Effects of dietary yeast nucleotides supplementation on intestinal barrier

function, intestinal microbiota, and humoral immunity in specific

pathogen-free chickens

Che Wu,∗,† Zhenwei Yang,∗ Cailiang Song,∗ Chao Liang,‡ Hongxin Li,∗ Weiguo Chen,∗
Wencheng Lin,∗,† and Qingmei Xie∗,†,1
∗
College of Animal Science, South China Agricultural University & Guangdong Provincial Key Laboratory of
Agro-Animal Genomics and Molecular Breeding, Guangzhou 510642, PR China; † Key Laboratory of Animal
Health Aquaculture and Environmental Control, Guangzhou 510642, PR China; and ‡ Guangdong Hinabiotech,
Co., Ltd, Guangzhou 510642, PR China

ABSTRACT Yeast nucleotides are a fine functional ited higher expression of zonula occludens-1 (ZO-1) and
additive in human and animals. The effects of dietary Occludin gene in ileum (P < 0.05), whereas groups 2
yeast nucleotides supplementation on intestinal devel- and 3 exhibited higher expression of Mucin 2 (MUC2)
opment, expression of intestinal barrier-related genes, and trefoil factor 2 (TFF2) gene (P < 0.05), group 2
intestinal microbiota, and infectious bronchitis virus showed lower expression of IFN-α gene (P < 0.05). Di-
(IBV) antibody titer of specific pathogen-free (SPF) etary yeast nucleotides increased intestinal bacterial di-
chickens were investigated. A total of 60 1-d-old chick- versity (P < 0.05), and the abundance of Lactobacil-
ens were divided into 4 groups, each of which included lus (P < 0.05). At day 10, 17, 24, 31, 38, and 45, the
3 replicates of 5 chickens. Group 1 served as a con- serum IBV antibody titers were tested. Group 2 exhib-
trol that was fed a basal diet. Groups 2 to 4 were fed ited higher IBV antibody titer at day 17 (P < 0.05), fur-
the basal diet supplemented with 0.1%, 0.3% and 0.5% thermore, groups 2 to 4 reached the effective levels 1 wk
yeast nucleotides, respectively. All chickens were inocu- earlier than control group. In conclusion, dietary yeast
lated intranasally with inactivated IBV vaccine at day nucleotides supplementation can help birds to mount
1 and day 10. At day 17, the intestinal development, a faster and stronger antibody response to IBV vac-
expression of intestinal barrier-related genes and micro- cine. In addition, dietary yeast nucleotides supplemen-
biota were evaluated. There was a significant increased tation can also promote the intestinal development and
ileal villus height and villus height to crypt depth ra- barrier-related genes expression, and diversity and rich-
tio in group 2 (P < 0.05). Moreover, group 4 exhib- ness of intestinal microbiota.
Key words: yeast nucleotide, intestinal barrier function, intestinal microbiota, IBV antibody, SPF chicken
2018 Poultry Science 97:3837–3846
http://dx.doi.org/10.3382/ps/pey268

INTRODUCTION used as functional feed ingredients and often supple-

mented to diets of livestock in the form of yeast extracts
Nucleotides are a group of bioactive agents with the or pure substance (Sauer et al., 2012a; Alizadeh et al.,
characteristic of low-molecular-weight and intracellular 2016).
compounds, which play an important role in physiolog- The intestinal mucosal barrier is crucial for ani-
ical activities of animals (Superchi et al., 2012). There mal health (Blikslager et al., 2007; Oshima and Miwa,
are 2 ways to synthetize nucleotides including endoge- 2016). Complete intestinal structure could guarantee
nous anabolism and food intake. In some conditions, the function of mucosal barrier reflected by the villus
endogenous synthesis of nucleotides is insufficient for length, villus width, and crypt depth (Hu et al., 2016).
performing physiological functions (Maldonado et al., Dietary nucleotides supplementation could improve the
2001). Dietary nucleotides are considered as essential development of porcine jejunum with higher villus to
nutrients to modulate immunological and gastrointesti- crypt ratio (Shen et al., 2009). Tight junctions are the
nal function and optimize intestinal microbiota (Gil, most important component of the intestinal mucosal
2002; Sauer et al., 2011). Therefore, nucleotides are barrier (Zihni et al., 2016). Expressions of tight junc-
tion genes, such as zonula occludens-1 (ZO-1), were

C 2018 Poultry Science Association Inc.
markedly increased in porcine ileum by feeding the nu-
Received March 18, 2018. cleotides diet (Che et al., 2016). Mucin 2 (MUC2) and
Accepted May 29, 2018. trefoil factor 2 (TFF2) are major component of the
1
Corresponding author: qmx@scau.edu.cn

3837
3838 WU ET AL.

protective mucus layer, both of which can protect Table 1. The composition and nutrition level of basal diet
the intestine against pathogen and promote the pro- (as air-dried basis).
cess of restitution (Hernandez et al., 2009; Zeinali et Ingredients (%) Nutrition level
al., 2017). Mucus synthesis and secretion are related
to host-derived inflammatory mediators, such as in- Corn 61.643 ME (MJ/kg)2 12.13
Soybean meal 30.250 Cp 20.50
terferon (Deplancke and Gaskins, 2001; Niv and Ko- Corn protein meal 3.000 EE 3.66
ren, 2003; Kang et al., 2005). Some studies have re- Soybean oil 1.050 Crude fibre 2.23
ported that dietary nucleotides supplementation im- CaHPO4 1.280 Ca 0.90
Limestone 1.290 TP 0.58
proved intestinal function and immunity in human and Nacl 0.340 AP 0.35
pig (Muhammad, 2013; Che et al., 2016). However, the Antiseptic 0.100 Lys 1.21
effect of nucleotides on the intestinal mucosal barrier in Choline chloride 0.080 DLys 1.10
Premix1 0.400 DMet+DCys 0.75
poultry is still uncertain. Lys 0.342
Previous studies have demonstrated that the intesti- DL-Met 0.170
nal microbiota plays an important role in growth per- Thr 0.55
Total 100.00
formance and intestinal health of broiler (Johansen
et al., 2006; Yang and Choct, 2009). Some factors, such 1
The premix provided the following per kilogram of diets: Cu:
as diet and antibiotics, can influence the species rich- 11 mg, Fe: 149 mg, Mn: 32 mg, Zn: 35 mg, I: 0.50 mg, Se: 0.35 mg,
vitamin A: 15,000 IU, vitamin D: 33,000 IU, vitamin E: 46 mg, vita-
ness and diversity of intestinal microbiota (Knarreborg min B1: 7 mg, vitamin B2: 11 mg, vitamin B6: 14 mg, vitamin B12:
et al., 2002; Scott et al., 2013). However, there is lim- 30 ug, nicotinic acid: 83 mg, D-pantothenic acid: 32 mg, folic acid:
ited information about the effect of yeast nucleotides 2 mg, and biotin: 190 ug.
2
ME was a calculated value and others were measured values.
on intestinal microbiota of chickens. There are some
evidences that dietary nucleotides could stimulate in-
nate immune responses and improve host resistance to Experimental Design and Diet
the damage of toxin in chickens (Frankic et al., 2006).
All chickens were randomly divided into 4 dietary
Moreover, dietary nucleotides can improve the humoral
treatments with 3 replicate isolators of 5 birds each.
immunity and intestinal function in humans and piglets
Dietary treatments consisted of a control diet (CON)
(Maldonado et al., 2001; Sauer et al., 2012b). There-
without yeast nucleotide, and basal diets supplemented
fore, the objective of this study was to determine the
with 0.1% (T1), 0.3% (T3), and 0.5% (T5) yeast nu-
effect of dietary supplementation of yeast nucleotides
cleotides (Guangdong Hinabiotech Co., Ltd., China.),
on small intestinal morphology, expression of intestinal
respectively. The basal diet was a corn–soybean meal-
barrier-related genes, intestinal microbiota of specific
based diet, which was formulated to approximately
pathogen-free (SPF) chickens and on their antibody
meet the nutrient requirements for chickens (Dale,
responses to routine vaccination with inactivated infec-
1994). All diets were prepared in a mash form. The
tious bronchitis virus (IBV) vaccine.
composition and nutrition level of basal diet are shown
in Table 1.

MATERIALS AND METHODS

Serum IBV Antibody Detection
Ethics Statement
At 10, 17, 24, 31, 38, and 45 d of age, all chickens were
The experimental protocol was approved by the Ani- selected, blood samples were collected from the wing,
mal Care and Welfare Committee at South China Agri- and the serums were used to determine the humoral im-
culture University (Guangzhou, China). mune response derived from vaccination against IBV.
An enzyme-linked immunosorbent assay kit (ELISA,
VDPro IBV AB ELISA Kit, MEDIAN Diagnostics Inc.,
Korea) was used to determine antibody titres of the
Animal Management
chickens against IBV according to the manufacturer’s
A total of 60 healthy 1-d-old SPF chickens with sim- protocol. The absorbance was measured at 450 nm us-
ilar body weight were obtained from Guangdong Wens ing an ELISA reader (Multiskan FC, Thermo Scientific,
Dahuanong Biotechnology Co., Ltd., China. They were Shanghai, China) and calculated according to the in-
raised in negative pressure isolators with enclosed and struction.
ventilated environment. A continuous lighting program
provided for the entire experimental period. Tempera- Animal euthanasia and Sample Collection
ture was controlled at 34◦ C to 36◦ C during the first 5 d,
then weekly reduced of 2◦ C to 3◦ C until reaching 24◦ C At 17 d of age, 3 chickens per group (1 chicken
to 26◦ C. Feed and water were supplied ad libitum. All per replication) were randomly chosen and euthanized
chickens were inoculated intranasally with inactivated by CO2 inhalation, then dislocated cervical vertebra.
IBV vaccine (IBV-D90) (Feng et al., 2015) at day 1 and The small intestines were quickly dissected in a ster-
day 10 with dosage at 104.5 EID50 /0.1 mL. ile environment. The jejunum was isolated and rinsed
 EFFECT OF NUCLEOTIDE ON GUT FUNCTION AND IMMUNITY 3839
Table 2. Primers used in the study.

Genes Primer sequences (5 -3 ) Product size (bp) GenBank accession
a
ZO-1 F : GCCTGAATCAAACCCAGCAA 197 XM 01,527,8980.1
Rb : TATGCGGCGGTAAGGATGAT
Occludin F: GATGGACAGCATCAACGACC 193 NM 205,128.1
R: CATGCGCTTGATGTGGAAGA
MUC2 F: AATGCTGAGTTCTTGCCTAA 154 NM 0,013,18434.1
R: GTTGCAGTTCATATCCTGGT
TFF2 F: CCCTGCTGATCCTCGTAT 189 XM 416,743.4
R: GCTGTTATTTCCCAGTTGA
IL-22 F: CAGGAATCGCACCTACACCT 119 NM 0,011,99614.1
R: TCATGTAGCAGCGGTTGTTC
IL-17A F: CCATTCCAGGTGCGTGAACT 130 NM 204,460.1
R: TTTCTTCTCCAGGCGGTACG
IFN-α F: CCAGCACCTCGAGCAAT 133 XM 01,527,7440.1
R: GGCGCTGTAATCGTTGTCT
β -actin F: CTGGCACCTAGCACAATGAA 123 NM 205,518.1
R: CTGCTTGCTGATCCACATCT
a
F stands for forward primer.
b
R stands for reverse primer.

gently with 0.9% saline solution, then put them into Kit with gDNA Eraser (Perfect Real Time) (Takara,
10% formalin solution fixed overnight for assessment Dalian, China) according to the protocol of the manu-
of morphology. The middle section of ileum was sep- facturer. The cDNA used as a template to amplify the
arated with sterile scissors. Half of ileal segment was gene using the specific primer for real-time quantita-
rinsed gently with 0.9% saline solution and then fixed tive PCR (qPCR). Primer sequences of ZO-1, occludin,
overnight in 10% formalin solution for assessment of MUC2, TFF2, Interleukin 22 (IL-22), Interleukin
morphology. The other half of ileal segment was opened 17A (IL-17A), interferon-α (IFN-α), and beta actin
for collecting intestinal mucosal tissue and contents. (β -actin) genes are shown in Table 2. The PCR reac-
The ileal contents were collected in sterile tubes pre- tion used a SYBR green qPCR mix (Selleck Chemicals,
frozen in liquid nitrogen and stored at −80◦ C for in- Shanghai, China) and performed on the CFX96 Touch
testinal microbiota analysis. The intestinal mucosal tis- Thermal Cycler (Bio-Rad, California, USA). Each sam-
sues were rinsed gently with 0.9% saline solution after ple was run in triple with thermocycling conditions for
collecting contents, then put in tubes pre-frozen in liq- real-time qPCR was 95◦ C for 10 min followed by 40 cy-
uid nitrogen and stored at −80◦ C for gene expression cles of 95◦ C for 15 s and 60◦ C for 60 s. The specificity of
assay. primers was examined by melting curve analysis. Quan-
tification of all gene expression was calculated using the
2–ΔΔCt method with normalization against the endoge-
Small Intestinal Morphology
nous reference gene (β -actin).
The segments of the jejunum and ileum were dehy-
drated with graded ethanol (50, 70, 80, 95, and 100%)
Intestinal Microbiome
and embedded in paraffin wax, then cut into thin slices
(4 μm) and attached to the slides. The slides were Microbial genomic DNA of each ileal content sam-
stained with hematoxylin and eosion (H&E). For in- ple was extracted by TIANamp Stool DNA Kit
testinal morphology, a slide per chicken was observed (Tiangen, Beijing, China) according to the man-
for cell infiltration, villi epithelial tissue and goblet cell ufacturer’s instruction. DNA samples were quan-
under a light microscope. For each staining section, 10 tified using a Qubit 2.0 Fluorometer (Invitrogen,
intact villi and their related crypt were selected to mea- Carlsbad, CA, USA). The V3 and V4 hypervari-
sure the villus height and crypt depth by Nikon digital able regions of prokaryotic 16S rDNA were selected
sight DS-FI2 image system attached to Nikon Eclipse for generating amplicons using the primers: forward
ci microscope (Nikon Corporation, Japan). primers: 5 -CCTACGGRRBGCASCAGKVRVGAAT-
3 and reverse primers: 5 -GGACTACNVGGGTWTC
Gene Expression Assay TAATCC-3 . The PCR amplification conditions were
94◦ C for 3 min, followed by 24 cycles of 94◦ C for 5 s,
Total RNA was extracted from ileum tissues ac- 57◦ C for 90 s and 72◦ C for 10 s, and finally 72◦ C
cording to the Trizol (Takara, Dalian, China) instruc- for 5 min. The PCR product was excised from 2%
tion. The integrity of total RNA was checked by agarose gel and purified with E.Z.N.A. Gel Extrac-
electrophoresis on 1.0% agarose gel and quantified tion Kit (Omega Bio-Tek Inc., Guangzhou, China). 30
by Micro-spectrophotometer (Nanodrop 100, allsheng, to 50 ng DNA was used to generate amplicons using
Hangzhou, China). Then total RNA was reverse tran- a MetaVx Library Preparation kit (GENEWIZ Inc.,
scribed into cDNA using the PrimeScript RT reagent South Plainfield, NJ, USA). Finally, DNA libraries were
3840 WU ET AL.

validated by Agilent 2100 Bioanalyzer (Agilent Tech- RESULTS

nologies, Palo Alto, CA, USA), and quantified by Qubit
2.0 Fluorometer (Invitrogen, Carlsbad, CA). DNA li- Small Intestinal Morphometry
braries were multiplexed and loaded on an Illumina
MiSeq instrument according to manufacturer’s instruc- The morphology changes of jejunum and ileum are
tions (Illumina, San Diego, CA, USA). Sequencing was shown in Figures 1A and 1B. Under experimental con-
performed using a 2 × 300 paired-end (PE) configura- ditions, no immune cell infiltration was observed in the
tion; image analysis and base calling were conducted by jejunum and ileum of all groups. In jejunum, the shed-
the MiSeq Control Software (MCS) embedded in the ding villi epithelial tissue can be seen in control and T1
MiSeq instrument. group (Figure 1A, red arrow). Goblet cells appear as
vacuoles when stained by H&E. In comparison to con-
trol group, there are more goblet cells in T1, T3, and
Sequence Analysis of 16 s rRNA T5 groups (Figure 1A, black arrow). The development
of jejunal villi in control group was not as good as other
The QIIME data analysis package (version 1.9.1) groups, which showed neatly lined villi (Figure 1A). In
was used for 16S rRNA data analysis (Caporaso et ileum, the control group appeared some shedding villi
al., 2010). The forward and reverse reads were joined epithelial tissue (Figure 1B, red arrow). The amount
and assigned to samples based on barcode and trun- of goblet cells was obviously increased in T3 group
cated by cutting off the barcode and primer sequence. (Figure 1B).
Quality filtering on joined sequences was performed The villus height, crypt depth, and the ratio of
and sequences which did not fulfill the following cri- villus height to crypt depth (V/C) of jejunum and
teria were discarded: sequence length <200 bp, no am- ileum are shown in Figures 1C–1E. There were no
biguous bases, mean quality score ≥20. Then the se- significant differences of the above indexes in the
quences were compared with the reference database jejunum among 4 treatment groups (P > 0.05)
(RDP Gold database) using UCHIME algorithm to de- (Figures 1C–1E). In ileum, both villus height and V/C
tect chimeric sequence, and then the chimeric sequences increased when comparing the T5 group with the con-
were removed. The effective sequences were used in trol group (P < 0.05) (Figures 1C and 1E), but there
the final analysis. Sequences were grouped into oper- was no significant difference in crypt depth (P > 0.05)
ational taxonomic units (OTUs) using the clustering (Figure 1D). However, the T1 and T3 groups had no
program VSEARCH (1.9.6) (Github, San Francisco, significant difference in the 3 indexes compared with
USA) against the Silva 119 database pre-clustered at the control group (P > 0.05) (Figures 1C–1E).
97% sequence identity. The Ribosomal Database Pro-
gram (RDP) classifier was used to assign taxonomic
category to all OTUs at confidence threshold of 0.8. Gene Expression of Ileum Mucosa
The RDP classifier uses the Silva 123 database which The fold change for ZO-1, Occludin, MUC2, TFF2,
has taxonomic categories predicted to the species level. IL-22, IL-17A, and IFN-α gene expression analysis ob-
Sequences were rarefied prior to calculation of alpha tained from qRT-PCR is shown in Figure 2. Yeast nu-
and beta diversity statistics. Alpha diversity indexes cleotides tended to increase expressions of ZO-1 and
were calculated in QIIME from rarefied samples us- Occludin genes, and it was markedly increased (P <
ing for diversity the Shannon index, for richness the 0.05) in SPF chickens fed T5 diet relative to CON diet
Chao1 index. Beta diversity was calculated using prin- (Figures 2A and 2B). Meanwhile, the expressions
cipal coordinate analysis (PCoA) performed based on of MUC2 and TFF2 were significantly upregulated
the Bray–Curtis distance metric. (P < 0.05) in ileum samples of birds of T1 and
T3 groups versus the control group (Figures 2C
Statistical Analysis and 2D). There were no significant differences in
the expression of IL-22 and IL-17A genes among
The experimental unit was the isolates, and 3 chick- 4 groups (P > 0.05) (Figures 2E and 2F). Com-
ens for intestinal analysis, 12 chickens for blood samples pared with the control group, the T1 group had
analysis. All the data were tested by normal distribu- a significant decreased expression of IFN-α gene
tion by the Shapiro–Wilk test using SPSS 22.0 (SPSS (P < 0.05), but there were no significant differences
Inc., Cary, NC, USA). The normalized data were an- in T3 and T5 groups (P > 0.05) (Figure 2G).
alyzed statistically with one-way analysis of variance
(ANOVA) by Duncan’s multiple comparison tests.
Non-normally distributed data were analyzed with the
Microbial Profile
Kruskal–Wallis test. Differences were considered to be A total of 2233,812 sequences obtained from all the
statistically significant at P < 0.05. Data were ex- samples with an average read length of 300 bp. After
pressed as means and standard error of mean (SEM). filtering for quality and checking chimera, 664,559 ef-
The column diagrams were made by with GraphPad fective reads remained with the number ranging from
Prism 7.0 (GraphPad Software, Inc. La Jolla, USA). 39,051 to 65,631 per sample and then clustering into a
 EFFECT OF NUCLEOTIDE ON GUT FUNCTION AND IMMUNITY 3841

Figure 1. Effects of yeast nucleotide on morphology changes of small intestinal in SPF chickens. The histological observation of jejunum (A)
and ileum (B) [H&E staining, 400 ×]. The light black arrow shows the shedding villi epithelial tissue and the black arrow shows the goblet cells
(vacuole). The villus height (C), crypt depth (D), and the ratio of villus height to crypt depth (V/C) (E) of jejunum and ileum were measured. Data
are shown as mean with SEM (N = 3) in (C to E). The asterisk superscripts on the bar mean significant difference compared with control group
(P < 0.05), whereas with no star superscripts mean no significant difference (P > 0.05). CON, control group. T1, 0.1% dose group. T3, 0.3%
dose group. T5, 0.5% dose group.

total of 154 OTUs for all samples at the 97% sequence represent a more rich and diverse microbial population,
similarity value for the final analysis. respectively. Consequently, these results demonstrated
The complexity of intestinal microbiota was evalu- that yeast nucleotides may increase the species richness
ated on the basis of alpha-diversity indices (Figure 3). and diversity to a certain extent. The PCoA plot indi-
The rarefaction curve for all samples tends to reach the cates the similarity between microbial communities as
plateau with the number of reads increased in each sam- shown in Figure 3D. The microbial communities from
ple, showing that sequences were large enough to cover each treatment were separated into a distinct cluster.
community in the SPF chickens’ intestinal microbiota The taxonomic analysis of OTU representative se-
(Figure 3A). The Chao1 was used to reflect the species quences of 97% similarity by using RDP classifier
richness. The Chao1 of T1, T3, and T5 groups are sig- Bayesian algorithm is shown in Figure 4. All OTU
nificantly higher than that of control group (P < 0.05) representative sequences were classified into 4 most
(Figure 3B). The Shannon’s index was used to reflect common phyla (>1%, at least in 1 group): Firmi-
the species diversity, and the Shannon’s index of all 3 cutes, Bacteroidetes, Proteobacteria, and Tenericutes
treatment groups was higher than that of control group (Figure 4A). However, there were no differences of the
(Figure 3C). The larger Chao1 and Shannon’s indexes microbial communities among 4 groups at phylum level.
3842 WU ET AL.

Figure 2. Effects of yeast nucleotides on gene expression of ileum mucosa in SPF chickens. Charts A to G show the expression of ZO-1,
Occludin, MUC2, TFF2, IL-17A, IL-22, and IFN-α genes in ileum by using real-time qPCR. Data are shown as mean with SEM (N = 3). The
asterisk superscripts on the bar mean significant difference compared with control group (P < 0.05), whereas with no star superscripts mean no
significant difference (P > 0.05). CON, control group. T1, 0.1% dose group. T3, 0.3% dose group. T5, 0.5% dose group.

At the genus level, the bacterial community composi- whereas the control group reached the positive value at
tion of different samples was performed in the form day 24. These results clearly demonstrated that yeast
of proportional columnar graph (Figure 4B). The se- nucleotides can enhance the immune response to IBV
quences from all samples were majorly identified into 39 vaccine.
genera whereas others were combined into “unclassed”.
We then selected the top 7 abundance genera for further
DISCUSSION
analysis. Compared with control group, the abundance
of Blautia and Ruminiclostriduium 5 in T1, T3 and T5 Some studies have demonstrated many benefits
groups and Selllimonas in T1 group was significantly drawn from nucleotides on the immune function and
decrease (P < 0.05). In addition, the abundance of Lac- intestinal health of humans and piglets (Maldonado
tobacillus in T1, T3 and T5 groups and [Ruminococ- et al., 2001; Godlewski et al., 2009). In mammals, pups
cus] torques group in T3 group was significantly higher can obtain high concentration nucleotides from milk
than control group (P < 0.05) (Figure 4C). (Thorell et al., 1996). However, chickens can only obtain
nucleotides from endogenous anabolism and food in-
take. The chickens are 1 of the major sources of valuable
Serum IBV Antibody Titers protein for human (Windhorst, 2006). The comprehen-
sion of physiological state in chickens is very important
The effect of yeast nucleotide supplement on IBV to prevent pathogenic infections and the occurrence of
antibody titers is presented in Figure 5. After vac- diseases. In this study, we used the yeast nucleotides as
cination, the antibody response to IBV vaccine was functional additives to investigate its effect on chickens.
higher in 3 treatment groups than in control group, Yeast nucleotides can be efficiently absorbed by the
and the IBV antibody titers of T5 group was signifi- small intestine, whereby enhances the function of in-
cantly higher than that of control group at day 17 (P < testinal epithelial cells (Seifert and Schultz, 1989). Gob-
0.05). From day 17 onward, the IBV antibody titers of let cells synthesize and secret lubricant mucus that
T1, T3, and T5 groups have reached the positive value, forms a mucus layer in the small intestine to protect the
 EFFECT OF NUCLEOTIDE ON GUT FUNCTION AND IMMUNITY 3843

Figure 3. The diversity of intestinal microbiota in SPF chickens. The vertical axis of rarefaction curves (A) shows the number of observe OTU
that would be expected to be found after sampling the number of sequences shown on the horizontal axis. The Chao 1 (B) was used to estimate
sample richness and Shannon indices (C) was used to estimate sample diversity. Data are shown as mean with SEM (N = 3) in (B) and (C).
Principal coordinate analysis (PCoA) plot (D) of the intestinal microbiota based on the Bray–Curtis distance metric. The percentages indicate
the relative contribution of the 2 principal coordinates (PC1–PC2). The asterisk superscripts on the bar mean significant difference compared
with control group (P < 0.05), whereas with no star superscripts mean no significant difference (P > 0.05). CON, control group. T1, 0.1% dose
group. T3, 0.3% dose group. T5, 0.5% dose group.

epithelial cells (Johansson et al., 2013). In this study, we 4 main components: the physical, chemical, immuno-
observed that the amount of goblet cells was obviously logical, and microbiological barriers (Anderson et al.,
increased in jejunum and ileum of chickens fed yeast 2012). The physical barrier is a layer of epithelial cells
nucleotides when compared with chickens fed the con- that connects each other with tight junctions (Farquhar
trol diet (Figures 1A and 1B). Complete small intesti- and Palade, 1963). In the present study, diets contain-
nal structure is important for digestive and absorptive ing yeast nucleotides tended to increase expressions of
function of small intestine and is closely related to the ZO-1 and Occludin genes compared to the control diet
morphological changes of small intestinal villus length (Figures 2A and 2B). This finding is consistent with the
and crypt depth (Brudnicki et al., 2017). Compared result of Che et al.(2016), who found that expressions
with chickens fed the control diet, an increase in villus of Claudin-1 and ZO-1 genes in ileum were markedly in-
height and villus height to crypt depth ratio in the ileum creased by feeding yeast nucleotides. MUC2 and TFF2
was observed in chickens fed 0.5% yeast nucleotides proteins are major component of the chemical barrier,
(T5 group) (Figures 1C and 1E). Similarly, a previous which play an important role in preventing against the
study reported that dietary yeast nucleotides have ben- bacteria and lubricating the small intestine to main-
eficial effects on ileal morphology in pigs (Domeneghini tain the mucosal barrier function (Johansson et al.,
et al., 2004). These findings are suggestive that yeast 2011). TFF3 (also TFF2 in chickens) could suppressed
nucleotides are useful to intestinal development. the secretion of proinflammatory cytokines in intesti-
The intestinal mucosal function is crucial for ani- nal mucosal, whereas the decrease of MUC2 induces
mal as a barrier to prevent pathogens and toxins. The inflammatory immune responses (Zhang et al., 2003;
intestinal barrier is a complex structure consisting of Van der Sluis et al., 2006). In this study, our results
3844 WU ET AL.

Figure 4. Relative abundance of intestinal microbiota in SPF chickens. The bacterial community composition at phylum level (A). Those
abundances below 1% were classified into “others”, including Actinobacteria and Acidobacteria. The bacterial community composition at the
genus level (B). Those abundances below 1% were classified into “others”. Lanes 1 to 12 represent each sample of ileal content in 4 treatment
groups. The top 7 abundances of genera in 4 treatment groups (C). Data are shown as mean with SEM (N = 3) in (A) and (C). The asterisk
superscripts on the bar mean significant difference compared with control group (P < 0.05), whereas with no star superscripts mean no significant
difference (P > 0.05). CON, control group. T1, 0.1% dose group. T3, 0.3% dose group. T5, 0.5% dose group.

showed that expression of MUC2 and TFF2 genes were diets supplemented with yeast nucleotides did not sig-
increased in T1 and T3 groups over the control group nificantly influence the expression of IL-22 and IL-17A
(Figures 2C and 2D). This finding demonstrated that genes (Figures 2E and 2F), and tended to decreased ex-
yeast nucleotides may enhance the immunity function of pression of IFN-α gene compared to the control group
intestinal mucosa. IFN-α is a proinflammatory cytokine (Figure 2G). These findings are suggestive that yeast
produced by virally infected cells and has a strong cor- nucleotides confer a moderate effect of intestinal mu-
relation with autoimmune diseases (Monteleone et al., cosal function on chickens.
2001). IL-17A and IL-22 are proinflammatory cytokine The microbiota is one of the essential components of
and positively correlated with the severity of the disease intestinal (Neish, 2009). The higher diversity of intesti-
(Li et al., 2012; Mizoguchi, 2012). In the present study, nal microbiota is beneficial to the intestinal ecosystem
 EFFECT OF NUCLEOTIDE ON GUT FUNCTION AND IMMUNITY 3845
vaccination with inactivated IBV vaccine was enhanced
by yeast nucleotides. Since we only focus on IBV im-
munization, further studies may need to be carried out
to evaluate the effects of yeast nucleotides on other vac-
cines, such as Newcastle disease virus and avian in-
fluenza virus vaccines. On the other hand, yeast nu-
cleotides will be used in normal chickens, due to differ-
ent genetic lineage having different physiological char-
acteristics and management.
In conclusion, our results confirmed that dietary
yeast nucleotides supplementation can help birds to
mount a faster and stronger antibody response to IBV
vaccine. Our findings are also suggestive that dietary
yeast nucleotides supplementation can promote the in-
Figure 5. Effects of yeast nucleotide on IBV antibody titers in SPF
chickens. The dotted line represents the log10 titer equal to 3.04. The testinal development and barrier-related genes expres-
log10 titer greater or equal to 3.04 mean the antibody was positive in sion, and diversity and richness of intestinal microbiota.
serum. The asterisk superscripts on the bar mean significant difference
compared with control group (P < 0.05), whereas no asterisk super-
scripts mean no significant difference (P > 0.05). Data are presented
as means with SEM (N = 12). CON, control group. T1, 0.1% dose
ACKNOWLEDGMENTS
group. T3, 0.3% dose group. T5, 0.5% dose group.
This study was supported by the National Modern
Agricultural Industry Technology System Project of
China ( CARS-41), Guangdong Province Agricultural
(Kühn et al., 1993). In this study, our results demon-
Industry Technology System Project ( 2016LM1112),
strated that diets supplemented with yeast nucleotides
and Science and Technology Program of Guangzhou
increase the diversity and richness of intestinal micro-
(201607010363).
biota (Figures 3B and 3C). Interestingly, the abundance
of genera Lactobacillus in yeast nucleotides treatment
groups was significantly higher than that of control CONFLICT OF INTEREST STATEMENT
group (Figure 4C), which are the major part of lac-
tic acid bacteria group. Genera Lactobacillus exhibits a The authors declare that there are no conflicts of
mutually beneficial relationship with the human body interest.
(Martin et al., 2013). Carbohydrates could be converted
to lactic acid by Lactobacillus and be further utilized
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