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Short communication

Effect of dietary inulin supplements on growth performance and intestinal

immunological parameters of broiler chickens
Qianqian Huang a, Yinan Wei a, Yajun Lv a, Yuxi Wang b, Tianming Hu a,n
a
b

College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, PR China
Agriculture and Agri-Food Canada, Lethbridge Research Centre, 5403 1st Avenue South Lethbridge, Alberta, Canada T1J4B1

art ic l e i nf o

a b s t r a c t

Article history:
Received 17 October 2014
Received in revised form
8 July 2015
Accepted 25 July 2015

The objective of this study was to assess the effects of dietary inulin supplementation on growth performance and intestinal immune parameters of broilers. A total of 280 one-day-old Cobb 500 male broilers
were randomly allocated into four groups of seven replicate pens and given a maize-soybean basal diet
supplemented with 0, 5, 10 and 15 g/kg of inulin during the 42 days of the experiment. Feed intake (FI),
body weight gain (BWG) and feed conversion ratio (FCR) were determined from d 1 to 21 (starter), and
from d 22 to 42 (grower). Intestinal T lymphocyte subpopulations, the production of immunoglobulin A
(IgA) and cytokines as well as mucin mRNA expression were measured at 21 d and 42 d. Feed intake was
increased quadratically (P0.001) as the dietary inulin level increasing during starter period only. However
BWG and FCR of broilers were not affected by inulin supplementation in either period. At d 21 and as the
dietary inulin concentration increasing, proportion of T CD4 T lymphocyte and CD4 /CD8 ratio in ileum
tissue tended (P 0.050.087) to be linearly increased, IgA concentration in cecal content and mucin mRNA
expression in jejunum tissue were linearly increased (P0.0060.01), whereas concentrations of interleuk6 and interferon- in ileum tissue quadratically (Po0.05) decreased. The effects of dietary inulin on these
intestinal immunological parameters were minimal at the 42-d age of broilers. These results indicated that
dietary inulin at the levels of 510 g/kg may have the benecial effects on enhancing intestinal immune
function of broiler chicken at younger age when the intestinal function is not fully developed.
& 2015 Elsevier B.V. All rights reserved.

Keywords:
Inulin
Growth
Intestinal immune function
Broilers

1. Introduction
Use of prebiotics and probiotics as alternatives to antibiotics in
poultry industry has become an increasing practice due to the
global trend of banning on the use of in-feed antibiotics as growth
promoters. Inulin-type fructans have attracted considerable attention from producers mainly because studies have showed various benecial effects on animal performance when they are used
as prebiotics (Flickinger and Fahey, 2002; Verdonk et al., 2005).
Moreover, the inulin can selectively stimulate the growth and/or
activity of the benecial ora thereby benet host wellbeing and
health (Roberfroid, 2007). However, variations in the effects of
inulin on growth performance (Ortiz et al., 2009; Rebol et al.,
2010) in poultry have been reported, possibly due to variations in
the products used, inclusion levels in the diet, diet composition,
animal characteristics and husbandry hygiene. Avian intestinal
immune function has received increasing attention because it is
closely associated with intestinal infectious diseases, such as coccidiosis and salmonellosis, which causes enormous losses in
n

Corresponding author. Fax: 86 29 87092164.

E-mail address: hutianming@126.com (T. Hu).

poultry industry (Lillehoj and Trout, 1996). It is regarded that avian

gut-associated lymphoid tissue plays an important role in host
defense against pathogenic invasion (Janardhana et al., 2009). Inulin and fructooligosccharides (FOS) have been shown to modulate
immunological processes of animals and humans (Seifert and
Watzl, 2007). However, there is little information in this area for
birds. Janardhana et al. (2009) reported that FOS had an immunosuppressant effect at both functional and phenotypic levels
in the cecal tonsil of broilers, but for inulin this effect is unknown.
Therefore, the objective of the current study was to assess the
effects of dietary supplementation of inulin on growth performance and on intestinal immune function of broiler chickens.

2. Materials and methods

2.1. Animal, experimental design and feeding management
A total of 280 one-d old Cobb 500 male broiler chickens
(average body weight 40.74 70.3 g) obtained from a local hatchery
plant were used in a 42-d feeding experiment. The sex of the
chicken was identied by an expert in the hatchery plant. All

http://dx.doi.org/10.1016/j.livsci.2015.07.015
1871-1413/& 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Huang, Q., et al., Effect of dietary inulin supplements on growth performance and intestinal immunological
parameters of broiler chickens. Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.07.015i

Q. Huang et al. / Livestock Science ()

experimental protocols were approved by the Northwest A&F

University Animal Care and Use Committee. The treatments were a
maize-soybean basal diet supplemented with 0, 5.5, 11.0 and
s
16.5 g/kg of an inulin product (Orafti GR, Tienen, Belgium). The
product was derived from chicory and contained 92% inulin with
an average degree of polymerization (DP) Z10 and 8% glucose/
fructose/sucrose. The actual inulin content in the diets were 0, 5,
10 and 15 g/kg. The treatments were arranged as completely
randomized design. The broilers were rst randomly allocated into
28 pens (10 birds per pen) that were then randomly divided into
4 groups (7 pens per group) and assigned to the treatments described above. The pens with plastic mesh oors (0.54 m2 oor
area/pen) were located in an environmentally controlled room.
The temperature of the room was set at 34 C for the initial 3 d and
was gradually reduced by 2 C per week. Birds received 24 h
constant light during the rst 3 weeks and then reduced to 16 h
per day for the last 3 weeks. The broilers were fed starter diet from
d 1 to d 21 (starter period) and grower diet from d 22 to d 42
(grower period) of the experiment. The basal diets (Table 1) for
both periods were free of antibiotics and coccidiostats and were
formulated according to the nutrient requirements of NRC (1994).
Inulin was incorporated into the basal diet weekly by mixing it
into respective diet to achieve the designated concentration.
Diets were offered twice daily (8:00 am and 6:00 pm) for ad
libitum intake and birds had free access to water for the entire
experimental period. Residual feed and birds were weighed
weekly to determine the feed intake (FI), body weight gain (BWG)
and feed conversion ratio (FCR).
2.2. Sample collection
On d 21 and d 42, two birds from each pen were randomly
picked out and the jejunum (from the entrance of bile ducts to
Meckel's diverticulum) and ileum (from Meckel's diverticulum to
the ileo-cecal junction) were immediately removed and dissected
after the birds were killed by cervical dislocation. A 3 cm long
ileum section before the ileo-cecal junction was separated and
kept at 4 C for cell isolation. The remaining ileum and the whole
Table 1
Composition (%, as-fed basis) of the starter (d 121) and grower (d 2242) basal
diets.
Item
Ingredients
Maize
Soybean meal
Soybean oil
Limestone
Dicalcium phosphate
L-Lys  HCl
DL-Met
Salt
Choline chloride
Mineral premixa
Vitamin premixb
Nutrient levels (Calculated)
Metabolizable energy (MJ/kg)
Crude protein (%)
Ca (%)
Available P (%)
Lysine (%)
Methionine (%)

Starter

Grower

56.8
36.6
3.00
1.00
1.70
0.10
0.15
0.30
0.15
0.10
0.10

62.1
31.8
3.00
1.00
1.50
0.05
0.05
0.30
0.05
0.10
0.05

12.7
21.3
0.96
0.43
1.22
0.52

13.0
19.59
0.89
0.39
1.05
0.41

jejunum were immediately placed into liquid nitrogen after being

washed with ice-cold phosphate buffered saline (PBS; 0.01 M,
pH 7.4) and stored at  80 C until assayed. In addition, the cecum with its content was also removed after being ligated at its
both ends and stored at  80 C.
2.3. Determination of intestinal lymphocyte subpopulations
The procedure used to isolate intestinal mucosal lymphocyte
was modied from published approaches (Davies and Parrott,
1981; Chai and Lillehoj, 1988). Briey, the mesentery-, fat- and
blood vessels-free ileum segments were cut into o0.5 cm pieces,
washed with PBS containing 100 IU/ml penicillin and 100 g/ml
streptomycin and incubated at 37 C with 20 ml digestive culture
medium (Yang and Guo, 2006) for 90 min. The suspensions were
ltered through 200 mm nylon cloth and the ltrates were centrifuged with an Allegra 64R Centrifuge (Beckman Coulter, Inc.,
CA) at 500g for 10 min. The resultant cell pellet was re-suspended
in 3.0-ml 40% (v/v) Percoll medium (Pharmacia, Piscataway, NJ),
which was then placed on 4.0 ml 70% (v/v) Percoll medium, and
the cells were recovered from the interface of the two solutions
after centrifuging at 800g for 30 min. The recovered cell fraction
was washed twice with PBS and then re-suspended in 1.0 ml of
RPMI-1640 culture medium to obtain a single lymphocyte cell
suspension.
The lymphocyte subpopulations (CD3 , CD4 and CD8 T
cells) in the lymphocyte cell suspension prepared above were
determined using the ow cytometric method (Holt et al., 2010;
Yang and Guo, 2006). Viable cell counts were determined by trypan blue exclusion and the cell concentrations were adjusted to
2  106 viable cells/ml. The cell suspension (100 l) was then incubated with mouse anti-chicken CD3-FITC, mouse anti-chicken
CD4-PE and mouse anti-chicken CD8-SPRD antibodies cocktails
(Southern Biotech, Birmingham, AL) at 4 C in the dark for 30 min.
After incubation, the cells were washed twice with PBS and then
re-suspended in 500 l PBS and enumerated by a ow cytometer
(Beckman).
2.4. Determination of intestinal IgA and cytokines
The cecal content was thawed at room temperature, thoroughly
mixed and 1.0 ml subsample was mixed with 2.0 ml of PBS. The
mixture was subsequently centrifuged at 5000g for 30 min and the
supernatant was collected for measuring the IgA concentration
with a commercial ELISA kit (KYM, Beijing, China) following the
manufacturer' instructions.
The cytokines were determined in ileum tissue by rst grinding
1.0 g of sample with 2.0 ml of PBS in mortar on ice, followed by
centrifuging the homogenized suspension at 10,000g for 15 min to
obtain the supernatant that was then used to determine the levels
of interleukin (IL)-2, IL-6, IL-10 and interferon- (IFN-) by commercial ELISA kits (KYM, Beijing, China).
2.5. Quantitative real-time polymer chain reaction (RT-qPCR)
s

a
Mineral premix supplied the following per kg of diet: Mn, 120 mg; Zn, 80 mg;
Fe, 80 mg; Cu, 8 mg; I, 0.35 mg, Se, 0.15 mg.
b
Vitamin mixture supplied the following per kg of diet: retinyl acetate,
103.2 mg; cholecalciferol, 1.5 mg; menadione, 2 mg; thiamine, 2 mg; riboavin,
7 mg; cyanocobalamin, 0.015 mg; DL--tocopheryl acetate, 7 mg; pyridoxine,
2.5 mg; niacin, 45 mg; pantothenic acid, 12 mg; folic acid, 0.1 mg; biotin, 0.11 mg.

Total RNA was extracted from jejunum tissue using the TRIzol
Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's
instructions. The integrity and concentration of total RNA were
determined by Epoch Microplate Spectrophotometer (BioTek Instruments, Inc., VT) at optical density (OD) 260/280. Reverse
transcription was performed by a PrimeScript RT reagent Kit with
gDNA Eraser (Takara, Dalian, China) following the manufacturers
protocol. The primers for mucin 2 gene (MUC2, Gallus gallus,
GI423101) were 5-AGGCCAGTTCTATGGAGCACAGTT-3 (forward),
and 5-TTGAGTGCCCAGAGGGACATTTCA-3 (reverse), and for actin (Beta-actin, Gallus gallus, GI: 396526 ) were 5-

Please cite this article as: Huang, Q., et al., Effect of dietary inulin supplements on growth performance and intestinal immunological
parameters of broiler chickens. Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.07.015i

Q. Huang et al. / Livestock Science ()

ACCCACACTGTGCCCATCTATGAA-3(forward), and 5- AATTTCTCT

CTCGGCTGTGGTGGT-3 (reverse). Each PCR was performed in triplicate using a CFX96 Real-Time System (Bio-Rad, CA) with SYBR
Premix Ex Taq II (Takara) according to manufacturer's instructions. The PCR reaction conditions included an initial denaturation
phase at 95 C for 30 s with 40 cycles of denaturation at 95 C for
5 s and annealing and extension at 60 C for 10 s. After amplication, the melting curve procedure was: 95 C for 15 s, 60 C for
30 s, and 95 C for 15 s. The results from RT-qPCR were analyzed
using 2CT method. Mucin 2 mRNA level was expressed relative to
-actin which was used as an endogenous control.
2.6. Statistic analysis
All data were statistically analyzed by One-way ANOVA using
SPSS Statistics Base 17.0 (SPSS Inc., 2007). Dietary inulin level was
the main effect with pen as statistical unit. Data obtained at d 21
and d 42 were analyzed separately. The effect of supplemental
levels of inulin was analyzed using orthogonal polynomials contrasts for linear and quadratic effects. Means were compared by
Duncan's multiple-range test and the differences among groups
were considered signicant at p o0.05, and as trend at p values
between 0.05 and 0.1.

3. Results
Broilers fed starter diets containing 5 g/kg of inulin had higher
(p o0.001) FI than Control broilers, but this was not observed with
10 or 15 g/kg of inulin nor in the grower period (Table 2). Inulin
had no effect on BWG and FCR in either period.
Percentages of CD3 and CD8 T cells of ileum were not affected by the treatments at either d 21 or d 42 of the experiment
(Table 3). However, the percentage of CD4 T cell tended
(p 0.087) to be linearly increased by supplemental inulin at d 21
but not at d 42. The ratio of CD4 /CD8 also tended to be linearly
increased by inulin supplementation at d 21 (P 0.05) and d 42
(P 0.073) (Data not shown). The concentration of IgA in cecal
content was linearly increased (p 0.01) at d 21 and quadratically
increased (p 0.035) at d 42 as the inulin supplementation levels
increasing. Compared to Control, higher IgA concentration in the
cecal content of all inulin supplemented groups was observed
(p o0.05) at d 21, whereas this was observed only for birds supplemented with 5 and 10 g/kg of inulin at d 42. The IgA concentration in cecal content was similar for all inulin groups at both

d 21 and d 42. Treatments did not affect the concentration of IL-2

and IL-10 in ileum tissue at either d 21 or d 42 (Data not shown).
However, the concentration of IL-6 in 5 g/kg inulin group was
lower than that of Control and the other inulin groups at d 21
(p o0.05) whereas this difference was not observed at d 42. The
IFN- concentration was lower (p o0.05) for 5 and 10 g/kg of inulin supplemented chickens than those for Control and 15 g/kg
groups at d 21, but all birds had similar IFN- concentration at d
42. Inulin supplementation linearly increased the mRNA expression jejunum mucin at both d 21 (p 0.006) and d 42 (p 0.019).
At d 21, chickens in 10 and 15 g/kg of inulin groups had higher
expression level of genes than those in the Control and the group
on 5 g/kg of inulin (p o0.05). At d 42, however, only birds fed 10 g/
kg of inulin had a higher expression level of mucin gene than the
Control (Po 0.05).

4. Discussion
The effects of inulin supplementation on growth performance
of broilers are inconsistent among studies. Improved BWG and FCR
of broilers were observed in some studies (Rebol et al., 2010;
Yusrizal and Chen, 2003) but not in others (Ortiz et al., 2009;
Rehman et al., 2008). The discrepancy among studies is likely due
to variations in inulin sources and dosages used animal characteristics, and feeding conditions (Verdonk et al., 2005). For example, Yusrizal and Chen (2003) showed that supplemental inulin
improved the growth performance of female but not of male
broilers. Rebol et al. (2010) reported that adding inulin at the
levels of 10 and 20 g/kg to a wheat-barely diet increased BWG of
broilers. In contrast, Nabizadeh (2012) found that adding 5 and
10 g/kg inulin to a soybean-corn diet did not affect broilers'
growth performance. This study showed that only 5 g/kg of dietary
inulin increased broilers' FI during starter (121 d), but not during
grower period and that other treatments had no effect on broilers
growth performance during both periods. This is consistent with
the observations of this study that the responses of most of the
immunological variables to inulin supplementation were greater
at d 21 than at d 42. Velasco et al. (2010) reported that broilers
supplemented inulin at 5 g/kg diet grew faster than those supplemented at 10 g/kg. The better FI of broilers at the dietary inulin
level of 5 g/kg diet than in the Control observed in this study is
also similar to the proposed optimal level of 2.55 g/kg for FOS in
poultry diet (Wu et al., 1999).
Although results among studies are not consistent, most of the

Table 2
Effect of inulin supplementation levels on growth performance of broiler chickens during 42-d experimental perioda.
Item

Days

SEMb

Inulin (g/kg)
0

10

15

p-Value
Linear

Quadratic

Body weight gain (g)

121
2242
142

775
1424
2199

778
1511
2289

797
1460
2257

749
1491
2218

9.05
20.22
19.65

0.453
0.420
0.891

0.169
0.493
0.113

Feed intake (g)

121
2242
142

922y
2537
3459

940x
2560
3500

922y
2539
3461

913y
2548
3462

2.49
15.40
15.57

0.008
0.946
0.817

0.001
0.828
0.537

Feed conversation ratio

121
2242
142

1.19
1.79
1.54

1.21
1.70
1.53

1.16
1.75
1.58

1.22
1.71
1.56

0.07
0.02
0.01

0.809
0.426
0.320

0.556
0.607
0.895

Values are the means of 7 pens of 10 birds per pen.

Standard error of means.
x,y
Means in a row without common superscripts differ (po 0.05).
b

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parameters of broiler chickens. Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.07.015i

Q. Huang et al. / Livestock Science ()

Table 3
Effect of inulin supplementation levels on ileal T lymphocyte subpopulations, cecal immunoglobulin A (IgA) concentrations, ileal cytokine production and jejunal mucin
mRNA expression in broiler chickens at 21 and 42 d of agea.
Items

Ileal T lymphocyte subpopulations

CD4 (%)
CD4 /CD8
Cecal IgA (g/ml)
Ileal cytokine concentration
Interleukin-6 (pg/g)
Interferon- (pg/g)
Jejunal mucin mRNA expression
Fold Change (  2^Ct)

x,y

Day

SEMb

Inulin (g/kg)
0

10

15

21
42
21
42
21
42

26.2
38.1
0.896
0.923
1.17y
1.33y

30.1
39.9
0.899
0.961
1.68x
2.06x

32.2
39.7
0.989
1.036
1.65x
2.02x

31.9
36.7
0.965
1.107
1.76x
1.89x,y

21
42
21
42

37.9x
38.8
80.9x
105.0

22.4y
38.4
62.9y
96.2

39.7x
38.3
56.4y
101.9

21
42

1.58y
1.47y

1.48y
2.43x,y

4.42x
4.20x

p-Value
Linear

Quadratic

1.22
2.00
0.017
0.038
0.080
0.107

0.087
0.822
0.050
0.073
0.010
0.071

0.386
0.573
0.692
0.828
0.158
0.035

38.9x
40.4
97.3x
96.2

1.89
0.625
4.21
3.18

0.109
0.412
0.13
0.487

0.013
0.330
o0.001
0.821

3.73x
3.48x,y

0.413
0.382

0.006
0.019

0.676
0.238

Means in a row without common superscripts differ (p o 0.05).

a
b
c

Values are the means of 7 pens of 2 birds per pen.

Standard error of means.
Results are expressed as the percentage of total lymphocytes counted.

data on humans and from animal models show that inulin-type

fructans possess direct and indirect immunomodulatory properties (Vogt et al., 2015). This study showed that the inulin at 5 and
10 g/kg increased the IgA concentration in cecal content of broilers
at both d 21 and d 42. Moreover, dietary levels of 10 and 15 g/kg
enhanced mucin mRNA expression in jejunum, and dietary levels
of 515 g/kg tended to increase the proportion of CD4 T cell and
the ratio of CD4 /CD8 in ileum tissue. IgA is a predominant
immunoglobulin antibody in intestinal secretion that plays an
important role in intestinal immunity (Lillehoj and Trout, 1996),
and mucins are the primary components of intestinal mucus layer
that are part of the innate immune system and act as a barrier
against luminal pathologies (Forstner et al., 1995). CD4 T cells
also play an important role in the immune system by acting as
coordinators of the immune response through producing upon
activation a variety of cytokines, soluble molecules secreted to the
extracellular space, which affect other cells of the immune system
(Arstila et al., 1994). The increased cecal IgA concentration and
enhanced mucin mRNA expression in jejunum and tendency of
increased proportion of CD4 T cells in ileum tissue observed in
this study suggest that diets supplemented with 515 g/kg inulin
improved intestinal immune functions in broilers. Ito et al. (2008;
2011) reported that rats fed inulin at 60 g/kg diet showed enhanced cecal IgA secretion, and Tako et al. (2008) found that inulin
at 40 g/kg diet increased duodenal mucin mRNA level in pigs. The
reasons why supplemented inulin enhanced intestinal immune
function of broilers were not investigated in this study, but are
likely related to the bidogenic effects of inulin on enhancing the
production of organic acid as observed in rat model (Ito et al.,
2011; Rehman et al., 2008; Rebol et al., 2010), and the direct effect of inulin on intestinal immune cell (Koo et al., 2003; Zenhom
et al., 2011). Further study should take into account the microbiota
as the composition of the intestinal immune system, aim to elucidate the mechanisms by which dietary inulin regulates the intestinal immune function in broiler chickens.
Interleukin-2, IL-6, IL-10 and IFN- measured in this study all
belong to a group of cytokines that mediate and orchestrate the
complex events in the immune reaction from the initiation of the
acute phase reaction to clonal expansion of effector T and B cells
(Lunney, 1998). Among these measured cytokines in ileum tissue,
only IL-6 and IFN- were decreased at d 21 with 510 g/kg inulin.

This indicated that effects of inulin on these intestinal cytokines

are associated with type of the cytokines, dietary concentration of
inulin and age of the broilers. Inulin may alleviate acute inammatory response or chronic inammation through decreasing
certain type of cytokines production. The observation that effects
of dietary inulin on these intestinal cytokines were mainly observed at d 21 is consistent with the effects of inulin on broilers
immune function observed in this study. The mechanisms by
which inulin modulate intestinal cytokines need to be elucidated.

5. Conclusion
The results obtained in this study demonstrate that broiler
diets supplemented with 510 g inulin/kg tended to increase FI at
starter period (d 0d 21) and enhanced intestinal immune function at d 21, but did not affect growth performance at grower
period or intestinal immunological parameters measured in this
study at d 42. This suggests that dietary inulin at the levels of 5
10 g/kg may have the benecial effects on enhancing intestinal
immune function of broiler chicken at younger age when the intestinal function is not fully developed.

Conict of interest statement

We conrm that there was no conict of interest.

Acknowledgments
This study was supported by the National Basic Research Program of China (Project 2011BAD17B05).

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parameters of broiler chickens. Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.07.015i

Q. Huang et al. / Livestock Science ()

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Please cite this article as: Huang, Q., et al., Effect of dietary inulin supplements on growth performance and intestinal immunological
parameters of broiler chickens. Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.07.015i