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Frontiers in Microbiology 01 frontiersin.org
Dried tea residue can alter the
blood metabolism and the
composition and functionality of
the intestinal microbiota in Hu
sheep
LiangyongGuo
1†, ShiqiangYu
2†, FangCao
3, KaizhiZheng
4,
ManmanLi
5, ZhenyingPeng
6, XingyunShi
1* and LipingLiu
1
*
1 Huzhou Key Laboratory of Innovation and Application of Agricultural Germplasm Resources, Huzhou
Academy of Agricultural Sciences, Huzhou, China, 2 Laboratory of Gastrointestinal Microbiology, Jiangsu
Key Laboratory of Gastrointestinal Nutrition and Animal Health, College of Animal Science and
Technology, Nanjing Agricultural University, Nanjing, China, 3 College of Life Science, Huzhou Teachers
College, Huzhou, China, 4 Institute of Animal Husbandry and Veterinary, Zhejiang Academy of
Agricultural Sciences, Hangzhou, China, 5 Key Laboratory of Animal Physiology and Biochemistry,
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China, 6 Beijing Jingmi Water
Diversion Management Oce, Beijing, China
Ruminant animals face multiple challenges during the rearing process, including
immune disorders and oxidative stress. Green tea by-products have gained
widespread attention for their significant immunomodulatory and antioxidant
eects, leading to their application in livestock production. In this study,
we investigated the eects of Dried Tea Residue (DTR) as a feed additive on
the growth performance, blood biochemical indicators, and hindgut microbial
structure and function of Hu sheep. Sixteen Hu sheep were randomly divided
into two groups and fed with 0 and 100 g/d of DTR, respectively. Data were
recorded over a 56-day feeding period. Compared to the control group, there
were no significant changes in the production performance of Hu sheep fed
with DTR. However, the sheep fed with DTR showed a significant increase in IgA
(p < 0.001), IgG (p = 0.005), IgM (p = 0.003), T-SOD (p = 0.013), GSH-Px (p = 0.005),
and CAT (p < 0.001) in the blood, along with a significant decrease in albumin
(p= 0.019), high density lipoprotein (p= 0.050), and triglyceride (p= 0.021). DTR
supplementation enhanced the fiber digestion ability of hindgut microbiota,
optimized the microbial community structure, and increased the abundance of
carbohydrate-digesting enzymes. Therefore, DTR can beused as a natural feed
additive in ruminant animal production to enhance their immune and antioxidant
capabilities, thereby improving the health status of ruminant animals.
KEYWORDS
dried tea residue, Hu sheep, serum, immune, antioxidant, intestinal microorganisms
Introduction
Ruminant animals, while having high production capacity, oen face issues such as oxidative
stress and metabolic disorders, primarily due to the high metabolic load and decreased immune
adaptability (Oh etal., 2017; Gonzalez-Rivas etal., 2020). Natural plants have been recognized
as potential remedies to improve animal health and maintain metabolic homeostasis (Besharati
and Taghizadeh, 2009; Ramdani etal., 2023). Due to increasing concerns regarding the side
eects of antibiotic drugs, their usage has been widely scrutinized and prohibited as feed
additives (Tang etal., 2017). Tea, a widely consumed plant, contains a signicant amount of
OPEN ACCESS
EDITED BY
Edoardo Pasolli,
University of Naples Federico II, Italy
REVIEWED BY
Asghar Kamboh,
Sindh Agriculture University, Pakistan
Maghsoud Besharati,
University of Tabriz, Iran
*CORRESPONDENCE
Xingyun Shi
shixingyunlove@163.com
Liping Liu
784751320@qq.com
†These authors have contributed equally to this
work
RECEIVED 06 September 2023
ACCEPTED 10 October 2023
PUBLISHED 03 November 2023
CITATION
Guo L, Yu S, Cao F, Zheng K, Li M, Peng Z,
Shi X and Liu L (2023) Dried tea residue can
alter the blood metabolism and the
composition and functionality of the intestinal
microbiota in Hu sheep.
Front. Microbiol. 14:1289743.
doi: 10.3389/fmicb.2023.1289743
COPYRIGHT
© 2023 Guo, Yu, Cao, Zheng, Li, Peng, Shi and
Liu. This is an open-access article distributed
under the terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in other forums is
permitted, provided the original author(s) and
the copyright owner(s) are credited and that
the original publication in this journal is cited,
in accordance with accepted academic
practice. No use, distribution or reproduction is
permitted which does not comply with these
terms.
TYPE Original Research
PUBLISHED 03 November 2023
DOI 10.3389/fmicb.2023.1289743
Guo et al. 10.3389/fmicb.2023.1289743
Frontiers in Microbiology 02 frontiersin.org
active compounds such as polyphenols. ese compounds possess
properties that can promote human and animal health (Yan et al.,
2020). In ruminant animals, feeding diets supplemented with
polyphenol-rich ingredients has shown signicant biological eects.
ese eects include improved animal productivity, enhanced quality
of livestock products, increased immune competence, and reduced
rumen methane emissions, among others (Jiménez-Ocampo etal.,
2019; Ku-Vera etal., 2020; Suresh etal., 2023). erefore, plants rich
in polyphenols and other active compounds have been used in the
production of ruminants (Maghsoud et al., 2008; Ramdani et al.,
2013). Tea, for example, generates a signicant amount of byproducts
during processing, which contain abundant active ingredients such as
polyphenols, polysaccharides, and catechins. As a result, tea extracts
and other derivatives are being increasingly applied in the
pharmaceutical and food industries (Duarah etal., 2023). Polyphenols,
such as epicatechin and epigallocatechin gallate, are major components
found in tea. ey exhibit various biological activities, including
antioxidant, anti-inammatory, and anti-stress properties (Teixeira
Oliveira etal., 2023).
ere have been previous reports indicating that tea and its
byproducts can serve as a source of protein, ber, secondary
metabolites, and minerals in ruminant diets. ey can beused as
natural feed additives for ruminant animals and have the advantage of
reducing methane emissions and minimizing resource wastage (Zebeli
and Ametaj, 2009; Sezmis etal., 2023). In the study conducted by
Chowdhury et al. (2022), it was reported that dried green tea
byproducts can improve protein digestibility in goats and increase
plasma glucose concentrations. Furthermore, the abundant
polyphenols found in green tea can reduce oxidative stress in ruminant
animals. For example, it signicantly lowers somatic cell counts in
periparturient cows and decreases concentrations of triglycerides,
reactive oxygen species, malondialdehyde, and hydrogen peroxide
(Ma etal., 2021). It also increases the concentrations of glutathione
peroxidase, superoxide dismutase, and total antioxidant capacity.
Additionally, it upregulates the concentrations of IL-6 and IL-10in
plasma while downregulating the concentrations of TNF-α, IL-1β,
IL-2, IL-8, and IFN-γ (Ma etal., 2021). ese eects help reduce
oxidative stress in cows and improve their lactation performance and
overall health status. e polyphenols present in green tea can also
inhibit the expression of TGF-β1in bovine mammary glands, thereby
reducing the phosphorylation of p38 and JNK. is leads to a
signicant decrease in the expression of inammatory cytokines
IL-1β, IL-6, and TNF-α (Xu et al., 2022). Additionally, green tea
polyphenols can alleviate oxidative stress, inammation, and cell
apoptosis in bovine mammary epithelial cells induced by hydrogen
peroxide. is eect is achieved through the activation of the ERK1/2-
NFE2L2-HMOX1 pathway (Ma et al., 2022). Indeed, green tea
compounds can also alter the fermentation in the rumen of ruminant
animals and the composition of their intestinal microbiota (Qiu etal.,
2021; Gao et al., 2022). ese reports indicate that green tea can
beutilized as an antioxidant additive and a microbial modulator in
ruminant animals production.
e active eects exerted by tea are mainly determined by its
major constituents and their metabolism within ruminant animals.
Tea polyphenols and EGCG are the most signicant active
components. For instance, tea polyphenols can have benecial eects
on the cellular redox balance of animals, reducing oxidative stress-
related damage and potentially serving as antioxidants in animal
antioxidant defense against oxidative stress (Ma etal., 2018; Xu etal.,
2021). On the other hand, it is documented that the interaction
between the gastrointestinal tract of ruminant animals and
polyphenols plays a crucial role in mediating the promotion of host
health by plant-derived polyphenols. For instance, these interactions
can inuence the structure and community of the gastrointestinal
microbiota, promoting benecial bacteria and inhibiting harmful
bacteria (Yu etal., 2023). e gut microbiota of ruminant animals can
further metabolize active substances such as polyphenols, thereby
enhancing their bioavailability and utilization (Bhat etal., 1998).
Consequently, the active substances produced through these
metabolisms may improve oxidative stress and inammatory
responses in ruminant animals, regulate gastrointestinal function, and
ultimately enhance microbial growth and the overall health status of
the animals.
e immune status and gut health of sheep signicantly inuence
their growth performance and milk production capacity. Previous
studies have indicated that tea leaves and tea waste have the ability to
regulate rumen fermentation, reduce methane emissions, and improve
immune status in animals (Qiu etal., 2021; Chowdhury etal., 2022).
However, there is limited research on the eects of tea-related
substances on blood metabolism and the composition and
functionality of the hindgut microbiota in sheep. Gaining a better
understanding of the microbial community and their functional
responses to tea components can help develop mechanisms for
manipulating the gut microbiota using natural plant compounds,
thereby improving the growth status and health of sheep.
Wehypothesize that adding dried tea waste to the diet can improve
the immune and antioxidant status of sheep by modulating the gut
microbiota. Weaim to study the eects of tea waste on sheep’s gut
functionality using metagenomics and other related methods, evaluate
its impact on blood antioxidant and immune indicators, and uncover
the mechanisms by which tea waste inuences the sheep’s hindgut and
improves their overall health status.
Materials and methods
Source of dried tea residue
e dried tea residue weselected is derived from the by-products
remaining from the production process of Anji white tea in Anji
County. e main components of this by-product are tea polyphenols
(18.10%), L-theanine (4.09%), catechin (14.75%), and epigallocatechin
gallate (EGCG) ester of gallic acid (13.00%).
Animals and treatments
e 16 male Hu sheep weighing 29.80 ± 0.91 kg at 3 months of age
were randomly divided into a control group and a treatment group, with
eight Hu sheep in each group. e control group was fed a basal diet
(CON), while the treatment group was fed 100 g/d of dried tea residue
(DTR). e dosage of DTR was determined based on the results of an in
vitro experiment (unpublished). e basal diet (Supplementary Table1)
was a complete mixed ration with a concentrate-to-roughage ratio of 7:
3, meeting the requirements of the Chinese Sheep Feeding Standards
(NY/T816-2004). e Hu sheep in the experiment were individually
housed in a pen and were fed twice a day (at 8:00 and 17:00 h). ey had
free access to feed and water, and daily feed intake was recorded. Initial
Guo et al. 10.3389/fmicb.2023.1289743
Frontiers in Microbiology 03 frontiersin.org
and nal body weights were recorded. e entire experiment lasted for
56 days, including a 14-day adaptation period and the formal
experimental period was 42-day. On the last day of the experiment, 2 h
before morning feeding, blood samples were collected via jugular vein
puncture, and feces were collected.
Serum sampling and analysis
Aer collecting the blood samples from the jugular veins of the
Hu sheep using non-anticoagulant vacuum tubes before morning
feeding, the samples were centrifuged at 3,000 × g for 10 min at 4°C to
collect the serum. Subsequently, the serum was frozen at −80°C until
analysis. e concentrations of total superoxide dismutase (T-SOD),
glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC),
catalase (CAT), malondialdehyde (MDA), total protein content (TP),
albumin (ALB), high density lipoprotein (HDL), low density
lipoprotein (LDL), glutamic pyruvic transaminase (GPT), glutamic-
oxalacetic transaminase (GOT), nonesteried fatty acid (NEFA),
triglyceride (TG) and total cholesterol (TCH) were determined using
the appropriate commercial assay kits (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China) and microplate reader
(Multiskan FC; ermo Fisher Scientic, Waltham, MA, USA)
analyzer. And were analyzed using commercial ELISA assay kits
(Nanjing Jiancheng Bioengineering Institute, Nanjing, China),
following the instructions provided by the supplier. All ELISA data
were recorded using a microplate reader (Multiskan FC; ermo
Fisher Scientic, Waltham, MA, USA).
Hindgut microbial analysis by
metagenomic sequencing
Microbial DNA was extracted from feces samples. e
concentrations of Immunoglobulin A (IgA), Immunoglobulin G
(IgG), and Immunoglobulin M (IgM) using the E.Z.N.A.® stool DNA
Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s
protocols. Metagenomic shotgun sequencing libraries were
constructed and sequenced at Shanghai Biozeron Biological
Technology Co. Ltd. In briey, for each sample, 1 μg of genomic DNA
was sheared by Covaris S220 Focused-ultrasonicator (Woburn, MA
USA) and sequencing libraries were prepared with a fragment length
of approximately 450 bp. All samples were sequenced in the Illumina
NovaSeq6000 instrument with pair-end 150 bp (PE150) mode.
e quality control of each dataset was performed using Fastp
(version 0.20.0, https://github.com/OpenGene/fastp). is involved
trimming the 3′-end and 5′-end of reads, cutting low-quality bases
(quality scores <20), and removing short reads (<50 bp) and “N”
records. e reads were then aligned to the host genome
1
using BWA
(version 0.7.17, http://bio-bwa.sourceforge.net/) to lter out host
DNA. e ltered reads were de novo assembled for each sample
using Megahit (Li etal., 2015; version 1.1.2, https://github.com/
voutcn/megahit). Prodigal
2
was employed to predict open reading
frames (ORFs) from the assembled contigs with a length > 100 bp. e
1 https://ensembl.org/index.html
2 https://github.com/hyattpd/Prodigal
assembled contigs were then pooled, and non-redundant sequences
were generated based on identical contigs using CD-HIT (Fu etal.,
2012; version v4.6.1, http://weizhongli-lab.org/cd-hit/) with 90%
identity. To determine the gene abundance information in each
corresponding sample, the high-quality reads of each sample were
compared with the non-redundant gene set using SOAPaligner (Li
etal., 2009; http://soap.genomics.org.cn/; default parameters: 95%
identity).
e non-redundant gene set was subjected to a comparison with the
NR database using DIAMOND (Buchnk etal., 2015) soware, with the
comparison type set to BLASTP. Species annotations were obtained from
the taxonomic information database corresponding to the NR database.3
e abundance of species in each samples were counted at each
taxonomic level, including domain, family, genus, and species, to
construct an abundance prole at the corresponding taxonomic level.
Principal Coordinate Analysis (PCoA) based on the Bray-Curtis
similarity matrix was conducted at the species level. Contigs were
annotated using DIAMOND against the KEGG database (Kanehisa,
2000; Kyoto Encyclopedia of Genes and Genomes, http://www.genome.
jp/kegg/) with an E-value of 1e-5. Furthermore, the non-redundant gene
set was compared with the CAZy database
4
using the corresponding tool
hmmscan from the CAZy database to obtain annotation information of
carbohydrate-active enzymes corresponding to the genes. e
abundances of KEGG Orthology (KO), pathway, KEGG enzyme, and
CAZymes were normalized into counts per million reads (cpm) for
further analysis. For downstream analysis, at least 50% of the animals in
each group were used. KEGG modules, pathways, KEGG enzymes, and
CAZymes with cpm > 5 were considered for the analysis.
e complete set of assembled and ltered raw sequence data has
been submitted to the NCBI Sequence Read Archive, and it is now
available under bioproject PRJNA1002066.
Statistical analysis
e data for growth performance and blood parameters were
analyzed using SPSS 21.0 soware (SPSS Inc., Chicago, IL,
UnitedStates). Aer testing for normal distribution, a double-tailed
t-tests was employed for analysis. e p-value ≤ 0.05 was considered
as indicating a signicant dierence, while p-value > 0.05 indicated no
signicant dierence.
Results
Growth performance
Table 1 reports the variations in the productive performance
indicators of Hu sheep in the experiment. ere were no signicant
dierences observed in the initial weight and terminal weight between
the CON and DTR groups during the course of the study (p > 0.05).
Additionally, no signicant dierences were found in average daily
feed intake, average daily gain, and Feed/Gain in this research
(p > 0.05).
3 https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/
4 http://www.cazy.org/
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Serum index
Table2 describes the dierences in biochemical indicators in the
serum of Hu sheep aer the addition of DTR. It can beobserved that
the concentrations of IgA (p < 0.001), IgG (p = 005), IgM (p = 0.003),
T-SOD (p = 0.013), GSH-Px (p = 0.005), and CAT (p< 0.001) in the
serum of Hu sheep were signicantly higher in the DTR group
compared to the CON group, indicating that DTR has the function
of enhancing the immunity and antioxidation of Hu sheep.
Additionally, the concentrations of ALB (p = 0.019), HDL (p = 0.050),
and TG (p = 0.021) in the serum were signicantly lower in the DTR
group compared to the CON group aer DTR supplementation.
Aer the addition of DTR, there were no signicant changes
observed in the indicators T-AOC, MDA, TP, LDL, GPT, GOT,
NEFA, and TCH (p > 0.05).
Metagenome profiling
Metagenomic sequencing of the total DNA from 16 rumen uid
samples generated a total of 1,866,654,428 reads, with an average of
116,665,901 ± 3,567,761 (mean ± SD) reads per sample. Aer quality
control and removal of host contamination, 1,850,381,554 high-
quality reads were generated, with 115,648,847 ± 35,201,440 reads
per sample. A total of 11,249,270 contigs were generated by the de
novo assembly (the N50 length of 1,561 ± 258 bp), with
703,079 ± 221,496 reads for each sample. e rumen metagenome
contains 98.39% bacteria, 0.97% eukaryota, 0.59% archaea, and
0.05% viruses. e PCoA plot visually showed the distinct separation
of bacteria between CON and DTR based on the Bray-Curtis
distance, eukaryota, archaea, and viruses have no signicantly
change (Figures1A–D). At the domain level, the relative abundance
TABLE1 Eects of DTR supplementation on growth performance of Hu sheep.
Item Treatments SEM p-value
CON DTR
Initial weight, kg 30.19 29.41 0.23 0.092
Terminal weight, kg 40.68 40.08 0.78 0.708
Average daily feed intake, g 1447.48 1514.57 69.11 0.644
Average daily gain, g 205.88 209.19 14.26 0.912
Feed/Gain 7.26 7.38 0.22 0.808
TABLE2 Eect of TEA on the serum index of sheep.
Item Treatments SEM p-value
CON DTR
IgA, μg/ml 1117.83b1566.26a293.24 <0.001
IgG, mg/ml 4.02b4.99a0.75 0.005
IgM, μg/ml 142.57b296.4a40.33 0.003
T-SOD, U/ml 59.31b76.18a3.61 0.013
GSH-Px, U/ml 111.51b128.75a3.34 0.005
T-AOC, U/ml 3.24 3.62 0.14 0.190
CAT, U/ml 2.68b2.92a0.04 <0.001
MDA, nmol/ml 3.23 3.29 0.09 0.757
TP, g/L 68.49 69.50 0.49 0.317
ALB, g/L 27.70a23.90b0.85 0.019
HDL, mmol/L 0.45a0.30b0.04 0.050
LDL, mmol/L 1.00 1.01 0.02 0.889
GPT, U/L 29.65 29.37 1.77 0.941
GOT, U/L 126.77 127.27 0.14 0.072
NEFA, μmol/L 138.50 142.27 5.46 0.743
TG, mmol/L 0.47a0.37b0.02 0.021
TCH, mmol/L 1.26 1.10 0.09 0.414
T-SOD, total superoxide dismutase; GSH-Px, glutathione peroxidase; T-AOC, total antioxidant capacity; CAT, catalase; MDA, malondialdehyde; TP, total protein content; ALB, Albumin;
HDL, high density lipoprotein; LDL, low density lipoprotein; GPT, glutamic pyruvic transaminase; GOT, glutamic-oxalacetic transaminase; NEFA, nonesteried fatty acid; TG, triglyceride;
TCH, total cholesterol.
Values in the same row with the same or no small letter superscripts mean no signicant dierence (p > 0.05), while with dierent letter superscripts mean signicant dierence (p≤ 0.05).
Guo et al. 10.3389/fmicb.2023.1289743
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of bacteria, eukaryota, archaea, and viruses were no signicantly less
in the hindgut of DTR sheep compared with CON (Figure2). At the
level of microbial phylum, Firmicutes, Bacteroidota, Proteobacteria,
Spirochaetota, Cyanobacteria, Verrucomicrobiota, Fibrobacterota,
Methanobacteriota, Campylobacterota, and Desulfobacterota,
Evosea are the main phylum (Supplementary Figure S1). At the
genus level, the dominant microbiota were Cryptobacteroides,
followed by Succiniclasticum Alistipes, Faecousia, Phocaeicola,
RF16, Treponema, Succinivibrio, HGM04593, HGM20899 and
UBA4372 (Supplementary Figure S2).
e comparison of the hindgut microbial taxa at the phylum and
genus levels between the CON and DTR groups was focused on
bacteria and archaea. e analytical results of the top10 bacterial
phyla obtained by the Wilcoxon rank-sum test are shown in Figure2.
e phyla BSAR324 exhibited higher abundances (p = 0.041) in the
hindgut of the DTR sheeps (Figure3A). No dierences were observed
in the top ve phyla within archaea between two groups (Figure3B).
e top50 dierential bacterial genera are shown in Figure4A. e
relative abundances of 51 genera including Polymorphum,
Amylolactobacillus, Abiotrophia, Phyllobacterium, Desulfoscipio,
Schneewindia, Ethanoligenens, Lawsonibacter, and Sporosarcina were
greater (p < 0.05) in the DTR sheeps, whereas the relative abundances
of nine genera, including UBA2922, 43-108, UBA3839, and RGIG8745
were greater (p < 0.05) in the CON sheep. e archaea genera of
JAHIMK01, FT1-020, Aciduliprofundum, and Halovenus showed a
high abundance (p < 0.05) in the DTR sheep, whereas the genera
Hydrothermarchaeum, JAHLMNO1, and BIN-L-1 were low abundant
(p < 0.05) in the CON sheep (Figure 4B). e virus genera of
Svunavirus and Vieuvirus showed a low abundance (p < 0.05) in the
DTR sheep, whereas the genera Copernicusvirus was more abundant
(p < 0.05) in the DTR sheep compared with the CON sheep
(Figure4C).
FIGURE1
Hindgut microbial structure analysis at the domain level. The compositional profiles of bacteria (A), eukaryota (B), archaea (C), and viruses (D) based on
PCoA.
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FIGURE2
Comparison of microbial domains between CON and DTR sheep. Significantly dierent domains were tested by Wilcoxon rank-sum test.
FIGURE3
Comparison of the main hindgut microbial taxa at the phylum level between the CON and DTR sheeps based on the Wilcoxon rank-sum test. (A) The
top10 phyla within bacteria. (B) The top five phyla within archaea.
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Comparisons of the taxa at the dierent levels were performed
using LEfSe with the non-parametric factorial Kruskal–Wallis and
Wilcoxon rank-sum tests. A total of seven species, four genes and one
phylum were more abundant in the CON sheep compared to the DTR
sheep (LDA > 2.5 and p < 0.05; Figure5), whereas 32 species, 9 genes
and 3 phyla were signicantly enriched in the CON sheep (LDA > 2.5
and p < 0.05; Figure5).
Functional analysis of the microbiome
Due to the fact that carbohydrates are degraded by multiple
enzymes, wefocused on the dierences in the proles of CAZymes
between the CON and DTR sheep. As shown in Figure 6, the
CAZymes community consisted of glycoside hydrolases (GH;
48.85%), glycosyltransferases (GT; 23.26%), carbohydrate esterases
(CE; 12.77%), carbohydrate-binding modules (CBM; 8.67%),
polysaccharide lyases (PL; 2.16%), Cellulosome (3.19%), and exhibited
auxiliary activities (AA; 1.12%). At the class level, there is no
signicant variation of CAZy between the CON and DTR groups
(Supplementary Figure S3), and PCoA also shows no apparent
separation at the Class level (Supplementary Figure S4). Among the
CAZymes that participated in degrading carbohydrates, three families
(2 of CBMs, and 1 of GT) were enriched in the CON sheep, whereas
13 families (4 of CBMs, 7 of GHs and 2 of PLs) were enriched in the
CON cows (Supplementary Table2).
Figure7 displays the main functions of KEGG in 16 sheep, which
primarily include ve pathways: Cellular Processes, Environmental
Information Processing, Genetic Information Processing, Metabolism,
and Organismal Systems. e PCoA plot based on the Bray-Curtis
distance showed a clear separation of two groups at pathway level 2
(Supplementary Figure S5). Weconsidered 348 endogenous third-
level metabolic pathways as hindgut microbial pathways in the KEGG
proles for the further analysis. As shown in Supplementary Table3,
compared to the CON group, there were 31 upregulated metabolic
pathways (including methane metabolism, propanoate metabolism,
microbial metabolism in diverse environments, purine metabolism,
carbon metabolism, starch and sucrose metabolism, pentose
phosphate pathway, and glycerolipid metabolism, among others,
p < 0.05) and 23 downregulated metabolic pathways (including Dorso-
ventral axis formation, bile secretion, sphingolipid signaling pathway,
growth hormone synthesis, secretion and action, cGMP-PKG
signaling pathway, fcepsilon RI signaling pathway, and calcium
signaling pathway, among others, p < 0.05).
Discussion
Under normal circumstances, adding food or industrial
by-products to an animal’s diet may not signicantly aect the
animal’s performance. is could bebecause the content of various
active or nutritional components in the by-products does not reach
concentrations that have a noticeable eect (Sarker etal., 2022;
Khurana etal., 2023). Previous studies have indicated that the use of
tea and its by-products in monogastric animals can signicantly
improve their production performance, such as increasing egg
production rate, egg weight, and other factors (Wang etal., 2018;
Chen etal., 2023). However, in this study, no signicant impact of
FIGURE4
Dierential hindgut microbial taxa at the genus level between the CON and DTR sheeps based on the Wilcoxon rank-sum test. (A) Heatmap of the
top50 dierential genera within bacteria. (B) All dierential genera within archaea. (C) All dierential genera within viruses.
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DTR on sheep’s production performance was observed, which may
beattributed to the unique rumen fermentation characteristic of
ruminant animals. Kolling etal. (2018) used green tea extract as an
additive in dairy cow production and found that it did not aect the
production performance of dairy cows. However, it promoted the
health and rumen fermentation pattern of the cows. However, in
Acharya etal.’s (2020) research report, it was indicated that adding
green tea extract could increase milk production in periparturient
cows. is may beattributed to the specic physiological state of
periparturient cows, and the enhanced production performance of
these cows aer adding green tea extract might bedue to the potent
antioxidant eects of green tea. In conclusion, the application of
green tea by-products as a feed resource or feed additive in sheep’s
diet will not have negative eects on sheep’s production.
Green tea is rich in various active compounds, which are high-
quality immune modulators. ese active compounds can stimulate
the activation of macrophages and B cells, promoting the formation
of antibodies (Ding etal., 2018). e active compounds in tea, such
as catechins and EGCG, can modulate the activity of immune cells,
promoting the activation of macrophages and lymphocytes, and
FIGURE5
Dierential rumen microbial taxa at the dierent levels between the CON and DTR sheep based on LEfSe.
Guo et al. 10.3389/fmicb.2023.1289743
Frontiers in Microbiology 09 frontiersin.org
enhancing their proliferation and secretion of immunoglobulins
(Yahfou etal., 2018). Immunoglobulins are antibody-active animal
proteins secreted by plasma cells and play a crucial role in both
specic and non-specic immunity. In this experiment, DTR
signicantly increased the concentrations of IgA, IgG, and IgM in
sheep, indicating that DTR exerted a signicant immunomodulatory
eect in sheep. is nding is consistent with the results of Yua n
etal.’s (2023) study. In production, oxidative stress is considered a
major factor leading to animal diseases. Green tea and its
by-products have been shown to enhance the antioxidant status in
animals (Lu etal., 2014). For example, in dairy cows (Ma etal., 2021)
and laying hens (Ling etal., 2022), the polyphenolic compounds in
green tea can scavenge various oxygen free radicals, including
superoxide anion, singlet oxygen, peroxynitrite, and hypochlorous
acid (Severino etal., 2009). ey can also achieve antioxidant eects
by reducing the expression of redox-sensitive transcription factors
such as NF-κB and activator protein-1, inhibiting the activity of
“pro-oxidant” enzymes, and increasing the activity of antioxidant
enzymes such as GSH-Px (Frei and Higdon, 2003). In this study, the
addition of DTR signicantly increased the concentrations of
T-SOD, GSH-Px, and CAT, indicating that DTR has signicant
antioxidant activity in sheep. is nding is similar to the research
results of Ma etal. (2021). is may beattributed to the abundant
polyphenols and EGCG content in DTR. However, the specic
mechanism by which DTR exerts antioxidant activity in sheep needs
further investigation. ALB and HDL are important “regulatory” and
“transport” functional indicators in animal blood. ey play crucial
roles in the metabolism of substances like glycerol. e decrease in
ALB and HDL aer the addition of DTR may bedue to the enhanced
metabolism of lipid substances in sheep, which is also related to the
strengthened Glycerolipid metabolism pathway observed in the
experiment. e signicant decrease in TG supports this
observation. e changes in these indicators are also signicantly
correlated with the enhancement of sheep’s immunity and
antioxidant capacity. However, the specic mechanisms have not
been studied yet.
In this study, the consumption of DTR by sheep had the greatest
impact on bacteria, with the most pronounced changes observed at
the phylum level for SAR324 and Eisenbacteria. Among them,
SAR324 is a widely distributed bacterial group on earth, and its
metabolic characteristics are mainly reected in genes that encode a
novel particulate hydrocarbon monooxygenase (pHMO), degradation
pathways for corresponding alcohols and short-chain fatty acids,
dissimilatory sulfur oxidation, formate dehydrogenase (FDH), and
nitrite reductase (NirK). It is primarily associated with lithotrophy,
heterotrophy, and alkane oxidation, among other metabolic functions
(Sheik etal., 2014; Boeuf etal., 2021). is also explains one of the
reasons why the addition of DTR leads to an increase in methane
metabolism pathways. However, there is limited knowledge about
Eisenbacteria and their potential involvement in carbohydrate
FIGURE6
Cazy composition diagram at class level. Glycoside hydrolases (GH), glycosyltransferases (GT), carbohydrate esterases (CE), carbohydrate-binding
modules (CBM), polysaccharide lyases (PL), and exhibited auxiliary activities (AA).
Guo et al. 10.3389/fmicb.2023.1289743
Frontiers in Microbiology 10 frontiersin.org
metabolism and methane metabolism (Poghosyan etal., 2020). At the
bacterial genus level, we observed a signicant increase in the
abundance of bacteria such as Ammylolactobacillus, Phyllobacterium,
Ethanoligenens, Lawsonibacter, Staphylococcus, Desulfosudis, etc.,
aer adding DTR. is increase may berelated to lactate fermentation
(Zheng et al., 2020), lipid metabolism (Zamlynska et al., 2017),
hydrogen and ethanol production fermentation (Li et al., 2019),
butyric acid production (Sakamoto et al., 2018), and immune
metabolism (Vaskevicius etal., 2023). However, the specic functions
associated with the signicantly decreased bacterial genera at the
genus level have not been reported yet. At the level of archaea and
viruses, the specic eects of DTR on microbial changes are yet to
befurther explored. However, overall, DTR does not have a negative
impact on the microbial structure in the sheep’s hindgut. is is
consistent with previously reported research ndings (Ramdani
etal., 2013).
e degradation of carbohydrates by gut microbiota requires
various enzymes, including GH, PL, CE, GT, AA, Cellulosome, and
CBM. e addition of DTR signicantly aects the distribution of
various CAZy in the gut, with most of these changes belonging to
the GH family, which are polysaccharide-degrading enzymes
produced by ber-degrading bacteria. e addition of DTR
signicantly aects the distribution of various CAZy in the gut, with
most of these changes belonging to the GH, which are
polysaccharide-degrading enzymes produced by ber-degrading
bacteria (Flint etal., 2012). e increase in GH abundance may
bedue to changes in bacterial composition. For instance, the GH1
family plays a crucial role in carbohydrate degradation within
organisms, breaking down complex polysaccharides into simpler
sugar molecules, and participating in the degradation of cellulose,
galactosides, polysaccharides, and oxalates to provide energy
metabolism and other biological processes (Cota et al., 2015;
Strazzulli et al., 2019). GH3 is involved in the degradation of
β-glucoside substrates and drug metabolism. Regarding GH, GH25,
GH27, GH112, GH120, and GH154 enzymes show higher
abundance aer the addition of DTR, indicating that supplementing
FIGURE7
Functional features profiling at KEGG pathway.
Guo et al. 10.3389/fmicb.2023.1289743
Frontiers in Microbiology 11 frontiersin.org
DTR can enhance microbial digestion and absorption of food.
Similarly, the changes of CBM (Arai etal., 2003) and PL (Yang etal.,
2021) are also related to ber digestion and starch digestion. In
general, the addition of DTR enhances the ability of ber digestion
in the hindgut.
With the changes in the hindgut microbial structure, there are
dierences in the KEGG functional proles between the CON
group and the DTR group. e addition of DTR signicantly
enhances pathways such as methane metabolism, propanoate
metabolism, purine metabolism, starch and sucrose metabolism,
and glycerolipid metabolism. e enhancement of methane
metabolism may berelated to the improved ber digestion, as
mentioned earlier in the changes in the abundance of ber-
digesting enzymes (Li etal., 2022). e alterations in propanoate
metabolism, purine metabolism, and glycerolipid metabolism
pathways also indicate the eectiveness of DTR in enhancing
carbohydrate metabolism (Hong et al., 2022). e changes in
glycerolipid metabolism may also berelated to the signicant
decrease in TG observed in this study. Aer the addition of DTR,
the sphingolipid signaling pathway signicantly decreases, which
may berelated to DTR’s regulation of animal lipid metabolism and
alteration of the animal’s gastrointestinal microbiota (Yua n
etal., 2023).
Conclusion
is study provides new insights into the application of DTR in
sheep production. Supplementing DTR signicantly improves the
sheep’s immune and antioxidant indicators and promotes their ber
digestion capability. is is of great signicance to the feeding system
for sheep. DTR is a promising natural additive that has positive eects
on animal health and the environment. Further exploration of DTR’s
eects on rumen fermentation, microbial communities, and ber
metabolism will contribute to its further development and application
in ruminant animals.
Data availability statement
e datasets presented in this study can befound in online
repositories. e names of the repository/repositories and accession
number(s) can befound in the article/Supplementary material.
Ethics statement
e animal study was approved by Animal Care and Use
Committee at Nanjing Agricultural University. e study was
conducted in accordance with the local legislation and institutional
requirements.
Author contributions
LG: Writing – original dra, Writing – review & editing. SY: Writing
– original dra, Data curation, Formal analysis. FC: Writing – original
dra, Data curation, Project administration, Soware. KZ: Writing –
original dra, Methodology, Formal analysis, Investigation, Soware.
ML: Writing – original dra, Data curation, Methodology, Supervision,
Conceptualization, Resources. ZP: Writing – original dra, Project
administration, Investigation, Visualization. XS: Writing – original dra,
Data curation, Supervision, Conceptualization, Investigation. LL:
Writing – review & editing.
Funding
e author(s) declare nancial support was received for the
research, authorship, and/or publication of this article. is work was
supported by the Regional Demonstration Project of the Alliance of
Municipal Academy of Agricultural Sciences of Zhejiang Province
(grant no. 2023SJLM07) and Key R&D project of Huzhou (grant no.
2021ZD2035).
Conflict of interest
e authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could
beconstrued as a potential conict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their aliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or claim
that may be made by its manufacturer, is not guaranteed or endorsed
by the publisher.
Supplementary material
e Supplementary material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1289743/
full#supplementary-material
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