Dairy cattle are typically fed a total mixed ration (TMR), which is prepared in an automated mixer wagon. On-farm, effective TMR mixing can often be neglected due to lack of time or training. This leads to a disbalance of intake and potentially detrimental effects on health and production. Using dietary treatments to simulate this effect, this study determined the response of rumen metabolism and microbiome to different concentrate allocations in combination with a live Saccharomyces cerevisiae supplement (yeast supplementation, YS). The 4 × 4 Latin square design consisted of four dairy cows fitted with permanent rumen cannulae, which were fed a partial mixed ration with dietary concentrates (4 kg per cow per day) in an even or an uneven pattern of allocation (concentrate allocation, CA). YS was included in the TMR at a rate of 10 g per cow per day. Rumen metabolism was determined by measuring the pH, volatile fatty acids (VFAs), and ammonia nitrogen (NH3–N). The rumen microbial community was characterised using 16S rRNA gene amplicon sequencing. Both CA and YS had no effect (p > 0.05) on the dry matter intake, milk yield, or composition. CA did not affect the rumen NH3–N and VFA concentrations (p > 0.05). YS inclusion tended to increase the rumen pH (p = 0.088), acetate (p = 0.076), and valerate (p = 0.091). YS significantly increased the total VFA (p = 0.033) and propionate concentrations (p < 0.016). CA had little overall effect on the rumen microbiome beta diversity. However, there was a reduction in the relative abundance of a Prevotellaceae feature associated with an uneven pattern of CA. Bray–Curtis clustering of the microbiome was observed with YS (p = 0.002), driven by a decrease of Gammaproteobacteria and Prevotellaceae features and an increase of a Christensenellaceae feature (LDA > 2.0).

Natural plant compounds can be used to supplement livestock diets, improving feed efficiency, production, and health, while also reducing environmental impact. In the present study, a Yucca schidigera (Mohave Yucca) extract was added at four rates of inclusion (ROI) of 0, 5, 15, or 30 g/day to a ryegrass and maize silage-based diet and fed to dairy cows in a 4 × 4 Latin square experimental design. Each period was 28 days in duration, with sampling undertaken during the final week of each period. Solid phase digesta (SPD) and liquid phase digesta (LPD) samples were collected via a rumen cannula and analyzed for volatile fatty acids (VFAs), ammonia N, and microbiome using 16S rRNA gene sequencing. Total fecal and urine collection was undertaken over a 3-day period. Rumen microbial diversity was not affected by ROI (LPD: P = 0.180; SPD: P = 0.059). However, discriminant analysis found a decrease in Methanobrevibacter millerae (linear discriminant analysis, LDA = 2.15) and an increase in an unclassified species of Proteobacteria (LDA = 2.10) associated with ROI. Univariate analysis also revealed differential abundance of operational taxonomic units classified as Prevotellaceae and Fibrobacteraceae by ROI (P < 0.05). Maximum rumen ammonia N concentration decreased linearly from 228 to 109 mg/L with ROI (P = 0.044). Rumen VFA concentration was unaffected with the exception of propionate, which showed a linear increase with ROI (P = 0.010). The diurnal rumen pH range (maximum-minimum) also decreased with ROI (P = 0.004). Dry matter intake and milk yield were not affected (P > 0.05) by ROI; however, there was a linear increase in milk fat content from 38.9 to 42.0 g/kg with ROI (P < 0.05).IMPORTANCEDomestic livestock such as dairy cows are inefficient utilizers of dietary nitrogen. This increases feed costs and reduces animal production efficiency. Excreted nitrogenous compounds are also an environmental hazard, such as when they enter water courses as nitrate or are lost to the atmosphere as ammonia or nitrous oxide. Dietary protein is degraded in the rumen via the activity of the microbial population, mainly into ammonia, which may then be utilized by the microbial population to synthesize microbial protein or absorbed into the blood and potentially excreted. Manipulation of the diet or altering the microbial population may increase the utilization of dietary protein, increasing animal performance, decreasing feed costs, and reducing the environmental impact of milk production. This study examines the effect of Yucca schidigera extract on the rumen microbiome and nitrogen utilization in dairy cows.

Abstract High yielding dairy cattle are typically fed a total mixed ration (TMR) to meet their energy requirements. However, inadequate mixing can lead to feed selection and dietary imbalance, negatively affecting rumen metabolism and the microbiome. To investigate the effect of TMR mixing, four dairy cows were fed a partial mixed ration with dietary concentrates (4 kg/cow/day) added separately in even and uneven patterns of allocation (CA) with a Saccharomyces cerevisiae yeast supplement (YS) (1 g/cow/day). Rumen digesta samples were taken to measure microbial metabolism and microbiome using16S rRNA amplicon sequencing. No effect of CA on rumen metabolism was detected. YS inclusion tended to increase rumen pH ( P = 0.088) reduce total VFA concentration ( P = 0.033) and propionate concentration ( P = 0.016). Clustering of the rumen microbiome was observed with YS supplementation, driven by a decrease in abundance of Gammaproteobacteria and Prevotellaceae OTUs respectively and an increase of a Christensenellaceae OTU. Pattern of concentrate allocation had no detectable effect on alpha diversity or Bray Curtis dissimilarity. However, reduction in relative abundance of a Prevotellaceae OTU biomarker was associated with uneven pattern of concentrate allocation . Pattern of concentrate allocation and YS supplementation did not adversely affect milk yield or composition.

Nitrogen (N) loss from livestock agriculture via ammonia and nitrous oxide can reduce feed efficiency, production and negatively affect the environment. One option to reduce N loss is to add dietary supplements such as Yucca schidigera extract which has ammonia-binding properties and contains antimicrobial steroidal saponins, or Saccharomyces cerevisiae yeast, which can stabilise rumen pH and promote fibre degradation, increasing microbial growth and demand for degradable N. To determine the effect of Yucca schidigera extract when fed alone or in combination with a live yeast on the performance, rumen metabolism, microbiome and N balance, six rumen cannulated dairy cows were fed a mixed ration (C), mixed ration with Y. schidigera extract (De-Odorase®, Alltech®; 5 g/cow/day; D), or mixed ration with Y. schidigera extract (5 g/day) and Saccharomyces cerevisiae (Yea-Sacc®, Alltech®, 1 g/cow per day; DY), in a 3 × 3 Latin rectangle design study with three periods of 49-day duration. Digesta samples were collected via the ruminal cannula during the final week of each period and separated into liquid (LPD) and solid (SPD) phases for microbiome analysis using 16S rRNA amplicon sequencing. DM intake was 0.8 kg/d lower (P < 0.05) in cows fed DY than C or D, with milk protein concentration 1.7 g/kg higher in C than D or DY. There was a beta diversity (Bray Curtis) clustering of the LPD in cows fed D or DY compared to C (P < 0.05), driven by an increase in Prevotella ruminicola-related operational taxonomic units (OTUs), and a decrease in P. brevis and P. bryantii OTUs. A methanogen OTU, Methanobrevibacter olleyae, was decreased in cows fed D or DY and an unclassified species of Gammaproteobacteria was increased in DY (LDA > 2.0, P < 0.05) compared to C. Rumen pH, ammonia and total VFA concentration were not affected by treatment (P > 0.05) but the concentration of propionate and iso-butyrate were lower at 1700 and 2000 h in cows fed DY compared to C (P < 0.05). Measurements of N balance were unaffected by supplementation with D or DY, and there was no effect of treatment on slurry pH. In conclusion, supplementing with an extract of Yucca schidigera either alone or in combination with a live yeast had only a small effect on performance, with Yucca schidigera altering species associated with carbohydrate and protein metabolism, and reduced Methanobrevibacter olleyae which is involved in methanogenesis.

This study investigated the effect of compaction, aeration and addition of a naturally mycotoxin contaminated ryegrass silage (MCS) containing 1803 µg/kg DM penicillic acid, on the nutritional value and mycotoxin content after ensiling and subsequent aerobic stability of ryegrass Lolium perenne silage (second-cut, June 2020, UK). Mini silos (30 L) were filled with differential compaction (500 kg FW/m 3 and 333 kg FW/m 3 ), aeration by injection of air (1L per 48h for 30d then 1L per 7d) and addition of MCS (1.5 g/kg FW ensiled forage) in a 2×2×2 factorial design. During ensilage, mean CO 2 % (kg FW) in the aerated silos increased with low compaction. Crude protein (CP) increased and ash decreased with aeration. Mean silage fermentation end products acetic (AA), lactic (LA) and propionic acid (PA) concentrations increased with MCS. PA concentration increased with aeration/low compaction. LA decreased and ethanol increased with low compaction. Mycotoxin profiles differed between the silages on opening and after 14-days incubation in aerobic conditions with disappearance of fusarenon X and penicillic acid and appearance of mycophenolic acid and roquefortine C (318 µg/kg DM and 890 µg/kg DM). Addition of MCS, increased the concentration of penicillic acid on opening with an interaction with aeration (80.6µg/kg DM MCS × aerated, 40.0 µg/kg DM in the MCS × sealed). Aerobic stability was affected by aeration and low compaction with reduced time taken to heat to +5°C and +10°C above ambient temperature, higher rate of increase in pH and higher cumulative temperatures to the first peak temperature. Higher mycotoxin concentration at opening had a similar effect increasing time to heat +5°C and +10°C above ambient temperatures in aerobic conditions. Regression analysis showed predominantly direct relationships between silage fermentation end-product concentrations and aerobic stability. This study revealed interactions between silage bacteria and fungi activity from the concentrations of fermentation end-products and mycotoxins during ensilage and subsequent aerobic spoilage. The results supported current best practice for silage making, promoting conditions for improved preservation and aerobic stability. The addition of MCS had unexpected positive effects. However, factors associated with the MCS benefiting aerobic stability were not determined.

High levels of supplementation with cereal increases production rates in cattle but can increase incidence of disease, ranging from mild indigestion to acute ruminal acidosis and death. Therefore, there is motivation to determine biological markers which can be used to identify whether animals have been, or are being fed, sufficient or excessive cereals. This study aimed to describe light microscopic findings from animals being fed diverse dietary cereal proportions and to test the performance of a novel rumen epithelial scoring system. Rumen wall tissue samples were obtained from the abattoir from 195 cattle from 11 Scottish farms and processed for histological examination. Light microscopic examination was used to characterise ruminal epithelial response to dietary challenge. Secondary objectives included describing the distribution of immune-related cells in bovine ruminal epithelium and assessing the use of a modified Elastin Martius Scarlet Blue stain (EMSB) for histological examination of the rumen epithelium. Cells staining positive for cluster of differentiation 3 were distributed mainly in the lower layers of the stratum basale and were found in higher densities in animals offered lower cereal proportion diets. Cells staining positive for major histocompatibility complex class 2 (MHCII) were most common in perivascular locations and in the junction between the lower stratum basale and the propria-submucosa. The density of MHCII positive staining cells was higher in animals on lower cereal diets. The level of supplementation with cereal was also associated with the thickness of the stratum corneum (SCT) and stratum granulosum (SGT), the integrity of the stratum corneum and sloughing of cornified cells. There were no advantages in using EMSB stain over haematoxylin and eosin (H&E) in this scoring system. We concluded that a scoring system that included only SCT, SGT and a measure of the loss of appearance of intercellular space allowed differentiation of groups of animals according to the level of cereal supplementation.

Sub-acute ruminal acidosis (SARA) can reduce the production efficiency and impair the welfare of cattle, potentially in all production systems. The aim of this study was to characterise measurable postmortem observations from divergently managed intensive beef finishing farms with high rates of concentrate feeding. At the time of slaughter, we obtained samples from 19 to 20 animals on each of 6 beef finishing units (119 animals in total) with diverse feeding practices, which had been subjectively classified as being high risk (three farms) or low risk (three farms) for SARA on the basis of the proportions of barley, silage and straw in the ration. We measured the concentrations of histamine, lipopolysaccharide (LPS), lactate and other short-chain fatty acids (SCFAs) in ruminal fluid, LPS and SCFA in caecal fluid. We also took samples of the ventral blind sac of the rumen for histopathology, immunohistopathology and gene expression. Subjective assessments were made of the presence of lesions on the ruminal wall, the colour of the lining of the ruminal wall and the shape of the ruminal papillae. Almost all variables differed significantly and substantially among farms. Very few pathological changes were detected in any of the rumens examined. The animals on the high-risk diets had lower concentrations of SCFA and higher concentrations of lactate and LPS in the ruminal fluid. Higher LPS concentrations were found in the caecum than the rumen but were not related to the risk status of the farm. The diameters of the stratum granulosum, stratum corneum and of the vasculature of the papillae, and the expression of the gene TLR4 in the ruminal epithelium were all increased on the high-risk farms. The expression of IFN-γ and IL-1β and the counts of cluster of differentiation 3 positive and major histocompatibility complex class two positive cells were lower on the high-risk farms. High among-farm variation and the unbalanced design inherent in this type of study in the field prevented confident assignment of variation in the dependent variables to individual dietary components; however, the CP percentage of the total mixed ration DM was the factor that was most consistently associated with the variables of interest. Despite the strong effect of farm on the measured variables, there was wide inter-animal variation.

Ruminants contribute to the emissions of greenhouse gases, in particular methane, due to the microbial anaerobic fermentation of feed in the rumen. The rumen simulation technique was used to investigate the effects of the addition of different supplemental plant oils to a high concentrate diet on ruminal fermentation and microbial community composition. The control (CTR) diet was a high-concentrate total mixed ration with no supplemental oil. The other experimental diets were supplemented with olive (OLV), sunflower (SFL) or linseed (LNS) oils at 6%. Rumen digesta was used to inoculate the fermenters, and four fermentation units were used per treatment. Fermentation end-products, extent of feed degradation and composition of the microbial community (qPCR) in digesta were determined. Compared with the CTR diet, the addition of plant oils had no significant ( P > 0.05) effect on ruminal pH, substrate degradation, total volatile fatty acids or microbial protein synthesis. Gas production from the fermentation of starch or cellulose were decreased by oil supplementation. Methane production was reduced by 21–28% ( P < 0.001), propionate production was increased ( P < 0.01), and butyrate and ammonia outputs and the acetate to propionate ratio were decreased ( P < 0.001) with oil-supplemented diets. Addition of 6% OLV and LNS reduced ( P < 0.05) copy numbers of total bacteria relative to the control. In conclusion, the supplementation of ruminant diets with plant oils, in particular from sunflower or linseed, causes some favorable effects on the fermentation processes. The addition of vegetable oils to ruminant mixed rations will reduce methane production increasing the formation of propionic acid without affecting the digestion of feed in the rumen. Adding vegetable fats to ruminant diets seems to be a suitable approach to decrease methane emissions, a relevant cleaner effect that may contribute to alleviate the environmental impact of ruminant production.

Background Dietary intake is known to be a driver of microbial community dynamics in ruminants. Beef cattle go through a finishing phase that typically includes very high concentrate ratios in their feed, with consequent effects on rumen metabolism including methane production. This longitudinal study was designed to measure dynamics of the rumen microbial community in response to the introduction of high concentrate diets fed to beef cattle during the finishing period. A cohort of 50 beef steers were fed either of two basal diet formulations consisting of approximately 10:90 or 50:50 forage:concentrate ratios respectively. Nitrate and oil rich supplements were also added either individually or in combination. Digesta samples were taken at time points over ~ 200 days during the finishing period of the cattle to measure the adaptation to the basal diet and long-term stability of the rumen microbiota. Results 16S rRNA gene amplicon libraries were prepared from 313 rumen digesta samples and analysed at a depth of 20,000 sequences per library. Bray Curtis dissimilarity with analysis of molecular variance (AMOVA) revealed highly significant ( p < 0.001) differences in microbiota composition between cattle fed different basal diets, largely driven by reduction of fibre degrading microbial groups and increased relative abundance of an unclassified Gammaproteobacteria OTU in the high concentrate fed animals. Conversely, the forage-based diet was significantly associated with methanogenic archaea. Within basal diet groups, addition of the nitrate and combined supplements had lesser, although still significant, impacts on microbiota dissimilarity compared to pre-treatment time points and controls. Measurements of the response and stability of the microbial community over the time course of the experiment showed continuing adaptation up to 25 days in the high concentrate groups. After this time point, however, no significant variability was detected. Conclusions High concentrate diets that are typically fed to finishing beef cattle can have a significant effect on the microbial community in the rumen. Inferred metabolic activity of the different microbial communities associated with each of the respective basal diets explained differences in methane and short chain fatty acid production between cattle. Longitudinal sampling revealed that once adapted to a change in diet, the rumen microbial community remains in a relatively stable alternate state.

Recent advances in bioinformatics have enabled the rapid assembly of genomes from metagenomes (MAGs), and there is a need for reproducible pipelines that can annotate and characterise thousands of genomes simultaneously. Here we present MAGpy, a Snakemake pipeline that takes FASTA input and compares MAGs to several public databases, checks quality, assigns a taxonomy and draws a phylogenetic tree.

The rumen microbiome is essential for the biological processes involved in the conversion of feed into nutrients that can be utilized by the host animal. In the present research, the influence of the rumen microbiome on feed conversion efficiency, growth rate, and appetite of beef cattle was investigated using metagenomic data. Our aim was to explore the associations between microbial genes and functional pathways, to shed light on the influence of bacterial enzyme expression on host phenotypes. Two groups of cattle were selected on the basis of their high and low feed conversion ratio. Microbial DNA was extracted from rumen samples, and the relative abundances of microbial genes were determined via shotgun metagenomic sequencing. Using partial least squares analyses, we identified sets of 20, 14, 17, and 18 microbial genes whose relative abundances explained 63, 65, 66, and 73% of the variation of feed conversion efficiency, average daily weight gain, residual feed intake, and daily feed intake, respectively. The microbial genes associated with each of these traits were mostly different, but highly correlated traits such as feed conversion ratio and growth rate showed some overlapping genes. Consistent with this result, distinct clusters of a coabundance network were enriched with microbial genes identified to be related with feed conversion ratio and growth rate or daily feed intake and residual feed intake. Microbial genes encoding for proteins related to cell wall biosynthesis, hemicellulose, and cellulose degradation and host-microbiome crosstalk (e.g., aguA, ptb, K01188, and murD) were associated with feed conversion ratio and/or average daily gain. Genes related to vitamin B12 biosynthesis, environmental information processing, and bacterial mobility (e.g., cobD, tolC, and fliN) were associated with residual feed intake and/or daily feed intake. This research highlights the association of the microbiome with feed conversion processes, influencing growth rate and appetite, and it emphasizes the opportunity to use relative abundances of microbial genes in the prediction of these performance traits, with potential implementation in animal breeding programs and dietary interventions.

A 1000-cow study across four European countries was undertaken to understand to what extent ruminant microbiomes can be controlled by the host animal and to identify characteristics of the host rumen microbiome axis that determine productivity and methane emissions. A core rumen microbiome, phylogenetically linked and with a preserved hierarchical structure, was identified. A 39-member subset of the core formed hubs in co-occurrence networks linking microbiome structure to host genetics and phenotype (methane emissions, rumen and blood metabolites, and milk production efficiency). These phenotypes can be predicted from the core microbiome using machine learning algorithms. The heritable core microbes, therefore, present primary targets for rumen manipulation toward sustainable and environmentally friendly agriculture.

The cow rumen is adapted for the breakdown of plant material into energy and nutrients, a task largely performed by enzymes encoded by the rumen microbiome. Here we present 913 draft bacterial and archaeal genomes assembled from over 800 Gb of rumen metagenomic sequence data derived from 43 Scottish cattle, using both metagenomic binning and Hi-C-based proximity-guided assembly. Most of these genomes represent previously unsequenced strains and species. The draft genomes contain over 69,000 proteins predicted to be involved in carbohydrate metabolism, over 90% of which do not have a good match in public databases. Inclusion of the 913 genomes presented here improves metagenomic read classification by sevenfold against our own data, and by fivefold against other publicly available rumen datasets. Thus, our dataset substantially improves the coverage of rumen microbial genomes in the public databases and represents a valuable resource for biomass-degrading enzyme discovery and studies of the rumen microbiome.

Previous shotgun metagenomic analyses of ruminal digesta identified some microbial information that might be useful as biomarkers to select cattle that emit less methane (CH4), which is a potent greenhouse gas. It is known that methane production (g/kgDMI) and to an extent the microbial community is heritable and therefore biomarkers can offer a method of selecting cattle for low methane emitting phenotypes. In this study a wider range of Bos Taurus cattle, varying in breed and diet, was investigated to determine microbial communities and genetic markers associated with high/low CH4 emissions. Digesta samples were taken from 50 beef cattle, comprising four cattle breeds, receiving two basal diets containing different proportions of concentrate and also including feed additives (nitrate or lipid), that may influence methane emissions. A combination of partial least square analysis and network analysis enabled the identification of the most significant and robust biomarkers of CH4 emissions (VIP > 0.8) across diets and breeds when comparing all potential biomarkers together. Genes associated with the hydrogenotrophic methanogenesis pathway converting carbon dioxide to methane, provided the dominant biomarkers of CH4 emissions and methanogens were the microbial populations most closely correlated with CH4 emissions and identified by metagenomics. Moreover, these genes grouped together as confirmed by network analysis for each independent experiment and when combined. Finally, the genes involved in the methane synthesis pathway explained a higher proportion of variation in CH4 emissions by PLS analysis compared to phylogenetic parameters or functional genes. These results confirmed the reproducibility of the analysis and the advantage to use these genes as robust biomarkers of CH4 emissions. Volatile fatty acid concentrations and ratios were significantly correlated with CH4, but these factors were not identified as robust enough for predictive purposes. Moreover, the methanotrophic Methylomonas genus was found to be negatively correlated with CH4. Finally, this study confirmed the importance of using robust and applicable biomarkers from the microbiome as a proxy of CH4 emissions across diverse production systems and environments.

The rumen is a complex ecosystem composed of anaerobic bacteria, protozoa, fungi, methanogenic archaea and phages. These microbes interact closely to breakdown plant material that cannot be digested by humans, whilst providing metabolic energy to the host and, in the case of archaea, producing methane. Consequently, ruminants produce meat and milk, which are rich in high-quality protein, vitamins and minerals, and therefore contribute to food security. As the world population is predicted to reach approximately 9.7 billion by 2050, an increase in ruminant production to satisfy global protein demand is necessary, despite limited land availability, and whilst ensuring environmental impact is minimized. Although challenging, these goals can be met, but depend on our understanding of the rumen microbiome. Attempts to manipulate the rumen microbiome to benefit global agricultural challenges have been ongoing for decades with limited success, mostly due to the lack of a detailed understanding of this microbiome and our limited ability to culture most of these microbes outside the rumen. The potential to manipulate the rumen microbiome and meet global livestock challenges through animal breeding and introduction of dietary interventions during early life have recently emerged as promising new technologies. Our inability to phenotype ruminants in a high-throughput manner has also hampered progress, although the recent increase in "omic" data may allow further development of mathematical models and rumen microbial gene biomarkers as proxies. Advances in computational tools, high-throughput sequencing technologies and cultivation-independent "omics" approaches continue to revolutionize our understanding of the rumen microbiome. This will ultimately provide the knowledge framework needed to solve current and future ruminant livestock challenges.

The main aim of this study was to elucidate whether rumen microbial gene abundances (RMGA) can be used to predict residual feed intake (RFI), feed conversion ratio (FCR) and its component traits: daily feed intake (DFI), average daily gain (ADG), and thus RMGA could be applied as indirect trait for breeding of feed efficiency in beef cattle. RMGA were generated by whole metagenomic sequencing of rumen microbial DNA samples from 42 beef cattle, with extreme low and high FCR, selected from two feed efficiency trials. The results of the PLS analysis indicated that RMGA showed substantial potential to be used as predictors for RFI, FCR and its components DFI and ADG explaining 55 to 73% of their variation. While only 12 and 23 microbial genes were significantly associated with RFI and DFI, respectively, there were 166 and 167 affecting ADG and FCR. RFI and DFI were influenced partly by the same microbial genes and combined in the same microbial network clusters as was also the case for FCR and ADG. The results elucidate the likely potential of RMGA to predict the difficult and costly to measure trait feed efficiency, but have to be confirmed under the more challenging conditions of practical breeding programmes.