The rumen is a complex microbial ecosystem in which lactic acid bacteria contribute to fermentation and microbial interactions, partly through bacteriocin production. However, the diversity and functional potential of rumen-derived bacteriocins remain insufficiently explored. In this study, ten Lactococcus lactis strains were isolated from Hanwoo cattle and evaluated for bacteriocin stability and antimicrobial activity. The bacteriocins exhibited high stability across a broad range of temperatures (40–121°C) and pH conditions (2–10), while remaining sensitive to proteolytic enzymes, confirming their proteinaceous nature. All isolates showed inhibitory activity against Streptococcus equinus and Listeria monocytogenes , significantly reduced S. equinus biofilm formation, and disrupted established streptococcal biofilms. Comparative genomic analysis of 99 lactococcal strains revealed extensive bacteriocin diversity, with subspecies-specific enrichment of garvicin Q and lactococcin A in Lactococcus cremoris , and nisin and sactipeptides in L. lactis . These findings highlight rumen-derived lactococci as robust sources of stable bacteriocins with potential applications in controlling pathogenic bacteria and biofilms, supporting their relevance as functional antimicrobial agents and probiotic candidates.

Asia is the largest producer of cattle, buffalo, sheep, and goats in the world, and these species are integral to smallholder livestock production systems, contributing significantly to food security, rural livelihoods, and greenhouse gas (GHG), especially methane (CH4) across the region. Livestock production in the region has increased rapidly due to population growth and shifts in diet towards animal products. This review outlines the latest information on ruminant GHG emissions and mitigation policies across diverse regions of Asia, Livestock production is dominated by China and India, which make a significant contribution to global CH4 emissions. Extensive grazing systems dominate in Central Asia. National policies, including the Methane Emissions Control Action Plan (2023), which is part of the national policies in China, the National Livestock Mission in India, and the 2030 reduction strategy in South Korea aim at reducing CH4 by enhancing feeding and manure management, as well as digital technologies. Improving animal husbandry practices represents a fundamental and effective approach to reducing methane emissions while enhancing overall productivity. Strengthening feeding strategies, animal health management, and production efficiency can lower emission intensity and improve system sustainability. Plant extracts, direct-fed microbials and red seaweed (Asparagopsis taxiformis) have demonstrated promising outcomes in CH4 reduction through feed-based interventions. Despite technological developments, there are obstacles due to low-quality feed, indigenous breeds, and a lack of modernization. In order to achieve low-emission livestock production in Asia, more investment, policy assistance, and regional cooperation are crucial.

The rumen microbiome significantly affects host performance, influencing feed efficiency, nitrogen utilization, and methane emission. However, conventional DNA-based marker gene sequencing cannot distinguish between viable and non-viable microbes, leading to inaccurate microbiota analyses. Viability PCR (v-PCR) with propidium monoazide (PMA) can inhibit DNA amplification from membrane-compromised cells, allowing the detection of viable microbes in rumen cultures. Therefore, this study aims to identify conditions for applying PMA in rumen culture experiments using qPCR and to examine its effect on the rumen microbial community using 16S rRNA gene sequencing in standard in vitro experiments. PMA treatment conditions were applied using a fivefold inoculum dilution, 100  µM PMA concentration, 30 min dark incubation, and 20 min light exposure, validated by a decrease in absolute abundance in heat-treated samples. When applied to in vitro rumen experiments, PMA treatment reduced bacterial evenness and induced shifts in key bacterial and archaeal taxa. Additionally, it affected major functional profiles of the microbiota. PMA treatment increased the relative abundance of Ruminobacter [log fold change (LFC) = 0.52] and Succinivibrio (LFC = 0.68) at 0 h (no incubation), along with Ruminobacter (LFC = 0.83) after 24 h of incubation, while decreasing that of Xylanibacter (LFC = -0.39) at 24 h. These shifts align with those of RNA-based studies showing higher Succinivibrionaceae abundance than Prevotellaceae, supporting the effectiveness of PMA in capturing active microbial dynamics. PMA-based v-PCR offers a reliable alternative to RNA-based methods, improving microbial community assessments and facilitating the identification of viability-associated microbial biomarkers in rumen studies.IMPORTANCEThis study identifies the optimal conditions for applying propidium monoazide (PMA) in in vitro rumen experiments to selectively amplify DNA from viable microorganisms while suppressing amplification from nonviable ones. PMA-based viability PCR (v-PCR) improves the accuracy of microbial community analysis by selectively detecting viable microorganisms, addressing the limitations of conventional DNA-based methods. Additionally, this approach provides a potential cost-effective alternative to RNA-based analyses, offering a practical tool for studying rumen microbial ecology.

Previous research has demonstrated that applying taxonomic weights to shotgun metagenomic data can improve species identification in 16S rRNA gene-based microbiome analysis. However, such an approach does not allow for accurate analysis of samples collected from less studied habitats, such as rumen. In the present study, we developed a method to incorporate taxonomic weights based on relative abundance of species identified from shotgun sequencing and amplicon sequencing data derived from rumen. Using this weighting method, we evaluated latest versions of five prominent databases—SILVA, Greengenes2 (GG2), RDP, NCBI RefSeq, and GTDB—against the BLAST 16S rRNA database, assessing classification counts, fully classified ratios (proportion of ASVs classified to a known genus and species), and error rates. Our results indicated that providing taxonomic weights partially increased classification counts and fully classified ratios, although the extent of improvement varied across databases. A reduction in error rates was also observed compared to the unweighted taxonomy classifier (P < 0.05). While GG2 and SILVA struggled with accurate classification at the species level owing to their inherent database characteristics, GTDB consistently improved all metrics using the manually weighted taxonomy classifier, achieving up to an 8% error rate reduction at the species level. NCBI RefSeq and RDP also exhibited remarkable improvement in the classification counts and fully classified ratios, along with error rate reductions by up to 47% at the species level. These findings demonstrate that amplicon sequencing datasets can enhance rumen microbiome analyses through effective weighting methods. While SILVA is commonly used in metataxonomic analyses of the rumen microbiome, we recommend NCBI RefSeq for species-level classification due to its superior accuracy and minimal ambiguous classification (e.g., “uncultured” or “sp.“) in future metataxonomic studies.

Ruminal ciliates are linked to methane production and nitrogen utilization efficiency in ruminants due to their association with other ruminal microorganisms. However, research on the specific interplay between ruminal bacteria and ciliates is still limited, particularly in different dietary conditions. This study examines the effect of the forage-to-concentrate (F:C) ratio on the ruminal bacteriome in vitro, focusing on bacteria associated with Isotricha spp. and small entodinia. The rumen fluid used as the inoculum for this experiment was collected from two cannulated Hanwoo cows. Dietary treatments included high-forage (HF, F:C of 7:3), high-concentrate (HC, F:C of 3:7), and control (CON, F:C of 5:5). After 24-hour incubation, fractions for entodinia-associated bacteria (EAB), Isotricha-associated bacteria (IAB), and total bacteria (TB) were collected for bacteriome analysis using QIIME2 with full-length 16S rRNA gene sequences on the PacBio system. All fermentation parameters, except for NH3-N, showed linear changes with increasing F:C ratios (p ≤ 0.05). F:C ratio affected Isotricha spp. and Dasytricha spp. counts. Ciliate-associated bacterial fractions were significantly less diverse than the total bacterial group, as indicated by richness, phylogenetic diversity, and evenness indices. This suggests potential specific associations within ciliate-provided microhabitats. Both diet and ciliate fractions significantly influenced the overall bacteriome (p ≤ 0.05). More bacteriome features were differentially abundant due to the ciliate fraction effect rather than diet (q ≤ 0.05). Our newly proposed washing procedure, using higher ciliate cell counts and minimal bacterial contamination, effectively removed free-living or loosely associated bacteria. This allows focus on ciliate-associated bacterial populations, which may include potential symbionts or engulfed bacteria of host ruminal ciliates. Verifying these associations could provide insights into rumen microbiome dynamics, nitrogen utilization, hydrogen balance, and microbiome variation under different F:C ratios.

Ruminants rely on the rumen for the anaerobic fermentation of fibrous plant materials, facilitated by a complex microbial community of bacteria, archaea, fungi, and ciliates. Among them, ruminal ciliates significantly influence ruminal fermentation, methane production, nitrogen utilization efficiency, and microbial interactions. This study examined the impact of ciliate inoculation on ruminal fermentation, microbial composition, and functional profiles in fauna-free conditions. Six treatments were tested: control (no ciliates), small entodinia, Epidinium spp., isotrichids, Ophryoscolex spp., and a mixed inoculum. Using QIIME2 to analyze 16S rRNA gene sequences, the study revealed group-specific effects on methane production, volatile fatty acids (VFAs), and microbial diversity. Small entodinia inoculation increased Streptococcus abundance, while isotrichids enriched Megasphaera, enhancing butyrate production. Alpha diversity indices indicated reduced richness in the ciliate-inoculated groups, reflecting predation on prokaryotes. Beta diversity showed distinct microbial and functional profiles among the treatments. Functional analysis highlighted elevated glycerolipid metabolism in isotrichid groups, associated with Bacteroides and Megasphaera, suggesting roles in lipid metabolism and oxidative stress resistance. Despite limited ciliate cell counts, the study emphasizes ciliate-specific interactions and the role of lactic acid-associated bacteria in shaping ruminal fermentation.

The gastrointestinal microbiome is pivotal to nutrient utilization and productivity in dairy cattle. This study compared the ruminal and fecal microbiomes of Holstein, Jersey, and Jeju Black cattle using 16S rRNA gene sequencing and three taxonomic classification databases to identify microbial differences across breeds. A total of 25 samples were analysed to evaluate microbial composition, diversity, functional phenotypes, and interactions. Jeju Black cattle exhibited significantly higher ruminal diversity (p < 0.001), whereas Holstein and Jersey cows had more diverse fecal microbiomes. Prevotella, Ruminococcus and Succinivibrio dominated the rumen of commercial breeds, while Methanobrevibacter and Desulfovibrio were enriched in Jeju Black cattle. Amino acid prototrophs were abundant in Holstein and Jersey feces, while sulfur-metabolizing microbial phenotypes predominated in the Jeju Black rumen, with implications for nitrogen metabolism and methane production. Microbial network analysis revealed Mogibacterium as a high-centrality genus in Holstein and Jersey cows, and Anaerovibrio in Jersey cows, potentially linked to propionate production. Jeju Black cattle displayed distinct microbial interactions involving sulphur-reducing and methanogenic bacteria. These results might be showing breed-related microbiome variability, potentially influencing feed efficiency, fermentation, and emissions. However, future work should standardize diets, feeding conditions, sampling methods, and include performance metrics to clarify breed-specificity of microbiome-function relationships.

Manipulation of the rumen microbial ecosystem in early life may affect ruminal fermentation and enhance the productive performance of dairy cows. The objective of this experiment was to evaluate the effects of dosing three different types of microbial inoculum on the rumen epithelium tissue (RE) transcriptome and the rumen epimural metatranscriptome (REM) in dairy calves. For this objective, 15 Holstein bull calves were enrolled in the study at birth and assigned to three different intraruminal inoculum treatments dosed orally once weekly from three to six weeks of age. The inoculum treatments were prepared from rumen contents collected from rumen fistulated lactating cows and were either autoclaved (control; ARF), processed by differential centrifugation to create the bacterial-enriched inoculum (BE), or through gravimetric separation to create the protozoal-enriched inoculum (PE). Calves were fed 2.5 L/d pasteurized waste milk 3x/d from 0 to 7 weeks of age and texturized starter until euthanasia at 9 weeks of age, when the RE tissues were collected for transcriptome and microbial metatranscriptome analyses, from four randomly selected calves from each treatment. The different types of inoculum altered the RE transcriptome and REM. Compared to ARF, 9 genes were upregulated in the RE of BE and 92 in PE, whereas between BE and PE there were 13 genes upregulated in BE and 114 in PE. Gene ontology analysis identified enriched GO terms in biological process category between PE and ARF, with no enrichment between BE and ARF. The RE functional signature showed different KEGG pathways related to BE and ARF, and no specific KEGG pathway for PE. We observed a lower alpha diversity index for RE microbiome in ARF (observed genera and Chao1 (p < 0.05)). Five microbial genera showed a significant correlation with the changes in host gene expression: Roseburia (25 genes), Entamoeba (two genes); Anaerosinus, Lachnospira, and Succiniclasticum were each related to one gene. sPLS-DA analysis showed that RE microbial communities differ among the treatments, although the taxonomic and functional microbial profiles show different distributions. Co-expression Differential Network Analysis indicated that both BE and PE had an impact on the abundance of KEGG modules related to acyl-CoA synthesis, type VI secretion, and methanogenesis, while PE had a significant impact on KEGGs related to ectoine biosynthesis and D-xylose transport. Our study indicated that artificial dosing with different microbial inocula in early life alters not only the RE transcriptome, but also affects the REM and its functions.

This study examined the effects of different forage sources on the ruminal bacteriome, growth performance, and carcass characteristics of Hanwoo steers during the fattening stage. In Korea, where high-concentrate feeding is common, selecting suitable forage is crucial for sustainable beef production. Fifteen 23-month-old Hanwoo steers, weighing an average of 679.27 ± 43.60 kg, were fed the following five different forage sources: oat hay (OAT), rye silage (RYE), Italian ryegrass (IRS), barley forage (BAR), and rice straw silage (RSS), alongside 1.5 kg of dry matter concentrate daily for five months. Carcass traits were evaluated post-slaughter, and rumen fluid samples were analyzed using full-length 16S rRNA gene sequencing to determine the bacteriome composition. The forage source significantly affected the alpha-diversity indices and bacteriome biomarkers linked to the feed efficiency and ruminal fermentation. Differences in the backfat thickness and meat yield index were noted, with alpha-diversity indices correlating with carcass traits. The phylum Planctomycetota, especially the family Thermoguttaceae, was linked to nitrogen fixation in high-protein diets like IRS, while the genus Limimorpha emerged as a biomarker for the meat yield. These findings highlight the importance of forage selection during late fattening to optimize beef production, considering diet and bacteriome shifts.

Methane production in ruminants is primarily due to the conversion of metabolic hydrogen (H2), produced during anaerobic microbial fermentation, into methane by ruminal methanogens. While this process plays a crucial role in efficiently disposes of H2, it also contributes to environmental pollution and eliminating methane production in the rumen has proven to be challenging. This study investigates the use of probiotics, specifically propionate-producing bacteria, to redirect accumulated H2 in a methane-mitigated environment. For this objective, we supplemented experimental groups with Lactiplantibacillus plantarum and Megasphaera elsdenii for the reinforced acrylate pathway (RA) and Selenomonas ruminantium and Acidipropionibacterium thoenii for the reinforced succinate pathway (RS), as well as a consortium of all four strains (CB), with the total microbial concentration at 1.0 × 1010 cells/mL. To create a methane-mitigated environment, 2-bromoethanesulfonate (BES) was added to all experimental groups at a dose of 15 mg/0.5 g of feed. BES reduced methane production by 85% in vitro, and the addition of propionate-producing bacteria with BES further decreased methane emission by up to 94% compared with the control (CON) group. Although BES did not affect the alpha diversity of the ruminal bacteriome, it reduced total volatile fatty acid production and altered beta diversity of ruminal bacteriota, indicating microbial metabolic adaptations to H2 accumulation. Despite using different bacterial strains targeting divergent metabolic pathways (RA and RS), a decrease in the dominance of the [Eubacterium] ruminantium group suggesting that both approaches may have a similar modulatory effect. An increase in the relative abundance of Succiniclasticum in the CB group suggests that propionate metabolism is enhanced by the addition of a propionate-producing bacterial consortium. These findings recommend using a consortium of propionate-producing bacteria to manage H2 accumulation by altering the rumen bacteriome, thus mitigating the negative effects of methane reduction strategies.

Hanwoo and Jeju Black cattle (Jeju Black) are native breeds of Korean cattle. Jeju Black cattle are recognized as natural monuments and are known to exhibit slower growth rates compared to Hanwoo. While several studies have analyzed the genetic characteristics of these cattle, there has been limited research on the differences in their microbiome. In this study, rumen fluid was obtained from three Hanwoo steers and three Jeju Black steers, and three different diets (total mixed rations [TMRs] for growing, early fattening, and late fattening periods) were used as substrates for in vitro fermentation. The in vitro incubation was conducted for 3 h and 24 h following a 2 × 3 factorial arrangement. After both incubation periods, fermentation characteristics were analyzed, and ruminal microbiome analysis was performed using 16S rRNA gene sequencing, employing both QIIME2 and PICRUSt2. The results revealed significant differences in the ruminal microbiota due to the inoculum effect. At the phylum level, Patescibacteria and Synergistota were found to be enriched in the Jeju Black inoculum-treated group. Additionally, using different inocula also affected the relative abundance of major taxa, including Ruminococcus, Pseudoramibacter, Ruminococcaceae CAG-352, and the [Eubacterium] ruminantium group. These microbial differences induced by the inoculum may have originated from varying levels of domestication between the two subspecies of donor animals, which mainly influenced the fermentation and microbiome features in the early incubation stages, although this was only partially offset afterward. Furthermore, predicted commission numbers of microbial enzymes, some of which are involved in the biosynthesis of secondary metabolites, fatty acids, and alpha amylase, differed based on the inoculum effect. However, these differences may account for only a small proportion of the overall metabolic pathway. Conversely, diets were found to affect protein biosynthesis and its related metabolism, which showed differential abundance in the growing diet and were potentially linked to the growth-promoting effects in beef cattle during the growing period. In conclusion, this study demonstrated that using different inocula significantly affected in vitro fermentation characteristics and microbiome features, mainly in the early stages of incubation, with some effects persisting up to 24 h of incubation.

Ruminal ciliates are a fundamental constituent within the rumen microbiome of ruminant animals. The complex interactions between ruminal ciliates and other microbial guilds within the rumen ecosystems are of paramount importance for facilitating the digestion and fermentation processes of ingested feed components. This review underscores the significance of ruminal ciliates by exploring their impact on key factors, such as methane production, nitrogen utilization efficiency, feed efficiency, and other animal performance measurements. Various methods are employed in the study of ruminal ciliates including culture techniques and molecular approaches. This review highlights the pressing need for further investigations to discern the distinct roles of various ciliate species, particularly relating to methane mitigation and the enhancement of nitrogen utilization efficiency. The promotion of establishing robust reference databases tailored specifically to ruminal ciliates is encouraged, alongside the utilization of genomics and transcriptomics that can highlight their functional contributions to the rumen microbiome. Collectively, the progressive advancement in knowledge concerning ruminal ciliates and their inherent biological significance will be helpful in the pursuit of optimizing rumen functionality and refining animal production outcomes.

Rumen cannulation is a surgical technique used to collect rumen contents from ruminants. However, rumen cannulation surgery may potentially impact the composition of the rumen microbiota. This study aimed to examine the longitudinal alterations in the rumen microbiota composition of Hanwoo steers after cannulation surgery. In this study, eight Hanwoo steers were used; four steers underwent rumen cannulation surgery (cannulation group), while the remaining four were left intact (control group). Rumen samples were collected from all eight steers using the stomach tubing method on the day before surgery (day 0) and on postoperative days 1, 4, 7, 10, 14, 17, 21, 24, and 28, resulting in 80 samples (10 timepoints × 8 animals). The microbiota of all 80 samples were analyzed using 16S rRNA gene amplicon sequencing with Quantitative Insights into Microbial Ecology version 2 (QIIME2). There were no significant differences (p > 0.05) in all major phyla and most major genera representing at least 0.5% of total sequences across all 80 samples between the control and cannulation groups on the preoperative and postoperative days. However, while the alpha diversity indices did not differ (p > 0.05) between the two groups on the preoperative day, they significantly differed (p < 0.05) between the two groups on the postoperative days. Further, the overall microbial distribution based on both unweighted and weighted principal coordinate analysis plots significantly differed (p < 0.05) between the two groups on both the preoperative and postoperative days. Orthogonal polynomial contrasts indicated that major genera and microbial diversity in the cannulation group decreased following surgery but returned to their initial states by postoperative day 28. In conclusion, this study demonstrates that rumen cannulation surgery affects some major taxa and microbial diversity, suggesting that the rumen cannulation method can alter the composition of rumen microbiota in Hanwoo steers.

Accumulating evidence suggests that the gut microbiome is associated with asthma. However, altered gut microbiome in adult asthma is not yet well established. We aimed to investigate the gut microbiome profiles of adult asthmatic patients with symptomatic eosinophilic inflammation.

The gut-liver axis is at the forefront of host-microbial interactions given the proximity of liver to the gut and connection via portal circulation. In recent years, many studies in human and mouse models have demonstrated the existence of a significant microbial community embedded in diverse tissue types, including blood and liver. Yet, in cattle, the rumen microbiome has been the primary focus. The liver microbiome and its metabolic role in host health and performance remain largely unexplored. While there has been considerable work focusing on the liver of diseased cattle, the objective of this study was to evaluate, through meta-analysis, the commensal liver microbiome in various cattle breeds. To our knowledge, this is the first study in which a core liver microbiome has been described in cattle without overt liver disease. We discovered abundant microbial taxa in the liver, varying by host age, species, and developmental stage. Eight bacterial phyla (Actinobacteria, Bacteroidetes, Cyanobacteria, Deinococcus-Thermus, Firmicutes, Fusobacteria, Proteobacteria, and Tenericutes) were found to be the core microbial taxa, representing almost half of the total liver bacterial population. Additionally, we identified several KEGG pathways with significant association with cattle age. This study provides a baseline knowledge of the liver microbiome as identified by metatranscriptome sequencing in cattle. Besides finding the microbial taxa previously reported by studies using DNA-based, 16S rRNA amplicon sequencing methods, this study identified several core phyla that have not been reported in cattle liver, highlighting the improved sensitivity or ability in detecting microbes by RNA-over DNA-based methods.