Effect of Dietary Non-phytate Phosphorus Levels on the Diversity and

Structure of Cecal Microbiota in Meat Duck from 1 to 21 d of age

S. J. Dai, K. Y. Zhang, X. M. Ding, S. P. Bai, Y. H. Luo, J. P. Wang, and Q. F. Zeng1

Institute of Animal Nutrition, Key Laboratory for Animal Disease-Resistance Nutrition of China, Ministry of
Education, Sichuan Agricultural University, Chengdu, Sichuan, China, 611130

ABSTRACT The study was conducted to distinguish
the effect of dietary non-phytate phosphorus (NPP) lev-
els on the community diversity and structure of the
cecal microbiota in meat duck based on 16S rDNA
high-throughput sequencing. In total, 525 1-d-old duck-
lings were fed diets (105 ducklings, 7 pens of 15 duck-
lings, on each diet) containing five levels of NPP (0.22,
0.34, 0.40, 0.46, and 0.58%) for 21 days. The results
showed that dietary NPP levels linearly and quadrat-
ically increased (P < 0.05) 21 d body weight, 1 to
21 d feed intake and NPP intake, and contrarily, lin-
early decreased (P < 0.05) β -diversity of cecal microbial
population in ducks. ß-diversity analyses showed that
Key words: cecal microbiota, dietary non-phytate phosphorus, meat duck, performance
microbiota clustering based on dietary NPP levels oc-
cured, with 0.22% NPP groups distinctly different from
the 0.46% and 0.58% NPP group samples. Moreover, di-
etary NPP levels could change the relative abundance
of the phylum Proteobacteria (linear, P < 0.05), genera
Eubacterium coprostanoligenes (quadratic, P < 0.05),
Ruminococcaceae UCG-014 (quadratic, P < 0.05)
and Subdoligrannulum (linear, P < 0.05), and Lach-
nospiraceae family (quadratic, P < 0.05) in cecal mi-
crobiota of ducks. Increasing the dietary NPP level in-
fluenced the cecal microbiota and positively affected the
growth of meat ducks.

2018 Poultry Science 97:2441–2450

http://dx.doi.org/10.3382/ps/pey090

INTRODUCTION species (Stanley et al., 2014), which play a crucial role

Dietary phosphorus (P) is an essential nutrient
not only for normal growth, skeletal system devel-
opment, and maintenance of animals (Shastak and
Rodehutscord, 2013) but also for bacteria, since it is
needed for a variety of metabolic processes in bacte-
rial cell (Durand and Komisarczuk, 1988). Moreover, it
was demonstrated that bacterial P and calcium (Ca)
assimilation and metabolic activity depend on P and
Ca availability in the large intestine of pigs (Mann et
al., 2014a). Mann et al. (2014b) found that high versus
adequate dietary Ca-P content significantly promoted
Lactobacillus by 14.9% units at the gastric Pars nong-
landularis of weaned pigs. An improved colonization re-
sistance against intestinal pathogens and promotion of
lactobacilli in ileal digesta and mucosa has been ob-
served with Ca-P rich diets in rats (Bovee-Oudenhoven
et al., 1999). These results suggested that dietary P con-
centration could affect gut health partly by modulating
the composition of gut microbiota.
The microbial community present in the broiler’s gas-
trointestinal tract (GIT) has more than 900 bacterial

C 2018 Poultry Science Association Inc.
Received November 21, 2017.
Accepted February 22, 2018.
1
Corresponding author: zqf@sicau.edu.cn
in feed digestion, toxins degradation, pathogens exclu-
sion, stimulation of the immune system, and endocrine
activity (Zhu et al., 2002). Current exciting research is
beginning to unravel how the composition of food mod-
ulates the gut microbiota (Rajoka et al., 2017). There
is a promising dietary strategy, which has recently re-
ceived much attention in human research, using a rat
or pig model with dietary Ca and P content to modu-
late the intestinal eubiosis. Changes in Ca and P sup-
plementation affected the composition and activity of
the microbial community in the digestive tract of broil-
ers (Ptak et al., 2015). Dietary higher P and Ca lev-
els led to a shift in relative abundance of Lactobacillus
(L.) reuteri and L. crispatus to L. salivarius and L. tai-
wanensis in broiler’s crop (Witzig et al., 2015). Daniel
et al. (2016) also found that diets supplemented with P
affected the cecal microbiota and improved the growth
of broilers. However, research on the interaction be-
tween dietary NPP levels and cecal microbiota of ducks
is poorly understood. In a qualitative comparison, there
were clear distinctions in the structure and composition
of gut microbiome between Pekin duck cecal contents
and those of broiler chickens or turkeys. Therefore, we
suppose that it is not advisable to extrapolate the re-
sults of studies on affecting the microbiomes from one
bird order to another (Best et al., 2017).

2441
2442 DAI ET AL

Therefore, in the present study, we investigated the Table 1. Ingredients and compositions of the
hypothesis that the diversity and composition of the basal diets (%, dry matter basis).
intestinal microbiota is linked with dietary NPP levels Ingredients contents
in ducks. The objective was to determine if there were
any differences in microbial diversity and/or relative Corn 59.50
Soybean Oil 1.87
abundance of bacterial taxa, at the phylum, class, order, Soybean meal 33.85
family, and genus levels, within the cecal microbiota in L-Lysine-HCL 0.024
meat ducks from 1 to 21 d of age. DL-Methionine 0.197
Limestone 1.905
Monocalcium phosphate 0.395
Tryptophan 0.061
Bentonite3 1.168
MATERIALS AND METHODS Sodium chloride 0.35
Choline chloride 0.15
The Institutional Animal Care and Use Committee Vitamin premix1 0.03
of Sichuan Agricultural University approved all proce- Mineral premix2 0.50
dures used in the study. Total 100.00
Calculated nutrients levels (%)
Metabolizable energy (Kcal/kg) 2,900.00
Crude protein 19.5
Calcium 0.90
Birds, Diets, and Management non-phytate phosphorus 0.22
Digestible lysine 0.96
A total of 525 1-d-old ducks (Cherry Valley, Sichuan Digestible methionine 0.46
Mianying Breeding Duck Co. Ltd. Mianyang, China) Digestible threonine 0.66
Digestible tryptophan 0.26
were randomly allocated to one of five treatments with Total phosphorus (analyzed) 0.54
seven replicate cages of 15 birds per cage in a com- 1
Provided per kilogram of diet vitamin A, 8,000 IU;
pletely randomized design and fed a basal corn-soybean cholecalciferol, 2,000 IU; vitamin E, 5 IU; vitamin K,
meal diet (NPP 0.13%) supplemented with 0.09, 0.21, 1 mg; thiamine, 0.4 mg; riboflavin, 3.2 mg; pyridoxine,
0.27, 0.33, or 0.45% of inorganic phosphorus in the form 1.2 mg; vitamin B12 , 6 μ g; folic acid, 100 μ g; niacin,
7 mg; calcium pantothenate, 5 mg.
of monobasic sodium phosphate (CaH2 PO4 ·2H2 O). The 2
Provided per kilogram of diet: Fe (FeSO4 ·H2 O)
analyzed total P (TP) content in experimental diets 80 mg, Cu (CuSO4 ·5H2 O) 8 mg, Mn (MnSO4 ·H2 O)
was 0.54, 0.72, 0.79, 0.84, or 0.92%, and the correspond- 70 mg, Zn (ZnSO4 ·H2 O) 90 mg, I (KI) 0.4 mg, Se
(Na2 SeO3 ) 0.3 mg.
ing dietary non-phytate phosphorus (NPP) levels were 3
Regulation of limestone, monocalcium and ben-
0.22, 0.34, 0.40, 0.46, and 0.58%, respectively. Each diet tonite to formulate the other 4 diets containing 0.34,
contained a constant calcium content of approximately 0.40, 0.46, and 0.58% NPP. The other ingredients in all
diets keep the same.
0.9%. The period of the experiment was from 1 to 21 d
of age.
The basal diet (1 to 21 d) (Table 1, 2-mm-diameter- DNA extraction and high-throughput
pellet) was formulated under digestible amino acid basis sequencing and analysis of 16S rDNA gene
to meet the nutrient requirements of Pekin ducks sug-
amplicons
gested in the NRC (1994) and Han et al. (2017). Ducks
were reared in pens (2.2 m × 1.2 m × 0.9 m) in a DNA extraction and high-throughput sequencing and
temperature- and humidity-controlled room with analysis of 16S rDNA gene amplicons were performed
24-h constant light schedule and free access to water using the Illumina Hiseq platform Novo gene (Novo
and feed. gene Bioinformation Technology, Beijing, China). The
total genome DNA from the cecal samples was ex-
tracted using the sixteen alkyl three ethyl ammo-
Data Collection and Sampling nium bromide (CTAB)/ sodium alkyl sulfonate twelve
(SDS) method (Bo et al., 2017). DNA was quantified in
On d 21, the body weight (BW) and feed consump- a NanoDrop ND-1000 spectrophotometer (Thermo Sci-
tion of ducks were recorded on basis. Average daily gain entific, Waltham, MA) and stored at −20◦ C (Stanley
(ADG), average daily feed intake (ADFI), feed con- et al., 2012).
version ratio (FCR), NPP, and TP intake were calcu- According to the concentration, DNA was diluted
lated. Feed waste was recorded daily, and the data were to 10 ng/μ L using sterile water. The 16S rDNA
used in the calculations of feed consumption. Then, one genes of distinct regions (16S V4) were amplified us-
bird per pen was euthanized by carbon dioxide asphyx- ing specific primer (515F GTGCCAGCMGCCGCG-
iation following anesthesia in a gas mixture (35%CO2 , GTAA; 806R GGACTACHVGGGTWTCTAAT) with
35%N2 , and 30% O2 ) (Zeller et al., 2015). The GIT was the unique barcodes. All PCR reactions were car-
immediately dissected after euthanization, the two ceca ried out with Phusion High-Fidelity PCR Master Mix
were opened longitudinally and digesta samples were (New England Biolabs). PCR products were mixed
collected with a sterile spoon. Samples were stored at in equal density ratios. Then, mixture PCR products
−80◦ C. were purified with Qiagen Gel Extraction Kit (Qiagen,
 DIETARY NON-PHYTATE PHOSPHORUS ON CECAL MICROBIOTA 2443
Table 2. Effect of dietary non-phytate phosphorus levels on growth performance and total phosphorus intake
in meat duck during 1 to 21 d of age.

Dietary non-phytate phosphorus levels, % P-Value

Parameter1 0.22 0.34 0.40 0.46 0.58 SEM2 ANOVA Linear Quadratic

ADG(g) 59.72a 68.6b 69.0b 70.8b 69.0b 0.85 0.001 0.001 0.001
ADFI(g) 102.8a 114.5b 116.6b 116.9b 116.9b 1.33 0.001 0.001 0.001
FCR(g/g) 1.72 1.67 1.69 1.65 1.68 0.02 0.066 0.079 0.061
NPP intake (g) 4.75a 8.18b 9.79c 11.3d 14.1e 0.10 0.001 0.001 0.001
TP intake(g) 11.7a 17.3b 19.3c 20.6d 22.4e 0.19 0.001 0.001 0.001
a-e
Means in the same row with no common superscript are significantly different (P < 0.05).
1
ADG: Average Daily Gain; ADFI: Average Daily Feed Intake; FCR: Feed Conversion Ratio; NPP: non-phytate phos-
phorus; TP: Total Phosphorus.
2
Means represent 7 pens of 15 ducks per pen.

Hilden, Germany). Sequencing libraries were generated Statistical Analysis

using TruSeq DNA PCR-Free Sample Preparation Kit
(Illumina, San Diego, CA) following the manufacturer’s
recommendations, and index codes were added. The li-
brary quality was assessed on the Qubit 2.0 Fluorom-
eter (Thermo Scientific) and Agilent Bioanalyser 2100
system. Finally, the eligible libraries were sequenced on
an Illumina HiSeq 2500 platform and 250 bp paired-end
reads were generated.
Data Analysis
Paired-end reads were assigned to samples based on
their unique barcode and truncated by cutting off the
barcode and primer sequence. Paired-end reads were
merged using FLASH (V1.2.7), a very fast and accurate
analysis tool, which was designed to merge paired-end
reads when at least some of the reads overlap the read
generated from the opposite end of the same DNA frag-
ment, and the splicing sequences were called raw tags.
Quality filtering on the raw tags was performed under
specific filtering conditions to obtain the high-quality
clean tags, according to the QIIME (V1.7.0) quality-
control process. Reads were filtered by QIIME quality
filters. Sequences with ≥97% similarity were assigned
to the same optimal taxonomic units (OTUs). Then,
a representative sequence was chosen for each OTU to
annotate the taxonomic information of that unit. All
OTUs were subsequently analyzed for abundance and
diversity. For α-diversity analysis, rarefaction curves
and rank abundance curves were generated by R project
(Version 2.15.3).
Sequences (clean data) were analyzed using
the Quantitative Insights into Microbial Ecology
(QIIME, Version 1.7.0) software package, and in-
house Perl scripts were used to analyze α-diversity
(Observed-species, Shannon, Simpson, Chao I, ACE,
Goods coverage, and PD whole tree). A jackknifed
β -diversity analysis was conducted to assess the
statistical variation of sample location in principal
coordinate analysis (PCoA) plots based on unweighted
UniFrac distances (Lozupone et al., 2011).
The effects of dietary NPP levels on BW, FI, FCR,
TP intake, NPP intake, and the structure and diver-
sity of the cecal microbiota were determined by a one-
way ANOVA using the GLM procedure in SAS software
(SAS Institute Inc., Cary, NC, 2004). When the dietary
NPP levels effect was significant (P < 0.05), polynomial
contrasts and the linearity of response were examined to
analyze dietary NPP levels using linear and quadratic
regressions. The R2 was provided to compare these re-
gressions when the linear or quadratic effect was signif-
icant (P < 0.05) (Pesti et al., 2009). Probability values
< 0.05 were considered significant.
RESULTS
Growth Performance
Dietary NPP levels linearly and quadratically in-
creased (P < 0.05) ADG, ADFI, TP intake, and NPP
intake of ducks during 1 to 21 d of age (Table 2). When
compared to other groups, ducks fed diets containing
0.22% NPP had lower (P < 0.05) ADG and ADFI. Di-
etary NPP levels significantly affected (P < 0.05) TP
intake and NPP intake among all treatments. However,
dietary NPP levels had no marked effect on FCR (P >
0.05).
Alpha-Diversity
Illumina Hiseq high-throughput sequencing was per-
formed to evaluate the bacterial abundance. On aver-
age, we obtained 81,488 total reads. Reads were then
merged based on the overlapped sequences to gen-
erate tags. On average, 75,767 total tags were ob-
tained. We clustered the tags using a 97% similarity
cutoff to obtain an average of 1,319 OTUs. Rarefac-
tion curves were analyzed together to test the suffi-
ciency of the sequence collection (Figure 1). In rarefac-
tion curves, the curve of the observed species number
plateaued with the increase in samples/sequences num-
ber, which indicated that enough samples/sequences
2444 DAI ET AL

Figure 1. Alpha diversity analysis of different groups. A, Rarefaction curve is generated by setting the number of sequence as x-axis, and
the number of observed species as y-axis. The curve reflected the relationship between the quantity of observed species and sequences. The
“plateaued” shape of the curve indicated that enough samples/sequences were obtained to cover the majority of species; B, each circle in the
Venn diagram represented one group noted by the name of same color. The numbers located in the overlapping area represented the number of
OTUs share with respective groups. The numbers located in the individual area represented the number of OTUs peculiar to the representative
group. Caecum 1, Caecum 2, Caecum 3, Caecum 4, and Caecum 5 represent dietary non-phytate phosphorus contents were 0.22, 0.34, 0.40, 0.46,
and 0.58%, respectively.

were obtained to cover the majority of species. A Venn
diagram was generated based on the OTU classifica-
tion (Figure 1). The five groups shared 570 OTUs in
common.
Multiple α-diversity metrics revealed clear differences
among microbial populations in ducks fed diets with dif-
ferent NPP levels with respect to richness and evenness.
In general, there was a significant linear decrease (P <
0.05) in the diversity of the microbial populations by
all metrics as dietary NPP levels increased (Table 3).
When considering the Shannon diversity metric, which
takes into account the richness and evenness of species,
a pattern of dietary NPP levels emerges. This was sup-
ported by statistically significant differences between
 DIETARY NON-PHYTATE PHOSPHORUS ON CECAL MICROBIOTA 2445
Table 3. Effects of dietary non-phytate phosphorus levels on alpha diversity metrics of cecal microbiota in meat
duck at 21 d of age.

Dietary non-phytate phosphorus levels, % P-Value

Parameters 0.22 0.34 0.40 0.46 0.58 SEM ANOVA Linear Quadratic

Observed species 743.21 802.8 797.0 513.2 496.8 97.15 0.077 0.019 0.251
Shannon 6.33a 6.39a 6.80a 5.80a,b 5.68b 5.39 0.014 0.001 0.783
Simpson 0.97 0.96 0.93 0.94 0.92 0.02 0.262 0.053 0.632
Chao1 817.0 941.0 881.5 548.5 551.3 111.9 0.056 0.017 0.232
ACE 830.5 944.1 888.7 558.4 559.3 113.6 0.064 0.018 0.253
Goods coverage 0.998 0.997 0.997 0.999 0.999 0.0004 0.079 0.032 0.237
PD whole tree 55.70a 58.90a 55.41a 34.88b 36.88b 6.21 0.028 0.005 0.413
a,b
Means in the same row with no common superscript are significantly different (P < 0.05).
1
Means represent 5 ducks per pen.

the 0.22%, 0.34%, or 0.40% NPP groups and the 0.58%
NPP group (Table 2, ANOVA, P < 0.05), the same as
PD whole tree metrics.
Beta-Diversity
To deeply illustrate the differences in cecal flora be-
tween these groups, β -diversity analysis was performed
to analyze the phylogenetic relationship and relative
abundance of species. The structure of the duck ce-
cal microbial populations is distinct and clearly corre-
lated with dietary NPP levels on β -diversity measures.
In the unweighted UniFrac principal coordinates anal-
ysis (PCoA), the first principal coordinate (PC1) ex-
plained 29.89% of the variation among samples, with
PC2 explaining 12.32% of the variation. The PCoA
can estimate the main discrepancies in the OTUs be-
tween groups using the distance of sample dots. Then,
PCoA was calculated based on samples’ distance matri-
ces, which were generated based on their group species
phylogenic and evolutionary relationships. In our study,
unweighted UniFrac PCoA showed that the species dis-
crepancy was obvious between the high dietary NPP
contents (0.46% and 0.58%) and the low dietary NPP
contents (0.22% and 0.34%) (Figure 2, A). According
to weighted UniFrac PCoA (Figure 2, B), the species
discrepancy was also distinguished between the high di-
etary NPP contents (0.46% and 0.58%) and the low
dietary NPP contents (0.22%).
The Relative Abundance of Some Bacteria
The relative abundance of bacteria was analyzed at
the levels of phylum, class, order, family, and genus. The
structure and stability of the gut microenvironment can
be denoted by the relative abundance of different types
of bacteria. The heat map was generated using the rela-
tive abundance of different phylum (Figure 3). In meat
ducks at 21 d of age, the cecal microbiota was mainly
composed of Firmicutes (55%), Bacteroidetes (33.4%),
Actinobacteria (3.3%), Tenericutes (4.38%), Proteobac-
teria (2.89%), and unclassified bacteria (1.03%). At the
class, order, family, and genus levels, the Clostridia
(phylum Firmicutes, 50.7%), Clostridiales (50.7%),
Ruminococcaceae (phylum Firmicutes, 37.5%), and
Bacteroides (28.8%) were dominant.
Certain taxa were identified as differentially abun-
dant, according to dietary NPP levels (Figure 4).
First, we found that the relative abundance of the
phylum Proteobacteria linearly decreased (P < 0.05)
with increasing dietary NPP levels. Other remarkable
distinctions occurred in the genera Eubacterium
coprostanoligenes, Ruminococcaceae UCG-014, and
Subdoligrannulum. The increase of dietary NPP
levels, quadratically increased (P < 0.05) the rela-
tive abundance of Eubacterium Coprostanoligenes, and
linearly increased (P < 0.05) the relative abundance
of Subdoligrannulum, while Ruminococcaceae UCG-014
quadratically decreased (P < 0.05). Ducks fed diets
with 0.46% and 0.58% NPP had a higher (P < 0.05)
abundance of Subdoligrannulum than that of ducks fed
the other three experimental diets. Some species also
showed some interesting variations influenced by di-
etary condition. The abundance of the family Lach-
nospiraceae present a quadratic increase (P < 0.05)
with the increase of dietary NPP levels.
DISCUSSION
The actual NPP requirement of meat ducks aged 1
to 21 d of age was 0.40% (NRC, 1994). A lower level
of NPP in the diet (0.22%) reduced ADG and ADFI
in ducks aged 1 to 21 d in the current study. Simi-
lar to the result of the present study, Rama Rao et al.
(2006) found that depression in weight gain and FI was
observed at higher levels of Ca (0.8% and 0.9%) with
lower levels of NPP in diet (0.3% and 0.35%) of broil-
ers. Zeng et al. (2015) also observed that meat ducks
fed diet with 0.23% NPP had a lower FI than ducks fed
diet with 0.28% NPP.
One major finding of the present study was that
higher dietary NPP levels (0.46% and 0.58%) decreased
α-diversity, and the species discrepancy was obvious
when compared to a lower dietary NPP level (0.22%).
These results agreed with Palacios et al. (2008), who
observed that in the ileal and cecal sections, the
more diverse microbial communities are responsible for
2446 DAI ET AL

Figure 2. Beta diversity analysis (PCoA) plot (based on OTUs) according to dietary non-phytate phosphorus levels. A, unweighted unifrac
PCoA; B, weighted unifrac PCoA. PC1 and PC2 in x-and y-axis represented two principle discrepancy components between groups, and the
percentage in bracket means contribution value to the discrepancies by the component. Dots represent samples. Samples in same group share
same color. Caecum 1, Caecum 2, Caecum 3, Caecum 4, and Caecum 5 represent dietary non-phytate phosphorus contents were 0.22, 0.34, 0.40,
0.46, and 0.58%, respectively.

phytate degrading activities. This might be due to the
lack of sufficient enzymes (such as endogenous mucosal
phytase and phosphatase) in the proximal GIT, which
lead to an incomplete hydrolysis of phytate in the body
of non-ruminant animals (Maenz and Classen, 1998;
Onyango and Adeola, 2009; Selle et al., 2012). This
phenomenon caused most of the phytate-P in the diet
to enter the hindermost ileum and cecum to change
bacterial structure and metabolic processes. Therefore,
when ducks are fed a diet with available P deficiency,
their gut microbial communities need to obtain P from
the degradation of phytate. Moreover, according to
Wood and Clark (1988), a surplus of P can be stored
as polyphosphates in bacterial cells and may be used
as an energy and P source for metabolic processes.
Furthermore, P is important for bacterial proliferation,
and contributes considerably to bacterial structure and
metabolic processes (Lengeler et al., 1999). According
to the results of the present study, ducks fed diets with
0.22% NPP had poor growth performance but had a
higher α-diversity of cecal microbial population. One
reason for this result may be because the ducks fed di-
ets with 0.22% NPP need to spend more energy to com-
pete with the gut microbial population to obtain more
P and other nutrients. In addition, increasing diversity
in cecal microbiota in ducks fed diets with 0.22% NPP
may imply the increase of several enteric pathogens.
Some enteric pathogens, for example, have developed
strategies to metabolically utilize myo-inositol from
phytate-P degradation as a carbon and energy source.
 DIETARY NON-PHYTATE PHOSPHORUS ON CECAL MICROBIOTA 2447

Figure 3. The heat map of relative abundance of different phylum. Samples information is transverse listed, and species annotations are
longitudinal shown. The left clustering tree is species-related clustering tree, and the upper tree is sample-related clustering tree. The heat map
was performed by discrepancies of species-relative abundance between samples, with colors gradually changed from deep red to deep blue, in
accordance with high relative abundance to low. Caecum 1, Caecum 2, Caecum 3, Caecum 4, and Caecum 5 represent dietary NPP contents were
0.22, 0.34, 0.40, 0.46, and 0.58%, respectively.

This feature applies to Gram-positive enteropathogens
such as Enterococcus faecalis, Bacillus cereus, Listeria
monocytogenes, and Clostridium perfringens, as well
as Gram-negative pathogens, such as Salmonella ty-
phimurium, which may under certain circumstances
cause infections in pigs (Staib and Fuchs, 2014).
Therefore, intervention studies are required to further
study dietary P deficiency or the increase of dietary
phytate-P to determine whether they increase intestinal
pathogenic bacterial colonization and impair gut health
of poultry.
As previously noted, at the phylum level, Firmicutes
and Bacteroidetes (more than 80% of sequences) were
major bacteria in the duck gut microbiota (Vasaı̈ et al.,
2014). Best et al. (2017) also found barn-raised ducks
contain four major phyla found most often in other ani-
mal systems as follows: Firmicutes (57%), Bacteroidetes
(30%), Proteobacteria (6%), and Actinobacteria (3%).
Consistently, in the current study, majority of the mi-
croorganisms colonizing the cecum of ducks belonged to
the phyla Firmicutes and Bacteroidetes (more than 80%
of sequences), which were commonly described in pre-
vious studies that characterized the microbial commu-
nities of the chicken GIT (Stanley et al., 2013; Deusch
et al., 2015). These results indicated that dietary NPP
levels did not change the composition of gut microbiota
in meat ducks, suggesting that the complexity and the
anaerobic environment of the ceca did not allow impor-
tant changes in the bacterial community (Ley et al.,
2008).
Moreover, in the current study, we first found that di-
etary NPP levels could change the relative abundance
of the phylum Proteobacteria, the genera Eubacterium
coprostanoligenes, Subdoligrannulum, and Ruminococ-
caceae UCG-014, and the family Lachnospiraceae in the
cecal digesta of ducks. Individuals fed on the Mediter-
ranean diet (a balanced intake of fruits, grains, monoun-
saturated fat, vegetables, and polyunsaturated fat) had
lower numbers of Bacillaceae, Proteobacteria and acute
phase C-reactive proteins (Marlow et al., 2013; De
Filippis et al., 2016), suggesting the decrease of the rela-
tive abundance of the phylum Proteobacteria represents
a healthier condition. Ruminococcaceae UCG-014 is a
common genus reported in the chicken ceca (Bjerrum
et al., 2006; Mohd et al., 2015), which has been asso-
ciated with the maintenance of gut health and has the
enzymatic capability to degrade cellulose and hemicel-
lulose (Biddle et al., 2013). The family Lachnospiraceae
2448 DAI ET AL

Figure 4. Significant difference in relative abundance analysis of some bacterial species according to dietary non-phytate phosphorus levels.
A, relative abundance of Phylum Proteobacteria; B, relative abundance of Genera Ruminococcaceae UCG-014; C, relative abundance of Genera
Eubacterium Coprostanoligenes; D, relative abundance of Genus Subdoligrannulum; E, relative abundance of Family Lachnospiraceae.

was reported to be associated with corn-based diets and
is mainly composed by anaerobes and some Clostrid-
ium members (Munyaka et al., 2015). OTUs belong-
ing to Lachnospiraceae are known to degrade complex
polysaccharides to SCFA. In the Lachnospiraceae fam-
ily, OTUS accounted for almost 60% of the mucin-
adhered microbiota (Roos et al., 2000), and there were
more abundant in the diet supplementation with P
(Daniel et al., 2016). Supplementation of Ca in the diet
enhanced the presence of an uncultured Subdoligran-
ulum sp., which was previously found in the ceca
of turkeys (Scupham, 2007) and can produce butyric
acid. Heyer et al. (2015) considered that P deficiency
caused a reduction in SCFA synthesis due to reduce
fermentation of cellulose in pig, which suggested that
the activity of bacterial fibrolytic enzymes is modulated
by available P of the surrounding medium (Metzler and
Mosenthin, 2008). Moreover, several authors have con-
cluded from the results of in vitro studies (Francis et al.,
1978; Lee et al., 1985) that P, as a coenzyme, is essential
for bacterial degradation of dietary fiber. Also, the bac-
terial synthesis of fibrolytic enzymes is strongly depen-
dent on a sufficient P supply. These results were in ac-
cordance with current study. According to the findings
 DIETARY NON-PHYTATE PHOSPHORUS ON CECAL MICROBIOTA
above, dietary P content could affect the abundance of
bacteria related with the production of SCFA and the
maturation of gut microbiota, which suggested that
regulation of the dietary P supply could improve
growth performance of meat ducks by regulating gut
health.
CONCLUSIONS
In conclusion, this study uses a high-throughput py-
rosequencing based on the 16S rDNA gene in cecal sam-
ples from ducks and provides a first inventory of the
microbial community and the effect of dietary NPP lev-
els on the abundance of major different groups. Higher
(0.58%) or lower (0.22%) dietary NPP contents affected
the diversity of cecal samples and had a significant ef-
fect in modifying the bacterial community in the ceca.
The increase in the dietary NPP supply influenced the
ceca microbiota and positively affected the growth per-
formance of meat ducks. Further investigations are nec-
essary to understand the functional microbiota in ducks
to improve the gut health and the availability of dietary
P, and to thus reduce P loss by excretion.
ACKNOWLEDGMENTS
This research was supported by grants from
the National Key R & D Program of China
(2017YFD0502004), and Sichuan Agricultural Univer-
sity 211 Foundation of China.
REFERENCES
Best, A. A., A. L. Porter, S. M. Fraley, and G. S. Fraley. 2017.
Characterization of Gut Microbiome Dynamics in Developing
Pekin Ducks and Impact of Management System. Front. Micro-
biol. 7:2125.
Biddle, A., L. Stewart, J. Blanchard, and S. Leschine. 2013. Un-
tangling the genetic basis of fibrolytic specialization by Lach-
nospiraceae and Ruminococcaceae in diverse gut communities.
Diversity 5:627–640.
Bjerrum, L., R. M. Engberg, T. D. Leser, B. B. Jensen, K. Finster,
and K. Pedersen. 2006. Microbial community composition of the
ileum and cecum of broiler chickens as revealed by molecular and
culture-based techniques. Poult. Sci. 85:1151–1164.
Bo, T., S. Shao, D. Wu, S. Niu, J. Zhao, and L. Gao. 2017. Relative
variations of gut microbiota in disordered cholesterol metabolism
caused by high-cholesterol diet and host genetics. Microbiology-
Open 00:e491.
Bovee-Oudenhoven, I. M., M. L. Wissink, J. T. Wouters, and R. Van
der Meer. 1999. Dietary calcium phosphate stimulates intestinal
lactobacilli and decreases the severity of a Salmonella infection in
rats. J. Nutr. 129:607–612.
Daniel, B.-M., V. Marius, S. Vera, R. Markus, and C.-S. Amélia
2016. Insights into Broilers Gut Microbiota Fed with Phospho-
rus, Calcium, and Phytase Supplemented Diets. Front. Microbiol.
7:2033.
De Filippis, F., N. Pellegrini, L. Vannini, I. B. Jeffery, A. La Sto-
ria, and L. Laghi. 2016. High-level adherence to a Mediter-
ranean diet beneficially impacts the gut microbiota and associ-
ated metabolome. Gut 65:1812–1821.
Deusch, S., B. Tilocca, A. Camarinha-Silva, and J. Seifert. 2015.
News in livestock research-use of Omics-technologies to study the
microbiota in the gastrointestinaltract of farm animals. Comput.
Struct. Biotechnol. J. 13:55–63.
2449
Durand, M., and S. Komisarczuk. 1988. Influence of major minerals
on rumen microbiota. J. Nutr. 118:249–260.
Francis, G. L., J. M. Gawthorne, and G. B. Storer. 1978. Factors
affecting the activity of cellulases isolated from the rumen digesta
of sheep. Appl. Environ. Microbiol. 36:643–649.
Han, H. Y., K. Y. Zhang, X. M. Ding, S. P. Bai, Y. H. Luo, J.
P. Wang, and Q. F. Zeng. 2017. Effect of dietary fiber levels
on performance, gizzard development, intestinal morphology, and
nutrient utilization in meat ducks from 1 to 21 days of age. Poult.
Sci. 96:4333–4341
Heyer, C. M., E. Weiss, S. Schmucker, M. Rodehutscord, L. E.
Hoelzle, R. Mosenthin, and V. Stefanski. 2015.The impact of
phosphorus on the immune system and the intestinal microbiota
with special focus on the pig. Nutr. Res. Rev. 28:67–82
Lee, S. F., C. W. Forsberg, and L. N. Gibbins. 1985
Cellulolytic activity of Clostridium acetobutylicum. Appl.
Environ. Microbiol. 50:220–228.
Lengeler, J. W., G. Drews, and H. G. Schlegel. 1999. Biology of the
Prokaryotes, Stuttgart: Georg Thieme.
Ley, R. E., M. Hamady, C. Lozupone, P. J. Turnbaugh, R. R. Ramey,
and J. S. Bircher. 2008. Evolution of mammals and their gut
microbes. Science 320:1647–1651.
Lozupone, C., M. E. Lladser, D. Knights, J. Stombaugh, and R.
Knight. 2011. UniFrac: an effective distance metric for microbial
community comparison. ISME J 5:169–172.
Maenz, D. D., and H. L. Classen. 1998. Phytase activity in the
small intestinal brush border membrane of the chicken. Poult.
Sci. 77:557–563.
Mann, E., M. Dzieciol, B. U. Metzler-Zebeli, M. Wagner, and S.
Schmitz-Esser. 2014. Microbiomes of unreactive and patholog-
ically altered ileocaecal lymph nodes of slaughter pigs. Appl.
Environ. Microbiol. 80:193–203.
Mann, E., S. Schmitz-Esser, Q. Zebeli, M. Wagner, M. Ritzmann,
and B. U. Metzler-Zebeli. 2014b. Mucosa-associated bacterial mi-
crobiome of the gastrointestinal tract of weaned pigs and dynam-
ics linked to dietary calcium-phosphorus. PLoS One 9:e86950.
Marlow, G., S. Ellett, I. R. Ferguson, S. Zhu, N. Karunasinghe, and
A. C. Jesuthasan. 2013. Transcriptomics to study the effect of a
Mediterranean-inspired diet on inflammation in Crohn’s disease
patients. Hum Genomics. 7:24.
Metzler, B. U., and R. Mosenthin. 2008. A review of interactions
between dietary fiber and the gastrointestinal microbiota and
their consequences on intestinal phosphorus metabolism in grow-
ing pigs. Asian Australas. J. Anim. Sci Sci. 21:603–615.
Mohd, S. M. A., C. C. Sieo, C. W. Chong, H. M. Gan, and Y. W.
Ho. 2015. Deciphering chicken gut microbial dynamics based on
high-throughput 16S rRNA metagenomics analyses. Gut Pathog.
7:4.
Munyaka, P. M., N. K. Nandha, E. Kiarie, C. M. Nyachoti, and E.
Khafipour. 2016. Impact of combined β -glucanase and xylanase
enzymes on growth performance, nutrients utilization and gut mi-
crobiota in broiler chickens fed corn or wheat-based diets. Poult.
Sci. 95:528–540.
National Research Council. 1994. Nutrient Requirements of Poultry.
9th rev. ed. Natl. Acad. Press,Washington, DC.
Onyango, E. M., and O. Adeola. 2009. Dietary phytate (inositol
hexaphosphate) regulates the activity of intestinal mucosa phy-
tase. J. Anim. Physiol. Anim. Nutr. 93:639–646.
Palacios, M. C., M. Haros, Y. Sanz, and C. M. Rosell. 2008.Selection
of lactic acid bacteria with high phytate degrading activity for
application in whole wheat breadmaking. LWT - Food Science
and Technology. 41:82–92.
Pesti, G. M., D. Vedenov, J. A. Cason, and L. Billard. 2009. A
comparison of methods to estimate nutritional requirements from
experimental data. Br. Poult. Sci. 50:16–32.
Ptak, A., M. R. Bedford, S. Światkiewicz, K. Żyla, and D. Józefiak.
2015. Phytase modulates ileal microbiota and enhances growth
performance of the broiler chickens. PLoS One. 10:e0119770.
Rajoka, M. S. R., J. L. Shia, M. M. Hafiza, J. Zhua, Q. Lia, D. Y.
Shao, Q. S. Huang, and Y. K. Hui. 2017. Interaction between diet
composition and gut microbiota and its impact on gastrointesti-
nal tract health. Food Sci. Hum. Wellness. 6:121–130.
Rama Rao, S. V., M. V. L. N. Raju, M. R. Reddy, and P. Pa-
vani. 2006. Interaction between dietary calcium and non-phytate
2450 DAI ET AL
phosphorus levels on growth, bone mineralization and mineral
excretion in commercial broilers. Anim. Feed Sci. Technol. 131:
135–150.
Roos, S., F. Karner, L. Axelsson, and H. Jonsson. 2000. Lactobacil-
lus mucosae sp. nov., a new species with in vitro mucus-binding
activity isolated from pig intestine. Int. J. Syst. Evol. Microbiol.
50:251–258.
SAS Institute Inc. 2004. SAS User’s Guide. Statistics. Version 9.1ed.
SAS Institute Inc., Cary, NC.
Scupham, A. J. 2007.Succession in the intestinal microbiota of pread-
olescent turkeys. FEMS Microbiol. Ecol. 60:136–147.
Selle, P. H., A. J. Cowieson, and N. P. Cowieson. 2012. Protein
phytate interactions in pig and poultry nutrition: a reappraisal.
Nutr. Res. Rev. 25:1–17.
Staib, L., and T. M. Fuchs. 2014. From food to cell: nutrient exploita-
tion strategies of enteropathogens. Microbiology. 160:1020–1039.
Stanley, D., S. E. Denman, R. J. Hughes, M. S. Geier, T. M. Crow-
ley, and H. Chen. 2012. Intestinal microbiota associated with
differential feed conversion efficiency in chickens. Appl Microbiol
Biotechnol. 96:1361–1369.
Stanley, D., M. S. Geier, S. E. Denman, V. R. Haring, T. M. Crowley,
and R. J. Hughes. 2013. Identification of chicken intestinal micro-
biota correlated with the efficiency of energy extraction from feed.
Vet. Microbiol. 164:85–92.
Stanley, D., R. J. Hughes, and R. J. Moore. 2014. Microbiota of the
chicken gastrointestinal tract: influence on health, productivity
and disease. Appl Microbiol Biotechnol. 98:4301–4310.
Shastak, Y., and M. Rodehutscord. 2013. Determination and
estimation of phosphorus availability in growing poultry
and their historical development. Worlds Poult. Sci. J. 69:
569–586.
Vasaı̈, F., K. Brugirard Ricaud, M. D. Bernadet, L. Cauquil, O.
Bouchez, and S. Combes. 2014. Overfeeding and genetics affect
the composition of intestinal microbiota in Anasplatyrhynchos
(Pekin) and Cairinamoschata (Muscovy) ducks. FEMS Microbiol.
Ecol. 87:204–216.
Witzig, M., A. CamarinhaDaSilva, R. Green-Engert, K. Hoelzle, E.
Zeller, and J. Seifert. 2015. Spatial variation of the gut microbiota
in broiler chickens as affected by dietary available phosphorus and
assessed by T-RFLP analysis and 454 pyrosequencing. PLoS One
10:e0143442.
Wood, H. G., and J. E. Clark. 1988. Biological aspects of inorganic
polyphosphates. Annu. Rev. Biochem. 57:235–260.
Zeller, E., M. Schollenberger, M. Witzig, Y. Shastak, I. Kühn, and
L. E. Hoelzle. 2015. Interactions between supplemented mineral
phosphorus and phytase on phytate hydrolysis and inositol phos-
phates in the small intestine of broilers. Poult. Sci. 94:1018–1029.
Zeng, Q., X. Huang, Y. Luo, X. Ding, S. Bai, J. Wang, X. Yue,
Z. Su, Y. Liu, and K. Zhang. 2015. Effects of a Multi-enzyme
Complex on Growth Performance, Nutrient Utilization and Bone
Mineralization of Meat Duck. J Animal Sci Biotechnol. 6:12–20.
Zhu, X. Y., T. Zhong, Y. Pandya, and R. D. Joerger. 2002. 16S
rRNA-based analysis of microbiota from the cecum of broiler
chickens. Appl. Environ. Microbiol. 68:124–137.