In this study, beeswax was used to coat nitrate salts, and its utilization as a partial substitute for plant protein sources in ruminant diets was tested in vitro. The study compared the effects of dietary supplementation with beeswax-coated nitrate salts, uncoated nitrate, and a nitrate-free diet (control) on gas production kinetics, methane (CH4) production, fiber digestibility, nitrate metabolism, and ruminal fermentation characteristics. The study consisted of a 2 (source) × 2 (dose) + 1 factorial arrangement. Each nitrate source was used at two levels of 9 g and 18 g nitrate/kg dry matter (DM). The beeswax coating resulted in the gradual release of nitrate into the buffer medium, with up to 69.6% released after 24 h of incubation. The inclusion of nitrate sources in the diet decreased the proportion of CH4 in total produced gas (mL/L), CH4 production (mL/g incubated DM), and CH4 emission (mL/g truly degraded substrate) in a dose-dependent manner compared to the control group (P < 0.05). There was no effect (P > 0.10) of treatments on total gas production, asymptotic gas production, rate constant of gas production, and lag time. No significant differences (P > 0.10) were observed among treatments in ruminal pH, ammonia-N concentration, volatile fatty acids (VFA) profiles, protozoa populations, microbial biomass production, and diet degradability. However, ammonia-N concentration tended to increase (P = 0.087) with the coated nitrate source compared with the uncoated nitrate source. Concentrations of nitrate and nitrite in the incubation media tended to be dose-dependent (P = 0.058 and P = 0.051, respectively). Nitrate concentration was lower in the beeswax-coated nitrate source than in the uncoated source (P = 0.036), whereas nitrite concentration tended to be lower in the coated source (P = 0.054). These findings suggest potential for beeswax-coated nitrate as a slow-release non-protein nitrogen source; however, in vivo studies are required to confirm its effects on animal performance, nitrogen utilization, and safety.

Enteric methane emissions from cattle pose a significant environmental concern and represent a substantial energy loss for the animal, necessitating the development of effective mitigation strategies. The gastrointestinal microbiota plays a crucial role in determining both feed efficiency and methane production. Still, the specific microbial signatures that predict these traits across different production systems remain poorly understood. This study aimed to identify common predictive microbial biomarkers for feed efficiency and methane emissions using co-association network analysis across contrasting cattle production systems. Rumen liquid and faecal microbiota from 55 Charolais heifers (beef) and 56 Holstein cows (dairy) were analysed using 16S rRNA gene amplicon sequencing. Phenotypic data included feed efficiency, methane yield and acetate/propionate ratio. Co-association networks were constructed using Partial Correlation and Information Theory to identify amplicon sequence variants (ASVs) directly connected to phenotypes. Multiple regression analysis determined the minimal ASV sets required to achieve optimal predictive accuracy. Rumen microbiomes consistently showed superior predictive performance compared to faecal communities across all traits. Network-selected ASVs explained substantial phenotypic variance across traits and production systems (R²=0.45-0.84), consistently outperforming randomly selected ASVs by 0.13-0.44 R² units. The ACET:PROP ratio showed the highest predictive accuracy (R²=0.84 in Charolais rumen, 0.77 in Holstein rumen), while minimal ASV sets achieved 93-97% of the full model's performance using 33-65% fewer ASVs (6-17 ASVs). Bacteroidaceae was consistently enriched across phenotypes in rumen networks, regardless of production system. Contrary to expectations, most associations were production system-specific, with notable exceptions including negative correlations between the ACET:PROP ratio and Prevotella/Ruminococcus genera and negative associations with members of the Succinivibrionaceae family for methane-related traits. The anatomical site-specific and production system-specific nature of most associations underscores the importance of context-specific approaches.

IntroductionMethane, a greenhouse gas, is produced in the rumen microbiome of ruminants. Various nutritional strategies can reduce enteric methane (CH4) emissions from livestock, but it is unclear whether diet affects rumen microbiome similarly across ruminant species. The objective of this study was to determine whether cows and goats differ in their rumen microbial functional responses to dietary strategies based on starch and/or lipid supplementation, and whether these differences explain diet-associated variation in CH4 emissions under comparable experimental conditions.MethodsExperiments were conducted simultaneously, and both species received the same diet based on grassland hay and concentrate as the Control diet (CTL) or supplemented with corn oil and wheat starch (COS), marine algae powder (MAP) or hydrogenated palm oil (HPO). To identify biologically relevant features from the rumen microbial metatranscriptomes, we followed a five-step integrative statistical and network analysis pipeline, combining a network-based approach with clustering and supervised model fitting to associate differentially expressed genes and taxa with CH4 emissions in the rumen.ResultsThe COS diet lowered CH4 emissions in both cows and goats and altered their rumen microbiomes. However, the number of differentially expressed KEGG orthologs and differentially abundant OTUs identified in the COS vs CTL comparison was greater in cows than in goats. Moreover, clustering analysis revealed differences in network topology between ruminant species. In goats, CH4 emission reduction was strongly associated with genes involved in carbohydrate metabolism and methylotrophic and hydrogenotrophic pathways of methanogenesis; whereas in cows, hydrogenotrophic pathways were prominent. Additionally, sparse Partial Least Squares (sPLS) analysis identified species-specific discriminant microbial features.DiscussionOverall, these results show that host species modulates rumen microbial responses to diet and suggest that microbial interactions underlying CH₄ mitigation differ between cows and goats, although validation in independent studies involving larger sample sizes is required.

The cattle rumen microbiota represents a complex and dynamic ecosystem whose organization and relationship to host phenotypes are important for food security and environmental sustainability. We analyzed rumen microbiota profiles from 2,496 cattle representing five breeds and production systems across five countries, identifying microbial co-abundance groups termed Ruminosignatures. We detected fourteen distinct Ruminosignatures, including two consistently observed across all populations dominated by Prevotella and UBA2810. Additional Ruminosignatures showed breed- and diet-specific patterns and collectively explained 96-99% of variance in rumen microbial composition. Integrative cross-country analysis confirmed 10 out of 14 Ruminosignatures identified in cohort-specific analyses. Several Ruminosignatures were associated with methane emissions and feed efficiency traits and were partially under host genetic control, with heritability estimates ranging from 0.09 to 0.58. Structural equation modelling revealed consistent negative genetic and phenotypic correlations between the UBA2810-dominated Ruminosignature (RS_UBA2) and methane emissions across cohorts (rg = -0.40 to -0.65), with structural coefficients concordant in sign across all populations, supporting the expected direction of phenotypic response to selection on RS_UBA2. Meta-analysis confirmed positive associations of RS_UBA2 with average daily gain and negative associations with methane-related traits and feed conversion ratio. Functional genome-based predictions suggested RS_UBA2 may reduce methanogenesis through alternative hydrogen utilization pathways competing with methanogenic archaea. Production system type influenced both Ruminosignature occurrence and relationships with host phenotypes, emphasizing the relevance of context-specific strategies for microbiome modulation. Our findings highlight the potential of the Ruminosignatures framework for microbiome-informed breeding programs aimed at improving feed efficiency while reducing the environmental impact of cattle production.

La réduction des émissions de méthane entérique associées aux systèmes d’élevage de ruminants est l’un des objectifs de lutte contre le changement climatique pour de nombreux pays. Si les mesures directes des émissions de méthane entérique sont limitées dans leur déploiement à un petit nombre d’animaux, des méthodes indirectes de prédiction via le lait ou les fèces peuvent être appliquées à un grand nombre d’animaux dans des contextes variés. Ces approches permettent de développer de nouveaux modèles de sélection génétique ou de conduites d’élevage des petits et gros ruminants associées à des niveaux d’émissions de méthane entériques plus faibles. Les recherches pour comprendre les relations entre la diversité microbienne du rumen et les phénotypes des ruminants contribuent à l’identification de nouveaux leviers pour moduler les populations microbiennes et les flux d’hydrogène dans le rumen, avec l’ambition de réduire les émissions de méthane tout en préservant la production et la santé de l’animal. L’alimentation et la conduite des troupeaux constituent aussi des leviers d’intérêt, mais leurs effets sur la réduction des émissions de méthane entérique sont variables selon les contextes pédoclimatiques, notamment en régions chaudes. Des stratégies combinant différents leviers doivent être identifiées pour réduire efficacement les émissions de méthane sans compromettre la santé de l’animal et les services écosystémiques rendus par leurs systèmes d’élevage.

Enteric methane emission from dairy cattle is an environmental challenge. The most efficient mitigation strategies nowadays include the use of methanogenesis inhibitors that specifically target the rumen methanogens. Specific inhibitors, such as 3-nitrooxypropanol (3-NOP), reduce methane emissions without negative effects on the products of fermentation that serve as energy metabolites for the host. However, the concomitant effects of methanogenesis inhibition on rumen microbiota and host metabolism are poorly characterized. Thus, the objective of this study was to explore the association between rumen microbiota and host metabolism when methanogenesis is inhibited. Thirteen dairy cows were used as controls, and 12 were supplemented with 3-NOP for 6 weeks. Rumen microbiota composition and activity were characterized using metagenomics and metatranscriptomics. The host metabolism was assessed in a previous publication by a metabolomic analysis of the plasma. Microbiota data were used as explanatory variables of the metabolome data in a multiblock sparse partial least squares analysis. Overall, the association between rumen microbiota and host metabolism was moderate. Notwithstanding this, a few downregulated transcripts related to glycolysis, hydrogen transfer, and protein synthesis, together with a decrease in the proportion of taxa of the Oscillospirales order, showed a correlation with host one-carbon metabolites (|r| > 0.6). These associations raised novel hypotheses that remain to be elucidated, especially with regard to the effects of dihydrogen on the accumulation of microbial glycolysis and methanogenesis metabolite intermediates.IMPORTANCEDairy cattle produce a substantial amount of methane, a potent greenhouse gas. Several strategies have been designed to reduce methane production by targeting the rumen microbiota. One such strategy specifically inhibits methanogens with a molecule called 3-nitrooxypropanol. This study uses an integrative data analysis approach, combining rumen microbiota and host metabolome information, to explore the consequences of inhibiting methanogenesis on the holobiont. This provides additional holistic insight into the effect of methane mitigation strategies on dairy cattle.

Introduction:Separating calves from their mothers at birth is linked to calf welfare issues and disturbances in the mother-calf relationship. It can also disrupt the maturation of the digestive tract, affecting calf health. It has been demonstrated that separation at weaning allows for the optimal establishment of the ruminal microbiota, whereas separation at birth alters colonization dynamics. We postulated that 4 weeks of cow-calf contact, a potentially more socially acceptable, and economically pragmatic, management practice, would induce a similar development of ruminal microbiota to that observed with separation at weaning, thereby conferring benefits on calf health and growth. Methods:We studied three groups of 14 cow-calf pairs (Holstein and Montbéliarde breeds) with different cow-calf separation times: 4 weeks of contact with the mother (Mixed group), immediate separation (at birth, Control group) and delayed separation at weaning (11 weeks, Dam group). Rumen microbial colonization was monitored in 9 calves per group at 3, 10, 13, and 20 weeks of age using a metataxonomic approach. Body weight, diarrhea and respiratory disease were recorded to assess the calves' overall health. Serum IgG concentrations were also monitored. Results:No differences were observed between the groups in diarrhea or IgG concentration. The incidence of respiratory disease was lower in calves that remained in contact with their dams until weaning. After separation, the Mixed group exhibited an increased average daily gain. The metataxonomic analysis demonstrated that as calves aged, there was an increase in richness, accompanied by a corresponding increase in the number of shared microbial species over time between all groups. Nevertheless, three discrete development pathways were identified in the rumen bacterial communities, as evidenced by the differences in beta diversity between the groups over time. Additionally, the presence of the mother had a favorable effect on the transfer of beneficial microbiota during the early stages of life. However, this was offset by the elevated detection of potential pathogens at a later age in the Dam group. Conclusion:In this study, the rearing method exerted a profound and enduring influence on the gastrointestinal microbiota, with no discernible negative impact on health.

Microbial communities play critical roles in various ecosystems. Despite extensive research on the taxonomic and functional diversity of microbial communities, effective approaches to regulate targeted microbial functions remain limited. Here, we present an innovative methodology that integrates core enzyme identification, protein structural characterization, regulator virtual screening, and functional validation to achieve precise microbiome functional regulation. As a proof of concept, we focused on the regulation of urea decomposition by the rumen microbiota in ruminants. Through metagenomic analysis, we identified the core urease gene and its corresponding microbial genome (MAG257) affiliated with the unclassified Succinivibrionaceae, and reconstructed its complete gene cluster. Structural analysis of the urease catalytic subunit (UreC) via cryo-electron microscopy (cryo-EM) revealed detailed features of its active site, guiding molecular docking studies that identified epiberberine, a natural compound with potent urease inhibitory activity. Validation in a rumen simulation system demonstrated that epiberberine significantly reduced urea decomposition and enhanced nitrogen utilization. This study establishes a robust framework that combines structural biology and computational screening to achieve targeted microbiome functional regulation, offering a promising tool for microbiome engineering and broader applications in animal productivity, human health, environmental improvement, and biotechnology.

Given the role of the rumen microbiome in providing nutrients to the host ruminant, it is expected that rumen microbes contribute to inter-animal variations in feed efficiency. However, the link between microbial structure and an “efficient” host phenotype is unclear. We hypothesized that extreme residual feed intake (RFI) phenotypes would display distinctive microbiome features regardless of the diet. In this study, we selected the 32 most extreme RFI Charolais bulls from a cohort of 100 animals fed corn-silage (CS; n = 50) or grass-silage (GS; n = 50) based diets. Rumen samples were obtained 3 h after feeding, at slaughter, for fermentation and metataxonomic and metatranscriptomic microbial analysis. Volatile fatty acid profiles showed no differences between diets and between extreme RFI phenotypes (P > 0.05). Total bacteria and methanogen populations did not differ between extreme RFI phenotypes (P > 0.05), although methanogens expressed per liquid rumen digesta weight tended to decrease in the most efficient bulls compared to the least efficient ones (P = 0.10). The rumen microbial community structure differed between diets (P < 0.001), and between extreme RFI phenotypes in the GS diet. In the whole dataset, we identified Succiniclasticum, Saccharofermentans, Clostridia_258483 and CAG-238 as bacteria discriminant between extreme RFI phenotypes (q < 0.10). Within diets, these four genera were also discriminant in the GS diet and were all associated with the least efficient bulls. In contrast, in bulls fed the CS diet, only Saccharofermentans and Succiniclasticum were discriminant but they were positively associated with the most efficient bulls. Rumen microbial functional features did not differ between extreme RFI phenotypes but did differ between diets. In conclusion, the rumen microbiome was mainly influenced by diet, with the RFI phenotype being a marginal effector. Succiniclasticum, Saccharofermentans, Clostridia_258483, and CAG-238 were discriminant between extreme RFI phenotypes regardless of diet. However, the direction of the association with RFI was diet dependent, indicating a diet-RFI interaction and suggesting that these discriminating microbes may be suitable microbial indicator species for RFI only when considered in conjunction with the diet information.

Ruminants play an important role in global food security and nutrition. The rumen microbial community provides ruminants with a unique ability to convert human indigestible plant matter, into high quality edible protein. However, enteric CH4 produced in the rumen is both a potent GHG and a metabolizable energy loss for ruminants. As the rumen microbiome constitutes 15–40% of the inter-animal variation in enteric CH4 emissions, understanding the microbiological mechanisms underpinning ruminal methanogenesis and its interaction with the host animal, is crucial for developing CH4 mitigation strategies. Variation in the relative abundance of different microbial species has been observed in cattle with contrasting residual CH4 emission and CH4 yield with up to 20% of the variation in inter-animal CH4 emissions attributable to the presence of a small number of microbial species. The demonstration of ruminotypes associated with high or low CH4 emissions suggests that interactions within complex microbial consortia and with their host are a major source of variation in CH4 emissions. Consequently, microbiome-assisted genomic approaches are being developed to select low CH4 emitting cattle, with breeding values for enteric CH4 being included as part of national breeding programmes. Generating rumen microbiome data for use in selection programs is expensive, therefore, identifying microbial biomarkers in milk or plasma to develop predictive models which include microbial predictors in equations based on animal related data, is required. A better understanding of the rumen microbiome has also aided the development and refinements of anti-methanogenic feed additives. However, these strategies, which increase the amount of reducing equivalents in the rumen ecosystem, do not generally result in an enrichment of propionate or an improvement in animal performance. Current research aims to provide alternative sinks to reducing equivalents and to stimulate activity of commensal microbes or the supplementation of direct fed microbials to capture lost energy. Furthering our knowledge of the rumen microbiome and its interaction with the host, will aid in the development of CH4 mitigation strategies for ruminant livestock.

The aim of this study was to test the effect of 3-nitrooxypropanol (3-NOP) on methane emissions, animal performance, milk composition, and rumen fermentation in early-lactation dairy cows fed a forage-rich diet. A second objective was to assess the 13C isotopic signature of milk as a potential proxy for methane-emission status. Twenty-seven cows with similar BW and age in early lactation (7-11 DIM) and fed a 75% forage-based diet were selected and distributed to 2 balanced groups in a randomized block design. The treatment group (n = 13) received 3-NOP (60 mg/kg DM basis) in a TMR for 105 d and the control group (n = 14) received a placebo. The 3-NOP additive was included in a supplement formulated with propylene glycol and adsorbed on silicon dioxide. The control group received the same supplement without the additive. Individual daily methane emissions were quantified using the GreenFeed system throughout the study. Intake and milk production were recorded daily, and milk composition (fat, protein, lactose, urea) twice a week. Natural 13C abundance (δ13C) in milk and feed samples was determined using isotope-ratio MS coupled with elemental analysis for each cow at wk 3, 7, 11, and 15. Methane emissions, methane yield, and methane intensity were lower in the treatment group throughout the 105 d (on average -31% g/d, -24% g/kg DMI, and -30% g/kg ECM). By monitoring methane emissions throughout the day, we found that the main effect occurred after feeding, preventing the postprandial peak in methane emissions. Intake was reduced by ∼7% with 3-NOP, whereas milk production was similar between groups (34.7 kg ECM/d). Consequently, feed conversion efficiency in animals tended to increase with 3-NOP (1.39 vs. 1.48 kg milk/kg DMI on average). Milk composition did not vary between groups. Although no differences were found in dietary δ13C across the treatments, milk from the treatment group had lower δ13C than the control group throughout the experimental period. The results of this work with medium-producing dairy cows confirm the efficacy of the additive on diets relatively high in NDF, which broadens its applicability to less intensive production systems. The results for milk δ13C, suggesting its potential as a qualitative proxy for methane emissions, merit further investigation, as it could be used in breeding and in monitoring, reporting, and validation systems.

This publication aims to provide guidelines of the knowledge required and the potential research to be conducted in order to understand the mode of action of antimethanogenic feed additives (AMFA). In the first part of the paper, we classify AMFA into 4 categories according to their mode of action: (1) lowering dihydrogen (H2) production; (2) inhibiting methanogens; (3) promoting alternative H2-incorporating pathways; and (4) oxidizing methane (CH4). The second part of the paper presents questions that guide the research to identify the mode of action of an AMFA on the rumen CH4 production from 5 different perspectives: (1) microbiology; (2) cell and molecular biochemistry; (3) microbial ecology; (4) animal metabolism; and (5) cross-cutting aspects. Recommendations are provided to address various research questions within each perspective, along with examples of how aspects of the mode of action of AMFA have been elucidated before. In summary, this paper offers timely and comprehensive guidelines to better understand and reveal the mode of action of current and emerging AMFA.

Background Plant cell walls are the main carbon sources for ruminal bacteria, which have evolved to produce sophisticated multi-functional enzyme cocktails in response to the structural diversity of lignocelulloses. Since a large proportion of ruminal bacteria are not yet cultured, we developed a high-throughput activity-based metagenomic approach to gain insight into this enzymatic diversity. Results A multi-step screening methodology was implemented to identify metagenomic clones acting on polysaccharides and polyaromatic compounds. This approach was used to explore the functional potential of two different microbial consortia derived from in vivo and in vitro enrichments of the bovine rumen microbiome on wheat straw. One hundred and sixty-eight fosmid clones were isolated from libraries. Five to seven times more β-mannanase and β-glucanase clones, and seven times less xylanase clones were obtained from the in vitro enrichment compared to the in vivo one. The sequencing of 51 fosmids, covering in total 1.4 Gb of metagenomic DNA, enabled the identification of various novel glycoside-hydrolases, esterases and oxidoreductases mostly encoded by unknown bacterial genera. Functional analysis showed that most of the identified xylanases belonged to Firmicutes members that were not enriched in the fermenter, while most cellulases and mannanases originate from Bacteroidetes. Conclusion These enzymes, that, for most of them, had not been previously identified by in depth-metagenome sequencing, present a high potential for biotechnological applications, as they could be used alone or in cocktails to break down plant cell walls. The relationships established between enzyme function and taxonomy highlight the complementary roles played by ruminal Firmicutes and Bacteroidetes in plant cell wall degradation. ### Competing Interest Statement The authors have declared no competing interest.

Monascus-fermented cereals reduce methane production from the rumen. The identification of the metabolites responsible of the antimethanogenic effect is important to assess the potential of this strategy as a mitigation option in ruminant production. This study highlights metabolites from Monascus ruber associated to methane inhibition. An in vitro rumen screening test was used to rank solid-state fermented wheat samples for their ability to inhibit methane. Four active and four less-active samples were selected for metabolomics analysis using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) and the identity of discriminant variables responsible for this group distinction was assigned thanks to tandem mass spectrometry (MS/MS) experiments. A total of 28 discriminating metabolites were putatively identified based on their accurate m/z values, fragmentation pathways and information from databases. The chemical structure (identification level 1) was confirmed for 9 of them thanks to the available authentic chemical standards. Most of these metabolites belong to the chemical class of statins and their derivatives (n=13), four of them annotated as statin-like molecules were observed here for the first time. A targeted approach using LC-MS/MS was performed to measure the levels of known metabolites and showed that the lovastatin concentration in active samples was 16-fold greater than in least-active samples. Whereas lovastatin was the major metabolite, up to 40% of the total statins were represented by other statin molecules. Comparison of the functional capability of lovastatin lactone and lovastatin acid demonstrates that the acid form is responsible for the antimethanogenic activity in the rumen environment. This study shows that Monascus-fermented feeds contain a wide variety of statins in both lactone and acid forms. Information from this work provides insight for improving the antimethanogenic efficacy of diets containing bioactive Monascus metabolites in ruminants

Many strategies for mitigating enteric methane (CH4) emissions in ruminants have focused on suppressing the activity of rumen methanogens, but this often leads to excess dihydrogen (H2) accumulation in the rumen, which is subsequently expelled and represents a potential energy loss. We hypothesized that phloroglucinol could act as a H2 acceptor when rumen methanogenesis is inhibited and be potentially transformed into beneficial compounds for the animal. Eight adult goats were randomly assigned to a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments: two levels of Asparagopsis taxiformis as CH4 inhibitor [0 vs. 5g/kg on a dry matter (DM) basis; AT- and AT+, respectively] and two levels of phloroglucinol as alternative H2 acceptor (0 vs. 20g/kg DM, PG- and PG+, respectively). Therefore, four dietary treatments were considered: i) basal diet (AT-PG-); ii) A. taxiformis alone (AT+PG-); iii) phloroglucinol alone (AT-PG+); and iv) the combination of A. taxiformis and phloroglucinol (AT+PG+). Animals were fed a maintenance diet with a 70:30 forage-to-concentrate ratio. After 10 d of adaptation to the diet, enteric gas emissions were measured in respiration chambers during 3 d prior to rumen content sampling on d 14. Dietary supplementation with A. taxiformis decreased CH4 production (-33.9%) and increased H2 emissions (+3465%), along with greater rumen propionate concentration. In contrast, phloroglucinol supplementation alone did not impact CH4 emissions or the rumen concentration of the main microbial groups but substantially increased acetate molar proportion (+10.2%) which could act as an alternative H2 acceptor. Moreover, when A. taxiformis was combined with phloroglucinol, it resulted in a decrease in H2 emissions (-68.1%). However, this decrease in H2 emissions was not fully explained by the increase in the acetate as phloroglucinol led to an increase in acetate both when methanogenesis was inhibited and when it was not. These findings suggest that the rumen fermentation of phloroglucinol may capture some of the additional H2 arising from the inhibition of methanogenesis by A. taxiformis through pathways other than acetate formation. Moreover, H2 emissions were not eliminated and most of the decrease occurred during the post-prandial stage, suggesting that the efficiency of H2 redirection could be further improved.

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The ruminant microbiome plays a key role in the health, feed utilization and environmental impact of ruminant production systems. Microbiome research provides insights to reduce the environmental footprint and improve meat and milk production from ruminants. However, the microbiome composition depends on the ruminant species, habitat and diet, highlighting the importance of having a good representation of ruminant microbiomes in their local environment to translate research findings into beneficial approaches. This information is currently lacking. In this study, we examined the metadata of farmed ruminant microbiome studies to determine global representativeness and summarized information by ruminant species, geographic location, body site, and host information. We accessed data from the International Nucleotide Sequence Database Collaboration via the National Center for Biotechnology Information database. We retrieved 47,628 sample metadata, with cattle accounting for more than two-thirds of the samples. In contrast, goats, which have a similar global population to cattle, were underrepresented with less than 4% of the total samples. Most samples originated in Western Europe, North America, Australasia and China but countries with large ruminant populations in South America, Africa, Asia, and Eastern Europe were underrepresented. Microbiomes from the gastrointestinal tract were the most frequently studied, comprising about 87% of all samples. Additionally, the number of samples from other body sites such as the respiratory tract, milk, skin, reproductive tract, and fetal tissue, has markedly increased over the past decade. More than 40% of the samples lacked basic information and many were retrieved from generic taxonomic classifications where the ruminant species was manually recovered. The lack of basic information such as age, breed or sex can limit the reusability of the data for further analysis and follow-up studies. This requires correct taxonomic assignment of the ruminant host and basic metadata information using accepted ontologies adapted to host-associated microbiomes. Repositories should require this information as a condition of acceptance. The results of this survey highlight the need to encourage studies of the ruminant microbiome from underrepresented ruminant species and countries worldwide. This shortfall in information poses a challenge for the development of microbiome-based strategies to meet sustainability requirements, particularly in areas with expanding livestock production systems.

Most mitigation strategies to reduce enteric methane (CH4) production in the rumen induce an excess of rumen dihydrogen (H2) that is expelled and consequently not redirected to the synthesis of metabolites that can be utilised by the ruminant. We hypothesised that phenolic compounds can be potential H2 acceptors when added to the diet, as they can be degraded to compounds that may be beneficial for the animal, using part of the H2 available when ruminal methanogenesis is inhibited. We performed four in vitro incubation experiments using rumen inoculum from Murciano-Granadina adult goats: Experiment 1 examined the inhibitory potential of Asparagopsis taxiformis (AT) at different concentrations (0, 1, 2, 3, 4 and 5% of the substrate on a DM basis) in 24 h incubations; Experiment 2 investigated the effect of a wide range of phenolic compounds (phenol, catechol, resorcinol, hydroquinone, pyrogallol, phloroglucinol, gallic acid and formic acid) at different doses (0, 2, 4, and 6 mM) on rumen fermentation for 24 h; Experiment 3 evaluated the combined effect of each phenolic compound at 6 mM with AT at 2% DM in sequential batch cultures for 5 days; and Experiment 4 examined the dose-response effect of phloroglucinol at different concentrations (0, 6, 16, 26 and 36 mM) combined with AT in sequential batch cultures for 5 days. Results from Experiment 1 confirmed that AT at 2% DM substantially inhibited CH4 production while significantly increasing H2 accumulation and decreasing the acetate:propionate ratio. Results from Experiment 2 showed that phenolic compounds did not negatively affect rumen fermenta-tion at any dose. In Experiment 3, each phenolic compound at 6 mM combined with AT at 2% DM inhib-ited CH4 production. Phloroglucinol numerically decreased H2 accumulation and significantly increased total gas production (TGP), volatile fatty acid (VFA) production and the acetate:propionate ratio. In Experiment 4, phloroglucinol at increasing doses supplemented with AT at 2% DM significantly decreased H2 accumulation and the abundances of archaea, protozoa and fungi abundances, and increased TGP, total VFA production and the acetate:propionate ratio in a dose-dependent way. In conclusion, combined treatment with AT and phloroglucinol was successful to mitigate CH4 production while preventing the accumulation of H2, leading to an increase in acetate and total VFA production and therefore an improve-ment in rumen fermentation in goats.(c) 2023 Published by Elsevier B.V. on behalf of The Animal Consortium. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Nitrogen use efficiency is an important index in ruminants and can be indirectly evaluated through the N isotopic discrimination between the animal and its diet (Δ15Nanimal-diet). The concentration and source of N may determine both the extent of the N isotopic discrimination in bacteria and N use efficiency. We hypothesised that the uptake and release of ammonia by rumen bacteria will affect the natural 15N enrichment of the bacterial biomass over their substrates (Δ15Nbacteria-substrate) and thereby further impacting Δ15Nanimal-diet. To test this hypothesis, two independent in vitro experiments were conducted using two contrasting N sources (organic vs inorganic) at different levels either in pure rumen bacteria culture incubations (Experiment #1) or in mixed rumen cultures (Experiment #2). In Experiment #1, tryptone casein or ammonium chloride were tested at low (1 mM N) and high (11.5 mM N) concentrations on three rumen bacterial strains (Fibrobacter succinogenes, Eubacterium limosum and Xylanibacter ruminicola) incubated in triplicate in anaerobic batch monocultures during 48h. In Experiment #2 mixed rumen cultures were incubated during 120 h with peptone or ammonium chloride at five different levels of N (1.5, 3, 4.5, 6 and 12-mM). In experiment #1, Δ15Nbacteria-substrate was lowest when the ammonia-consumer bacterium Fibrobacter succinogenes was grown on ammonium chloride, and highest when the proteolytic bacterial strain Xylanibacter ruminicola was grown on tryptone. In experiment #2, Δ15Nbacteria-substrate was lower with inorganic (ammonium chloride) vs organic (peptone) N source. A strong negative correlation between Δ15Nbacteria-substrate and Rikenellaceae_RC9_gut_group, a potential fibrolytic rumen bacterium, was detected. Together, our results showed that Δ15Nbacteria-substrate may change according to the balance between synthesis of microbial protein from ammonia versus non-ammonia N sources and confirm the key role of rumen bacteria as modulators of Δ15Nanimal-diet.