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Introduction: The rumen microbial community plays a crucial role in the digestion and metabolic processes of ruminants. Although sequencing-based studies have helped reveal the diversity and functions of bacteria in the rumen, their physiological and biochemical characteristics, as well as their dynamic regulation along the digestion process in the rumen, remain poorly understood. Addressing these gaps requires pure culture studies to demystify the intricate mechanisms at play. Bacteria exhibit morphological differentiation associated with different species. Based on the difference in size or shape of microorganisms, size fractionation by filters with various pore sizes can be used to separate them., Methods: In this study, we used polyvinylidene difluoride filters with pore sizes of 300, 120, 80, 40, 20, 8, 6, 2.1, and 0.6 μm. Bacterial suspensions were successively passed through these filters for the analysis of microbial population distribution using 16S rRNA gene sequences., Results: We found that bacteria from the different pore sizes were clustered into four branches (> 120 μm, 40-120 μm, 6-20 μm, 20-40 μm, and < 0.6 μm), indicating that size fractionation had effects on enriching specific groups but could not effectively separate dominant groups by cell size alone. The species of unclassified Flavobacterium, unclassified Chryseobacterium, unclassified Delftia , Methylotenera mobilis , unclassified Caulobacteraceae, unclassified Oligella , unclassified Sphingomonas , unclassified Stenotrophomonas , unclassified Shuttleworthia , unclassified Sutterella , unclassified Alphaproteobacteria, and unclassified SR1 can be efficiently enriched or separated by size fractionation., Discussion: In this study, we investigated the diversity of sorted bacteria populations in the rumen for preliminary investigations of the relationship between the size and classification of rumen bacteria that have the potential to improve our ability to isolate and culture bacteria from the rumen in the future., Competing Interests: 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., (Copyright © 2024 Liu, Zheng, Wang and Zhao.)

The rumen microbiota and metabolites play an important role in energy metabolism and immune regulation of the host. However, the regulatory mechanism of rumen microbiota and metabolite interactions with host on Tibetan sheep's plateau adaptability is still unclear. We analyzed the ruminal microbiome and metabolome, host transcriptome and serum metabolome characteristics of Tibetan sheep at different ages. Biomarkers Butyrivibrio , Lachnospiraceae&#95;XPB1014&#95;group , Prevotella , and Rikenellaceae&#95;RC9&#95;gut&#95;group were found in 4 months, 1.5 years, 3.5 years, and 6 years Tibetan sheep, respectively. The rumen microbial metabolites were mainly enriched in galactose metabolism, unsaturated fatty acid biosynthesis and fatty acid degradation pathways, and had significant correlation with microbiota. These metabolites further interact with mRNA, and are co-enriched in arginine and proline metabolism, metabolism of xenobiotics by cytochrome P450, propanoate metabolism, starch and sucrose metabolism, gap junction pathway. Meanwhile, serum metabolites also have a similar function, such as chemical carcinogenesis - reactive oxygen species, limonene and pinene degradation, and cutin, suberine and wax biosynthesis, thus participating in the regulation of the body's immune and energy-related metabolic processes. This study systematically revealed that rumen microbiota, metabolites, mRNA and serum metabolites of Tibetan sheep were involved in the regulation of fermentation metabolic function and immune level of Tibetan sheep at different ages, which provided a new perspective for plateau adaptability research of Tibetan sheep at different ages., Competing Interests: 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., (Copyright © 2024 Sha, Liu, He, Zhao, Hu, Wang, Li, Shao, Wang, Chen, Yang and Xie.)

Introduction: Milk fat is the most variable nutrient in milk, and recent studies have shown that rumen bacteria are closely related to milk fat. However, there is limited research on the relationship between rumen bacteria and milk fatty. Fatty acids (FAs) are an important component of milk fat and are associated with various potential benefits and risks to human health., Methods: In this experiment, forty-five healthy Holstein dairy cows with alike physiological and productive conditions were selected from medium-sized dairy farms and raised under the same feeding and management conditions. The experimental period was two weeks. During the experiment, raw milk and rumen fluid were collected, and milk components were determined. In this study, 8 high milk fat percentage (HF) dairy cows and 8 low milk fat percentage (LF) dairy cows were selected for analysis., Results: Results showed that the milk fat percentage in HF group was significantly greater than that of the dairy cows in the LF group. 16S rRNA gene sequencing showed that the rumen bacterial abundance of HF dairy cows was significantly higher than that in LF dairy cows; at the genus level, the bacterial abundances of Prevotellaceae&#95;UCG-001, Candidatus&#95;Saccharimonas, Prevotellaceae&#95;UCG-003 , Ruminococcus &#95;1, Lachnospiraceae&#95;XPB1014&#95;group , Lachnospiraceae&#95;AC2044&#95;group , probable&#95;genus&#95;10 and U29-B03 in HF group were significantly higher than those in the LF group. Spearman rank correlation analysis indicated that milk fat percentage was positively related to Prevotellaceae&#95;UCG-001, Candidatus&#95;Saccharimonas, Prevotellaceae&#95;UCG-003, Ruminococcus &#95;1, Lachnospiraceae&#95;XPB1014&#95;group , Lachnospiraceae&#95;AC2044&#95;group , probable&#95;genus&#95;10 and U29-B03 . Furthermore, Prevotellaceae&#95;UCG-001 was positively related to C14:0 iso, C15:0 iso, C18:0, Ruminococcus &#95;1 with C18:1 t9, Lachnospiraceae&#95;AC2044&#95;group with C18:1 t9 and C18:1 t11, U29-B03 with C15:0 iso., Discussion: To sum up, rumen bacteria in dairy cows are related to the variation of milk fat, and some rumen bacteria have potential effects on the deposition of certain fatty acids in raw milk., Competing Interests: 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., (Copyright © 2023 Si, Liu, Huang, Chen, Yang, Wu, Li, Tang, Zhao, Zheng, Zhang and Wang.)

Introduction: Urea is an important non-protein nitrogen source for ruminants. In the rumen, ureolytic bacteria play critical roles in urea-nitrogen metabolism, however, a few ureolytic strains have been isolated and genomically sequenced. The purpose of this study was to isolate a novel ureolytic bacterial strain from cattle rumen and characterize its genome and function., Methods: The ureolytic bacterium was isolated using an anaerobic medium with urea and phenol red as a screening indicator from the rumen fluid of dairy cattle. The genome of isolates was sequenced, assembled, annotated, and comparatively analyzed. The pan-genome analysis was performed using IPGA and the biochemical activity was also analyzed by test kits., Results: A gram-positive ureolytic strain was isolated. Its genome had a length of 4.52 Mbp and predicted genes of 4223. The 16S rRNA gene and genome GTDB-Tk taxonomic annotation showed that it was a novel strain of Enterobacter hormaechei , and it was named E. hormaechei Z129. The pan-genome analysis showed that Z129 had the highest identity to E. hormaechei ATCC 49162 with a genome average nucleotide identity of 98.69% and possessed 238 unique genes. Strain Z129 was the first E. hormaechei strain isolated from the rumen as we know. The functional annotation of the Z129 genome showed genes related to urea metabolism, including urea transport ( urt A -urt E), nickel ion transport ( ure J, ton B, nix A, exb B, exb D, and rcn A), urease activation ( ure A-ureG) and ammonia assimilation ( gdh A, gln A, gln B, gln E, gln L, gls A, glt B, and glt D) were present. Genes involved in carbohydrate metabolism were also present, including starch hydrolysis ( amy E), cellulose hydrolysis ( cel B and bgl X), xylose transport ( xyl F -xyl H) and glycolysis ( pgi , pgk , fba A, eno , pfk A, gap , pyk , gpm L). Biochemical activity analysis showed that Z129 was positive for alkaline phosphatase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-glucosidase, β-glucosidase, and pyrrolidone arylaminase, and had the ability to use D-ribose, L-arabinose, and D-lactose. Urea-nitrogen hydrolysis rate of Z129 reached 55.37% at 48 h of incubation., Discussion: Therefore, the isolated novel ureolytic strain E. hormaechei Z129 had diverse nitrogen and carbon metabolisms, and is a preferred model to study the urea hydrolysis mechanism in the rumen., Competing Interests: 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., (Copyright © 2023 Zhong, Zheng, Wang and Zhao.)

Biochanin A (BCA), an isoflavone phytoestrogen, is a secondary metabolite produced mainly in leguminous plants. The objective of this study was to evaluate the effect of BCA on lactation performance, nitrogen metabolism, and the health of dairy goat. Thirty mid-lactation Saanen dairy goats were divided into three groups randomly: control, 2 g/d BCA group, and 6 g/d BCA group. After 36 days of feeding, 30 dairy goats were transferred to individual metabolic cages. Subsequently, milk yield, feed intake, total feces, and urine excretion were recorded and samples were collected continuously for 3 days. Blood and ruminal fluid samples were collected over the subsequent 4 days. Milk yield, milk protein, fat content, and the feed conversion ratio of dairy goat were significantly increased by the BCA treatment. The levels of serum 17β-estradiol, growth hormone, insulin-like growth factor 1, glutathione peroxidase activity, and total antioxidant capacity were also increased significantly by BCA, indicating that BCA enhanced the antioxidant capacity of dairy goat. Amino acid degradation was significantly inhibited, while the ammonia nitrogen content was reduced significantly by BCA. Total volatile fatty acids was significantly increased by BCA supplementation. In addition, the relative abundance of Verrucomicrobiota was decreased significantly. However, the growth of nitrogen metabolism and cellulolytic bacteria was significantly increased under BCA treatment, including Prevotella sp ., Treponema sp. , Ruminococcus flavefaciens , and Ruminobacter amylophilus . In conclusion, supplementation with BCA improved the milk production performance, nitrogen metabolism, rumen fermentation and antioxidant capacity, and regulated the rumen microbiome of dairy goat., Competing Interests: 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., (Copyright © 2023 Xu, Li, Du, Zheng, Wang and Zhao.)

High nitrogen utilization efficiency (NUE) is important for increasing milk protein production and decreasing the feed nitrogen cost and nitrogen emission to the environment. Currently, there is a limited whole picture of the relationship between ruminal bacteriome and the NUE of dairy cows, even though some information has been revealed about the bacteriome and milk or milk protein production of dairy cows. The purpose of this study was to compare the rumen bacterial community in dairy cows with different nitrogen utilization efficiency under the same diet. The natural abundance of 15 N between the animal proteins and diet (Δ 15 N) was used as a simple, non-invasive, and accurate biomarker for NUE in ruminants to mark the individual variation. Dairy cows with high NUE (HE&#95;HP, n = 7), medium NUE (ME&#95;MP, n = 7), and low NUE (LE&#95;LP, n = 7) were selected from 284 Holstein dairy cows with the same diet. Measurement of the rumen fermentation indices showed that the proportion of propionate was higher in HE&#95;HP cows and ME&#95;MP cows than in LE&#95;LP cows ( P < 0.05). The diversity of rumen bacterial community was higher in LE&#95;LP cows than in ME&#95;MP cows and HE&#95;HP cows by 16S rRNA sequencing analysis ( P < 0.05). Moreover, at the genus level, the relative abundances of Succinivibrionaceae &#95;UCG&#95;001, uncultured&#95; Selenomonadaceae , and Acidaminococcus were higher in HE&#95;HP cows than in LE&#95;LP cows ( P < 0.05). Interestingly, we found that these bacteria were positively correlated with milk protein yield and negatively correlated with Δ 15 N ( P < 0.05). However, Clostridia &#95;UCG&#95;014, Saccharofermentans , Bacilli &#95;RF39, and Desulfovibrio were lower in HE&#95;HP cows and ME&#95;MP cows than in LE&#95;LP cows ( P < 0.05), which were negatively correlated with milk protein yield and positively correlated with Δ 15 N ( P < 0.05). In conclusion, the study showed that the diversity and relative abundances of rumen bacteria differed among different NUE cows, indicating that rumen bacteriome contributes to nitrogen metabolism in dairy cows., Competing Interests: 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., (Copyright © 2022 Li, Zhong, Li, Zheng, Wang and Zhao.)

In this study, we investigated the species composition and diversity of psychrotrophic bacteria in raw milk from Heilongjiang, Inner Mongolia, Gansu, Henan, Anhui, Jiangsu, Chongqing, and Hunan provinces in China using traditional cultivation and PacBio Single Molecule Real-Time sequencing methods. The isolated psychrotrophic bacteria were highly diverse, which composed of 21 genera and 59 species. Pseudomonas accounted for 58.9% of the total genera while Stenotrophomonas and Enterococcus were also highly represented (above 5.0%). In particular, P. azotoformans occurred at a level of 16.9% and P. paralactis, P. lactis, E. faecalis, and P. marginalis were present in relatively high proportions (above 4.0%). Regional differences were found significantly among the test regions except samples from Heilongjiang and Inner Mongolia were similar. Additionally, differences were observed between days in Henan, Anhui, and Jiangsu samples. Therefore, control strategies must be implemented on regional and season basis., Competing Interests: 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., (Copyright © 2022 Du, Meng, Liu, Zheng, Zhang, Zhao and Wang.)

Tibetan pigs show a widespread distribution in plateau environments and exhibit striking physiological and phenotypic differences from others pigs for adaptation to hypoxic conditions. However, the regulation of mRNAs and metabolites as well as their functions in the alveolar type II epithelial (ATII) cells of Tibetan pigs remain undefined. Herein, we carried out integrated metabolomic and transcriptomic profiling of ATII cells between Tibetan pigs and Landrace pigs across environments with different oxygen levels to delineate their signature pathways. We observed that the differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs) profiles displayed marked synergy of hypoxia-related signature pathways in either Tibetan pigs or Landrace pigs. A total of 1,470 DEGs shared between normoxic (TN, ATII cells of Tibetan pigs were cultured under 21% O 2 ; LN, ATII cells of Landrace pigs were cultured under 21% O 2 ) and hypoxic (TL, ATII cells of Tibetan pigs were cultured under 2% O 2 ; LL, ATII cells of Landrace pigs were cultured under 2% O 2 ) groups and 240 DAMs were identified. Functional enrichment assessment indicated that the hypoxia-related genes and metabolites were primarily involved in glycolysis and aldosterone synthesis and secretion. We subsequently constructed an interaction network of mRNAs and metabolites related to hypoxia, such as guanosine-3', 5'-cyclic monophosphate, Gly-Tyr, and phenylacetylglycine. These results indicated that mitogen-activated protein kinase (MAPK) signaling, aldosterone synthesis and secretion, and differences in the regulation of MCM and adenosine may play vital roles in the better adaptation of Tibetan pigs to hypoxic environments relative to Landrace pigs. This work provides a new perspective and enhances our understanding of mRNAs and metabolites that are activated in response to hypoxia in the ATII cells of Tibetan pigs., Competing Interests: 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., (Copyright © 2022 Yang, Yuan, Liu, Wang, Li, Ren, Gao, Jiao, Cai and Zhao.)

Flaxseed is rich in α-linolenic acid (ALA) and can increase omega-3 polyunsaturated fatty acid in the milk of dairy cows. However, the response of rumen fermentation to different forms of flaxseed supplementation is unknown. This study aimed to investigate the effect of different forms of flaxseed on the fatty acid profile, fermentation, and composition of bacteria in the rumen of dairy cows. In total, 30 Holstein dairy cows were selected and randomly assigned into three groups (10/group). Cows were fed a basal diet (control check; CK) or basal diets supplemented with either 1,500 g per day whole flaxseed (WF) or 1,500 g per day ground flaxseed (GF). The WF group had the highest ALA content in rumen fluid, whereas no difference was found between the CK and GF groups. However, the molar proportion of acetate increased in the WF and GF groups and was the highest in the GF group, and a similar trend was shown by propionate, isobutyrate, butyrate, isovalerate, and valerate (CK < WF < GF). The abundance of Ruminococcaceae&#95; NK4A214 &#95;group , Christensenellaceae&#95; R-7 &#95;group , and Eubacterium&#95;coprostanoligenes&#95;group also showed the same trend (CK < WF < GF). Different forms of flaxseed release ALA by different mechanisms in the rumen, and the molar proportions of volatile fatty acids and the bacterial composition were potentially influenced mainly by the amount of ALA released into the rumen., Competing Interests: 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., (Copyright © 2021 Huang, Guo, Chang, Liu, Tang, Zheng, Zhao, Zhang and Wang.)

To adapt to a low-oxygen environment, Tibetan pigs have developed a series of unique characteristics and can transport oxygen more effectively; however, the regulation of the associated processes in high-altitude animals remains elusive. We performed mRNA-seq and miRNA-seq, and we constructed coexpression regulatory networks of the lung tissues of Tibetan and Landrace pigs. HBB, AGT, COL1A 2, and EPHX 1 were identified as major regulators of hypoxia-induced genes that regulate blood pressure and circulation, and they were enriched in pathways related to signal transduction and angiogenesis, such as HIF -1, PI 3 K - Akt, mTOR , and AMPK . HBB may promote the combination of hemoglobin and oxygen as well as angiogenesis for high-altitude adaptation in Tibetan pigs. The expression of MMP 2 showed a similar tendency of alveolar septum thickness among the four groups. These results indicated that MMP 2 activity may lead to widening of the alveolar wall and septum, alveolar structure damage, and collapse of alveolar space with remarkable fibrosis. These findings provide a perspective on hypoxia-adaptive genes in the lungs in addition to insights into potential candidate genes in Tibetan pigs for further research in the field of high-altitude adaptation., Competing Interests: 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., (Copyright © 2021 Yang, Yuan, Yang, Li, Gao, Jiao, Cai and Zhao.)

In the rumen of cattle, urease produced by ureolytic bacteria catalyzes the hydrolysis of urea to ammonia, which plays an important role in nitrogen metabolism and animal production. A high diversity of rumen bacterial urease genes was observed in our previous study; however, information on urease protein diversity could not be determined due to technical limitations. Here, we developed a targeted meta-proteomic pipeline to analyze rumen urease protein diversity. Protein extraction (duration of cryomilling in liquid nitrogen), protein digestion state (in-solution or in-gel), and the digestion enzyme used (trypsin or Glu-C/Lys-C) were optimized, and the digested peptides were analyzed by LC-MS/MS. Four minutes was the best duration for cryomilling and yielded the highest urease activity. Trypsin digestion of in-gel proteins outperformed other digestion methods and yielded the greatest number of identifications and superior peptide performance in regards to the digestion efficiency and high-score peptide. The annotation of peptides by PEAKS software revealed diversity among urease proteins, with the predominant proteins being from Prochlorococcus , Helicobacter , and uncultured bacteria. In conclusion, trypsin digestion of in-gel proteins was the optimal method for the meta-proteomic pipeline analyzing rumen microbial ureases. This pipeline provides a guide for targeted meta-proteomic analyses in other ecosystems., (Copyright © 2020 Zhang, Zhao, He, Zheng, Yan and Wang.)

The unclassified Succinivibrionaceae lineages are abundant in high yielding multiparous cows, and their presence is positively correlated with milk yield and fat percentage and reduces methane emissions. However, it is still unclear which species are associated with the most efficient feed nutrient utilization and productivity. Here, we used integrated whole genome sequencing and matrix-assisted laser desorption/ionization mass spectrometry, coupled with phenotypic and chemotaxonomic analysis, to characterize S. dextrinosolvens Z6, a species in Succinivibrionaceae isolated from the rumen. To assess the role of S. dextrinosolvens Z6 in nitrogen metabolism, cells grown in different nitrogen sources were analyzed by RNA sequencing. The whole genome sequence result revealed a genome size of 3.47 Mbp with 38.9% of G + C content. A total of 2993 encoding sequences account for 98%. The genes for regulating carbohydrate (10.6%) and amino acid (9%) transport and metabolism were the most abundant. ANI (Average nucleotide identity) showed that SD-Z6 was most closely related to SD-22B (99.96%). The whole genome alignment of SD-Z6 with SD-22B showed a more than 0.34 Mb nucleotide difference. Growth of SD-Z6 occurred at a temperature 36-42°C with an optimum at 39.7°C, pH 6-8; the optimum pH was 6.9 and with 0-1% (w/v) NaCl. The maximum growth (OD 600 0.825 ± 0.12) and microbial crude protein (MCP) (178.2 μg/ml) were observed in cells grown in amino acid. The maximum concentration of ammonia (3.96 ± 1.2) was observed in urea containing media and 1.06 mM (26.7% of the produced) remained after 24 h incubation. Activities of urease and glutamine synthase ( P < 0.01) and glutamate dehydrogenase ( P < 0.05) were significantly different in nitrogen and growth phase. Glutamate synthetase ( P < 0.01) was significantly different only at different growth phases. In total, 1246 differentially expressed genes (DEGs) were identified in all nitrogen. Among DEGs, 33 were related to nitrogen metabolism. Their expression correlated with nitrogen sources and the intensity of enzyme activity. This result enhances our understanding of the roles of Succinivibrionaceae in the efficient nitrogen utilization and on environmental protection., (Copyright © 2020 Hailemariam, Zhao and Wang.)

Psychrotrophic bacteria in raw milk are most well known for their spoilage potential and the economic losses they cause to the dairy industry. Food-related psychrotrophic bacteria are increasingly reported to have antibiotic resistance features. The aim of this study was to evaluate the resistance patterns of Pseudomonas spp. isolated from bulk-tank milk. In total, we investigated the antibiotic susceptibility profiles of 86 Pseudomonas spp. isolates from raw milk. All strains were tested against 15 antimicrobial agents. Pseudomonas isolates were most highly resistant to imipenem (95.3%), followed by trimethoprim-sulfamethoxazole (69.8%), aztreonam (60.5%), chloramphenicol (45.3%), and meropenem (27.9%). Their multiple antibiotic resistance (MAR) index values ranged from 0.0 to 0.8. Whole-genome sequencing revealed the presence of intrinsic resistance determinants, such as BcI, amp C-09, bla CTX-M, opr D, sul 1, dfr E, cat A1, cat B3, cat I, flo R, and cml V. Moreover, resistance-nodulation-cell division (RND) and ATP-binding cassette (ABC) antibiotic efflux pumps were also found. This study provides further knowledge of the antibiotic resistance patterns of Pseudomonas spp. in milk, which may advance our understanding of resistance in Pseudomonas and suggests that antibiotic resistance of Pseudomonas spp. in raw milk should be a concern., (Copyright © 2020 Meng, Liu, Lan, Dong, Hu, Zhao, Zhang, Zheng and Wang.)

Milk spoilage is caused by the presence of proteolytic enzymes produced by Pseudomonas spp. during storage at low temperatures. The aim of this study was to identify Pseudomonas spp. in raw milk and investigate their associated proteolytic properties at low temperatures. Raw milk samples ( n = 87) were collected from 87 bulk tanks in Shaanxi Province in China. Pseudomonas spp. were identified using Pseudomonas specific 16S, universal 16S rRNA sequencing, and rpo B gene sequencing. The proteolytic properties of Pseudomonas spp. were examined using milk agar, quantitative trinitrobenzenesulfonic acid assay, and by the presence of alkaline metallopeptidase gene ( apr X). A total 143 isolates from all 87 samples were confirmed as Pseudomonas , and were identified as belonging to 14 Pseudomonas species. Of these, 40 (28.0%) isolates revealed proteolysis on milk agar at 2°C, 74 (51.8%) at 4°C, 104 (72.7%) at 7°C, and 102 (71.3%) at 10°C. However, proteolytic activity of 45 (31.5%) isolates exceeded 2 μmol of glycine equivalents per mL at 7°C, followed by 43 (30.1%) at 10°C, 18 (12.6%) at 4°C, and 7 (4.9%) at 2°C. The results reveal proteolytic activity of Pseudomonas spp. present in milk and their spoilage potential at different temperatures.

Diarrhea is a leading cause of increased mortality in neonatal and young piglets. Aberration of the gut microbiota is one important factor in the etiology of piglet diarrhea. However, information regarding the structure and function of the gut microbiome in diarrheic neonatal piglets is limited. To investigate the composition and functional potential of the fecal microbiota in neonatal piglets, we performed 16S rRNA gene sequencing on 20 fecal samples from diarrheic piglets and healthy controls, and metagenomics sequencing on a subset of six samples. We found striking compositional and functional differences in fecal microbiota between diarrheic and healthy piglets. Neonatal piglet diarrhea was associated with increases in the relative abundance of Prevotella , Sutterella , and Campylobacter , as well as Fusobacteriaceae . The increased relative abundance of Prevotella was correlated with the reduction in Escherichia coli and the majority of beneficial bacteria that belonging to the Firmicutes phylum (e.g., Enterococcus , Streptococcus , Lactobacillus, Clostridium , and Blautia ) in diarrheic piglets. The differentially functional gene abundances in diarrheic piglets were an increase in bacterial ribosome, and contributed primarily by the genera Prevotella , this indicates a growth advantage of the Prevotella in diarrheic conditions. Additional functional gene sets were associated with the reduction of polyamine transport, monosaccharide and sugar-specific PTS transport, amino acid transport, and two-component regulatory system. These profiles likely impact the ability to transport and uptake nutrients, as well as the ability to fight microbial infections in the piglet gut ecosystem. This work identifies a potential role for Prevotella in the community-wide microbial aberration and dysfunction that underpins the pathogenesis of piglet diarrhea. Identification of these microbial and functional signatures may provide biomarkers of neonatal piglet diarrhea.

Ureolytic bacteria are key organisms in the rumen producing urease enzymes to catalyze the breakdown of urea to ammonia for the synthesis of microbial protein. However, little is known about the diversity and distribution of rumen ureolytic microorganisms. The urease gene ( ureC ) has been the target gene of choice for analysis of the urea-degrading microorganisms in various environments. In this study, we investigated the predominant ureC genes of the ureolytic bacteria in the rumen of dairy cows using high-throughput sequencing. Six dairy cows with rumen fistulas were assigned to a two-period cross-over trial. A control group ( n = 3) were fed a total mixed ration without urea and the treatment group ( n = 3) were fed rations plus 180 g urea per cow per day at three separate times. Rumen bacterial samples from liquid and solid digesta and rumen wall fractions were collected for ureC gene amplification and sequencing using Miseq. The wall-adherent bacteria (WAB) had a distinct ureolytic bacterial profile compared to the solid-adherent bacteria (SAB) and liquid-associated bacteria (LAB) but more than 55% of the ureC sequences did not affiliate with any known taxonomically assigned urease genes. Diversity analysis of the ureC genes showed that the Shannon and Chao1 indices for the rumen WAB was lower than those observed for the SAB and LAB ( P < 0.01). The most abundant ureC genes were affiliated with Methylococcaceae, Clostridiaceae, Paenibacillaceae, Helicobacteraceae, and Methylophilaceae families. Compared with the rumen LAB and SAB, relative abundance of the OTUs affiliated with Methylophilus and Marinobacter genera were significantly higher ( P < 0.05) in the WAB. Supplementation with urea did not alter the composition of the detected ureolytic bacteria. This study has identified significant populations of ureolytic WAB representing genera that have not been recognized or studied previously in the rumen. The taxonomic classification of rumen ureC genes in the dairy cow indicates that the majority of ureolytic bacteria are yet to be identified. This survey has expanded our knowledge of ureC gene information relating to the rumen ureolytic microbial community, and provides a basis for obtaining regulatory targets of ureolytic bacteria to moderate urea hydrolysis in the rumen.

Urea, a non-protein nitrogen for dairy cows, is rapidly hydrolyzed to ammonia by urease produced by ureolytic bacteria in the rumen, and the ammonia is used as nitrogen for rumen bacterial growth. However, there is limited knowledge with regard to the ureolytic bacteria community in the rumen. To explore the ruminal ureolytic bacterial community, urea, or acetohydroxamic acid (AHA, an inhibitor of urea hydrolysis) were supplemented into the rumen simulation systems. The bacterial 16S rRNA genes were sequenced by Miseq high-throughput sequencing and used to reveal the ureoltyic bacteria by comparing different treatments. The results revealed that urea supplementation significantly increased the ammonia concentration, and AHA addition inhibited urea hydrolysis. Urea supplementation significantly increased the richness of bacterial community and the proportion of ureC genes. The composition of bacterial community following urea or AHA supplementation showed no significant difference compared to the groups without supplementation. The abundance of Bacillus and unclassified Succinivibrionaceae increased significantly following urea supplementation. Pseudomonas, Haemophilus, Neisseria, Streptococcus, and Actinomyces exhibited a positive response to urea supplementation and a negative response to AHA addition. Results retrieved from the NCBI protein database and publications confirmed that the representative bacteria in these genera mentioned above had urease genes or urease activities. Therefore, the rumen ureolytic bacteria were abundant in the genera of Pseudomonas, Haemophilus, Neisseria, Streptococcus, Actinomyces, Bacillus, and unclassified Succinivibrionaceae. Insights into abundant rumen ureolytic bacteria provide the regulation targets to mitigate urea hydrolysis and increase efficiency of urea nitrogen utilization in ruminants.