Viruses contribute to microbial diversification in the rumen ecosystem and are associated with certain animal production traits.

BACKGROUND: The rumen microbiome enables ruminants to digest otherwise indigestible feedstuffs, thereby facilitating the production of high-quality protein, albeit with suboptimal efficiency and producing methane. Despite extensive research delineating associations between the rumen microbiome and ruminant production traits, the functional roles of the pervasive and diverse rumen virome remain to be determined. RESULTS: Leveraging a recent comprehensive rumen virome database, this study analyzes virus-microbe linkages, at both species and strain levels, across 551 rumen metagenomes, elucidating patterns of microbial and viral diversity, co-occurrence, and virus-microbe interactions. Additionally, this study assesses the potential role of rumen viruses in microbial diversification by analyzing prophages found in rumen metagenome-assembled genomes. Employing CRISPR-Cas spacer-based matching and virus-microbe co-occurrence network analysis, this study suggests that the viruses in the rumen may regulate microbes at strain and community levels through both antagonistic and mutualistic interactions. Moreover, this study establishes that the rumen virome demonstrates responsiveness to dietary shifts and associations with key animal production traits, including feed efficiency, lactation performance, weight gain, and methane emissions. CONCLUSIONS: These findings provide a substantive framework for further investigations to unravel the functional roles of the virome in the rumen in shaping the microbiome and influencing overall animal production performance. Video Abstract.

Effects of replacing of alfalfa hay with Plantago lanceolata hay on digestibility, methane production and microbial protein production of total mixed diet.

The aim of current experiment was to determine the effect of replacement of alfalfa hay with ribwort plantain (Plantago lanceolata) hay in ruminant diets on the fermentation parameters such as gas production, methane (CH4) production, true digestible dry matter (TDDM), true digestibility (TD), partitioning factor, microbial protein, and efficiency of microbial protein using in vitro gas production technique. The alfalfa hay was replaced with P. lanceolata hay in a diets isocaloric (2650 kcal/kg DM) and nitrogenic (17% CP kg DM) at the ratio of 0, 5, 10 and 15%. Partial substitution of alfalfa hay with P. lanceolata hay had no significant effect on gas and methane (ml/incubated substrate or %) production whereas the partial substitution had a significant effect on TDDM, TD, gas (ml/digested DM), CH4 (ml ml/digested DM) and microbial MP of diets. The replacement of alfalfa hay with ribwort plantain hay shifted the fermentation pattern from gas and methane production to microbial protein production. Therefore alfalfa hay can be replaced with ribwort plantain hay with high digestibility and anti-methanogenic potential in ruminant diets up to 15% to decrease methane production and improve microbial protein production. However further in vivo experiments are required to determine the effect of replacement on feed intake and animal production.

Differential responses of rumen and fecal fermentation and microbiota of Liaoning cashmere goats after 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester supplementation.

The 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester (HMBi), a rumen protective methionine, has been extensively studied in dairy cows and beef cattle and has been shown to regulate gastrointestinal microbiota and improve production performance. However, knowledge of the application of HMBi on cashmere goats and the simultaneous study of rumen and hindgut microbiota is still limited. In this study, HMBi supplementation increased the concentration of total serum protein, the production of microbial protein in the rumen and feces, as well as butyrate production in the feces. The results of PCoA and PERMANOVA showed no significant difference between the rumen microbiota, but there was a dramatic difference between the fecal microbiota of the two groups of Cashmere goats after the HMBi supplementation. Specifically, in the rumen, HMBi significantly increased the relative abundance of some fiber-degrading bacteria (such as Fibrobacter) compared with the CON group. In the feces, as well as a similar effect as in the rumen (increasing the relative abundance of some fiber-degrading bacteria, such as Lachnospiraceae FCS020 group and ASV32), HMBi diets also increased the proliferation of butyrate-producing bacteria (including Oscillospiraceae UCG-005 and Christensenellaceae R-7 group). Overall, these results demonstrated that HMBi could regulate the rumen and fecal microbial composition of Liaoning cashmere goats and benefit the host.

The Discovery of a Multidomain Mannanase Containing Dual-Catalytic Domain of the Same Activity: Biochemical Properties and Synergistic Effect.

In recent years, the wide application of mannan has driven the demand for the exploration of mannanase. As one of the main components of hemicellulose, mannan is an important polysaccharide that ruminants need to degrade and utilize, making rumen a rich source of mannanases. In this study, gene mining of mannanases was performed using bioinformatics, and potential dual-catalytic domain mannanases were heterologously expressed to analyze their properties. The hydrolysis pattern and enzymatic products were identified by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). A dual-catalytic domain mannanase Man26/5 with the same function as the substrate was successfully mined from the genome of cattle rumen microbiota. Compared to the single-catalytic domain, its higher thermal stability (≤50 °C) and catalytic efficiency confirm the synergistic effect between the two catalytic domains. It exhibited a unique "crab-like" structure where the CBM located in the middle is responsible for binding, and the catalytic domains at both ends are responsible for cutting. The exploration of its multidomain structure and synergistic patterns could provide a reference for the artificial construction and molecular modification of enzymes.

Understanding the Diversity and Roles of the Ruminal Microbiome.

The importance of ruminal microbiota in ruminants is emphasized, not only as a special symbiotic relationship with ruminants but also as an interactive and dynamic ecosystem established by the metabolites of various rumen microorganisms. Rumen microbial community is essential for life maintenance and production as they help decompose and utilize fiber that is difficult to digest, supplying about 70% of the energy needed by the host and 60-85% of the amino acids that reach the small intestine. Bacteria are the most abundant in the rumen, but protozoa, which are relatively large, account for 40-50% of the total microorganisms. However, the composition of these ruminal microbiota is not conserved or constant throughout life and is greatly influenced by the host. It is known that the initial colonization of calves immediately after birth is mainly influenced by the mother, and later changes depending on various factors such as diet, age, gender and breed. The initial rumen microbial community contains aerobic and facultative anaerobic bacteria due to the presence of oxygen, but as age increases, a hypoxic environment is created inside the rumen, and anaerobic bacteria become dominant in the rumen microbial community. As calves grow, taxonomic diversity increases, especially as they begin to consume solid food. Understanding the factors affecting the rumen microbial community and their effects and changes can lead to the early development and stabilization of the microbial community through the control of rumen microorganisms, and is expected to ultimately help improve host productivity and efficiency.

Cloning, expression and application of a novel laccase derived from water buffalo ruminal lignin-degrading bacteria.

Water buffalo is the only mammal found to degrade lignin so far, and laccase plays an indispensable role in the degradation of lignin. In this study, multiple laccase genes were amplified based on the water buffalo rumen derived lignin-degrading bacteria Bacillus cereus and Ochrobactrum pseudintermedium. Subsequently, the corresponding recombinant plasmids were transformed into E. coli expression system BL21 (DE3) for induced expression by Isopropyl-ß-D-thiogalactopyranoside (IPTG). After preliminary screening, protein purification and enzyme activity assays, Lac3833 with soluble expression and high enzyme activity was selected to test its characteristics, especially the ability of lignin degradation. The results showed that the optimum reaction temperature of Lac3833 was 40 °C for different substrates. The relative activity of Lac3833 reached the highest at pH 4.5 and pH 5.5 when the substrates were ABTS or 2,6-DMP and guaiacol, respectively. Additionally, Lac3833 could maintain high enzyme activity in different temperatures, pH and solutions containing Na+, K+, Mg2+, Ca2+ and Mn2+. Importantly, compared to negative treatment, recombinant laccase Lac3833 treatment showed that it had a significant function in degrading lignin. In conclusion, this is a pioneering study to produce recombinant laccase with lignin-degrading ability by bacteria from water buffalo rumen, which will provide new insights for the exploitation of more lignin-degrading enzymes.

Impact of rumen microbiome on cattle carcass traits.

Rumen microbes are crucial in the anaerobic fermentation of plant polysaccharides to produce volatile fatty acids. However, limited information exists about the specific microbial species and strains in the rumen that affect carcass traits, and it is unclear whether there is a relationship between rumen metabolic functions and these traits. This study investigated the relationship between the rumen microbiome and carcass traits in beef cattle using 16S rRNA amplicon and shotgun sequencing. Metagenomic sequencing was used to compare the rumen microbiome between high-carcass weight (HW) and low-carcass weight (LW) cattle, and high-marbling (HM) and low-marbling (LM) cattle. Prokaryotic communities in the rumen of HW vs. LW and HM vs. LM were separated using 16S rRNA amplicon sequencing. Notably, shotgun metagenomic sequencing revealed that HW cattle had more methane-producing bacteria and ciliate protozoa, suggesting higher methane emissions. Additionally, variations were observed in the abundances of certain glycoside hydrolases and polysaccharide lyases involved in the ruminal degradation of plant polysaccharides between HW and LW. From our metagenome dataset, 807 non-redundant metagenome-assembled genomes (MAGs) of medium to high quality were obtained. Among these, 309 and 113 MAGs were associated with carcass weight and marbling, respectively.

Predicting feed efficiency of Angus steers using the gastrointestinal microbiome.

Microbial composition of the gastrointestinal tracts is an important factor affecting the variation in feed efficiency in ruminants. Several studies have investigated the composition of the ruminal and fecal microbiotas, as well as their impacts on feed efficiency and digestion. In addition, next-generation DNA sequencing techniques have allowed us to gain a better understanding of such microbiomes. In this study, the beef cattle microbiome data were analyzed using both a multivariate and a univariate approach and the results were compared. Moreover, a statistical procedure to classify calves in two groups with extreme Residual Feed Intake (RFI) values, using their microbiota profile, was developed. Both fecal and ruminal samples were collected from 63 Angus steers at two different time points for evaluation of their microbiomes: at the beginning and at the end of the feedlot. An additional fecal sample was collected at weaning. A total of 149 and 119 bacterial families (BFs) were retrieved from the ruminal and fecal samples, respectively. A Canonical Discriminant Analysis (CDA) was used to investigate whether BFs were able to distinguish between rumen and fecal samples. A sub-sample of 28 steers was divided in two groups based on their feed efficiency status: positive or negative for RFI. Fecal samples collected at weaning were used to assign the positive and negative RFI animals to their corresponding groups using both Stepwise Discriminant Analysis and CDA. Results revealed that CDA was able to distinguish between rumen and fecal samples. Peptostreptococcaceae was the family most associated with the fecal samples, whereas Prevotellaceae the most associated with the ruminal samples. The CDA using 19 BFs selected from the stepwise was able to correctly assign all animals to the proper RFI groups (negative or positive). Rhizobiaceae was the family most associated with negative RFI, whereas Comamonadacea was the family most linked with positive RFI. The results from this study showed that the multivariate approach can be used to improve microbiome data analysis, as well as to predict feed efficiency in beef cattle using information derived from the fecal microbiome.

Development of an experimental model for liver abscess induction in Holstein steers using an acidotic diet challenge and bacterial inoculation.

Holstein steers (n = 40; initial BW = 84.9 ±â€…7.1 kg) were used to study the genesis of liver abscesses (LA) using an acidotic diet challenge with or without intraruminal bacterial inoculation. Steers were housed in individual pens inside a barn and randomly assigned to one of three treatments: (1) low-starch control diet comprised primarily of dry-rolled corn and wet corn gluten feed (CON); (2) high-starch acidotic diet with steam-flaked corn (AD); or (3) acidotic diet plus intraruminal inoculation with Fusobacterium necrophorum subsp. necrophorum (9.8 × 108 colony forming units [CFU]/mL), Trueperella pyogenes (3.91 × 109 CFU/mL), and Salmonella enterica serovar Lubbock (3.07 × 108 CFU/mL), previously isolated from LA (ADB). Steers in AD and ADB were fed the acidotic diet for 3 d followed by 2 d of the CON diet, and this cycle was repeated four times. On day 23, ADB steers were intraruminally inoculated with the bacteria. At necropsy, gross pathology of livers, lungs, rumens, and colons was noted. Continuous data were analyzed via mixed models as repeated measures over time with individual steer as the experimental unit. Mixed models were also used to determine the difference in prevalence of necropsy scores among treatments. Ruminal pH decreased in AD and ADB steers during each acidotic diet cycle (P ≤ 0.05). LA prevalence was 42.9% (6 of 14) in ADB vs. 0% in AD or CON treatments (P < 0.01). Ruminal damage was 51.1% greater in ADB than in AD (P ≤ 0.04). Culture of LA determined that 100% of the abscesses contained F. necrophorum subsp. necrophorum, 0% contained T. pyogenes, 50% contained Salmonella, and 50% contained a combination of F. necrophorum subsp. necrophorum and Salmonella. The F. necrophorum subsp. necrophorum was clonally identical to the strain used for the bacterial inoculation based on phylogenetic analysis of the whole genome. This experimental model successfully induced rumenitis and LA in Holstein steers and confirms the central dogma of LA pathogenesis that acidosis and rumenitis lead to the entry of F. necrophorum into the liver to cause abscesses. Our findings suggest that an acidotic diet, in conjunction with intraruminal bacterial inoculation, is a viable model to induce LA. Further research is needed to determine the repeatability of this model, and a major application of the model will be in evaluations of novel interventions to prevent LA.

Liver abscesses (LA) in feedlots are costly to the beef industry. At harvest, LA cause an increase in liver condemnations, carcass trimming, and a decrease in quality grade. The objective of this research was to develop an experimental LA model in Holstein steers using an acidotic diet with and without intraruminal inoculation of bacteria involved in LA formation. These data suggest acidotic diet challenges in conjunction with bacterial inoculation were able to induce LA in Holstein steers. The acidotic diet alone caused reduced rumen content pH and caused rumen wall inflammation and damage, observed at harvest. Nonetheless, the addition of bacteria had a compounding effect on rumen damage. Both bacteria inoculated were isolated from 57% of LA suggesting they may work in synergy to form LA.

Diversity and community structure of anaerobic gut fungi in the rumen of wild and domesticated herbivores.

The rumen houses a diverse community that plays a major role in the digestion process in ruminants. Anaerobic gut fungi (AGF) are key contributors to plant digestion in the rumen. Here, we present a global amplicon-based survey of the rumen AGF mycobiome by examining 206 samples from 15 animal species, 15 countries, and 6 continents. The rumen AGF mycobiome was highly diverse, with 81 out of 88 currently recognized AGF genera or candidate genera identified. However, only six genera (Neocallimastix, Orpinomyces, Caecomyces, Cyllamyces, NY9, and Piromyces) were present at >4% relative abundance. AGF diversity was higher in members of the families Antilocapridae and Cervidae compared to Bovidae. Community structure analysis identified a pattern of phylosymbiosis, where host family (10% of total variance) and species (13.5%) partially explained the rumen mycobiome composition. As well, diet composition (9%-19%), domestication (11.14%), and biogeography (14.1%) also partially explained AGF community structure; although sampling limitation, geographic range restrictions, and direct association between different factors hindered accurate elucidation of the relative contribution of each factor. Pairwise comparison of rumen and fecal samples obtained from the same subject (n = 13) demonstrated greater diversity and inter-sample variability in rumen versus fecal samples. The genera Neocallimastix and Orpinomyces were present in higher abundance in rumen samples, while Cyllamyces and Caecomyces were enriched in fecal samples. Comparative analysis of global rumen and feces data sets revealed a similar pattern. Our results provide a global view of AGF community in the rumen and identify patterns of AGF variability between rumen and feces in herbivores Gastrointestinal (GI) tract.IMPORTANCERuminants are highly successful and economically important mammalian suborder. Ruminants are herbivores that digest plant material with the aid of microorganisms residing in their GI tract. In ruminants, the rumen compartment represents the most important location where microbially mediated plant digestion occurs, and is known to house a bewildering array of microbial diversity. An important component of the rumen microbiome is the anaerobic gut fungi (AGF), members of the phylum Neocallimastigomycota. So far, studies examining AGF diversity have mostly employed fecal samples, and little is currently known regarding the identity of AGF residing in the rumen compartment, factors that impact the observed patterns of diversity and community structure of AGF in the rumen, and how AGF communities in the rumen compare to AGF communities in feces. Here, we examined the rumen AGF diversity using an amplicon-based survey targeting a wide range of wild and domesticated ruminants (n = 206, 15 different animal species) obtained from 15 different countries. Our results demonstrate that while highly diverse, no new AGF genera were identified in the rumen mycobiome samples examined. Our analysis also indicate that animal host phylogeny, diet, biogeography, and domestication status could play a role in shaping AGF community structure. Finally, we demonstrate that a greater level of diversity and higher inter-sample variability was observed in rumen compared to fecal samples, with two genera (Neocallimastix and Orpinomyces) present in higher abundance in rumen samples, and two others (Cyllamyces and Caecomyces) enriched in fecal samples. Our results provide a global view of the identity, diversity, and community structure of AGF in ruminants, elucidate factors impacting diversity and community structure of the rumen mycobiome, and identify patterns of AGF community variability between the rumen and feces in the herbivorous GI tract.

Assessing the impact of three feeding stages on rumen bacterial community and physiological characteristics of Japanese Black cattle.

In Japan, Japanese Black cattle, known for their exceptional meat quality owing to their abundant intramuscular fat, undergo a unique three-stage feeding system with varying concentrate ratios. There is limited research on physiological and rumen microbial changes in Japanese Black cattle during these stages. Therefore, this study aimed to examine Japanese Black steers in these three stages: early (T1, 12-14 months), middle (T2, 15-22 months), and late (T3, 23-30 months). The rumen bacteria of 21 cattle per phase was analyzed using 16S rRNA gene sequencing. Rumen bacterial diversity was significantly higher in T1, with a distinct distribution, than in T2 and T3. Specific phyla and genera were exclusive to each stage, reflecting the shifts in feed composition. Certain genera dominated each stage: T1 had Flexilinea, Streptococcus, Butyrivibrio, Selenomonas, and Kandleria; T2 had Bifidobacterium, Shuttleworthia, and Sharpea; and T3 had Acetitomaculum, Mycoplasma, Atopobium, and Howardella. Correlation analysis revealed significant associations between certain microbial populations and physiological parameters. These findings indicate that changes in energy content and feed composition are associated with physiological and ruminal alterations. This study may guide strategies to improve rumen health and productivity in Japanese Black cattle by modifying diets to specific fattening stages.

Effects of the alpine meadow in different phenological periods on rumen fermentation and gastrointestinal tract bacteria community in grazing yak on the Qinghai-Tibetan Plateau.

BACKGROUND: In this study, we investigated the effects of alpine meadow in different phenological periods on ruminal fermentation, serum biochemical indices, and gastrointestinal tract microbes in grazing yak on the Qinghai-Tibetan Plateau. A total of eighteen female freely grazing yaks with an average age of 3 years old and a body weight of 130 ± 19 kg were selected. According to the plant phenological periods, yaks were randomly allocated to one of three treatments: (1) regreen periods group (RP, n = 6); (2) grassy periods group (GP, n = 6); and (3) hay periods group (HP, n = 6). At the end of the experiment, the blood, rumen fluids, and rectal contents were collected to perform further analysis. RESULTS: The concentrations of total volatile fatty acid (TVFA), acetate, glucose (GLU), triglyceride (TG), cholesterol (CHO), high density lipoprotein (HDL), and low density lipoprotein (LDL) were higher in the GP group than in the HP group (P < 0.05). However, compared with the RP and GP groups, the HP group had higher concentrations of isobutyrate, isovalerate, valerate, and creatinine (CREA) (P < 0.05). The abundance of Prevotella in the rumen, and the abundances of Rikenellaceae_RC9_gut_group, Eubacterium_coprostanoligenes_group, and Prevotellaceae_UCG-004 in the gut were higher in the GP group compared with the HP group (P < 0.05). The HP had higher abundance of Eubacterium_coprostanoligenes_group in the rumen as well as the abundances of Romboutsia and Arthrobacter in the gut compared with the RP and GP groups (P < 0.05). CONCLUSIONS: Based on the results of rumen fermentation, serum biochemical, differential biomarkers, and function prediction, the carbohydrate digestion of grazing yak would be higher with the alpine meadow regreen and grassy due to the gastrointestinal tract microbes. However, the risk of microbe disorders and host inflammation in grazing yak were higher with the alpine meadow wither.

Bacterial community and diversity in the rumen of 11 Mongolian cattle as revealed by 16S rRNA amplicon sequencing.

Through microorganism in the rumen of ruminant, plant fiber can be converted to edible food such as meat and milk. Ruminants had a rich and complex microbial community within the rumen, and the bacteria comprised the dominant proportion of the ruminal microbes. High-throughput sequencing offered a viable solution for the study of rumen microbes. In this study, rumen fluid samples were taken from 11 cattle from Inner Mongolian, the DNA of 11 rumen fluid samples were extracted and bacterial amplicons of the V4 regions of 16S rRNA were subjected to Illumina sequencing. More than 90,000 raw reads and 60,000 effect Tags per sample were obtained. 28,122 operational taxonomic units (OTUs) were observed from 11 samples, in average 2557 ± 361 OTUs for each sample. Bacteroidetes (44.41 ± 7.31%), Firmicutes (29.07 ± 3.78%), and Proteobacteria (7.18 ± 5.63%) were the dominant phyla among the bacteria of rumen, accounting for 82%. At the genus level, the highest relative abundance was Prevotella. Their functions were predicted using the Kyoto Encyclopedia of Genes and Genomes (KEGG). The results showed that they included metabolism, genetic information processing, environmental information processing and cellular processes. It explored the bacterial community diversity and composition of the rumen of Mongolian cattle. On the whole, our research showed that there was a high diversity as well as rich bacterial flora function of rumen bacteria in Mongolian cattle. Meanwhile, these findings provided information for further studies on the relationship between the community, diversity, functions of rumen bacteria and the nutritional physiological functions of the host.

Isolation and characterization of tannin-degrading bacteria from the rumen of wild Hokkaido sika deer (Cervus nippon yezoensis).

We isolated tannin-degrading bacteria from the rumen of wild Hokkaido sika deer and characterized their phylogeny and tannase activity in relation to sample sources. The condensed tannin level was higher in all deer rumen samples (n = 20) than in forage-fed cattle rumen samples (n = 6), whereas no hydrolyzable tannins were detected in any of the rumen samples. Rumen bacteria were enumerated on nonselective brain heart infusion (BHI) agar medium and then transferred onto tannic acid-containing BHI agar plates to screen for bacteria only showing growth (tannin-resistant bacteria) and those showing both growth and a clear zone (tannin-degrading bacteria). Summer samples provided only tannin-resistant bacteria, none of which showed tannin-degrading activity. Although winter samples also provided tannin-resistant bacteria, most isolates exhibited tannin-degrading activity. A total of 70 isolates exhibiting tannin-degrading activity were classified as Streptococcus bovis group based on 16S rRNA gene sequencing and further classified into two groups, either group A or group B. Group A consisted of isolates showing weak tannase activity, whereas group B included a majority of the isolates exhibiting high tannase activity. These results suggest that wild Hokkaido sika deer develop tannin-degrading Streptococcus in the rumen during winter, which allows access to woody food materials rich in tannins.

TMT-based quantitative proteomics reveals the nutritional and stress resistance functions of anaerobic fungi in yak rumen during passage at different time intervals.

OBJECTIVES: Anaerobic fungi are critical for nutrient digestion in the yak rumen. Although studies have reported the effects of passage at different time intervals on the community structure of yak rumen anaerobic fungi, it is unknown whether passage culture at different time intervals affects the microbial proteins of rumen anaerobic fungi and their functions. METHODS: Mycelium was obtained using the anaerobic continuous batch culture (CBC) of yak rumen fluid at intervals of 3 d, 5 d and 7 d. Quantitative analysis of fungal proteins and functional analysis was performed using tandem mass tagging (TMT) and bioinformatics. RESULTS: A total of 56 differential proteins (DPs) were found in 5 d vs. 3 d and 7 d vs. 3 d. Gene ontology (GO) enrichment indicated that the up-regulated proteins were mainly involved in biological regulation, cellular process, metabolic process, macromolecular complex, membrane, cell part, organelle, binding, catalytic activity and transporter activity. The downregulated proteins were mainly enriched in metabolic process, cell part, binding and catalytic activity. Furthermore, the downregulated proteins in 7 d vs. 3 d were related to membrane and organelle. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results indicated that DPs were enriched in 14 pathways in 5 d vs. 3 d and 7 d vs. 3 d, mainly including terpenoid backbone biosynthesis, alaine, aspartate and glutamate metabolism, arginine biosynthesis, hypotaurine, cyanoamino acid, glutathione, ß-alanine, pyrimidine, purine, galactose and propanate metabolism, steroid biosynthesis, ribosome biogenesis in eukaryotes and aminoacyl tRNA biosynthesis. The DPs were enriched in only 2 pathways in 5 d vs 3 d, lysine biosynthesis and cysteine and methionine metabolism. N-glycan biosynthesis and retinol metabolism are only found in the metabolism of DPs in 7 d vs 3 d. CONCLUSIONS: Yak rumen anaerobic fungal proteins are involved in nutrition and stress tolerance during passage at different time intervals.

Altered microbiota, antimicrobial resistance genes, and functional enzyme profiles in the rumen of yak calves fed with milk replacer.

IMPORTANCE: Yaks, as ruminants inhabiting high-altitude environments, possess a distinct rumen microbiome and are resistant to extreme living conditions. This study investigated the microbiota, resistome, and functional gene profiles in the rumen of yaks fed milk or milk replacer (MR), providing insights into the regulation of the rumen microbiome and the intervention of antimicrobial resistance in yaks through dietary methods. The abundance of Prevotella members increased significantly in response to MR. Tetracycline resistance was the most predominant. The rumen of yaks contained multiple antimicrobial resistance genes (ARGs) originating from different bacteria, which could be driven by MR, and these ARGs displayed intricate and complex interactions. MR also induced changes in functional genes. The enzymes associated with fiber degradation and butyrate metabolism were activated and showed close correlations with Prevotella members and butyrate concentration. This study allows us to deeply understand the ruminal microbiome and ARGs of yaks and their relationship with rumen bacteria in response to different milk sources.

Response of rumen microorganisms to pH during anaerobic hydrolysis and acidogenesis of lignocellulose biomass.

Rumen microorganisms can efficiently degrade lignocellulosic wastes to produce volatile fatty acids (VFAs). pH is a key factor in controlling the type and yield of VFAs by affecting the microorganisms involved in rumen fermentation. However, the effects of different pH on rumen microbial diversity, communities, and mechanisms are unclear. In this study, the hydrolysis and acidogenesis of corn straw and diversity, communities, and mechanisms of rumen microorganisms were explored at different initial pHs. Results showed that the highest hemicellulose, cellulose, and lignin degradation efficiency of corn straw was 55.2 %, 38.3 %, and 7.01 %, respectively, and VFA concentration was 10.2 g/L at pH 7.0. Low pH decreased the bacterial diversity and increased the fungal diversity. Rumen bacteria and fungi had different responses to initial pHs, and the community structure of bacteria and fungi had obviously differences at the genus level. The core genera Succiniclasticum, Treponema, and Neocallimastix relative abundance at initial pH 7.0 samples were significantly higher than that at lower initial pHs, reaching 6.01 %, 1.61 %, and 5.35 %, respectively. The bacterial network was more complex than that of fungi. pH, acetic acid, and propionic acid were the main factors influencing the bacterial and fungal community structure. Low pH inhibited the expression of functional genes related to hydrolysis and acidogenesis, explaining the lower hydrolysis and acidogenesis efficiency. These findings will provide a better understanding for rumen fermentation to produce VFAs.

A rumen virosphere with implications of contribution to fermentation and methane production, and endemism in cattle breeds and individuals.

Viruses have a potential to modify the ruminal digestion via infection and cell lysis of prokaryotes, suggesting that viruses are related to animal performance and methane production. This study aimed to elucidate the genome-based diversity of rumen viral communities and the differences in virus structure between individuals and cattle breeds and to understand how viruses influence on the rumen. To these ends, a metagenomic sequencing of virus-like particles in the rumen of 22 Japanese cattle, including Japanese Black (JB, n = 8), Japanese Shorthorn (n = 2), and Japanese Black sires × Holstein dams crossbred steers (F1, n = 12) was conducted. Additionally, the rumen viromes of six JB and six F1 that were fed identical diets and kept in a single barn were compared. A total of 8,232 non-redundant viral genomes (≥5-kb length and ≥50% completeness), including 982 complete genomes, were constructed, and rumen virome exhibited lysogenic signatures. Furthermore, putative hosts of 1,223 viral genomes were predicted using tRNA and clustered regularly interspaced short palindromic repeat (CRISPR)-spacer matching. The genomes included 1 and 10 putative novel complete genomes associated with Fibrobacter and Ruminococcus, respectively, which are the main rumen cellulose-degrading bacteria. Additionally, the hosts of 22 viral genomes, including 2 complete genomes, were predicted as methanogens, such as Methanobrevibacter and Methanomethylophilus. Most rumen viruses were highly rumen and individual specific and related to rumen-specific prokaryotes. Furthermore, the rumen viral community structure was significantly different between JB and F1 steers, indicating that cattle breed is one of the factors influencing the rumen virome composition.IMPORTANCEHere, we investigated the individual and breed differences of the rumen viral community in Japanese cattle. In the process, we reconstructed putative novel complete viral genomes related to rumen fiber-degrading bacteria and methanogen. The finding strongly suggests that rumen viruses contribute to cellulose and hemicellulose digestion and methanogenesis. Notably, this study also found that rumen viruses are highly rumen and individual specific, suggesting that rumen viruses may not be transmitted through environmental exposure. More importantly, we revealed differences of viral communities between JB and F1 cattle, indicating that cattle breed is a factor that influences the establishment of rumen virome. These results suggest the possibility of rumen virus transmission from mother to offspring and its potential to influence beef production traits. These rumen viral genomes and findings provide new insights into the characterizations of the rumen viruses.

Combined effects of nitrate and medium-chain fatty acids on methane production, rumen fermentation, and rumen bacterial populations in vitro.

This study investigated the combined effects of nitrate (NT) and medium-chain fatty acids (MCFA), including C8, C10, C12, and C14, on methane (CH4) production, rumen fermentation characteristics, and rumen bacteria using a 24 h batch incubation technique. Four types of treatments were used: control (no nitrate, no MCFA), NT (nitrate at 3.65 mM), NT + MCFA (nitrate at 3.65 mM + one of the four MCFA at 500 mg/L), and NT + MCFA/MCFA (nitrate at 3.65 mM + a binary combination of MCFA at 250 and 250 mg/L). All treatments decreased (P < 0.001) methanogenesis (mL/g dry matter incubated) compared with the control, but their efficiency was dependent on the MCFA type. The most efficient CH4 inhibitor was the NT + C10 treatment (- 40%). The combinations containing C10 and C12 had the greatest effect on bacterial alpha and beta diversity and relative microbial abundance (P < 0.001). Next-generation sequencing showed that the family Succinivibrionaceae was favored in treatments with the greatest CH4 inhibition at the expense of Prevotella and Ruminococcaceae. Furthermore, the relative abundance of Archaea decreased (P < 0.05) in the NT + C10 and NT + C10/C12 treatments. These results confirm that the combination of NT with MCFA (C10 and C12 in particular) may effectively reduce CH4 production.

Enhancing the Conventional Culture: the Evaluation of Several Culture Media and Growth Conditions Improves the Isolation of Ruminal Bacteria.

The rumen microbiota is critical in cattle digestion. Still, its low cultivability makes it difficult to study its ecological function and biotechnological potential. To improve the recovery of ruminal microorganisms, this study combined the evaluation of several cultivation parameters with metabarcoding analysis. The parameters tested comprised eight media cultures, three sample dilutions (10-2, 10-6, 10-12), and two incubation times (3 and 7 days). Bacterial populations were determined through Illumina sequencing of 16S rRNA from three biological replicates. The results indicate that none of the culture media recovered all rumen populations and that there was an altered relative abundance of the dominant phyla. In the rumen, Bacteroidetes and Firmicutes comprised 75% and 15% of the relative abundance, respectively, while in the culture media, these were 15% and 60%, respectively. Principal coordinate analysis (PCoA) of the bacterial community revealed significant shifts in population composition due to dilution, with 10-2 and 10-6 dilutions clustered closely while the 10-12 dilution differed markedly. In contrast, incubation duration did not influence population diversity. According to the results, two media, CAN and KNT, were selected based on their ability to recover more similar populations compared to the rumen sample. The metataxonomic study showed that CAN media had consistent reproducibility over time, while KNT showed enrichment of different taxa due to the use of rumen fluid as a substrate. From these, 64 pure cultures were obtained and 54 were identified through 16S rRNA gene sequencing. Being Streptococcus the most frequently isolated genus, this prevalence contrasts with the liquid media composition, underscoring the importance of refining single colony isolation strategies. Although no culture medium could replicate the native rumen bacterial population perfectly, our findings highlight the potential of CAN and KNT media in recovering populations that are more closely aligned to natural rumen conditions. In conclusion, our study emphasizes the importance of integrating molecular approaches in selecting suitable cultivation media and parameters to depict rumen bacteria accurately.