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<journal-id journal-id-type="publisher-id">Front. Microbiol.</journal-id>
<journal-title>Frontiers in Microbiology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Microbiol.</abbrev-journal-title>
<issn pub-type="epub">1664-302X</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
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<article-meta>
<article-id pub-id-type="doi">10.3389/fmicb.2024.1342804</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Microbiology</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Age-related compositional and functional changes in the adult and breastfed buffalo rumen microbiome</article-title>
</title-group>
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<name><surname>Xu</surname> <given-names>Yixue</given-names></name>
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<name><surname>Feng</surname> <given-names>Tong</given-names></name>
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<name><surname>Ding</surname> <given-names>Zixu</given-names></name>
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<name><surname>Li</surname> <given-names>Ling</given-names></name>
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<name><surname>Li</surname> <given-names>Zhipeng</given-names></name>
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<name><surname>Cui</surname> <given-names>Kuiqing</given-names></name>
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<name><surname>Chen</surname> <given-names>Weihua</given-names></name>
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<name><surname>Pan</surname> <given-names>Hongping</given-names></name>
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<name><surname>Zhu</surname> <given-names>Peng</given-names></name>
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<contrib contrib-type="author" corresp="yes">
<name><surname>Liu</surname> <given-names>Qingyou</given-names></name>
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<aff id="aff1"><sup>1</sup><institution>State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University</institution>, <addr-line>Nanning</addr-line>, <country>China</country></aff>
<aff id="aff2"><sup>2</sup><institution>Department of Bioinformatics and Systems Biology, Key Laboratory of Molecular Biophysics of the Ministry of Education, Hubei Key Laboratory of Bioinformatics and Molecular-imaging, Center for Artificial Biology, College of Life Science and Technology, Huazhong University of Science and Technology</institution>, <addr-line>Wuhan</addr-line>, <country>China</country></aff>
<aff id="aff3"><sup>3</sup><institution>Guangxi Key Laboratory of Buffalo Genetics</institution>, <addr-line>Nanning</addr-line>, <country>China</country></aff>
<aff id="aff4"><sup>4</sup><institution>Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University</institution>, <addr-line>Foshan</addr-line>, <country>China</country></aff>
<aff id="aff5"><sup>5</sup><institution>Guangxi Key Laboratory of Beibu Gulf Marine Biodiversity Conservation, Beibu Gulf Marine Ecological Environment Field Observation and Research Station of Guangxi, Beibu Gulf University</institution>, <addr-line>Qinzhou</addr-line>, <country>China</country></aff>
<author-notes>
<fn fn-type="edited-by" id="fn0002">
<p>Edited by: Thi Thu Hao Van, RMIT University, Australia</p>
</fn>
<fn fn-type="edited-by" id="fn0003">
<p>Reviewed by: Sara Fareed Mohamed Wahdan, Suez Canal University, Egypt</p>
<p>Rahul Bodkhe, University of California, Berkeley, United States</p>
</fn>
<corresp id="c001">&#x002A;Correspondence: Qingyou Liu, <email>qyliu-gene@gxu.edu.cn</email></corresp>
<corresp id="c002">Peng Zhu, <email>yijianrudi@163.com</email></corresp>
<corresp id="c003">Hongping Pan, <email>panhp65@163.com</email></corresp>
<fn fn-type="equal" id="fn0001"><p><sup>&#x2020;</sup>These authors have contributed equally to this work</p></fn>
</author-notes>
<pub-date pub-type="epub">
<day>30</day>
<month>05</month>
<year>2024</year>
</pub-date>
<pub-date pub-type="collection">
<year>2024</year>
</pub-date>
<volume>15</volume>
<elocation-id>1342804</elocation-id>
<history>
<date date-type="received">
<day>22</day>
<month>11</month>
<year>2023</year>
</date>
<date date-type="accepted">
<day>07</day>
<month>05</month>
<year>2024</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#x00A9; 2024 Xu, Feng, Ding, Li, Li, Cui, Chen, Pan, Zhu and Liu.</copyright-statement>
<copyright-year>2024</copyright-year>
<copyright-holder>Xu, Feng, Ding, Li, Li, Cui, Chen, Pan, Zhu and Liu</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>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.</p>
</license>
</permissions>
<abstract>
<sec>
<title>Introduction</title>
<p>The buffalo is an important domestic animal globally, providing milk, meat, and labor to more than 2 billion people in 67 countries. The rumen microorganisms of buffaloes play an indispensable role in enabling the healthy functionality and digestive function of buffalo organisms. Currently, there is a lack of clarity regarding the differences in the composition and function of rumen microorganisms among buffaloes at different growth stages.</p>
</sec>
<sec>
<title>Methods</title>
<p>In this study, metagenomics sequencing technology was applied to examine the compositional and functional differences of rumen microorganisms in adult and breastfed buffaloes.</p>
</sec>
<sec>
<title>Results</title>
<p>The results revealed that the rumen of adult buffaloes had significantly higher levels of the following dominant genera: Prevotella, UBA1711, RF16, Saccharofermentans, F23-D06, UBA1777, RUG472, and Methanobrevibacter_A. Interestingly, the dominant genera specific to the rumen of adult buffaloes showed a significant positive correlation (correlation&#x003E;0.5, p-value&#x003C;0.05) with both lignocellulose degradation-related carbohydrate-active enzymes (CAZymes) and immune signaling pathways activated by antigenic stimulation. The rumen of breastfed buffaloes had significantly higher levels of the following dominant genera: UBA629, CAG- 791, Selenomonas_C, Treponema_D, Succinivibrio, and RC9. Simultaneously, the rumen-dominant genera specific to breastfed buffaloes were significantly positively correlated (correlation&#x003E;0.5, p-value&#x003C;0.05) with CAZymes associated with lactose degradation, amino acid synthesis pathways, and antibiotic-producing pathways.</p>
</sec>
<sec>
<title>Discussion</title>
<p>This indicates that rumen microorganisms in adult buffaloes are more engaged in lignocellulose degradation, whereas rumen microorganisms in breastfed buffaloes are more involved in lactose and amino acid degradation, as well as antibiotic production. In conclusion, these findings suggest a close relationship between differences in rumen microbes and the survival needs of buffaloes at different growth stages.</p>
</sec>
</abstract>
<kwd-group>
<kwd>buffalo</kwd>
<kwd>rumen</kwd>
<kwd>microorganisms</kwd>
<kwd>metagenomics</kwd>
<kwd>immunity</kwd>
<kwd>lignocellulose</kwd>
</kwd-group>
<contract-num rid="cn1">U20A2051</contract-num>
<contract-num rid="cn2">AA22068099</contract-num>
<contract-num rid="cn3">AA18118041</contract-num>
<contract-num rid="cn4">2019B030301010</contract-num>
<contract-num rid="cn4">2019KSYS011</contract-num>
<contract-sponsor id="cn1">National Natural Science Fund</contract-sponsor>
<contract-sponsor id="cn2">Guangxi Science and Technology Major Project</contract-sponsor>
<contract-sponsor id="cn3">Major Science and Technology Projects in Guangxi</contract-sponsor>
<contract-sponsor id="cn4">Open Foundation of Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding</contract-sponsor>
<counts>
<fig-count count="6"/>
<table-count count="0"/>
<equation-count count="0"/>
<ref-count count="51"/>
<page-count count="13"/>
<word-count count="7669"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-at-acceptance</meta-name>
<meta-value>Microorganisms in Vertebrate Digestive Systems</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec sec-type="intro" id="sec1">
<title>Introduction</title>
<p>The buffalo, a globally significant domestic animal of great value to humans, like other ruminants, possesses a specialized four-chambered stomach consisting of the rumen, reticulum, omasum, and abomasum. This unique digestive system enables the transformation of low-nutritional-value forage into high-quality animal protein, allowing the buffalo to adapt to the life cycle of forage grass (<xref ref-type="bibr" rid="ref20">Lei et al., 2018</xref>). The ability of buffaloes to digest plant feeds is primarily attributed to the function of the microbial community within their gastrointestinal tract (<xref ref-type="bibr" rid="ref31">Mizrahi et al., 2021</xref>). The structure of the rumen microbial community is community-based and can be influenced by various factors, such as host species (<xref ref-type="bibr" rid="ref21">Li et al., 2019</xref>), gender (<xref ref-type="bibr" rid="ref9">Guo et al., 2022</xref>), genetic factors (<xref ref-type="bibr" rid="ref50">Zhang et al., 2020</xref>), diet (<xref ref-type="bibr" rid="ref36">Scharen et al., 2017</xref>), age, and early life microbial colonization (<xref ref-type="bibr" rid="ref7">Furman et al., 2020</xref>). The rumen microbiota plays a pivotal role in the utilization of plant material by ruminants (<xref ref-type="bibr" rid="ref42">Su et al., 2022</xref>). The efficiency of plant feed utilization by rumen flora has a direct impact on host animal feed efficiency (<xref ref-type="bibr" rid="ref21">Li et al., 2019</xref>), energy acquisition efficiency (<xref ref-type="bibr" rid="ref35">Sasson et al., 2017</xref>), methane emissions (<xref ref-type="bibr" rid="ref31">Mizrahi et al., 2021</xref>), antibiotic resistance genes (<xref ref-type="bibr" rid="ref34">Sabino et al., 2019</xref>), milk yield, and milk protein content (<xref ref-type="bibr" rid="ref48">Xue et al., 2020</xref>).</p>
<p>Dietary changes have a significant effect on the digestive flora of animals (<xref ref-type="bibr" rid="ref36">Scharen et al., 2017</xref>; <xref ref-type="bibr" rid="ref32">Morais and Mizrahi, 2019</xref>; <xref ref-type="bibr" rid="ref7">Furman et al., 2020</xref>; <xref ref-type="bibr" rid="ref4">Cao et al., 2023</xref>). Previous studies have demonstrated that diet influences the development of rumen flora in dairy cows (<xref ref-type="bibr" rid="ref7">Furman et al., 2020</xref>), and the composition of the gut flora of humans (<xref ref-type="bibr" rid="ref40">Stewart et al., 2018</xref>), pigs (<xref ref-type="bibr" rid="ref22">Lim et al., 2019</xref>), and goats (<xref ref-type="bibr" rid="ref4">Cao et al., 2023</xref>) varies considerably between the period of breastfeeding and the composition of adulthood. This shift occurs as neonates consume solid foods and cease breastfeeding, leading to a microbiome that resembles a more adult-like state and exhibits increased microbial diversity. Beef cattle (<xref ref-type="bibr" rid="ref41">Stewart et al., 2018</xref>), dairy cows (<xref ref-type="bibr" rid="ref49">Xue et al., 2022</xref>), sheep (<xref ref-type="bibr" rid="ref18">Lane et al., 2000</xref>), goats (<xref ref-type="bibr" rid="ref4">Cao et al., 2023</xref>), and camels (<xref ref-type="bibr" rid="ref8">Gharechahi et al., 2022</xref>) have been found to have important roles in rumen microbial functioning for the health of the host animal. However, little attention has been given to differences in rumen microbial composition and functioning between calf buffaloes during the breastfeeding stage and adult buffaloes.</p>
<p>In this study, we analyzed the rumen microbial metagenomic data from 18 breastfed buffaloes and 29 adult female buffaloes fed with feed. We found that adult buffaloes had a higher abundance of genera (Prevotella, Saccharofermentans, Methanobrevibacter_A, UBA1711, RF16, F23-D06, UBA1777, and RUG472), lignocellulose degradation-related CAZymes (AA1, AA6, AA10, GH5, etc.), and antigenic stimulus-activated immune signaling pathways. In contrast, breastfed buffaloes had a higher abundance of genera (Selenomonas_C, Treponema_D, Succinivibrio, UBA629, CAG-791, and RC9), lactose degradation-related CAZymes (GH35, GH42, etc.), amino acid synthesis pathways, and antibiotic production pathways.</p>
</sec>
<sec sec-type="materials|methods" id="sec2">
<title>Materials and methods</title>
<sec id="sec3">
<title>Sample collection</title>
<p>A total of 47 samples were collected for metagenome sequencing. Among these samples, 29 were obtained from adult female buffaloes fed with feed (90&#x2009;&#x00B1;&#x2009;36sd months), while 18 were obtained from breastfed buffaloes (&#x003C;1&#x2009;month). To ensure diversity, adult buffaloes with no history of intestinal disease and calves in the breastfeeding period were individually confined in cleaned pens. After a 12-h fasting period, rumen samples were collected using a stomach tube. All samples were immediately frozen in liquid nitrogen and stored at &#x2212;80&#x00B0;C until DNA extraction. Most samples were processed for DNA extraction within a week of collection.</p>
</sec>
<sec id="sec4">
<title>DNA extraction, library construction, and metagenomics sequencing</title>
<p>Three grams of each sample were used for DNA extraction. DNA was extracted using a mini-bead beater (BioSpec Products; Bartlesville, United States), followed by phenol&#x2013;chloroform extraction through a bead-beating method. After precipitation with ethanol, the pellets were suspended in 50&#x2009;&#x03BC;L of Tris&#x2013;EDTA buffer. DNA quantification was performed using a NanoPhotometer&#x00AE; (IMPLEN, CA, USA) after staining with a Qubit&#x00AE; 2.0 Fluorometer (Life Technologies, CA, USA). DNA samples were stored at &#x2212;80&#x00B0;C until they were further processed. We followed the TruSeq DNA sample preparation guide (Illumina, 15,026,486 Rev. C) method and procedure, using 500&#x2009;ng DNA as the template. We selected qualified libraries for paired-end sequencing on the Illumina NovaSeq 6,000 platform, with a read length of 150 base pairs (PE150).</p>
</sec>
<sec id="sec5">
<title>Quality control and removal of host- and food-associated genomes</title>
<p>The paired-end raw sequencing reads were first trimmed using Trimmomatic (<xref ref-type="bibr" rid="ref2">Bolger et al., 2014</xref>) (v.0.35) to eliminate vectors and low-quality bases. Sequences longer than 110 bases and with an average base quality greater than 30 after trimming were retained for subsequent analysis. To eliminate potential reads from the host or food, genomic sequences of buffalo (<xref ref-type="bibr" rid="ref27">Luo et al., 2020</xref>), <italic>Glycine max</italic> (<xref ref-type="bibr" rid="ref38">Shen et al., 2019</xref>), <italic>Zea mays</italic> (<xref ref-type="bibr" rid="ref14">Jiao et al., 2017</xref>), and <italic>Medicago truncatula</italic> (<xref ref-type="bibr" rid="ref44">Tang et al., 2014</xref>) were downloaded from the National Center for Biotechnology Information (NCBI) and used as references in Bowtie2 (<xref ref-type="bibr" rid="ref19">Langmead and Salzberg, 2012</xref>) (v.2.3.3) analysis with the options &#x201C;-p 10-very-sensitive&#x201D;. Reads that aligned concordantly with references were removed as contamination.</p>
</sec>
<sec id="sec6">
<title>Annotation and functional characterization of MAG-encoded proteins</title>
<p>To enhance the accuracy of our results, we used the gut microbiota genome catalog of the buffalo species from a prior study as a reference (<xref ref-type="bibr" rid="ref45">Tong et al., 2022</xref>). For predicting MAG-encoded proteins, we utilized Prodigal (<xref ref-type="bibr" rid="ref13">Hyatt et al., 2010</xref>) (v.2.6.3). Additionally, we used CD-HIT (<xref ref-type="bibr" rid="ref6">Fu et al., 2012</xref>) (v.4.8.1) to cluster the predicted proteins, employing the following options: &#x201C;-c 0.95 -n 10 -d 0 -M 16000 -T 8&#x201D;. These non-duplicate proteins were further scrutinized against the CAZy database through the usage of dbCAN2 (<xref ref-type="bibr" rid="ref51">Zhang et al., 2018</xref>) and the EggNOG database using eggnog-mapper (v.4.5) (<xref ref-type="bibr" rid="ref11">Huerta-Cepas et al., 2016</xref>). For the KEGG annotation, we use the metagenome pipeline (<xref ref-type="bibr" rid="ref24">Liu et al., 2021</xref>) designed by <xref ref-type="bibr" rid="ref23">Liu et al. (2023)</xref>, in conjunction with its requisite common software, script files, and database annotation files (EasyMicrobiome).</p>
</sec>
<sec id="sec7">
<title>Identification of differential taxa between groups</title>
<p>To establish the microbial strains exhibiting marked differences in abundance among the selected sample groups, we initially evaluated the relative redundancy of the MAGs in each sample. We subsequently mapped the clean reads from each sample to all MAGs and calculated the proportional abundance of a given MAG as a percentage of the total number of reads mapped to all MAGs in that sample. Therefore, the total relative abundance of all MAGs in each sample invariably amounts to 100%. Relative abundances at higher taxonomic levels, including genera, families, and orders, were determined by summing the abundances of their respective daughter clades based on the GTDB-TK phylogenetic tree.</p>
<p>The LEfSe tool (<xref ref-type="bibr" rid="ref37">Segata et al., 2011</xref>) was utilized to identify differential taxa among groups of samples, selecting those with LDA scores &#x003E;2 as the significant taxa. The statistical significance of the differential taxa abundance between groups was revealed using the Wilcoxon test. Subsequently, we picked taxa that have a crucial role in the physiological functions linked with the context of grouping.</p>
</sec>
<sec id="sec8">
<title>Statistics</title>
<p>The alpha diversity of the microbial community was obtained using the Shannon index obtained from the R package vegan analysis, and the beta diversity was obtained from non-metric multidimensional scaling (NMDS) analysis using the metaMDS function of the R package vegan. Spearman&#x2019;s rank correlation was used for all correlation analyses, with R&#x2009;&#x003E;&#x2009;0.5 or&#x2009;&#x003C;&#x2009;&#x2212;0.5 being considered strong correlations and <italic>p</italic>-values &#x003C;0.05 being considered significant.</p>
</sec>
</sec>
<sec sec-type="results" id="sec9">
<title>Results</title>
<sec id="sec10">
<title>Structure of microbial communities in the rumen of adult and breastfed buffaloes</title>
<p>In this investigation, we collected rumen contents from 29 adult female buffaloes that were provided with feed, along with 18 breastfed buffaloes, followed by sequencing and bioinformatic analysis (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). The Shannon index was higher in the adult buffalo group than in the breastfed buffalo group (<xref ref-type="fig" rid="fig1">Figure 1B</xref>, <italic>p</italic> &#x003C; 0.0001), indicating that the diversity of rumen microorganisms was higher in adult buffaloes than in breastfed buffaloes. Adult buffaloes are exposed to a more complex environment than breastfed buffalo, with more time and opportunity for microbes to colonize. Forage is more difficult to digest compared to milk, so adult buffaloes need more microbes to assist in digesting forage. This results in a higher diversity of microbes colonizing the rumen of adult buffaloes. NMDS analyses showed that the rumen microbial communities of adult and breastfed buffaloes were significantly different (<xref ref-type="fig" rid="fig1">Figure 1C</xref>), with a higher degree of aggregation among microbial samples in adult buffaloes and a greater degree of disaggregation in breastfed calves, which was also seen in other species (<xref ref-type="bibr" rid="ref10">Huang et al., 2018</xref>; <xref ref-type="bibr" rid="ref22">Lim et al., 2019</xref>; <xref ref-type="bibr" rid="ref15">Jin et al., 2021</xref>; <xref ref-type="bibr" rid="ref47">Xiao et al., 2022</xref>).</p>
<fig position="float" id="fig1">
<label>Figure 1</label>
<caption>
<p>Compositional analysis of rumen microorganisms in adult and breastfed buffaloes. <bold>(A)</bold> Experimental design, macro genomic analysis of rumen contents of adult female buffaloes fed with feed (29), and breastfed buffaloes (18). <bold>(B)</bold> Shannon index. <bold>(C)</bold> NMDS analysis. <bold>(D)</bold> Percentage stacking of the phylum of rumen microorganisms in adult and breastfed buffaloes. <bold>(E)</bold> Sankey diagram of dominant rumen communities in adult and breastfed buffaloes. The diagram shows the dominant genera and the families and phyla to which they belong that are greater than 1% in the rumen of adult and breastfed buffaloes. The green dots represent that the genus, family, and phylum are greater than 1% in adult buffaloes only, the yellow dots represent that the genus, family, and phylum are greater than 1% in breastfed buffaloes only, and the unspecialized markings indicate that it is greater than 1% in both groups.</p>
</caption>
<graphic xlink:href="fmicb-15-1342804-g001.tif"/>
</fig>
<p>We adopted the approach of <xref ref-type="bibr" rid="ref16">Jin et al. (2022)</xref>, designating that relative abundance greater than 1% is the dominant bacterial group. In this study, we counted the phyla, families, and genera of organisms in &#x003E;1% of the rumen of adult and breastfed buffaloes. There were 6 phyla (97.50%), 14 families (93.05%), and 15 genera (84.87%) belonging to the dominant flora in the rumen of adult buffaloes. A total of 8 phyla (97.49%), 13 families (88.51%), and 13 genera (76.52%) were dominant in the rumen microflora of breastfed buffaloes (<xref ref-type="supplementary-material" rid="SM1">Supplementary Table S1</xref>). At the phylum level (<xref ref-type="fig" rid="fig1">Figure 1D</xref>), Bacteroidota (79.03, 64.96%), Firmicutes_A (11.75, 17.05%), Firmicutes_C (2.34, 4.48%), and Firmicutes (1.37, 1.93%) were present in both adult and breastfed buffalo. Methanobacteriota (1.85%) and Verrucomicrobiota (1.16%) were only dominant in adult buffalo, while Proteobacteria (2.86%), Spirochaetota (2.86%), Synergistota (1.72%), and Actinobacteriota (1.63%) were only dominant in breastfed buffalo. At the family level (<xref ref-type="supplementary-material" rid="SM1">Supplementary Figure S1B</xref>), Bacteroidaceae (56.28, 36.15%), UBA932(7.68, 12.52%), Muribaculaceae (4.82, 8.78%), F082 (4.54, 5.50%), Lachnospiraceae (2.61, 10.37%), Acidaminococcaceae (2.11, 2.27%), and Oscillospiraceae (2.41, 3.38%) were present in both adult and breastfed buffalo. Saccharofermentanaceae (2.21%), Acidaminococcaceae (2.11%), CAG-138(2.10%), P3(2.09%), Methanobacteriaceae (2.06%), Paludibacteraceae (1.64%), WCHB1-69 (1.62%), and CAG-74 (1.09%) were only dominant in adult buffalo, while Succinivibrionaceae (2.51%), Selenomonadaceae (1.68%), Treponemataceae (1.62%), Atopobiaceae (1.40%), Sphaerochaetaceae (1.23%), and P3 (1.08%) were only dominant in breastfed buffaloes.</p>
<p>The relative abundance of rumen flora in both adult buffaloes and breastfed buffaloes was greater than 1% in eight genera (<xref ref-type="fig" rid="fig1">Figure 1E</xref>), of which five, Prevotella (51.98,32.52%), RC9 (6.36,11.46%), C941 (4.70,8.58%), F082 (4.54,5.50%), and UBA4372 (1.94,1.28%), were the major contributors to Bacteroidota, while the remaining three, CAG-110(1.02, 2.19%), Succiniclasticum (2.09,1.95%), and unclassified GWE2-31-10_(1.68,1.71%), were derived from Firmicutes_A, Firmicutes_C, and Spirochaetota, respectively. There were seven genera with relative abundance greater than 1% in adult buffaloes only, of which four UBA1711 (1.91%), RF16 (1.40%), Saccharofermentans (1.96%), and F23-D06(1.19%) were derived from Bacteroidota, two UBA1777(1.05%), RUG472(1.19%) from Firmicutes_A, and Methanobrevibacter_A (1.85%) from Methanobacteriota. There were five genera with relative abundance greater than 1% in breastfed buffaloes only, two of them UBA629 (3.36%), CAG-791 (3.23%) were derived from Firmicutes_A, Selenomonas_C (1.55%), Treponema_D (1.62%), Succinivibrio (1.56%) from Firmicutes_C (4.48%), Spirochaetota (2.86%), and Proteobacteria (2.86%), respectively. In addition, the top 20 family-level and 30 genus-level microorganisms with the highest abundance per buffalo were enumerated (<xref ref-type="supplementary-material" rid="SM1">Supplementary Figures S3, S4</xref>).</p>
</sec>
<sec id="sec11">
<title>Analysis of differences in rumen microbiology between adult and breastfed buffaloes</title>
<p>Significant difference analysis was conducted for phylum, dominant family, and genus using STAMP software (<xref ref-type="supplementary-material" rid="SM1">Supplementary Figure S2</xref>). In adult buffaloes, seven phyla (all including Bacteroidota, Riflebacteria, UBP6, Methanobacteriota, UBA3054, Fibrobacterota, Planctomycetota), six dominant families (all including Bacteroidaceae, Saccharofermentanaceae, Methanobacteriaceae, Paludibacteraceae, CAG-138, WCHB1-69), and eight dominant genera (all including Prevotella, RC9, UBA1711, RF16, Saccharofermentans, F23-D06, UBA1777, RUG472) were found to be significantly higher (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). While, breastfed buffaloes showed significantly higher levels of eight phyla (Proteobacteria, Spirochaetota, Desulfobacterota, Firmicutes_C, Firmicutes_B, Actinobacteriota, Campylobacterota, Firmicutes_A), eight dominant families (Lachnospiraceae, UBA932, Muribaculaceae, Succinivibrionaceae, Selenomonadaceae, Treponemataceae, Atopobiaceae, Sphaerochaetaceae), and six dominant genera (Selenomonas_C, Treponema_D, Succinivibrio, RC9, UBA629, CAG-791) (<xref ref-type="fig" rid="fig2">Figure 2A</xref>).LEfSe analysis of variance was conducted to obtain an overall view of the differential rumen microbes in adult and breastfed buffaloes. Linear Discriminant Analysis (LDA) was used to identify microorganisms that were statistically different between the adult buffalo and breastfed buffalo groups, with an LDA score &#x003E;2 (<xref ref-type="fig" rid="fig2">Figure 2BC</xref>). Microorganisms that are higher in adult buffalo have Alphaproteobacteria, Saccharofermentanales, Acetobacterales, RF32, Saccharofermentanaceae, Paludibacteraceae, Acetobacteraceae, CAG-302, CAG-433, CAG-239, Saccharofermentans, RF16, UBA3792, g_Firm-16, UBA3766, UBA1786, RUG658, RUG705, Acetobacter, CAG-95, UBA1712_A, UBA6382, RUG410, Kandleria. Breastfed buffaloes had higher levels of Anaerotignaceae, Anaerotignum, Megasphaeraceae, Megasphaera, Pyramidobacter. In summary, genera with a relative abundance of over 1%, which exhibit no difference in both adult and breastfed buffalo (C941, F082, UBA4372, CAG-110, Succiniclasticum, unclassified GWE2-31-10_), may be the fundamental genera in the buffalo rumen. Significantly higher dominant genera in adult buffaloes (UBA1711, RF16, Saccharofermentans, F23-D06, UBA1777, RUG472, Methanobrevibacter_A) may be associated with degradation of lignocellulose, and significantly higher genera in breastfed buffaloes (UBA629, CAG-791, Selenomonas_C, Treponema_D, Succinivibrio) may be associated with degradation of high-protein, high-energy diets. These genera may have distinct roles in diverse dietary patterns at varying ages.</p>
<fig position="float" id="fig2">
<label>Figure 2</label>
<caption>
<p>Composition and differential analysis of rumen microorganisms in adult and breastfed buffaloes. <bold>(A)</bold> Violin plots of the significant difference analysis of the dominant genus, with the color block where the genus name is located being green for significantly higher in adult buffalo and yellow for significantly higher in breastfed buffaloes. <bold>(B)</bold> Bar chart of the distribution of LDA values: demonstrates species that differed at LDA scores &#x003E;2, with statistically different biomarkers, and the length of the histogram represents the magnitude of the influence of the significantly different species. <bold>(C)</bold> Species evolutionary branching diagram of the differing species, with circles radiating from inside to outside representing taxonomic levels from phylum to genus (or species). Each small circle at a different taxonomic level represents a taxon at that level, and the size of the circle diameter is proportional to the size of the relative abundance. Coloring principle: species with no significant differences are colored white, different species are colored following the group, yellow nodes indicate microbial taxa that play an important role in the breastfed buffalo group, and green nodes indicate microbial taxa that play an important role in the adult buffalo group. The full names of the species represented by the letters in the figure are shown in the legend.</p>
</caption>
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</fig>
</sec>
<sec id="sec12">
<title>Differences in the functional composition of rumen microorganisms in adult and breastfed buffaloes</title>
<p>In order to explore the functions of the excavated microbiota, we functionally annotated the predicted acquired genes for CAZyme, COG, and KEGG (<xref ref-type="supplementary-material" rid="SM1">Supplementary Table S1</xref>). Overall, glycoside hydrolases (GH) and glycosyl transferases (GT) were the major dominant CAZymes in both adult and breastfed buffaloes, with relatively higher GH, carbohydrate-binding module (CBM), and auxiliary activities (AAs) in adult buffaloes and higher relative abundance of GT in breastfed buffaloes (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). We counted a high abundance of CAZymes (<xref ref-type="fig" rid="fig3">Figure 3B</xref>; <xref ref-type="supplementary-material" rid="SM1">Supplementary Table S1</xref>), and GH35 (<xref ref-type="bibr" rid="ref46">Wang et al., 2020</xref>) with lactose degradation capacity had a relative abundance greater than 1% in breastfed buffaloes. In the COG database, significantly higher in breastfed buffaloes than in adult buffaloes C-Energy production and conversion (<italic>p</italic> &#x003C; 0.001) enables the bacteria to access and utilise energy for survival and function. Adult buffalo rumen microorganisms had significantly higher G-Carbohydrate transport and metabolism to take up carbohydrates from the rumen and convert them into energy supply and metabolites (<xref ref-type="fig" rid="fig3">Figure 3C</xref>; <xref ref-type="supplementary-material" rid="SM1">Supplementary Table 1</xref>).</p>
<fig position="float" id="fig3">
<label>Figure 3</label>
<caption>
<p>Functional composition and differential analysis of rumen microbes in adult and breastfed buffaloes. <bold>(A)</bold> Percentage CAZy stacking plot of rumen microbes of adult buffalo and breastfed calves. <bold>(B)</bold> CAZymes of rumen microbes greater than 1% in adult buffalo and breastfed calves. Green CAZymes numbers represent greater than 1% in adult buffalo only, yellow CAZymes numbers represent greater than 1% in breastfed buffalo only, and black CAZymes numbers indicate greater than 1% in both groups. <bold>(C)</bold> Analysis of rumen microbial COG function in adult and breastfed buffalo; on the left is a radar plot of the relative abundance of rumen microbial COG function in buffaloes and breastfed calves; and in the center is a STAMP difference analysis of rumen microbial COG in adult buffaloes and breastfed calves. The yellow color represents breastfed buffaloes, and the green color represents adult buffaloes. <bold>(D)</bold> Heatmap of differential KEGG tertiary pathways related to immunity in rumen microbes of adult buffalo and breastfed calves and the secondary pathways to which they belong.</p>
</caption>
<graphic xlink:href="fmicb-15-1342804-g003.tif"/>
</fig>
<p>We also compared the KEGG database (<xref ref-type="fig" rid="fig3">Figure 3D</xref>; <xref ref-type="supplementary-material" rid="SM1">Supplementary Table S1</xref>). We found that amino acid metabolic pathways including alanine, aspartate, and glutamate metabolism; glycine, serine, and threonine metabolism; cysteine and methionine metabolism; valine, leucine, and isoleucine degradation; valine, leucine, and isoleucine biosynthesis; lysine biosynthesis; arginine and proline metabolism; tyrosine metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis were significantly higher in breastfed buffaloes than in adult buffaloes in terms of nutrient utilization. In immunity (<xref ref-type="fig" rid="fig3">Figure 3D</xref>; <xref ref-type="supplementary-material" rid="SM1">Supplementary Table S1</xref>), pathways related to antibiotic synthesis (carbapenem biosynthesis, monobactam biosynthesis, novobiocin biosynthesis, penicillin, cephalosporin biosynthesis, phenazine biosynthesis, and streptomycin biosynthesis) were significantly higher in breastfed buffalo than in adult buffalo, and immune signaling pathways (Toll-like receptor signaling pathway, Toll-like receptor signaling pathway, natural killer cell-mediated cytotoxicity, mTOR signaling pathway, leukocyte transendothelial migration, antigen processing and presentation, B cell receptor signaling pathway, T cell receptor signaling pathway, IL-17 signaling pathway, and Th17 cell differentiation) were significantly higher in adult buffalo than in breastfed buffalo. Adult buffalo exhibited greater binding to immune functions than breastfed buffalo, while the latter displayed higher amino acid metabolism and antibiotic synthesis. These findings suggest that rumen microbes play varying roles in immunity between adult and breastfed buffalo.</p>
<p>Overall, in terms of nutrition, rumen microbes in adult buffaloes are proficient in degrading lignocellulose and carbohydrates, while rumen microbes in suckling buffaloes are adept at degrading lactose and generating energy and amino acid metabolism. Regarding immunity, adult buffalo microbes rely on established immune signaling pathways, while rumen microbes in suckling buffaloes rely on self-produced antibiotics to combat pathogenic microbes.</p>
</sec>
<sec id="sec13">
<title>Spearman&#x2019;s correlation analysis of dominant microorganisms and functions in adult and breastfed buffaloes</title>
<p>We further explored the potential microbiota functional relationship with an analysis of all identified genera to assess their correlations, and genera exhibiting significant positive correlations were visually represented on a co-occurrence diagram (correlation&#x003E;0.5, <italic>p</italic>-value&#x003C;0.05, <xref ref-type="fig" rid="fig4">Figure 4</xref>). We found a clear trend of segregation of significantly different genera between the two groups, with stronger aggregation of significantly higher genera (green nodes) in the rumen of adult buffaloes and stronger aggregation among significantly higher genera (yellow nodes) in the rumen of breastfed calves, suggesting that we have a community effect and symbiotic relationship between microorganisms in the rumen of buffaloes at different growth stages (<xref ref-type="fig" rid="fig4">Figure 4</xref>).</p>
<fig position="float" id="fig4">
<label>Figure 4</label>
<caption>
<p>Plot of significant positive correlations between genera in the rumen of adult and breastfed buffaloes. The green dots represent all genera that were more abundant in adult buffaloes, the yellow dots represent all genera that were significantly more abundant in the rumen of breastfed buffaloes, the pink dots represent genera that did not differ between the two groups, and all the lines represent significant positive correlations between the two genera that are connected (correlation&#x003E;0.5, <italic>p</italic>-value&#x003C;0.05).</p>
</caption>
<graphic xlink:href="fmicb-15-1342804-g004.tif"/>
</fig>
<p>We correlated dominant genera and CAZymes and found that CAZymes associated with lignocellulose degradation (AA1, AA6, AA10, GH5, GH8, GH9, GH10, GH44, GH48, GH51, and GH74) were significantly positively correlated with dominant genera in adult buffaloes (correlation &#x003E;0.5, <italic>p</italic>-value&#x003C;0.05, <xref ref-type="fig" rid="fig5">Figure 5A</xref>), and CAZymes associated with lactose degradation (GH1, GH35, GH42, GH59, and GH39) were significantly positively correlated with dominant genera in breastfed buffaloes (correlation &#x003E;0.5, <italic>p</italic>-value&#x003C;0.05). This suggests that microorganisms in the rumen of adult and breastfed buffaloes have a clear preference for substrate digestion. A large proportion of CAZymes was significantly positively correlated both with dominant genera in the rumen of adult buffaloes and with dominant genera shared by both groups (Prevotella, UBA4372, RUG472, and F082). Many of these CAZymes were lignocellulose degradation-related; for example, AA3, GH6, and GH12 were significantly positively correlated with Prevotella and UBA4372. This implies that the rumen microorganisms of our breastfed buffaloes also have some lignocellulose degrading ability, and the pre-weaning colonization of rumen microorganisms provides sufficient preparation for the calves to feed on forage and pasture later on.</p>
<fig position="float" id="fig5">
<label>Figure 5</label>
<caption>
<p>Correlation analysis of rumen microorganisms and function in adult and breastfed buffaloes. <bold>(A)</bold> Network diagram of significant positive correlation (correlation&#x003E;0.5, <italic>p</italic>-value&#x003C;0.05) between rumen microorganisms and CAZymes in adult and breastfed buffaloes. The green dots represent the dominant genera in adult buffaloes, the yellow dots represent the dominant genera in breastfed buffaloes, the red dots represent the co-dominant genera in both groups, and the blue dots represent the CAZymes. The green line connects the dominant genera specific to adult buffaloes with the CAZymes that are significantly positively correlated with them, and the yellow line connects the dominant genera specific to breastfed buffaloes with the CAZymes that are significantly positively correlated with them. CAZymes, the red lines connect the dominant genera, and CAZymes significantly positively correlated with both groups &#x003E;1%. <bold>(B)</bold> Bubble heatmap of rumen microbial and immune-related KEGG tertiary pathway significantly positively correlated (correlation&#x003E;0.5, <italic>p</italic>-value&#x003C;0.05) in adult and breastfed buffaloes. Horizontal coordinates represent genera, and vertical coordinates are KEGG level 3 pathways. The size of the bubbles represents the level of significance; the smaller the <italic>p</italic>-value, the larger the bubbles, and the redder the color of the bubbles, the higher the correlation coefficient.</p>
</caption>
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</fig>
<p>Then, we found out that pathways associated with amino acid metabolism, energy production, and antibiotic production were significantly positively correlated with the dominant genus of bacteria in the rumen of breastfed buffaloes. Interestingly, antibiotic production appeared to be dependent on amino acid metabolism and energy production. For example, the intermediate chorismate of phenylalanine, tyrosine, and tryptophan biosynthesis is required as an initial substance for phenazine biosynthesis, and both pathways are significantly positively correlated with Succinivibrio and UBA629. The dominant genera, specific to adult buffaloes, were significantly positively correlated with the previously mentioned immune-related pathways (correlation &#x003E;0.5, <italic>p</italic>-value&#x003C;0.05, <xref ref-type="fig" rid="fig5">Figure 5B</xref>). The positive correlation between UBA4372 (correlation &#x003E;0.5, <italic>p</italic>-value&#x003C;0.05, <xref ref-type="fig" rid="fig5">Figure 5B</xref>), which is the dominant genus in the rumen of both adult and breastfed buffaloes, and the immune signaling pathways (Toll-like receptor signaling pathway, Toll-like receptor signaling pathway, natural killer cell-mediated cytotoxicity, mTOR signaling pathway, and leukocyte transendothelial migration) that do not require antigenic stimulation suggests that UBA4372 may play an important role in the rumen immunity of buffaloes at different stages of growth.</p>
<p>Based on the above analysis, the rumen microorganisms of breastfed buffaloes have their own unique physiological functions, and the synthesis of amino acids and antibiotics is genus-specific. However, we did not find any genus that was significantly positively correlated with all metabolic pathways in buffaloes. We speculate that the rumen microorganisms of breastfed buffaloes may need to cooperate with each other to provide energy, produce and metabolize amino acids, and produce antimicrobial substances. This suggests that the nutritional requirements of breastfed buffaloes should be high in energy and protein, which is not only important for the growth and development of buffaloes but also for their resistance to diseases during the breastfeeding period.</p>
</sec>
</sec>
<sec sec-type="discussion" id="sec14">
<title>Discussion</title>
<p>In this study, we analyzed the microbiome data of rumen contents from 29 adult female buffaloes that were provided with feed, along with 18 breastfed buffaloes. The abundance (Shannon index) and similarity (NMDS) of rumen microorganisms were significantly higher in adult buffaloes than in breastfed buffaloes, and this pattern was also observed in dairy cows (<xref ref-type="bibr" rid="ref7">Furman et al., 2020</xref>) and goats (<xref ref-type="bibr" rid="ref4">Cao et al., 2023</xref>). This can be due to the fact that the microorganisms are still in the colonization stage at early life stages and are influenced by various factors such as the mode of delivery pregnancy diet (<xref ref-type="bibr" rid="ref25">Lundgren et al., 2018</xref>), feeding mode (<xref ref-type="bibr" rid="ref39">Stearns et al., 2017</xref>), and environment (<xref ref-type="bibr" rid="ref5">Depner et al., 2020</xref>), so there is a wide variation within the group. Aging and highly similar diets contribute to richer and more similar rumen microorganisms in the rumen of adult buffaloes. This could be linked to the nutritional and immunological needs of adult and nursing buffaloes during this stage of their growth, as well as their strong association with the primary diet of adult and nursing buffaloes.</p>
<p>In terms of nutritional requirements, dietary differences have resulted in dominant rumen microbes specific to buffaloes at different growth stages. Significantly higher dominant microbes in the rumen of breastfed buffaloes were found to be positively correlated with lactose degradation-related CAZymes, amino acid metabolism pathways, and pathways of energy synthesis and utilization, and a low-fiber, high-energy, high-protein diet has been reported to elevate the levels of Succinivibrio (<xref ref-type="bibr" rid="ref43">Tang et al., 2019</xref>) in abundance. Selenomonas_C, CAG-791, Treponema_D, RC9, and UBA629. We speculate that these five genera also have the potential to degrade high-energy, high-protein substrates such as milk. Significantly higher dominant microorganisms in the rumen of adult buffaloes were all significantly positively correlated with CAZymes associated with lignocellulose degradation (<xref ref-type="fig" rid="fig6">Figure 6</xref>), with Prevotella (<xref ref-type="bibr" rid="ref29">Maus et al., 2020</xref>) and Saccharofermentans (<xref ref-type="bibr" rid="ref33">Perea et al., 2017</xref>) reported to be associated with lignocellulose degradation. UBA1711, F23-D06, UBA1777, Methanobrevibacter_A, RF16, Prevotella, and Saccharofermentans have co-associated and lignocellulose degradation-related CAZymes, so we suggest that these five genera may have lignocellulose degradation potential.</p>
<fig position="float" id="fig6">
<label>Figure 6</label>
<caption>
<p>Physiological functions assumed for rumen execution in adult and breastfed buffaloes.</p>
</caption>
<graphic xlink:href="fmicb-15-1342804-g006.tif"/>
</fig>
<p>In terms of immune requirements, a higher abundance of antibiotic synthesis pathways has been annotated in the rumen microbes of breastfed buffaloes. The products of these pathways, including penicillin, cephalosporin, carbapenem, monobactam, novobiocin, phenazine, and streptomycin (<xref ref-type="bibr" rid="ref28">Majumdar and Kutzner, 1962</xref>; <xref ref-type="bibr" rid="ref3">Bush and Bradford, 2016</xref>; <xref ref-type="bibr" rid="ref30">May et al., 2017</xref>; <xref ref-type="bibr" rid="ref12">Huigens et al., 2019</xref>; <xref ref-type="bibr" rid="ref1">Armstrong et al., 2021</xref>) are antibiotics known to have activity against pathogenic microorganisms. The immune system of breastfed buffaloes is not well developed, and the microorganisms in the rumen have not formed a strong bond with the organism, so they rely on antibiotics produced by some genera in the rumen to defend themselves against pathogenic microorganisms. Adult buffalo rumen microbes are more closely linked to the body&#x2019;s immune system, and higher abundances of antigen processing and presentation, the B cell receptor signaling pathway, the T cell receptor signaling pathway, the IL-17 signaling pathway, and Th17 cell differentiation contribute to the capture and presentation of antigens in the rumen (<xref ref-type="bibr" rid="ref17">Kaufmann and Schaible, 2005</xref>) and stimulate the activation of immune cells and immune factors, resulting in a series of immune responses.</p>
<p>Remarkably, amino acid metabolic pathways significantly positively correlated with the dominant rumen microorganisms of breastfed buffaloes, which appeared to have a significant impact on antibiotic synthesis (<xref ref-type="fig" rid="fig6">Figure 6</xref>). The intermediate chorismate of phenylalanine, tyrosine, and tryptophan biosynthesis is required for phenazine biosynthesis. Additionally, the intermediate L-proline (C00148) of arginine and proline metabolism serves as the initial compound for novobiocin biosynthesis. All four pathways were significantly positively correlated with UBA629, while UBA629 was significantly positively correlated with the most antibiotic synthesis pathways, suggesting that UBA629 may be the main antibiotic-producing genus in the rumen of breastfed buffaloes. Arginine enhances streptomycin production (<xref ref-type="bibr" rid="ref28">Majumdar and Kutzner, 1962</xref>), and in our results, we also found that streptomycin biosynthesis and arginine biosynthesis were both significantly positively correlated with Treponema_D. This suggests that our amino acid synthesis and metabolism are important for buffaloes during the breastfeeding period, in addition to maintaining their immunological development. Meanwhile, Luo et al. found that Treponema_D and goat immunity are positively correlated (<xref ref-type="bibr" rid="ref26">Luo et al., 2022</xref>), thus confirming our view from the side. Therefore, breastfed buffaloes can be fed a high protein diet to enhance immunity and maintain their growth needs and should not be supplemented with antibiotics to avoid damaging the rumen environment of breastfed buffaloes.</p>
<p>In conclusion, the composition and function of rumen microbes differed significantly between adult feed-fed and breastfed buffaloes. Adult buffaloes exhibited more similar rumen microbial compositions, with specific dominant genera that possess greater abilities to degrade lignocellulose and carbohydrates, as well as established immune pathways. The rumen microbial composition of breastfed buffaloes was more different, with particular dominant genera exhibiting greater benefits in lactose degradation, amino acid metabolism, and antibiotic synthesis. The contrasting dietary patterns of adult and breastfed buffaloes gave rise to differing dominant rumen microbes, which meet the nutritional and immune necessities of buffaloes at diverse stages of growth.</p>
</sec>
<sec sec-type="conclusions" id="sec15">
<title>Conclusion</title>
<p>In our study, we explored the composition and function of rumen microorganisms in adult and breastfed buffaloes. We discovered distinctive differences in the community and function of these microorganisms. Rumen microorganisms in adult buffalo primarily contribute to lignocellulose degradation and exhibit close associations with immune responses when exposed to antigens. In contrast, rumen microbes in breastfed buffaloes focus on galactose conversion and synthesizing antibiotics to resist pathogens. These variations arise from differences in growth stages and diets, offering valuable insights into the gut microorganisms of domestic animals.</p>
</sec>
<sec sec-type="data-availability" id="sec16">
<title>Data availability statement</title>
<p>Publicly available datasets were analyzed in this study. This data can be found here: the raw sequencing data used in this study are available in the NCBI SRA database under accession code PRJNA656389. The 4960 strain-level MAG data used for species annotation in this study are available in the figshare database under access code 17000302.</p>
</sec>
<sec sec-type="ethics-statement" id="sec17">
<title>Ethics statement</title>
<p>The animal studies were approved by Experimental Animal Ethics Committee, College of Animal Science and Technology, Guangxi University. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.</p>
</sec>
<sec sec-type="author-contributions" id="sec18">
<title>Author contributions</title>
<p>YX: Visualization, Validation, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization, Writing &#x2013; review &#x0026; editing, Writing &#x2013; original draft. TF: Methodology, Formal analysis, Data curation, Writing &#x2013; review &#x0026; editing. ZD: Investigation, Formal analysis, Data curation, Writing &#x2013; review &#x0026; editing. LL: Resources, Writing &#x2013; review &#x0026; editing. ZL: Supervision, Writing &#x2013; review &#x0026; editing. KC: Supervision, Writing &#x2013; review &#x0026; editing. WC: Supervision, Writing &#x2013; review &#x0026; editing. HP: Supervision, Project administration, Writing &#x2013; review &#x0026; editing. PZ: Supervision, Funding acquisition, Writing &#x2013; review &#x0026; editing. QL: Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization, Writing &#x2013; review &#x0026; editing.</p>
</sec>
</body>
<back>
<sec sec-type="funding-information" id="sec19">
<title>Funding</title>
<p>The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The present study was granted and supported by the Guangxi Science and Technology Major Project (Guike AA22068099), National Natural Science Fund (U20A2051), and Major Science and Technology Projects in Guangxi (AA18118041), and the Open Foundation of Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding (2019B030301010, 2019KSYS011).</p>
</sec>
<sec sec-type="COI-statement" id="sec20">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec sec-type="disclaimer" id="sec21">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated 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.</p>
</sec>
<sec sec-type="supplementary-material" id="sec22">
<title>Supplementary material</title>
<p>The Supplementary material for this article can be found online at: <ext-link xlink:href="https://www.frontiersin.org/articles/10.3389/fmicb.2024.1342804/full#supplementary-material" ext-link-type="uri">https://www.frontiersin.org/articles/10.3389/fmicb.2024.1342804/full#supplementary-material</ext-link></p>
<supplementary-material xlink:href="Image_1.TIF" id="SM1" mimetype="image/tiff" xmlns:xlink="http://www.w3.org/1999/xlink">
<label>SUPPLEMENTARY FIGURE S1</label>
<caption>
<p>Stacked plots of dominant rumen microorganisms in adult and breastfed buffaloes. <bold>(A)</bold> Phylum-level. <bold>(B)</bold> Family-level. <bold>(C)</bold> Genus-level.</p>
</caption>
</supplementary-material>
<supplementary-material xlink:href="Image_2.TIF" id="SM2" mimetype="image/tiff" xmlns:xlink="http://www.w3.org/1999/xlink">
<label>SUPPLEMENTARY FIGURE S2</label>
<caption>
<p>Differential microbiology in adult and breastfed buffaloes. <bold>(A)</bold> Phylum-level. <bold>(B)</bold> Family-level &#x003E;1%.</p>
</caption>
</supplementary-material>
<supplementary-material xlink:href="Image_3.TIF" id="SM3" mimetype="image/tiff" xmlns:xlink="http://www.w3.org/1999/xlink">
<label>SUPPLEMENTARY FIGURE S3</label>
<caption>
<p>Percentage stacking of family of rumen microorganisms of adult and breastfed buffaloes.</p>
</caption>
</supplementary-material>
<supplementary-material xlink:href="Image_4.TIF" id="SM4" mimetype="image/tiff" xmlns:xlink="http://www.w3.org/1999/xlink">
<label>SUPPLEMENTARY FIGURE S4</label>
<caption>
<p>Percentage stacking of genus of rumen microorganisms of adult and breastfed buffaloes.</p>
</caption>
</supplementary-material>
<supplementary-material xlink:href="Image_5.tif" id="SM5" mimetype="image/tiff" xmlns:xlink="http://www.w3.org/1999/xlink">
<label>SUPPLEMENTARY FIGURE S5</label>
<caption>
<p>Heatmap of the level 2 kegg pathway of differences between adult and breastfed buffaloes.</p>
</caption>
</supplementary-material>
<supplementary-material xlink:href="Image_6.TIF" id="SM6" mimetype="image/tiff" xmlns:xlink="http://www.w3.org/1999/xlink">
<label>SUPPLEMENTARY FIGURE S6</label>
<caption>
<p>Heatmap of correlation bubbles of dominant bacterial genera and KEGG class 3 pathways in adult and breastfed buffaloes.</p>
</caption>
</supplementary-material>
<supplementary-material xlink:href="Table_1.XLSX" id="SM7" mimetype="application/vnd.openxmlformats-officedocument.spreadsheetml.sheet" xmlns:xlink="http://www.w3.org/1999/xlink"/>
<supplementary-material xlink:href="Table_2.XLSX" id="SM8" mimetype="application/vnd.openxmlformats-officedocument.spreadsheetml.sheet" xmlns:xlink="http://www.w3.org/1999/xlink"/>
</sec>
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