal morphology, and digestive
enzyme activities of aflatoxinB1 challenged broilers. Anim Sci
J. 2021;92:e13540. https://doi.org/10.1111/asj.13540