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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., (© 2023. The Author(s).)

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., (© 2022. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Background: Preweaned rumen development is vital for animal health and efficient fermentation. In this study, we integrated ruminal transcriptomic and metagenomic data to explore the dynamics of rumen functions, microbial colonization, and their functional interactions during the first 8 weeks of life in goats., Results: The dynamic rumen transcriptomic and microbial profiles both exhibited two distinct phases during early rumen development. The differentially expressed genes of the rumen transcriptome between the two phases showed that the immune-related response was enriched in the first phase and nutrient-related metabolism was enriched in the second phase, whereas the differentially expressed genes of the rumen microbiome were enriched in bacteriocin biosynthesis and glycolysis/gluconeogenesis activities. The developmental shift in the rumen transcriptome (at d 21) was earlier than the feed stimulus (at d 25) and the shift in the rumen microbiome (at d 42). Additionally, 15 temporal dynamic rumen gene modules and 20 microbial modules were revealed by coexpression network analysis. Functional correlations between the rumen and its microbiome were primarily involved in rumen pH homeostasis, nitrogen metabolism and the immune response. Rumen gene modules associated with the microbial alpha diversity index were also enriched in the immune response process., Conclusions: The present study touched the critical developmental process of rumen functions, microbial colonization and their functional interactions during preweaned development. Taken together, these results demonstrated that rumen development at the first phase is more likely a programmed process rather than stimulation from feed and the microbiome, while the shift of rumen metagenomes was likely regulated by both the diet and host. The intensive functional correlations between rumen genes and the microbiome demonstrated that synergistic processes occurred between them during early rumen development.

The rumen is the hallmark organ of ruminants and hosts a diverse ecosystem of microorganisms that facilitates efficient digestion of plant fibers. We analyzed 897 transcriptomes from three Cetartiodactyla lineages: ruminants, camels and cetaceans, as well as data from ruminant comparative genomics and functional assays to explore the genetic basis of rumen functional innovations. We identified genes with relatively high expression in the rumen, of which many appeared to be recruited from other tissues. These genes show functional enrichment in ketone body metabolism, regulation of microbial community, and epithelium absorption, which are the most prominent biological processes involved in rumen innovations. Several modes of genetic change underlying rumen functional innovations were uncovered, including coding mutations, genes newly evolved, and changes of regulatory elements. We validated that the key ketogenesis rate-limiting gene (HMGCS2) with five ruminant-specific mutations was under positive selection and exhibits higher synthesis activity than those of other mammals. Two newly evolved genes (LYZ1 and DEFB1) are resistant to Gram-positive bacteria and thereby may regulate microbial community equilibrium. Furthermore, we confirmed that the changes of regulatory elements accounted for the majority of rumen gene recruitment. These results greatly improve our understanding of rumen evolution and organ evo-devo in general.

The objective of this experiment was to characterize the relationship among rumen fermentation variables, milk fatty acid profile, and dietary physically effective neutral detergent fiber (peNDF) content in a study that controlled for the potential confounding effects of dissimilar dry matter intake among treatments. Ten multiparous Xinong Saanen dairy goats were divided into 2 groups with 2 ruminally cannulated goats per group. Goats in each group were assigned to 1 of 2 dietary treatments (high and low peNDF) according to a 2×2 crossover design with 2 periods. The peNDF content of alfalfa hay (proportion of neutral detergent fiber retained on an 8.0-mm screen) was 42.1% for the high-peNDF and 14.5% for the low-peNDF group. To ensure similar dry matter intake, each morning the amount of alfalfa hay consumed on the prior day by the high-peNDF group was determined (amount offered minus morning refusals), and this was the amount of hay offered to the low-peNDF group that day. Each adaptation period consisted of 21d, followed by a 9-d sampling period. Dry matter intake and milk production and composition were similar between treatments. Milk energy efficiency increased with low dietary peNDF. Duration of pH below 5.60 was longer for goats fed the low-peNDF ration compared with the high-peNDF ration (4.08 vs. 0.41h/d); however, mean rumen pH (6.05 vs. 6.13) was not different between treatments. Reducing dietary peNDF increased rumen total volatile fatty acids (114.6 vs. 95.1mM) and decreased chewing time (404 vs. 673min/d), but did not affect the ratios of acetate, propionate, and butyrate. The relative abundance of Fibrobacter succinogenes and Ruminococcus flavefaciens increased with reduced dietary peNDF, but Ruminococcus albus proportions were not influenced by treatment. Reducing dietary peNDF decreased the proportion of iso C14:0, iso C15:0, and trans-11 C18:1 in milk fat, whereas the iso C17:0 and trans-10 C18:1 increased. This study demonstrated that low dietary peNDF in dairy goats increases rumen volatile fatty acids, reduces chewing time, and is correlated to the amount of F. succinogenes and R. flavefaciens., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)