TagedAPRH1Chitosan oligosaccharide improves intestinal function by promoting intestinal
development, alleviating intestinal inflammatory response, and enhancing
antioxidant capacity in broilers aged d 1 to 14TagedAPTREn
TagedAPRRuixia Lan ,1 Fan Wu,1 Yuchen Wang , Ziwei Lin, Haoxuan Wang, Jia Zhang, and Zhihui Zhao2TagedAPREn
TagedAPRDepartment of Animal Science, College of Coastal Agriculture Sciences, Guangdong Ocean University, Zhanjiang
524-088, Guangdong, PR ChinaTagedAPTREn
TAGEDPRABSTRACT This study was conducted to investigate
the effects of chitosan oligosaccharide (COS) supple-
mentation on intestinal development and functions,
inflammatory response, antioxidant capacity and the
related signaling pathways in broilers aged d 1 to 14. A
total of 240 one-day old male Arbor Acres broilers
(40.47 § 0.30 g) were randomly allotted to 4 groups,
and each group consisted of 6 replicate pens with 10
broilers per replicate. Broilers fed a basal diet supple-
mentation with COS at 0 (CON group), 200 (COS200
group), 400 (COS400 group), and 800 mg/kg
(COS800 group) for 14 d, respectively. Broilers in the
COS supplementation groups had no significant effects
on growth performance. Compared to the CON group,
dietary COS supplementation increased (P < 0.05) the
relative weight of duodenum, jejunal lipase activity,
duodenal and ileal villus surface area, and lower (P <
0.05) ileal amylase and alkaline phosphatase activity,
and crypt depth. The expression level of duodenal glu-
cose transporter 1 (GLUT1), Na+-glucose cotrans-
porter 1 (SGLT1), peptide transporter 1 (PepT1),
occludin, zonula occludens-1 (ZO-1), toll-like receptor 4
TagedAPREn
TagedAPRKey words: chitosan oligosaccharide, intestinal function, inflammatory response, antioxidant capacity, broilerTagedAPTREn
TAGEDPRH1INTRODUCTIONTAGEDPTRN
TagedAPRIntestinal health is associated with general health sta-
tus, which is crucial for optimum feed efficiency and
growth performance, as well as maintains intestinal
function, including digestive and absorption, metabo-
lism, barrier, and immune functions (Schokker et al.,
2015; Oviedo-Rondon, 2019; Liu et al., 2023). However,
TagedAPREnÓ 2023 The Authors. Published by Elsevier Inc. on behalf of Poultry
Science Association Inc. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/).
TagedAPREnReceived November 8, 2023.
TagedAPREnAccepted December 12, 2023.
1
TagedAPREn The authors contribute the same.
TagedAPREn2Corresponding author: zhzhaogdou@163.com
1
(TLR4), myeloid differentiation factor 88 (MyD88),
and interleukin-10 (IL-10), jejunal SGLT1, PepT1,
occludin, tumor necrosis factor-a (TNF-a), and ileal
SGLT1, PepT1, and fatty acid binding protein 1
(FABP1) was upregulated by COS. However, the
expression level of duodenal FABP1 and TNF-a, jejunal
GLUT1, ZO-1, TLR4, MyD88, nuclear factor kappa-B
p65 (NF-kB p65), and IL-1b, and ileal GLUT1, NF-kB
p65, and IL-1b was downregulated by COS. Further-
more, dietary COS supplementation increased duodenal
catalase (CAT), glutathione peroxidase (GSH-Px),
and total superoxide dismutase (T-SOD) activity, jeju-
nal CAT and T-SOD activity, upregulated the expres-
sion level of duodenal nuclear factor-erythroid 2-related
factor 2 (Nrf2), CAT, glutathione peroxidase 1
(GPX1), and copper and zinc superoxide dismutase
(Cu/Zn SOD), jejunal CAT, and ileal Nrf2, CAT, and
GPX1. These results suggested that COS could promote
intestinal development and functions in broilers aged d 1
to 14, which might be mediated by alleviating intestinal
inflammatory response and enhancing antioxidant
capacity.
2024 Poultry Science 103:103381
https://doi.org/10.1016/j.psj.2023.103381
it must be mentioned that the intestinal function is
immature and the functional maturation occurs mainly
at the first 2 wk of life in broilers (Iji et al., 2001a,b;
Schokker et al., 2009; Prabakar et al., 2016). During this
stage, various environmental factors, such as bacterial
infections, oxidative stress and inflammation, can
change intestinal function, manifesting by intestinal oxi-
dative damage and inflammatory response, decreasing
growth performance, villus height, and digestive enzyme
activity, increasing intestinal permeability and damage
intestinal barrier function by overproduction of reactive
oxygen species (ROS) and proinflammatory cytokines,
when compares with the later stage of life (Jha et al.,
2019; El Jeni et al., 2021; Liu et al., 2023). Therefore,
broilers aged d 1 to 14 is a key phase, which leaves a
TagedAPREn2 LAN ET AL.
critical stage for permanent remodeling of the intestinal
development and intestinal health (Liu et al., 2023;
Zheng et al., 2023). Taking into the consideration of the
global prohibition of antibiotics as growth promoters, as
well as the public health concern (Adedokun and Olo-
jede, 2019; Jha et al., 2019), it is urgent to find nutri-
tional strategies to regulate the early intestinal
development to maintain the intestinal function and
subsequently maximize the growth performance in
broilers.TagedAPTREn
TagedAPRChitosan oligosaccharide (COS) is an oligosaccharide
from chitin or chitosan, which is an environmentally
friendly feed additive with beneficial effects on animal
nutrition (Chen et al., 2022b; Uyanga et al., 2023). For-
mer studies illustrated that dietary COS supplementa-
tion had beneficial effects on growth performance,
intestinal function, immune response, antioxidant
capacity and lipid metabolism in broilers (Lan et al.,
2019, 2020b, 2021, 2023; Chang et al., 2022). These ben-
eficial effects are related to its biological properties, such
as low toxicity, biodegradability, biocompatibility,
immune modulation, antioxidant capacity, and intesti-
nal microflora modulation functions (Naveed et al.,
2019). However, little is known about the effects of COS
on intestinal development and functions in broilers aged
d 1 to 14. We hypothesized that COS will exert benefi-
cial effects on intestinal development and functions by
alleviating intestinal inflammatory response and
enhancing antioxidant capacity in broilers aged d 1 to
14. Therefore, this study was conducted to verify the
aforementioned hypothesis by investigating the effects
of COS on the development of small intestine, intestinal
morphology, digestion, absorption, and barrier func-
tions, inflammatory response, antioxidant capacity and
the related signaling pathways in broilers aged d 1 to 14.TagedAPTREn
TAGEDPRH1MATERIALS AND METHODSTAGEDPTRN
TagedAPRH2Experimental Design, Diets, and
ManagementTagedAPTREn
TagedAPRThe use of broilers and experimental protocols were
approved by the Animal Care and Use Committee of
Guangdong Ocean University, China (SYXK-2018-0147).TagedAPTREn
TagedAPRA total of 300 one-day-old male Arbor Acres broilers
were purchased from the local commercial hatchery
(Suixi, China). After body weight (BW) check, 240
broilers with similar BW (40.47 § 0.30 g) were selected
and randomly allotted to 4 groups, and each group con-
sisted of 6 replicate pens with 10 broilers per replicate.
Broilers fed a corn-soybean meal basal diet with COS at
a graded level of 0 (CON group), 200 (COS200
group), 400 (COS400 group), and 800 mg/kg
(COS800 group) for 14 d (the dose level based on our
preliminary experiment), respectively. The basal diet
composition and broilers’ management followed the
instruction of Production technique criterion for com-
mercial broilers in China (GB/T 19664-2005). The basal
diet composition and nutrient level on a dry matter basis
and the detailed formula was listed in Supplemental
Table S1. The COS used in this study was purchased
from Qindao Songtian Biotechnology Co. Ltd. (Qindao,
PR China) with deacetylation degree ≥90.00%, and
average molecular weight ≤3,000 Da. To minimize nutri-
ent variation among experimental groups, the basal diet
was firstly prepared and subsequently divided into 5
equal portions and to facilitate the COS supplementa-
tion. Broilers were raised in a 3-level battery cages and
environmentally controlled room. The indoor tempera-
ture was maintained at 34°C § 1°C during d 1 to 10.
During d 11 to 14, the temperature was gradually
reduced by 0.5°C per d. The relative humidity of chicken
house was maintained at approximately 70% during d 1
to 10, then maintained at approximately 65% during d
11 to 14. Artificial light schedule was 23 h of light and 1
h of dark during the experimental period.TagedAPTREn
TagedAPRH2Growth PerformanceTagedAPTREn
TagedAPROn d 1 and 14, broilers were weighted and feed con-
sumption was recorded on pen basis to calculate growth
parameters, including average daily feed intake
(ADFI), average daily gain (ADG), and feed conver-
sion ratio (FCR).TagedAPTREn
TagedAPRH2Sample CollectionTagedAPTREn
TagedAPROn d 14, 6 broilers per treatment (1 broiler per cage)
were randomly selected and fasted for 12 h before body
weight check. Then blood samples were collected from
the brachial vein, centrifugation at 3,000 £ g for 15 min
at 4°C to collect the serum and stored at 4°C for later
analysis. Then the broilers were euthanized by cervical
dislocation and the abdomen was immediately opened
to collect the intestinal samples. The duodenum, jeju-
num, and ileum were dissected and individually to mea-
sure their length and weight to calculate the relative
length and weight as our former described methods (Lan
et al., 2020a). For intestinal morphology analysis,
approximately 2 cm segment from the middle part of the
duodenum, jejunum and ileum were collected and fixed
in 4% paraformaldehyde solution at room temperature
for 24 h. After that, the rest of the duodenum, jejunum
and ileum were longitudinally opened to collect the
intestinal mucosal samples, quickly flash-freezing in liq-
uid nitrogen and stored at 80°C to later examination
of digestive enzyme activity and real-time polymerase
chain reaction (RT-PCR) analysis.TagedAPTREn
TagedAPRH2Intestinal MorphologyTagedAPTREn
TagedAPRThe intestinal morphology examination followed the
method described in our former study (Lan et al.,
2020a). Briefly, serial tissue sections of 4 mm were cut
and mounted on slide. Then deparaffinized, rehydrated,
rinsed in distilled water, stained with hematoxylin and
eosin, dehydrated and mounted. Morphological parame-
ters were measured using the Image-Pro Plus software
(Version 6.0). The villus height (Vh), crypt depth (Cd),
 TAGEDPRNCOS PROMOTES INTESTINAL DEVELOPMENT AND FUNCTIONS 3
Table 1. TagedAPRCptionEffects of chitosan oligosaccharide supplementation on the growth performance in broilers. TagedAPREn

Items1TagedAPREn CONTagedAPTREn COS200TagedAPREn COS400TagedAPREn COS800TagedAPREn SEM2TagedAPREnboy

ADG, gTagedAPTREn 24.30 § 1.02TagedAPTREn 23.73 § 1.78TagedAPTREn 23.78 § 2.37TagedAPTREn 23.38 § 1.39TagedAPTREn 0.7660TagedAPTREn
ADFI, gTagedAPTREn 32.32 § 0.97TagedAPTREn 32.69 § 1.43TagedAPTREn 32.71 § 1.69TagedAPTREn 30.91 § 2.21TagedAPTREn 0.7324TagedAPTREn
FCRTagedAPTREn 1.33 § 0.08TagedAPTREn 1.38 § 0.07TagedAPTREn 1.39 § 0.12TagedAPTREn 1.32 § 0.03TagedAPTREn 0.0361TagedAPTREn
1
COS, chitosan oligosaccharide; CON, COS200, COS400, COS800, basal diet with 0, 200, 400, or 800 mg/kg COS, respectively.
2
SEM, standard error of mean.

and the villus width (calculated as the average width of cDNA, and the RT-PCR was performed as our previ-
tip, middle and bottom of the villus, Vw) were measured ously described method (Lan et al., 2014). The primer
and the villus surface area were calculated following the sequences used were listed in Supplemental Table S3.
formula described by Li et al. (2020).TagedAPTREn The 2 DDCt method was used to calculate the relative
TagedAPRVillus surface area = pVwVh + p(Vw/2)2, Vh, villus gene expression, and b-actin used as internal reference
height; Cd, crypt depth; Vw, villus width.TagedAPTREn gene (Livak and Schmittgen, 2001).TagedAPTREn

TagedAPRH2Intestinal Permeability Parameter, Digestive TagedAPRH2Statistical AnalysisTagedAPTREn

Enzyme Activity and Antioxidant Related
TagedAPRThe pen was used as the experiment unit and all data
Parameters MeasurementTagedAPTREn were analyzed with SAS 2003 (v. 9.1, SAS Institute Inc.,
TagedAPRThe serum diamine oxidase (DAO) activity was mea- Cary, NC) using the mixed procedure. Duncan’s multi-
sured using a commercial kit. The intestinal mucosal ple range test was used to analyze the differences among
samples were homogenized with an ice-cold physiological the treatments. P < 0.05 indicated statistically signifi-
saline solution at a weight to volume ratio of 1: 9. The cantly difference.TagedAPTREn
homogenate supernatant was collected after being cen-
trifuged at 3,000 £ g for 15 min at 4°C to detect the TAGEDPRH1RESULTSTAGEDPTRN
digestive enzyme activity, including amylase, lipase,
sucrase, maltase, and alkaline phosphatase (AKP), and TagedAPRH2Growth PerformanceTagedAPTREn
the antioxidant related parameters, including malon-
dialdehyde (MDA), catalase (CAT), glutathione per- TagedAPRDietary COS supplementation had no significant
oxidase (GSH-Px), and total superoxide dismutase (T- effects on growth performance parameters, including
SOD). The assays were performed using corresponding ADG, ADFI, and FCR (Table 1).TagedAPTREn
kits purchased from Nanjing Jiancheng Bioengineering
Co., Ltd. (Nanjing, China), and the procedures were TagedAPRH2Intestinal DevelopmentTagedAPTREn
conducted based on the manufacturer’s instruction. The
kits’ information was listed in Supplemental Table S2.TagedAPTREn TagedAPRThe relative weight of duodenum in the COS200 and
COS400 groups was higher (P < 0.05) than that in the
CON group (Table 2).TagedAPTREn
TagedAPRH2Quantitative Real-Time PCR AnalysisTagedAPTREn
TagedAPRTotal RNA extraction was done using the Trizol TagedAPRH2Intestinal MorphologyTagedAPTREn
reagent kit (Accurate Biotechnology Co., Ltd., Hunan,
China), the 1-step gDNA removal kit (TransGen Bio- TagedAPRThe effects of COS supplementation on small intesti-
tech Co., Ltd., Beijing, China) was used to synthesize nal morphology were shown in Figure 1. In the

Table 2. TagedAPRCptionEffects of chitosan oligosaccharide supplementation on the development of small intestine in broilers. TagedAPREn

Items1TagedAPREn CONTagedAPTREn COS200TagedAPREn COS400TagedAPREn COS800TagedAPREn SEM2TagedAPREnboy

Relative length3, cm/kgTagedAPTREn TagedAPREn
DuodenumTagedAPTREn 57.58 § 5.51abTagedAPREn 58.52 § 7.00aTagedAPREn 57.54 § 5.51abTagedAPREn 49.46 § 7.28bTagedAPREn 2.8526TagedAPTREn
JejunumTagedAPTREn 142.09 § 13.67TagedAPTREn 130.59 § 16.19TagedAPTREn 137.45 § 14.43TagedAPTREn 128.18 § 17.97TagedAPTREn 6.6183TagedAPTREn
IleumTagedAPTREn 105.25 § 13.67TagedAPTREn 111.17 § 15.87TagedAPTREn 112.89 § 13.97TagedAPTREn 103.53 § 11.76TagedAPTREn 6.2150TagedAPTREn
Relative weight4, g/kgTagedAPTREn TagedAPREn
DuodenumTagedAPTREn 12.82 § 0.89bTagedAPREn 16.23 § 2.20aTagedAPREn 15.81 § 2.95aTagedAPREn 12.81 § 2.15bTagedAPREn 0.9746TagedAPTREn
JejunumTagedAPTREn 21.91 § 2.42TagedAPTREn 21.53 § 3.17TagedAPTREn 21.41 § 3.78TagedAPTREn 20.46 § 2.85TagedAPTREn 1.3839TagedAPTREn
IleumTagedAPTREn 12.22 § 2.14abTagedAPREn 13.17 § 1.66abTagedAPREn 15.01 § 3.64aTagedAPREn 10.96 § 1.98bTagedAPREn 1.1057TagedAPTREn
1
COS, chitosan oligosaccharide; CON, COS200, COS400, COS800, basal diet with 0, 200, 400, or 800 mg/kg COS, respectively.
2
SEM, standard error of mean.
3
Calculated as small intestinal segment length (cm) to body weight (kg), cm/kg.
4
Calculated as small intestinal segment weight (g) to body weight (kg), g/kg.
a,b
Different superscript letters indicated significantly differences at P < 0.05.
TagedAPRFiur TagedAPREn4
Figure 1. Effects of chitosan oligosaccharide supplementation on intestinal morphology in broilers.TagedAPTREn
duodenum, the villus surface area in the COS200 group
was larger (P < 0.05) than that in the CON group
(Figure 2). In the ileum, the villus surface area in the
COS200 and COS400 groups were larger (P < 0.05) than
that in the CON group, and the crypt depth and villus
width in the COS800 group was decreased (P < 0.05)
when compared to the CON group.TagedAPTREn
TagedAPRH2Digestive Enzyme Activity of the Small
Intestinal MucosaTagedAPTREn
TagedAPRIn the jejunum, the COS supplementation groups had
higher (P < 0.05) lipase activity than that in the CON
group (Table 3). In the ileum, the amylase and AKP
activity in the COS800 group were lower (P < 0.05) than
that in the CON group.TagedAPTREn
TagedAPRH2Nutrient Transporters Related Gene
ExpressionTagedAPTREn
TagedAPRIn the duodenum, compared to the CON group, the
expression level of glucose transporter 1 (GLUT1) in
the COS200 and COS800 groups, Na+-glucose cotrans-
porter 1 (SGLT1) in the COS400 group, peptide trans-
porter 1 (PepT1) in the COS400 and COS800 groups
were higher (P < 0.05) than those in the CON group
(Figure 3). In the jejunum, compared to the CON group,
the expression level of SGLT1 and PepT1 in the COS400
and COS800 groups were higher (P < 0.05), while the
expression level of GLUT1 in the COS supplementation
groups was lower. In the ileum, compared to the CON
group, the expression level of SGLT1 and PepT1 in the
COS400 group, and fatty acid binding protein 1
(FABP1) in the COS400 and COS800 groups were
higher (P < 0.05), while the expression level of GLTU1
in the COS400 and COS800 groups was lower.TagedAPTREn
LAN ET AL.
TagedAPRH2Intestinal Permeability and Tight Junction
Protein Related Gene ExpressionTagedAPTREn
TagedAPRCompared to the CON group, serum DAO level in the
COS200 group was increased (P < 0.05), while decreased
(P < 0.05) in the COS800 group (Figure 4). In the duode-
num, the expression level of occludin and zonula occlu-
dens-1(ZO-1) in the COS400 and COS800 groups were
higher (P < 0.05) than those in the CON group
(Figure 5). In the jejunum, compared to the CON group,
the expression level of occludin in the COS800 group was
higher (P < 0.05), while the expression level of ZO-1 in
the COS supplementation groups was lower (P < 0.05).TagedAPTREn
TagedAPRH2Inflammatory Response Related Gene
ExpressionTagedAPTREn
TagedAPRIn the duodenum, compared to the CON group, the
expression level of toll-like receptor 4 (TLR4) and interleu-
kin-10 (IL-10) in the COS800 group, myeloid differentiation
factor 88 (MyD88) in the COS400 and COS800 groups were
higher (P < 0.05), while the expression level of tumor necro-
sis factor-a (TNF-a) in the COS400 group was lower (P <
0.05) (Figure 6). In the jejunum, compared to the CON
group, the expression level of TLR4, MyD88, nuclear factor
kappa-B p65 (NF-kB p65), and IL-1b in the COS supple-
mentation groups were lower (P < 0.05), while the expres-
sion level of TNF-a in the COS400 group was higher (P <
0.05). In the ileum, the expression level of NF-kB p65 and
IL-1b in the COS supplementation groups were lower (P <
0.05) than those in the CON groups.TagedAPTREn
TagedAPRH2Antioxidant Capacity of the Small Intestinal
MucosaTagedAPTREn
TagedAPRIn the duodenum, compared to the CON group, the
MDA concentration in the COS200 group was lower (P <
TagedAPRFiur TAGEDPRNCOS PROMOTES INTESTINAL DEVELOPMENT AND FUNCTIONS 5

Figure 2. Effects of chitosan oligosaccharide supplementation on intestinal morphology indexes in broilers. a, bDifferent superscript letters indi-
cated significantly difference at P < 0.05.TagedAPTREn

0.05), while higher (P < 0.05) in the COS400 and COS800
groups (Figure 7). The CAT, GSH-Px and T-SOD activ-
ity in the COS supplementation groups were higher (P <
0.05) than those in the CON group. In the jejunum,
compared to the CON group, the MDA concentration in
the COS200 group was lower (P < 0.05), while higher (P
< 0.05) in the COS400 group. The CAT activity in the
COS200 and COS800 groups was higher (P < 0.05) than
that in the CON group. The T-SOD activity in the COS
supplementation groups was higher (P < 0.05) than that
in the CON group. In the ileum, the MDA concentration
in the COS200 and COS400 groups were lower (P < 0.05)
than that in the CON group. The T-SOD activity in the
COS200 and COS800 groups were lower (P < 0.05) than
that in the CON group.TagedAPTREn
TagedAPRTo further evaluate the effects of dietary COS supple-
mentation on antioxidant capacity, the antioxidant
related genes expression level was detected (Figure 8).
In the duodenum, the expression level of nuclear factor-
erythroid 2-related factor 2 (Nrf2), CAT, glutathione
peroxidase 1 (GPX1) and copper and zinc superoxide
dismutase (Cu/Zn SOD) in the COS400 and COS800
groups were higher (P < 0.05) than those in the CON
group. In the jejunum, compared to the CON group, the
expression level of Nrf2 in the COS supplementation
groups, heme oxygenase 1 (HO-1) and Cu/Zn SOD in
TagedAPREn6 LAN ET AL.

Table 3. TagedAPRCptionEffects of chitosan oligosaccharide supplementation on digestive enzyme activity of broilers. TagedAPREn

Items1TagedAPREn CONTagedAPTREn COS200TagedAPREn COS400TagedAPREn COS800TagedAPREn SEM2TagedAPREnboy

Duodenum, U/mg proteinTagedAPTREn TagedAPREn

AmylaseTagedAPTREn 0.12 § 0.02TagedAPTREn 0.12 § 0.02TagedAPTREn 0.12 § 0.01TagedAPTREn 0.12 § 0.02TagedAPTREn 0.0085TagedAPTREn
LipaseTagedAPTREn 2.21 § 0.66TagedAPTREn 2.32 § 0.46TagedAPTREn 2.37 § 0.67TagedAPTREn 2.39 § 0.49TagedAPTREn 0.2590TagedAPTREn
SucraseTagedAPTREn 31.99 § 4.17TagedAPTREn 33.06 § 4.88TagedAPTREn 35.72 § 5.61TagedAPTREn 34.75 § 4.06TagedAPTREn 2.1115TagedAPTREn
MaltaseTagedAPTREn 60.26 § 7.80TagedAPTREn 60.30 § 7.71TagedAPTREn 63.94 § 8.08TagedAPTREn 65.95 § 7.24TagedAPTREn 3.4508TagedAPTREn
AKPTagedAPTREn 0.20 § 0.02TagedAPTREn 0.21 § 0.03TagedAPTREn 0.21 § 0.02TagedAPTREn 0.21 § 0.03TagedAPTREn 0.0215TagedAPTREn
Jejunum, U/mg proteinTagedAPTREn TagedAPREn
AmylaseTagedAPTREn 0.06 § 0.02TagedAPTREn 0.07 § 0.01TagedAPTREn 0.07 § 0.01TagedAPTREn 0.07 § 0.01TagedAPTREn 0.0061TagedAPTREn
LipaseTagedAPTREn 1.19 § 0.18bTagedAPREn 1.64 § 0.27aTagedAPREn 1.47 § 0.19aTagedAPREn 1.41 § 0.14aTagedAPREn 0.0899TagedAPTREn
SucraseTagedAPTREn 21.55 § 3.88TagedAPTREn 20.97 § 3.60TagedAPTREn 23.35 § 2.02TagedAPTREn 24.13 § 3.99TagedAPTREn 1.5500TagedAPTREn
MaltaseTagedAPTREn 63.26 § 6.08TagedAPTREn 61.03 § 4.15TagedAPTREn 64.18 § 7.99TagedAPTREn 65.22 § 4.96TagedAPTREn 2.6699TagedAPTREn
AKPTagedAPTREn 0.18 § 0.02TagedAPTREn 0.18 § 0.02TagedAPTREn 0.17 § 0.03TagedAPTREn 0.18 § 0.01TagedAPTREn 0.0203TagedAPTREn
Ileum, U/mg proteinTagedAPTREn TagedAPREn
AmylaseTagedAPTREn 0.12 § 0.01aTagedAPREn 0.11 § 0.01abTagedAPREn 0.12 § 0.02aTagedAPREn 0.10 § 0.02bTagedAPREn 0.0072TagedAPTREn
Lipase,TagedAPTREn 2.38 § 0.29TagedAPTREn 2.45 § 0.41TagedAPTREn 2.29 § 0.33TagedAPTREn 2.22 § 0.38TagedAPTREn 0.1591TagedAPTREn
SucraseTagedAPTREn 37.96 § 4.92TagedAPTREn 35.35 § 3.46TagedAPTREn 37.01 § 4.76TagedAPTREn 37.65 § 4.60TagedAPTREn 2.0014TagedAPTREn
MaltaseTagedAPTREn 91.05 § 6.58TagedAPTREn 94.71 § 5.38TagedAPTREn 95.93 § 5.18TagedAPTREn 94.64 § 8.06TagedAPTREn 2.8636TagedAPTREn
AKPTagedAPTREn 0.33 § 0.03aTagedAPREn 0.32 § 0.03abTagedAPREn 0.33 § 0.04aTagedAPREn 0.28 § 0.04bTagedAPREn 0.0357TagedAPTREn
1
COS, chitosan oligosaccharide; AKP, alkaline phosphatase; CON, COS200, COS400, COS800, basal diet with 0, 200, 400, or 800 mg/kg COS,
respectively.
2
SEM, standard error of mean.
a,b
Different superscript letters indicated significantly difference at P < 0.05.

TagedAPRFiur

Figure 3. Effects of chitosan oligosaccharide supplementation on nutrient transporters related gene expression in broilers. GLUT1, glucose
transporter-1; SGLT1, sodium glucose transport protein-1; PepT1, peptide transporter 1; EAAT3, excitatory amino acid transporters-3; FABP1,
fatty acid-binding proteins 1. a, bDifferent superscript letters indicated significantly difference at P < 0.05.TagedAPTREn
TagedAPRFiur TAGEDPRNCOS PROMOTES INTESTINAL DEVELOPMENT AND FUNCTIONS
Figure 4. Effects of chitosan oligosaccharide supplementation on
intestinal permeability parameter in broilers. DAO, diamine oxidase. a
−c
Different superscript letters indicated significantly difference at P <
0.05.TagedAPTREn
the COS200 group were lower (P < 0.05), while the
expression level of CAT in the COS400 group was higher
(P < 0.05). In the ileum, the expression level of Nrf2 in
the COS200 group was higher (P < 0.05) than that in the
CON group. The expression level of CAT and GPX1 in
the COS supplementation groups were higher (P < 0.05)
than those in the CON group.TagedAPTREn
TAGEDPRH1DISCUSSIONTAGEDPTRN
TagedAPRCOS was the degradation product of chitin, which was
the second abundant polymer after cellulose in nature
(Hahn et al., 2020). In recent decades, COS was widely
used to promote growth performance in broilers due to
its unique biological activities, such as antimicrobial,
antioxidant, anti-inflammation and immune regulation
TagedAPRFiur
Figure 5. Effects of chitosan oligosaccharide supplementation on tight junction protein related gene expression in broilers. ZO-1, zonula occlu-
dens-1. a−cDifferent superscript letters indicated significantly difference at P < 0.05.TagedAPTREn
7
functions (Elnesr et al., 2022; Uyanga et al., 2023).
Numerous studies were conducted to evaluate the func-
tionality of COS in improving poultry intestinal health
and production. For instance, dietary COS supplemen-
tation exerted beneficial effects on growth performance,
gut morphology, intestinal digestion and absorption
function, intestinal barrier function, immunological
function, antioxidant capacity, and gut microbiota bal-
ance (Ibitoye et al., 2019; Chang et al., 2020; Lan et al.,
2020b). In this study, dietary COS supplementation had
no significant effects on ADG, ADFI, or FCR in broilers
aged d 1 to 14. Consistent with our results, Tufan and
Arslan (2020) reported that dietary COS supplementa-
tion had no significant effects on ADG, ADFI, or FCR
in broilers aged d 1 to 14. Ayman et al. (2022) also
reported that dietary COS supplementation had no
effects on ADG in broilers aged d 1 to 35. However, Li et
al. (2019) and Lan et al. (2023) reported that dietary
COS supplementation improved growth performance in
broilers or yellow-feathered chicken aged d 1 to 21.
These variable results probably due to the differences in
COS molecular weight, deacetylation degree, polymeri-
zation, as well as the breed, age and health status of
broilers (Ayman et al., 2022; Wang et al., 2022).TagedAPTREn
TagedAPRThe small intestinal was the main site for nutrient
digestion and absorption (Nazem et al., 2017). Early
intestinal development had the greatest impact on intes-
tinal function and overall lifetime growth performance
in broilers (Iji et al., 2001a,b; Cheled-Shoval et al., 2011;
Zhang et al., 2022). The intestinal development was fast-
est in broilers aged d 1 and 7 (Iji et al., 2001a). The intes-
tinal function mainly depended on the integrity of the
intestinal morphology (Zheng et al., 2023). The intesti-
nal morphological characteristics, including villus
height, crypt depth, villus width, and villus surface area,
TagedAPRFiur TagedAPREn8 LAN ET AL.

Figure 6. Effects of chitosan oligosaccharide supplementation on inflammatory response related gene expression in broilers. TLR4, toll-like
receptor 4; MyD88, myeloid differentiation factor 88; NF-kB p65, nuclear factor-kB p65; TNF-a, tumor necrosis factor-a; IL, interleukin. a, bDiffer-
ent superscript letters indicated significantly difference at P < 0.05.TagedAPTREn

were all functional indicators, which reflected nutrient
absorption, immunity, and barrier functions (Montagne
et al., 2003; Pelicano et al., 2005). In this study, dietary
COS supplementation increased the relative weight of
duodenum, and the duodenal and ileal villus surface
area, as well as decreased ileal crypt depth. Consistent
with our results, Lan et al. (2020b) illustrated that die-
tary COS supplementation increased the relative length
of duodenum, jejunum, and ileum in heat-stressed
broilers. The increasing intestinal villus surface area is
directly associated with increasing nutrient absorption
ability (Li et al., 2020). Meanwhile, the decreasing crypt
depth is responsible for slow tissue turnover and the
decreasing demand by villus growth, which is beneficial
for nutrient absorption (Ayman et al., 2022). Consistent
with our results, Li et al. (2019) reported that dietary
COS supplementation decreased jejunal crypt depth in
broilers.TagedAPTREn
TagedAPRSmall intestine is the main site for nutrient digestion
and absorption, the increasing digestive activity and
content enhance the absorption ability (Lu et al., 2023).
Meanwhile, intestinal morphology changes accompany
with the alteration of intestinal digestive enzyme activ-
ity (Iji et al., 2001a,b). Amylase, sucrase, maltase, and
lipase are the main enzymes for starch, disaccharide,
and lipid digestion, and their activity partly reflect the
intestinal digestion and absorption capacity (Wan et al.,
2017; Lin et al., 2023). In this study, dietary COS
TagedAPRFiur TAGEDPRNCOS PROMOTES INTESTINAL DEVELOPMENT AND FUNCTIONS
Figure 7. Effects of chitosan oligosaccharide supplementation on the antioxidant capacity of the small intestinal mucosa in broilers. MDA,
malondialdehyde; CAT, catalase; GSH-Px, glutathione peroxidase; T-SOD, total superoxide dismutase. a−cDifferent superscript letters indicated
significantly difference at P < 0.05.TagedAPTREn
supplementation increased jejunal lipase activity, which
indicated that COS promoted the maturation of the
digestive system and had beneficial effects on lipid diges-
tion in broilers. Consistent with our results, Lu et al.
(2023) reported that rare earth chitosan chelates
increased duodenal lipase activity in laying hens. Similar
results were also reported by Zhang (2019), who indi-
cated that COS increased intestinal lipase activity in
loaches. To further illustrate the beneficial effects of
COS on small intestinal absorption, we also evaluated
the gene expression level of intestinal nutrient transport-
ers. The glucose transporters (GLTU1) and sodium-
glucose cotransporters (SGLT1) were responsible for
carbohydrates transport. In this study, dietary COS
supplementation increased the expression level of
SGLT1 in small intestine. However, the expression level
of GLTU1 exhibited a regional-specific effects, dietary
COS supplementation increased duodenal GLTU1
expression level, while decreased in the jejunum and
ileum. In broilers, SGLT1 is the major pathway for glu-
cose uptake in small intestine, and GLTU1 is responsible
for basal level of glucose uptake (Gilbert et al., 2007;
Habashy et al., 2017), these results suggested that
SGLT1 played a predominantly role in glucose assimila-
tion in broilers aged d 1 to 14. The assimilation of small
peptides and amino acids are mediated by PepT1 and
9
EAAT3 (Gilbert et al., 2007). In small intestine, the
absorption of protein digestion products is mainly in the
form of small peptides, rather than amino acids (Fernan-
dez-Alarcon et al., 2017). Therefore, COS increased the
expression level of PepT1 in small intestine could reflect
that COS had beneficial effects on protein digestion and
absorption. The lipid absorption is mediated by FABP1
(Orhan et al., 2019). In this study, COS increased ileal
FABP1 expression, which suggested that COS could
enhance the absorption of lipids. Till now, relatively lit-
tle was known about the underlying mechanisms of COS
supplementation modulating the expression level of
nutrient transporters in small intestine, further studies
should be conducted to evaluate the mechanism.TagedAPTREn
TagedAPRIntestinal barrier disturbance, characterized by
increasing intestinal permeability, resulted in bacteria,
toxins, and antigens translocation into intestinal epithe-
lium, affected intestinal health and barrier function, as
well as nutrient absorption (Vancamelbeke and Ver-
meire, 2017). Serum DAO activity was a commonly used
biomarker to evaluate the intestinal permeability
(Cheng et al., 2019). In this study, serum DAO activity
in COS800 group was lower than that in the CON group,
indicating that COS had beneficial effects on maintain
intestinal barrier function in broilers aged d 1 to 14. Sim-
ilarly, former studies also reported that dietary COS
TagedAPRFiur 0
TagedAPREn1 LAN ET AL.

Figure 8. Effects of chitosan oligosaccharide supplementation on antioxidant related gene expression in broilers. Nrf2, nuclear factor erythroid-
derived 2-related factor 2; HO-1, heme oxygenase-1; CAT, catalase; GPX1, glutathione peroxidase-1; Cu/Zn SOD, Cu/Zn superoxide dismutase. a
−c
Different superscript letters indicated significantly difference at P < 0.05.TagedAPTREn

supplementation decreased serum DAO activity in heat-
stressed broilers (Li et al., 2019; Lan et al., 2020b). At
the early age of broilers, the intestinal barrier function
was primarily maintained and regulated by tight junc-
tion proteins (occludin, claudin-1, and ZO-1) between
the epithelial cells, therefore, the expression level of
occludin, claudin-1, and ZO-1 was associated with intes-
tinal barrier function (Chen et al., 2018; Zheng et al.,
2023). Li et al. (2019) indicated that COS upregulated
jejunal and ileal claudin-3 expression in broilers. Osho
and Adeola (2020) also reported that COS increased
jejunal occludin and claudin-1 expression in broilers.
Consistent with these results, in this study, COS
increased duodenal and jejunal occludin and duodenal
ZO-1 expression, and accompanied with decreased
serum DAO activity. These results suggested that COS
could maintain intestinal barrier function by regulating
occludin and ZO-1 expression in broilers aged d 1 to 14.
Nevertheless, COS decreased jejunal ZO-1 expression,
further studies should be conducted to evaluate if the
increased expression of occludin could compensate the
decreased expression of jejunal ZO-1, and subsequently
maintain intestinal barrier function.TagedAPTREn
TagedAPRIncreasing studies reported that inflammatory
response was an important factor influencing intestinal
barrier function (Wen et al., 2019). Previous studies
indicated that proinflammatory cytokines produced dur-
ing intestinal inflammatory response, such as TNF-a
and IL-1b, directly disrupt the tight junction proteins
expression (Capaldo and Nusrat, 2009; Kaminsky et al.,
2021). Therefore, inhibiting the overproduction of intes-
tinal proinflammatory cytokines was an effective strat-
egy to maintain intestinal barrier function. In this
study, dietary COS supplementation decreased duode-
nal TNF-a, and jejunal and ileal IL-1b expression, how-
ever, increased duodenal IL-10, and jejunal TNF-a
expression. Further studies need be conducted to evalu-
ate if the decreased expression of IL-1b can compensate
the increased expression of TNF-a in the jejunum. Simi-
lar results also reported by Osho and Adeola (2020),
who indicated that dietary COS supplementation
increased the expression level of jejunal IL-10 and TNF-
 TAGEDPRNCOS PROMOTES INTESTINAL DEVELOPMENT AND FUNCTIONS
a in broilers. Fathi et al. (2023) reported that dietary
COS supplementation increased IL-10 level in serum
and liver of cold-stressed broilers, while decreasing IL-1b
and TNF-a level. To further explain the molecular
mechanism of dietary COS supplementation in alleviat-
ing intestinal inflammatory response, we also measured
the effects of COS on the activation of TLR4/NF-kB sig-
naling pathway. The intestinal TLR4 activation will
interact with MyD88 to activate NF-kB signaling path-
way, then results in inflammatory cytokines production
(Yu et al., 2022). In other words, suppressing TLR4/
NF-kB signaling pathway activation will alleviate
inflammatory response, and subsequently maintain
intestinal barrier function. Previous studies in heat-
stressed mice and IPEC-J2 cell model indicated that
COS could decrease inflammatory cytokines expression
by inhibition TLR4/NF-kB signaling pathway activa-
tion (Shi et al., 2019; Mohyuddin et al., 2021). Consis-
tently, in this study, dietary COS supplementation
decreased the expression level of jejunal TLR4, MyD88,
NF-kB p65, and IL-1b, and ileal NF-kB p65 and IL-1b,
suggesting that COS could inhibit the activation of
TLR4/NF-kB signaling pathway in intestine. Neverthe-
less, the expression level of duodenal TLR4, MyD88,
and IL-10 was increased with dietary COS supplementa-
tion, while the expression level of TNF-a was decreased,
further studies should be conducted to evaluate if the
increased expression level of IL-10 in the duodenum (not
detected in the jejunum and ileum) could compensate
the increased expression of TLR4 and MyD88, and sub-
sequently alleviate intestinal inflammatory response.TagedAPTREn
TagedAPROxidative stress is another factor inducing intestinal
barrier dysfunction (Wen et al., 2019; Chen et al.,
2022a). Numerous studies reported that COS had excel-
lent antioxidant and free radical scavenging capacity in
vitro and in vivo, due to it could denote amino and
hydroxyl groups (Naveed et al., 2019; Zhou et al., 2021).
Our previous studies reported that COS exerted effec-
tive capacity of free radical scavenging in jejunal mucosa
of oxidative-stressed rats, as well as in vitro study (Lan
et al., 2019; Lan et al., 2021). Meanwhile, COS could
directly inhibit ROS production, and activate Nrf2/HO-
1 signal pathway and subsequently elevate the antioxi-
dant related genes expression (Zhou et al., 2021). Chang
et al. (2022) reported that COS alleviated acute heat
stress-induced oxidative stress in broilers, which was evi-
denced by the decreased ROS content, the inhibited
lipid peroxidation (lower MDA content), the increased
free radical scavenging capacity, CAT activity, and the
upregulated expression of level HO-1 and GPX1 in
breast muscle. Lan et al. (2023) reported that COS could
improve antioxidant capacity in breast and thigh muscle
of broilers, which manifested by the increased free radi-
cal scavenging capacity, GSH-Px, SOD, and CAT activ-
ity, the upregulated expression of Nrf2, HO-1, SOD,
CAT, and GPX1, and decreased MDA content in
broilers. Li et al. (2019) reported that COS could
enhance antioxidant capacity in small intestine of
broilers, which was supported by increasing SOD activ-
ity and decreasing MDA content. Lan et al. (2021)
11
reported that COS alleviated H2O2-induced oxidative
stress in small intestine of Sprague-Dawley rats, which
was evidenced by increasing free radical scavenging
capacity, GSH-Px and T-AOC activity, the upregulated
expression level of Nrf2 and HO-1, and the decreased
MDA content. In consistent with these results, in this
study, dietary COS supplementation increased duodenal
CAT, GSH-Px, and T-SOD activity, jejunal CAT and
T-SOD activity, upregulated the expression level of duo-
denal Nrf2, CAT, GPX1, and Cn/Zn SOD, jejunal
CAT, and ileal Nrf2, CAT, and GPX1. These results
suggested that COS could activate the Nrf2/HO-1 sig-
naling pathway and subsequently elevate the antioxi-
dant related genes expression and antioxidant enzyme
activity. Meanwhile, the functional reactive groups
(amino and hydroxyl groups) of COS may also explain
the improvement of antioxidant capacity in broilers
aged d 14. Novelty, the MDA content was variable and
COS does-specific and regional-specific. Compared to
the CON group, the duodenal MDA content was lower
in the COS200 group, but higher in the COS400 and
COS800 groups, the jejunal MDA content was lower in
the COS200 group, but higher in the COS400 group, and
the ileal MDA content was lower in the COS200 and
COS40 groups. Inconsistent with our results, former
studies reported that dietary COS supplementation
decreased MDA content in small intestine of broilers or
laying hens (Li et al., 2017; Li et al., 2019; Lan et al.,
2020b; Gu et al., 2022). These discrepancies might be
attributed to the differences in COS molecular weight,
deacetylation degree, polymerization, as well as the
breed, age and health status of broilers (Ayman et al.,
2022; Wang et al., 2022).TagedAPTREn
TAGEDPRH1CONCLUSIONSTAGEDPTRN
TagedAPRIn conclusion, this study demonstrated that dietary
COS supplementation exerted beneficial effects on intes-
tinal development, digestion, absorption, and barrier
functions in broilers aged d 1 to 14, however, no signifi-
cant effects were observed in growth performance. The
excellent effects of COS on intestinal function were
adducible to its beneficial effects to improve small intes-
tinal development, morphology characters, digestion
and absorption function, tight junction proteins expres-
sion, inhibiting intestinal inflammatory cytokines pro-
duction by inhibiting the TLR4/NF-kB signaling
pathway, and alleviating oxidative stress by activating
Nrf2/HO-1 signaling pathway. Dietary COS supplemen-
tation at an inclusion level of 400 to 800 mg/kg provided
an optimal effect on intestinal function in broilers aged d
1 to 14.TagedAPTREn
TAGEDPRH1ACKNOWLEDGMENTSTAGEDPTRN
TagedAPRThis study was funded by Program for Scientific
Research Start-Up Funds of Guangdong Ocean Univer-
sity (R18005).TagedAPTREn
TagedAPREn12 LAN ET AL.
TAGEDPRH1DISCLOSURESTAGEDPTRN
TagedAPRThe authors declare the following financial interests/
personal relationships which may be considered as
potential competing interests: Ruixia Lan reports finan-
cial support was provided by Guangdong Ocean Univer-
sity.TagedAPTREn
TAGEDPRH1SUPPLEMENTARY MATERIALSTAGEDPTRN
TagedAPRSupplementary material associated with this article
can be found, in the online version, at doi:10.1016/j.
psj.2023.103381.TagedAPTREn
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