Age-associated changes in the growth development of abdominal fat and their
correlations with cecal gut microbiota in broiler chickens
Xiaoying Liu,*,1 Chenxu Wang,y,1 Yumeng Wang,* Chaohui Wang,* Xi Sun,* Yufei Zhu,z,x
Xiaojun Yang,* Lixin Zhang,y and Yanli Liu *,2
College of Animal Science and Technology, Northwest A&F University, Yangling, China; yCollege of Life Sciences,
*
Northwest A&F University, Yangling, China; zShanxi Dayu Biological Functions Co., Ltd., Yuncheng, Shanxi, China;
and xDAYU Bioengineering (Xi’an) Industrial Development Research Institute, Xi’an, Shaanxi, China
ABSTRACT Excess abdominal fat is a common phe-
nomenon in broiler chickens. Gut microbiota could regu-
late lipid metabolism through their effects on short-chain
fatty acids (SCFAs) production. This study was con-
ducted to investigate the potential relationship between
abdominal fat development and cecal microorganism pop-
ulations. Abdominal fat and cecum contents were col-
lected at 3, 7, 14, 21, 28, 35, and 42 d of age. The results
showed that abdominal fat weight increased with age. The
abdominal fat percentage was higher between 7 and 21 d
of age than at 3 d (P < 0.05), and it increased again at 28
to 42 d (P < 0.05). Morphological analysis showed that
adipocyte diameter and cross-sectional area (CSA)
increased significantly after 14 d of age (P < 0.05). More-
over, gut microbiota analysis indicated that the Chao1
and Shannon indices were higher between 14 and 28 d
than at 3 d of age (P < 0.05). Furthermore, LEfse analysis
revealed that Faecalibacterium, Anaerotruncus, Anaero-
plasma, Subdoligranulum, and Clostridium emerged to
become dominant at 14 d. A greater abundance of Bacter-
oides, Ruminococcus, Dehalobacterium, and Lactobacillus
Key words: abdominal fat, broiler chickens, cecal microbiota, development stage, 16S rRNA
INTRODUCTION
Improvements in broiler genetics have led to signifi-
cant advances in growth rates and efficiencies as well as
carcass yield, but one consequence is that in some cases
fat accumulation can be excessive (Liang et al., 2022).
Although abdominal fat is deposited later on in the life of
the broiler (Liu et al., 2023), the rate of fat accumulation
Ó 2023 The Authors. Published by Elsevier Inc. on behalf of Poultry
Science Association Inc. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
Received April 28, 2023.
Accepted June 24, 2023.
1
These authors contributed equally to this work.
2
Corresponding author: liuyanli@nwsuaf.edu.cn
1
were determined at 28 d when compared with 14 d of age.
Parabacteroides, Ochrobactrum, Lactobacillus, Blautia,
Alistipes, Dehalobacterium, Odoribacter, and Suuterella
were found to be predominant at 42 d. PICRUSt analysis
revealed that amino acid metabolism, lipid metabolism,
and terpenoids and polyketides metabolism were elevated
at 14 d; the immune and digestive systems were signifi-
cantly developed at 28 d. In addition, cecum propionic
acid and butyric acid contents gradually increased (P <
0.05), while the isobutyric acid contents gradually
decreased with advancing age (P < 0.05). Correlation
analysis among SCFAs, differential genera and abdominal
fat suggested that Coprobacillus, Shigella, and Butyrici-
coccus had negative correlations with propionic acid,
butyric acid, and abdominal fat weight, but positive corre-
lations with isobutyric acid. Isobutyric acid was identified
as being negatively associated with abdominal fat weight,
while the reverse was found for propionic acid and butyric
acid. In conclusion, abdominal fat development is corre-
lated with the emergence of specific microbes and d 14
may be a pivotal age for establishing this relationship.
2023 Poultry Science 102:102900
https://doi.org/10.1016/j.psj.2023.102900
increases in older birds to the point where it exceeds other
components and reduces efficiency and relative value
(Wang et al., 2021). Therefore, attention needs to be
focused on how to reduce abdominal fat accumulation.
However little is understood about the development of
the abdominal fat pad over the lifespan of the broiler,
highlighting the need for further investigation into the
mechanisms involved in controlling its accumulation.
The gut microbiota plays an important role in maintain-
ing the health of the host (Zhou et al., 2022; Fan et al.,
2023). It was reported that most chronic metabolic dis-
eases were associated with the disturbance of gut micro-
flora such as adiposity (Dabke et al., 2019), diabetes
mellitus type 2 (Bouter et al., 2017), and nonalcoholic
fatty liver disease (Lee et al., 2020). There is accumulating
2 LIU ET AL.
evidence in humans (Ley et al., 2005), mice (Ridaura et al.,
2013), and livestock (Huang et al., 2020) suggesting that
there is a strong relationship between gut microbiota and
body fat mass. Transplanting the gut microbiota from
obese donors to germ-free recipients significantly increased
fat mass accumulation and metabolic syndrome (Wang
et al., 2020a,b), and conversely a high-fat diet can lead not
only to obesity but also to changes in the gut microbiota
composition and function in mice (Cani et al., 2008; Wang
et al., 2020a). Akkermansia muciniphila treatment can
reverse high-fat diet-induced obesity, fasting blood glu-
cose, and adipose tissue metabolism in mice (Everard
et al., 2013). Excess abdominal fat deposition is usually
associated with altered gut microbiota composition and
metabolism functions (Ding et al., 2016; Wang et al.,
2020a). Moreover, the gut microbiota was reported to be
largely independent of host genetics in regulating fat depo-
sition in chickens (Zhao et al., 2013). Wen et al. (2019)
pointed out that the cecal microbiota had a significant
effect on fat deposition and might account for 21% of the
variance in the abdominal fat mass after correcting for
host genetic effects via genome wide association study
analysis. These points above imply that the gut microbiota
is closely related to fat deposition in humans and animals.
Obesity, a systemic lipodystrophic syndrome, has
become a common health problem throughout the world
(Asadi et al., 2022). Similar to humans, the liver is mainly
responsible for de novo lipogenesis in chickens and adipose
tissue is the main site for lipid storage. However, both the
liver and adipose tissue are considered to have equal
importance for lipogenesis in rodents and rabbits. There-
fore, the chicken is considered as a relevant biomedical
model to study human obesity. However, few studies have
focused on the relationship between abdominal fat and
gut microbiota in chickens. The current study was con-
ducted to analyze age-associated changes in the develop-
ment of abdominal fat and further reveal their
correlations with cecal gut microbiota in broiler chickens.
The aim of this work was to provide guidance on whether
nutritional strategies that alter fat deposition may be a
result of their effects on the gut microbiota.
MATERIALS AND METHODS
All broiler chickens and experimental protocols in the
study were approved by the Institution Animal Care
and Use Committee of Northwest A&F University (Per-
mit Number: DK202123).
Animals and Sample Collection
All broiler chickens were obtained from the Xian
Dacheng Poultry Industry Co., Ltd. (Xianyang, China).
A total of 300 one-day-old Arbor Acres broiler chickens
were raised with free access to a commercial diet and water
at the Experimental Teaching Center of Animal Science at
the Norwest A&F University. At the age of 3, 7, 14, 21, 28,
35, and 42 d, 12 birds (half male and half female) were
selected and killed by cervical dislocation and dissected,
and then abdominal fat was removed and weighed. The
abdominal fat percentage was expressed relative to body
weight (g of organ/g of body weight). Fresh cecum chymus
was collected for subsequent gut microbiota and short-
chain fatty acids (SCFAs) analysis. After sampling,
abdominal fat and cecum chymus were immediately frozen
in liquid nitrogen and stored at 80°C.
Abdominal Fat Morphology Analysis
Approximately 1 cm3 abdominal fat tissue pieces were
fixed in 4% formaldehyde for more than 48 h for hema-
toxylin and eosin (H&E) histological analysis, which
was performed by Wuhan Servicebio Technology Co.,
Ltd. (Wuhan, China). Abdominal adipose tissue sec-
tions were examined and photographed using an
inverted fluorescence microscope, and the diameter and
area of adipocytes were determined using Image-Pro
Plus 6.0 software (Media Cybernetics, Silver Spring,
MD) based on the previously reported method (Chen
and Farese, 2002).
Microbiota DNA Extraction and 16S Library
Construction
Microbiota genomic DNA of cecal contents was
extracted for quality detection by agarose gel electro-
phoresis and UV spectrophotometer and bacterial V3V4
regions were amplified using the following primers: F:
ACTCCTACGGGAGGCAGCA, R: GGAC-
TACHVGGGTWTCTAAT. The PCR amplification
program was as follows: denaturation at 94°C to 96°C
for 1 min, annealing at 50°C to 60°C for 30 s, and exten-
sion at 72°C for 1 min, repeated for 35 cycles. After
amplification, PCR product purification was performed
using the AxyPrep DNA Gel Extraction Kit (Axygen,
Phoenix, AZ), then purified DNA was quantified for
library construction and sequencing on the Illumina
platform, which was carried out by Shanghai Personal
Biotechnology Co., Ltd., Shanghai, China.
Bacterial Community Composition and
Diversity Analysis
After filtering, merging, removing impurities, and
pruning effective sequences, sequencing data were com-
pared using the Greengene database to analyze alpha-
and beta-diversity and detect the relative abundance at
the phylum, class, order, family, and genus levels accord-
ing to QIIME2 software (http://qiime.org/index.html).
Furthermore, LEfSe analysis was applied to determine
differential microbiota among different days based on
the following parameters: linear discriminant analysis
score >2 and P < 0.05.
Functional Prediction Analysis
Phylogenetic investigation of communities by recon-
struction of unobserved states (PICRUSt) was employed
 ABDOMINAL FAT AND CECAL MICROBIOTA IN BROILER CHICKENS
to predict microbial functional changes (Langille et al.,
2013). The sequencing information was automatically nor-
malized to known bacterial genomes from the Integrated
Microbial Genomes database. Predicted functions were
performed with the Kyoto Encyclopedia of Genes and
Genomes (KEGG) database based on 16S rRNA genes,
and STAMP software was used to analyze functional dif-
ferences along with the age in broiler chickens.
Short-Chain Fatty Acids
Cecal acetic acid, propionic acid, butyric, acid and iso-
butyric acid contents were detected based on our previ-
ous report (Liu et al., 2023). Briefly, cecal chymus was
homogenized in cold normal saline. Then the superna-
tant was collected and mixed with metaphosphoric acid
and crotonic acid sequentially following centrifugation
at 10,000 £ g for 15 min. After pretreatment, samples
were analyzed using the GC-MS method via the ratio of
the peak area with the standard solution.
3
was used to determine significance. A probability value
less than 0.05 was considered statistically significant.
RESULTS
Growth Performance
As shown in Figure 1A to C, there was no difference in
abdominal fat weight and abdominal fat percentage
between male and female chicks at the same age. How-
ever, body weight in male birds was higher than females
during 28 to 42 d. Figure 1D and E showed that the
body weight and abdominal fat weight increased gradu-
ally from D3 to D42 for both sexes. The abdominal fat
percentage increased significantly from 7 d of age (P <
0.05), and remained at approximately 0.75% until 21 d,
and then increased again to approximately 1% from 28
to 42 d as shown in Figure 1F (P < 0.05). As shown in
Figure 1G, there was little abdominal fat deposition at
D3 but this increased significantly with age.

Abdominal Fat Morphology Analysis

Statistical Analysis
All experimental data were analyzed by Statistical
software SPSS 23.0 (SPSS Inc., Chicago, IL) and plotted
by GraphPad Prism 8. The test data were expressed as
the mean with standard error and analyzed by the 1-
way ANOVA, and Duncan’s multiple comparisons test
To investigate the dynamic changes of abdominal adi-
pocytes during the whole life of broiler chickens we per-
formed morphology analysis for abdominal adipose
tissue at D3, D7, D14, D21, D28, D35, and D42. As dis-
played in Figure 2A, the adipocytes looked smaller at
D3, and enlarged with age. The proportion of cells with

Figure 1. The observation of abdominal fat phenotypes. (A−C) Body weight, abdominal weight, and abdominal percentage for male or female
broiler chickens. Data are expressed as mean with standard error, and the asterisk (*) indicated statistically significant differences (2-tailed unpaired
test, *P < 0.05). (D and E) Body weight, abdominal weight, and abdominal percentage in broiler chickens without considering genders. Data are
expressed as mean with standard error, and the different letters indicate significant differences and the same letter indicates no significant differences
(1-way ANOVA, P < 0.05). (F) Abdominal fat anatomy observation.
4 LIU ET AL.

Figure 2. Morphological analysis, adipocyte diameter, and area statistics for abdominal fat. (A) H&E staining for abdominal fat tissue (magnifi-
cation: 10 £ 40; the scale is 50 um). (B) and (C) adipocyte diameter and CSA statistics. The different letters indicate significant differences and the
same letter indicates no significant differences (1-way ANOVA, P < 0.05).

a diameter of 10 to 20 um decreased gradually while for
cells with a diameter of more than 30 um the proportion
increased (Figures S1 and S2). The results in Figure 2B
and C showed that the average diameter and cross-sec-
tional area (CSA) of adipocytes increased significantly
after D14 (P < 0.05).
Cecum Microbiota
Considering the results of abdominal fat weight and adi-
pocyte diameter in broiler chickens, we selected 4 stages
(D3, D14, D28, and D42) for gut microbiota analysis. As
shown in Figure 3A, the alpha-diversity indices including
Chao1 and Shannon were higher at D14, D28, and D42
than at D3. Principal coordinates analysis (PCoA) pre-
sented significant differences and separation for microbial
communities among 4 different days (Figure 3B). Fimi-
cutes, Proteobacteria, Bacteroidetes, and Tenericutes are
the most abundant phyla in broiler chickens. Proteobacte-
ria was higher at D3 and decreased along with the age,
while Bacteroidetes apparently increased from D28
(Figure 3C). At the genus level, the relative abundance of
Shigella accounted for about 30% at D3 but decreased sig-
nificantly from D14 (Figure 3D). Further, LEfSe analysis
was used to identify dominant microbiota between 2 adja-
cent days (Figure 4A−C) and results showed the follow-
ing: the dominant microflora were Faecalibacterium,
Anaerotruncus, Anaeroplasma, Subdoligranulum, and
Clostridium at 14 d when compared with those at 3 d; the
higher abundance of Bacteroides, Ruminococcus, Dehalo-
bacterium, and Lactobacillus were determined at 28 d in
comparison with 14 d; Parabacteroides, Ochrobactrum,
Lactobacillus, Blautia, Alistipes, Dehalobacterium, Odori-
bacter, and Suuterella were found to be predominant at 42
d. Meanwhile, as shown in Figure 5D to I, with the
advancing age, the relative abundances of Ruminococcus,
Coprobacillus, Shigella, and Butyricicoccus decreased
when compared with those at D3, while Oscillospira and
Faecalibacterium were found to increase first and then
decrease from D42 and D28, respectively.
Functional Prediction Analysis for Cecal
Microbiota
The functional prediction for the cecal microbiota
between 2 adjacent developmental stages was performed
using PICRUSt method. As shown in Figure 5A to C,
functional capacities involved in carbohydrate metabo-
lism, glycan biosynthesis, and metabolism, and metabo-
lism of cofactors and vitamins were enriched
significantly at D3. Amino acid metabolism, lipid
metabolism, terpenoids, and polyketides metabolism
were enhanced at D14 when compared with D3. Fur-
thermore, the immune and digestive systems were signif-
icantly developed at D28, and the gut microbiota were
predicted to have a significant involvement in lipid
metabolism again at D42.
Cecum SCFAs and Their Correlation
Analysis
As displayed in Figure 5D to G, cecal acetic acid con-
tent did not change with time. However, propionic acid
and butyric acid concentration increased from D14 (P <
 ABDOMINAL FAT AND CECAL MICROBIOTA IN BROILER CHICKENS 5

Figure 3. Cecum microbiota analysis in broiler chickens. (A) Chao1 and Shannon index. (B) Principal coordinates analysis (PCoA). (C and D)
Relative abundance of bacterial composition at phylum level and genus level. Every color means 1 bacteria and Y-axis showed the relative abun-
dance of the bacteria.

Figure 4. Differential microbiota analysis. (A−C) Differential microbiota based on LEfSe method between 2 adjacent stages. The default
parameters were LDA score >2 and P < 0.05. (D−I) Relative abundance analysis for selected differential bacteria. Data are expressed as mean with
standard error (n = 7). The different letters indicate significant differences and the same letter indicates no significant differences (1-way ANOVA,
P < 0.05).
6 LIU ET AL.

Figure 5. Functional predicted analysis of cecal microbiota, cecum SCFAs, and their correlation analysis. (A−C) Comparisons of functional
pathways. (D−G) Cecum SCFA concentrations of broiler chickens. Data are expressed as mean with standard error (n = 7). Different letter indi-
cates significant correlation (P < 0.05), the same letter or no letter indicates no significance (P > 0.05). (H and I) Heatmap of correlation analysis
among cecal microbiota, cecum SCFAs, diameter of adipocytes, and abdominal fat weight. * or ** means that there is a significant correlation
between 2 indices at the 0.05 or 0.01 level (2-tailed). Red and green colors represent positive and negative correlation, respectively, and color grada-
tion indicates the size of the correlation coefficient.

0.05) and reached an asymptote between 14 and 42 d,
while the inverse effect was noted for isobutyric acid.
Pearson correlation analysis among cecal microbiota,
SCFAs, and abdominal fat phenotypes was carried out
and results were shown in Figure 5H and I. The relative
abundances of Coprobacillus, Shigella, and Butyricicoc-
cus were significantly negatively correlated with pro-
pionic acid, butyric acid, and abdominal fat weight, but
positively correlated with isobutyric acid. There was a
negative relationship between bacteria (Coprobacillus
and Shigella) and adipocyte diameter. Isobutyric acid
was negatively correlated with abdominal fat weight,
while propionic acid and butyric acid were positively
related to abdominal fat weight.
DISCUSSION
Excess abdominal fat deposition is a common issue in
broilers, which not only reduces feed utilization effi-
ciency but also adversely affects host health. Recently,
numerous studies have reported that gut microbes are
inextricably linked to fat deposition (Everard et al.,
2013; Anto and Blesso, 2022). Our previous study indi-
cated that dietary folic acid could reduce abdominal fat
deposition by altering gut microbiota and SCFAs in
broiler chickens (Liu et al., 2023). Cecal microbiota was
considered to be involved in nonalcoholic steatohepatitis
in laying hens (Hamid et al., 2019). These results suggest
that there is a relationship between abdominal fat
accumulation and the development of the cecal micro-
biota. This work was therefore initiated to determine if
such a correlation exists between specific bacteria and
abdominal fat pad accumulation.
Adipose tissue is the main organ for fat storage in
poultry. The number and size of adipocytes are closely
related to fat synthesis and deposition (Wang et al.,
2017). The morphological study on abdominal adipose
tissue found that the number and size of adipocytes
increased with age in high-fat and low-fat line chickens
(Guo et al., 2011), which was consistent with our results
that adipocyte diameter and area increased along with
the age of broiler chickens from D14. In the current
study, abdominal fat weight gradually increased
between 3 and 14 d of age, but there was no difference in
the average diameter and mean area of adipocytes in
this period which implied that adipocyte proliferation (i.
e., number) might contribute more to the increase in
abdominal fat weight during this period. On the other
hand, the percentage of adipocytes with 30 to 50 um
diameter started to increase at D14 onward while cells
between 10 and 20 um diameter decreased in proportion.
In addition, abdominal fat weight reached a significant
inflection point at D14. Thus D14 might be a key physio-
logical stage for abdominal adipocyte hypertrophy. This
also explains the accelerated increase in abdominal fat
weight between 14 and 42 d, which might be due not
only to adipocyte proliferation but also to cell differenti-
ation. A previous study also reported that genes
involved in adipocyte differentiation were more highly
 ABDOMINAL FAT AND CECAL MICROBIOTA IN BROILER CHICKENS
expressed at D14 than at D4 such as PPARg, FABP4,
and LPL (Bai et al., 2015), indicating that cell differenti-
ation indeed took place in abdominal fat of broiler chick-
ens at D14.
Huang et al. (2018) reported that the gut microbiota
developed into a relatively mature community around
28 d of age in broiler chickens based on the metagenome
analysis. Considering the physiological characteristics of
fat deposition, we selected 4 stages (D3, D14, D28, and
D42) for further gut microbiota analysis. Our results
showed that more abundant cecal bacterial communities
were determined in broiler chickens from 28 d of age.
PCoA analysis revealed more similarity for cecum micro-
biota composition between 14, 28, and 42 d, indicating
that the establishment and development of the cecal
microbiota in broiler chickens was mainly completed
between 14 and 28 d. These findings were in accordance
with a previous study in which the gut microbiota
tended to be stable between 14 and 28 d in chickens
(Ballou et al., 2016). Further, function prediction of gut
microbiota revealed that more complex metabolic func-
tions such as amino acid and lipid metabolism were
enriched at D14 as well as that for terpenoid and polyke-
tide metabolism. Moreover, the immune and digestive
system were evolved up to D28, suggesting cecal micro-
biota could take part in regulating immune and digestive
function of hosts based on the mature of structure and
function on gut microbiota. These results implied that
gut microbiota reached maturity at approximately D28
in broiler chickens, which is also supported by the
SCFAs results in the current study because there was no
difference in acetic acid, propionic acid, butyric acid,
and isobutyric acid concentration in cecum at D28 when
compared with D14 or D42.
Cornejo-Pareja et al. (2019) noted that fat deposition
was related to changes in microbial community composi-
tion. It was reported that the relative abundance of Fir-
micutes is higher in the gut of obese animals, while the
relative abundance of Bacteroidetes is higher in lean
individuals (Walters et al., 2014). Firmicutes was con-
sidered to be positively correlated with fat deposition in
animals in a separate study (Patterson et al., 2016). To
reveal the potential genus associated with fat deposition
in the life of the broiler, different bacterial genera with
relatively higher abundance were picked and used to per-
form correlation analysis together with abdominal fat
phenotypes. The results showed that Coprobacillus and
Shigella were identified as being negatively associated
with the adipocyte diameter and abdominal fat weight.
The same phenomenon was found between abdominal
fat weight and Butyricicoccus. These results imply that
these 3 genera might regulate abdominal lipid deposi-
tion. In addition, the lipid metabolism pathway was sig-
nificantly enriched at both D14 and D42 based on
functional predictions of gut microbiota, suggesting the
cecal microbiota might indeed play a key role in fat
deposition in broiler chickens.
SCFAs are fermentation products from intestinal
microorganisms metabolizing dietary nutrients, which
bridges gut microbiota and host physiology. It was
7
reported that SCFAs could promote energy metabolism
and fatty acid synthesis, thus affecting the accumulation
of body fat (Cronin et al., 2021). In the current study,
the cecum contents of propionic acid and butyric acid
gradually increased with the advancing age, while the
opposite result was found for isobutyric acid. Correla-
tion analysis was performed to reveal the relationship
between SCFAs and differential genera as well as
abdominal fat phenotypes. Propionic acid and butyric
acid were positively related to abdominal fat weight. It
was possible that increased propionic acid and butyric
acid provided more energy for adipocyte hyperplasia
and hypertrophy. Recent studies have found that pro-
pionic acid can promote the increase of intracellular fat
content in mice preadipocytes (Yeganeh et al., 2016;
Wang et al., 2020a). It was reported that butyric acid
could regulate the acetylation level of lipid-related genes
by inhibiting histone deacetylase (HDAC) activity,
thereby affecting fat synthesis and deposition (Yan and
Ajuwon, 2015; Huang et al., 2017). However, as an
important butyric acid-producing bacteria, Butyricicoc-
cus was found to be positively associated with isobutyric
acid rather than butyric acid. In addition, isobutyric
acid was identified as being negatively correlated with
abdominal fat weight, indicating that Butyricicoccus
might be a potential bacterium for reducing fat deposi-
tion. The same relationship was found between Copro-
bacillus and Shigella and isobutyric acid. Thus,
isobutyric acid might be a marker reflecting abdominal
fat deposition in broiler chickens.
CONCLUSIONS
In conclusion, the current study revealed age-associ-
ated changes in the development of abdominal fat and
their correlations with cecal gut microbiota in broiler
chickens. Abdominal fat development is correlated with
the emergence of specific microbes and d 14 may be a
pivotal age for establishing this relationship. On the
other hand, Coprobacillus, Shigella, and Butyricoccus
were identified to be related to abdominal fat deposition
via regulating isobutyric acid production, which needs
to be validated in the future.
ACKNOWLEDGMENTS
This work was funded by the National Science Foun-
dation of China (32102567), the National Key R&D Pro-
gram for Young Scientists (2022YFF1001000), the
Program for Shaanxi Science & Technology (2023-
YBNY-144, 2022KJXX-13, and 2021TD-30). The
authors acknowledge members of the Innovative
Research Team of Animal Nutrition & Healthy Feeding
(Northwest A&F University, Shaanxi, China) for their
help in animal care and sample harvesting.
Author Contributions: Y. L. L. and X. Y. L. designed
the research; Y. M. W., C. H. W., X. S., and Y. F. Z. per-
formed the research and analyzed the data; X. Y. L. and
C. X. W. wrote the manuscript; X. J. Y., L. X. Z., and
8 LIU ET AL.
Y. L. L. have taken part in the revision of the manu-
script. All authors read and approved the final version of
the manuscript.
DISCLOSURES
The authors declare that they have no competing
interests.
SUPPLEMENTARY MATERIALS
Supplementary material associated with this article
can be found in the online version at doi:10.1016/j.
psj.2023.102900.
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