The diverse rumen virome can modulate the rumen microbiome, but it remains largely unexplored. Here, we mine 975 published rumen metagenomes for viral sequences, create a global rumen virome database (RVD), and analyze the rumen virome for diversity, virus-host linkages, and potential roles in affecting rumen functions. Containing 397,180 species-level viral operational taxonomic units (vOTUs), RVD substantially increases the detection rate of rumen viruses from metagenomes compared with IMG/VR V3. Most of the classified vOTUs belong to Caudovirales, differing from those found in the human gut. The rumen virome is predicted to infect the core rumen microbiome, including fiber degraders and methanogens, carries diverse auxiliary metabolic genes, and thus likely impacts the rumen ecosystem in both a top-down and a bottom-up manner. RVD and the findings provide useful resources and a baseline framework for future research to investigate how viruses may impact the rumen ecosystem and digestive physiology.

Protozoa comprise a major fraction of the microbial biomass in the rumen microbiome, of which the entodiniomorphs (order: Entodiniomorphida) and holotrichs (order: Vestibuliferida) are consistently observed to be dominant across a diverse genetic and geographical range of ruminant hosts. Despite the apparent core role that protozoal species exert, their major biological and metabolic contributions to rumen function remain largely undescribed in vivo. Here, we have leveraged (meta)genome-centric metaproteomes from rumen fluid samples originating from both cattle and goats fed diets with varying inclusion levels of lipids and starch, to detail the specific metabolic niches that protozoa occupy in the context of their microbial co-habitants. Initial proteome estimations via total protein counts and label-free quantification highlight that entodiniomorph species Entodinium and Epidinium as well as the holotrichs Dasytricha and Isotricha comprise an extensive fraction of the total rumen metaproteome. Proteomic detection of protozoal metabolism such as hydrogenases (Dasytricha, Isotricha, Epidinium, Enoploplastron), carbohydrate-active enzymes (Epidinium, Diplodinium, Enoploplastron, Polyplastron), microbial predation (Entodinium) and volatile fatty acid production (Entodinium and Epidinium) was observed at increased levels in high methane-emitting animals. Despite certain protozoal species having well-established reputations for digesting starch, they were unexpectedly less detectable in low methane emitting-animals fed high starch diets, which were instead dominated by propionate/succinate-producing bacterial populations suspected of being resistant to predation irrespective of host. Finally, we reaffirmed our abovementioned observations in geographically independent datasets, thus illuminating the substantial metabolic influence that under-explored eukaryotic populations have in the rumen, with greater implications for both digestion and methane metabolism.

Understanding the biodiversity and genetics of gut microbiomes has important implications for host physiology and industrial enzymes, whereas most studies have been focused on bacteria and archaea, and to a lesser extent on fungi and viruses. One group, still underexplored and elusive, is ciliated protozoa, despite its importance in shaping microbiota populations. Integrating single-cell sequencing and an assembly-and-identification pipeline, we acquired 52 high-quality ciliate genomes of 22 rumen morphospecies from 11 abundant morphogenera. With these genomes, we resolved the taxonomic and phylogenetic framework that revised the 22 morphospecies into 19 species spanning 13 genera and reassigned the genus Dasytricha from Isotrichidae to a new family Dasytrichidae. Comparative genomic analyses revealed that extensive horizontal gene transfers and gene family expansion provided rumen ciliate species with a broad array of carbohydrate-active enzymes (CAZymes) to degrade all major kinds of plant and microbial carbohydrates. In particular, the genomes of Diplodiniinae and Ophryoscolecinae species encode as many CAZymes as gut fungi, and ~80% of their degradative CAZymes act on plant cell-wall. The activities of horizontally transferred cellulase and xylanase of ciliates were experimentally verified and were 2–9 folds higher than those of the inferred corresponding bacterial donors. Additionally, the new ciliate dataset greatly facilitated rumen metagenomic analyses by allowing ~12% of the metagenomic sequencing reads to be classified as ciliate sequences.

Background: In recent years, nitrooxy compounds have been identified as promising inhibitors of methanogenesis in ruminants. However, when animals receive a nitrooxy compound, a high portion of the spared hydrogen is eructated as gas, which partly offsets the energy savings of CH₄ mitigation. The objective of the present study was to evaluate the long-term and combined effects of supplementation with N-[2-(nitrooxy)ethyl]-3-pyridinecarboxamide (NPD), a methanogenesis inhibitor, and fumaric acid (FUM), a hydrogen sink, on enteric CH₄ production, rumen fermentation, bacterial populations, apparent nutrient digestibility, and lactation performance of dairy goats. Results: Twenty-four primiparous dairy goats were used in a randomized complete block design with a 2 × 2 factorial arrangement of treatments: supplementation without or with FUM (32 g/d) or NPD (0.5 g/d). All samples were collected every 3 weeks during a 12-week feeding experiment. Both FUM and NPD supplementation persistently inhibited CH₄ yield (L/kg DMI, by 18.8% and 18.1%, respectively) without negative influence on DMI or apparent nutrient digestibility. When supplemented in combination, no additive CH₄ suppression was observed. FUM showed greater responses in increasing the molar proportion of propionate when supplemented with NPD than supplemented alone (by 10.2% vs. 4.4%). The rumen microbiota structure in the animals receiving FUM was different from that of the other animals, particularly changed the structure of phylum Firmicutes. Daily milk production and serum total antioxidant capacity were improved by NPD, but the contents of milk fat and protein were decreased, probably due to the bioactivity of absorbed NPD on body metabolism. Conclusions: Supplementing NPD and FUM in combination is a promising way to persistently inhibit CH₄ emissions with a higher rumen propionate proportion. However, the side effects of this nitrooxy compound on animals and its residues in animal products need further evaluation before it can be used as an animal feed additive.

Social interaction facilitates the horizontal transmission of the microbiota between different individuals. However, little is known about the level of microbiota transmission in different livestock animals and different digestive tracts. The Hainan black goat and Wuzhishan pig are typical tropical local breeds on Hainan Island in China. Thus, we sampled and analyzed the gut microbiome in Hainan black goats (cecum and rumen) and Wuzhishan pigs (cecum) to study horizontal transmission by rearing them in the same pen (six goats and six pigs) or separate pens (nine goats and nine pigs). De novo assembly and binning recovered 3,262 strain-level and 2,488 species-level metagenome-assembled genomes (MAGs) using ∼1.3 Tb sequencing data. Of these MAGs, 1,856 MAGs were identified as novel strain. Compared with goats living in separate pens, social interaction in the same pen promotes community homogeneity in the rumen microbiome (P < 0.05) and the cecum microbiome (P < 0.05), respectively. Notably, approximately 7.08% (231/3262) of the gut microbial population could transmit during cohousing, 12 strains only in inter-species transmission, versus 190 strains only in intra-species transmission, and 10 strains only in foregut and hindgut transmission. In addition, the social contact group has high transmitted strain abundance, which is correlated with community composition. This study provided a new insight into the influence of social interaction on the animal gut microbiota.

For the agroecosystems of the dairy cow industry, dietary carbohydrate (starch, neutral detergent fiber [NDF]) and fat could directly affect rumen methane emissions and host energy utilization. However, the relationships among diet, lactation performance, and methane emissions need to be further determined to assist dairy farms to adjust diet formulations and feeding strategies for environmental and production management. A meta-analysis was conducted in the current study to explore quantitative patterns of dietary fat and carbohydrate at different levels in balancing lactation performance and environment sustainability of dairy cows, and to establish a methane emission prediction model using the artificial neural network (ANN) model. The results showed that the regression relationship between dietary fat, carbohydrate and methane emissions could be shown by the following models: methane = 106.78 + (14.86 × DMI), R² = 0.80; methane = 443.17 – (46.41 × starch/NDF), R² = 0.76; and methane = 388.91 + (31.40 × fat) – (5.42 × fat²), R² = 0.80. The regression relationships between dietary fat, carbohydrate and lactation performance could be shown by the following models: milk fat yield = 1.08 + (0.43 × starch/NDF) – [0.34 × (starch/NDF)²], R² = 0.79; milk protein yield = 0.68 + (0.15 × fat) – (0.016 × fat²), R² = 0.82. In the structural equation model, we found that when formulating dietary carbohydrates and fats, it was necessary to balance the relationship between methane emissions and lactation performance. Specifically, dietary starch/NDF was lower than 0.63 (extremum point) and dietary fat was between 2.89% and 4.69% (extremum point), it could ensure that the aim of methane emission reduction (methane emissions decrease with increasing dietary starch/NDF and fat) was achieved without losing lactation performance of dairy cows (lactation performance increase with increasing dietary starch/NDF and fat). Finally, we established the ANN model to predict methane emissions (training set: R² = 0.62; validation set: R² = 0.61).