Background: Methanomassiliicoccales are a recently identified order of methanogens that are diverse across global environments particularly the gastrointestinal tracts of animals; however, their metabolic capacities are defined via a limited number of cultured strains.Results: Here, we profile and analyze 243 Methanomassiliicoccales genomes assembled from cultured representatives and uncultured metagenomes recovered from various biomes, including the gastrointestinal tracts of different animal species. Our analyses reveal the presence of numerous undefined genera and genetic variability in metabolic capabilities within Methanomassiliicoccales lineages, which is essential for adaptation to their ecological niches. In particular, gastrointestinal tract Methanomassiliicoccales demonstrate the presence of co-diversified members with their hosts over evolutionary timescales and likely originated in the natural environment. We highlight the presence of diverse clades of vitamin transporter BtuC proteins that distinguish Methanomassiliicoccales from other archaeal orders and likely provide a competitive advantage in efficiently handling B₁₂. Furthermore, genome-centric metatranscriptomic analysis of ruminants with varying methane yields reveal elevated expression of select Methanomassiliicoccales genera in low methane animals and suggest that B₁₂exchanges could enable them to occupy ecological niches that possibly alter the direction of H₂utilization.Conclusions: We provide a comprehensive and updated account of divergent Methanomassiliicoccales lineages, drawing from numerous uncultured genomes obtained from various habitats. We also highlight their unique metabolic capabilities involving B₁₂, which could serve as promising targets for mitigating ruminant methane emissions by altering H₂flow.

The newly proposed methanogenic order ‘Methanomassiliicoccales’ is the second largest archaeal population in the rumen, second only to the Methanobrevibacter population. However, information is limited regarding the community of this new order in the rumen. This study used real-time PCR and 454 pyrosequencing to explore the abundance and community composition of Methanomassiliicoccales in the rumen of Chinese goats fed a hay (0% grain, n = 5) or a high grain (65% grain, n = 5) diet. Real-time PCR analysis showed that the relative abundance of Methanomassiliicoccales (% of total archaea) in the goat rumen was significantly lower in the high-grain-diet group (0.5% ± 0.2%) than that in the hay-diet group (8.2% ± 1.1%, P < 0.05). The pyrosequencing results showed that a total of 208 operational taxonomic units (OTUs) were formed from ten samples at 99% sequence identity. All the sequences were identified as Methanomassiliicoccaceae at the family level, and most of the sequences (96.82% ± 1.64%) were further classified as Group 8, 9, and 10 at the Methanomassiliicoccales genus level in each sample based on the RIM-DB database. No significant differences were observed in the number of OTUs or Chao1’s, Shannon’s or Pielou’s evenness indexes between the hay- and high-grain-diet groups (P ≥ 0.05). PCoA analysis showed that diet altered the community of Methanomassiliicoccales. At the genus level, the relative abundances of Group 10 (67.25 ± 12.76 vs. 38.13 ± 15.66, P = 0.012) and Group 4 (2.07 ± 1.30 vs. 0.27 ± 0.30, P = 0.035) were significantly higher in the high-grain-diet group, while the relative abundance of Group 9 was significantly higher in the hay-diet group (18.82 ± 6.20 vs. 47.14 ± 17.72, P = 0.020). At the species level, the relative abundance of Group 10 sp. (67.25 ± 12.76 vs. 38.13 ± 15.66, P = 0.012) and Group 4 sp. MpT1 (2.07 ± 1.30 vs. 0.27 ± 0.30, P = 0.035) were significantly higher in the high-grain-diet group, while the relative abundance of Group 9 sp. ISO4-G1 was significantly higher in the hay-diet group (12.83 ± 3.87 vs. 42.44 ± 18.47, P = 0.022). Only a few highly abundant phylogenetic groups dominated within the Methanomassiliicoccales community in the rumens of Chinese goats, and these were easily depressed by high-grain-diet feeding. The relatively low abundance suggests a small contribution on the part of Methanomassiliicoccales to the rumen methanogenesis of Chinese goats.

The objective of this study was to investigate the effects of live yeast (LY, Saccharomyces cerevisiae) on the lactation performance, bacterial community, and functions in the rumen and hindgut of dairy cows under heat stress. Thirty-three multiparous (parity 3.9 ± 0.8) Holstein dairy cows (189.1 ± 6.6 d in milk at the beginning of the experiment) were randomly assigned to three groups (11 cows per treatment). Cows in the three groups were fed a diet without yeast (CON), with 10 g yeast/d/head (LY-10), and with 20 g yeast/d/head (LY-20). The yeast product contained 2.0 × 10¹⁰ CFU/g. Supplementing LY decreased the rectal temperature and respiratory rate of cows, and increased dry matter intake, milk yield, milk fat yield, milk protein yield, and milk lactose yield (P < 0.001), yet decreased milk urea nitrogen concentration (P = 0.035). Interaction effects of treatment × week were observed for rectal temperature (P < 0.05), respiratory rate (P < 0.05), milk yield (P = 0.015), milk urea nitrogen (P = 0.001), milk protein yield (P = 0.008), and milk lactose yield (P = 0.030). In rumen, LY increased the concentrations of acetate, isobutyrate, isovaterate, valerate, total volatile fatty acids (VFAs), and NH₃-N (P < 0.05). Miseq sequencing of the 16S rRNA genes showed that LY increased the relative abundance of Prevotella and Prevotellaceae UCG-003 at the genus level with a series of enriched pathways in the metabolism of carbohydrates and protein. In fecal samples, LY did not affect the profile of VFAs (P > 0.05). Clostridium sensu stricto 1 (P = 0.013) and Actinobacillus (P = 0.011) increased in the relative abundance by LY, whereas Bacteroides (P = 0.016) and Oscillospirales UCG-010 (P = 0.005) decreased with a series of enriched pathways in carbohydrate metabolism, secondary bile acid biosynthesis. In summary, LY supplementation altered the bacterial community’s composition and function in rumen and hindgut, and simultaneously alleviated the detrimental effects of heat stress on dairy cows. These findings provide extended insight into the effects of LY in the rumen and hindgut of dairy cows exposed to heat stress. Dairy cows are exposed to severe heat stress under hot and humid climates in summer in south China, resulting in a decline in feed intake and milk yield. Therefore, we investigated the effect of live yeast (LY, Saccharomyces cerevisiae) supplementation on the milk performance, bacterial community, and functions in the rumen and hindgut of dairy cows under heat stress. Thirty-three dairy cows were randomly assigned to control (CON, without yeast addition), treatment 1 (LY-10, with 10 g yeast/d/head) and treatment 2 (LY-20, with 20 g yeast/d/head). Supplementing LY decreased the rectal temperature and respiratory rate of the dairy cows and increased feed intake and milk performance. Live yeast enhanced fermentation in the rumen but did not affect it in the hindgut. Live yeast altered the microbiota in the rumen and hindgut, with an enrichment of bacteria in the pathways of the metabolism of carbohydrates, protein, and other substances. In all, LY supplementation had beneficial effects on dairy cows under heat stress by affecting the microbiota and fermentation in the rumen and hindgut.

Yeast strains are widely used in ruminant production. However, knowledge about the effects of rumen native yeasts on ruminants is limited. Therefore, this study aimed to obtain a rumen native yeast isolate and investigate its effects on growth performance, nutrient digestibility, rumen fermentation and microbiota in Hu sheep. Yeasts were isolated by picking up colonies from agar plates, and identified by sequencing the ITS sequences. One isolate belonging to Pichia kudriavzevii had the highest optical density among these isolates obtained. This isolate was prepared to perform an animal feeding trial. A randomized block design was used for the animal trial. Sixteen Hu sheep were randomly assigned to the control (CON, fed basal diet, n = 8) and treatment group (LPK, fed basal diet plus P. kudriavzevii, CFU = 8 × 10⁹ head/d, n = 8). Sheep were housed individually and treated for 4 weeks. Compared to CON, LPK increased final body weight, nutrient digestibility and rumen acetate concentration and acetate-to-propionate ratio in sheep. The results of Illumina MiSeq PE 300 sequencing showed that LPK increased the relative abundance of lipolytic bacteria (Anaerovibrio spp. and Pseudomonas spp.) and probiotic bacteria (Faecalibacterium spp. and Bifidobacterium spp.). For rumen eukaryotes, LPK increased the genera associated with fiber degradation, including protozoan Polyplastron and fungus Pichia. Our results discovered that rumen native yeast isolate P. kudriavzevii might promote the digestion of fibers and lipids by modulating specific microbial populations with enhancing acetate-type fermentation.