The rumen was a complex anaerobic fermentation system in which the microbial species cooperated with each other. They included bacteria (up to 1,011 cells/mL), anaerobic fungi (103–105 cells/mL), methanogens (106 cells/mL), ciliates (104–107 cells/mL), and bacteriophages (107–109 cells/mL) [24]. In this complex and diverse ecosystem, bacteria were the dominant microbial population. It could digest and degrade fibrous materials in plants that could not be directly used by humans and converted them into VFAs. It is thus absorbed by the rumen epithelium as a major carbon source for animal growth [25]. Rumen microbiota was an important protein source in ruminants and the main energy source for ruminants to produce VFAs through fiber fermentation. Therefore, rumen bacteria were a huge biological resource bank. It was very important to actively explore some functional genes of rumen microbiota closely related to important nutritional and physiological functions, such as carbohydrate transport and metabolism, Amino acid transport and metabolism, VFA production, etc. Recently, studies had shown that these microbes exhibit some niche specialization in nutrient utilization and could remodel the rumen ecosystem for subsequent microbial colonization and nutrient utilization [26][27].
In the GenBank Database, the vast majority of sequences of bacteria in the rumen belong to the Firmicutes and Bacteroidetes, followed by Proteobacteria [28]. Regarding the degradation of plant cell walls, both functions are distinct, with the degradation properties of Firmicutes mainly at the cell surface, whereas the degradation of Bacteroidetes is mainly at the cytosol or pericyte [29][30]. Firmicutes are mainly composed of three Bacilli: Clostridia, Bacilli, and Erysipelotrichi. The dominant families of Firmicutes are Lachnospiraceae, Ruminococcaceae, and Veillonellaceae. The dominant genera include Butyrivibrio, Acetivibrio, Ruminococcus, Succiniclasticum, Pseudobutyrivibrio, and Mogibacterium. Among the Bacilli, the dominant genera are Lactobacillus, such as Streptococcus and Carnobacterium [30]. For Bacteroidetes, most of the sequences belong to Bacteroidia and Sphingobacteria. Prevotella is the dominant genus, accounting for 60–70% of the sequences. Prevotella in the rumen is an important protein-degrading and starch-utilizing bacterium [31][32]. The dominant family of Proteobacteria is Succinivibrionaceae [33].
Koike [34] described the composition of sheep rumen bacteria and that Bacteroidota and Firmicutes were the predominant flora. The phylogenetic analysis of 16S rRNA gene sequences in sheep showed that Prevotella-related and Butyrivibrio fibrisolvens-related sequences formed large clusters in the phylogenetic tree [34]. Li [28] found 1,049 genera of 41 phyla in the digestive tract of goats. The intestinal flora was dominated by Bacteroidetes, and the Firmicutes were dominated in the intestine. When studying the effect of dietary energy and protein levels on rumen bacteria in Tan sheep, the results showed that the bacteria distributed in 16 phyla of 33 genera, as Firmicutes and Bacteroidetes were the main phyla. Moreover, the abundance of Firmicutes increased, and the number of Bacteroidetes and Proteobacteria decreased as the nutrient level increased. Meanwhile, the numbers of Prevotella, Ruminococcus, and Vibrio succinosus increased linearly, but the numbers of Weisseria mirabilis and Selenomonasruminantium did not increased (P > 0.05) [35]. Through metagenomes the effects of dietary energy on rumen fermentation and rumen microorganisms in fattening sheep were studied [36]. The results showed that the relative abundance of Prevotella, Succiniclasticum, Prevotellaceae, and Lachnospiraceae was closely related to rumen fermentation [36].
The 11 sheep used in this study were from the Xilin Gol League grassland of Plain Blue Banner, Inner Mongolia. The local sheep in the pastoral area of Xilin Gol grassland had a long history and were once the main mutton Inner Mongolia in the northern Xilin Gol grassland of China. The Xilin Gol grassland was a part of the Inner Mongolia Plateau, with an elevation of 800-1,200 meters. It was cold, windy and dry, with an average annual temperature of 1–2℃ and a frost-free period of 90–120 days. The annual precipitation of the Xilin Gol grassland was 150–400 mm. It belonged to the semi-arid and arid continental monsoon in the temperate zone. The local sheep are characterized by their medium size, firm meat, disease resistance, their ability to live in cold and semi-arid regions. And they had wide range of herbivorous types, such as Leymus chinensis, Festuca ovina, Agropyrun Cristatum, Clinelymus dahuricus, Hordeum brevisubulatum, and Achnatherum splendens, which contains 80.42% of total cellulose. The rumen of this kind of sheep had the ability to degrade cellulose efficiently. The study on the population structure and abundance of rumen bacteria in the sheep will lay a foundation for future research.
In the study, 16S rDNA amplicon sequencing technology was used to detect the composition of rumen bacteria in Mongolian sheep from Inner Mongolia Xilin Gol League, Plain Blue Banner pasture. The function of rumen bacteria was predicted according to the sequencing results. Through the dilution curve and Shannon, Simpson, chao1, ACE, good’s coverage, and PD-whole-tree index, it was indicated the diversity of rumen microorganisms in Mongolian sheep. The results of rumen flora structure analysis showed that the phylum was Bacteroides, Sclerochaeta, Proteobacteria, and Spirochaete in order of abundance. At the genus level, the microbial content of Prevost in the rumen fluid of Mongolian sheep in the two groups was the highest. At the phylum level, Bacteroidota and Firmicutes were the dominant flora in all samples.
Through the relative abundance analysis of each level, Bacteroidetes and Firmicutes showed higher relative abundance, accounting for 64.0%-85.0% together. Firmicutes and Bacteroidetes were mainly phyla reported in most studies, which could hydrolyze and acidify lignocellulose biomass [37]. Among them, Bacteroides could produce acetic acid, propionic acid or succinic acid as the final product of fermentation. This relative abundance at the phylum level was consistent with previous studies. Bacteroidetes and Firmicutes contain a variety of hydrolytic and acidogenic bacteria such as Prevotella, Clostridium, Bacteroides, and Ruminococcus, etc. It could be rapidly enriched in the environment with high soluble organic matter content. They degraded cellulose and hemicellulose to sugars, which were further converted into VFAs. Based on the family level of Bacteroide, Prevotellaceae had the highest relative abundance. The genera Prevotella was efficient hemicellulose and starch degrader, which could degrade hemicellulose to produce sugars and further convert the sugars to VFA[38]. The members of Prevotella have the ability to glycolysis, for example the metabolism of sugars to acetate, propionate, and succinate [39]. Prevotella is involved in the degradation of wheat straw in the stomach [40]. In addition, Clostridium is an important acid-producing bacteria in anaerobic fermentation system [41]. Notably, some members in Lachnospiraceae group can produce butyrate to prevent inflammation [40], while Ali stipes is also an important genus of anti-inflammatory bacilli [42]. Prevotella, Fibrobacter, Bacteroidetes, Clostridium, Bacillus, and Lachnospiraceae could secrete cellulase to digest cellulose [41]. At the same time, it is now widely believed that Fibrobacter succinogenes, Ruminococcus favefaciens and Ruminococcus albus were the main fiber-degrading bacteria in rumen [43]. In this study, it could be seen that the aforementioned bacteria, including Prevotella, Fibrobacter, Bacteroidetes, Clostridium, Bacillus, and Lachnospiraceae, were distributed in 11 samples, but their distribution patterns and relative abundance were different. At the genus level, Prevotella was widely distributed in all 11 samples. Especially in Y7, Prevotella had the highest relative abundance at 19%. It was worth mentioning that in Y5, the relative abundance of Rikenellaceae-RC9-gut group at the genus level was 6% higher than that of Prevotella. However, the relative abundance of Rikenellaceae-RC9-gut group was relatively high at 9% in Y8 and Y10, and slightly higher in Y10 than in Y8. Fibrobacter had a relatively low abundance in all samples, with relative abundance ranging from 0.7–1% at the genus level. The content of Bacteroides was high in the sample, with a relative abundance ranging from 33–47%, and the relative abundance was highest in sample Y7 and lowest in sample Y5. The relative abundance of Clostridia at the class level ranged from 20–29%, with the main genus being Christensenellaceae_R-7_group NK4A214_group Saccharofermentans and Ruminococcaceae. Lachnospirales existed in all sample and belongs to Clostridia, but their relative abundance was very low at the order level, ranging from 4–7%, with the highest in Y5 at 7%. The relative abundance of Bacillus was relatively low, and the relative abundance of each sample was similar at the class level, ranging from 7–10%, with the main genus being Lactobacillus. It could be seen that rumen fluid had a complex microbial system, and the degradation of cellulose did not depend on the action of a single bacterium, but rather on the interaction of many bacterial species. However, the main bacterial genera were Prevotella, Fibrobacterium, and Bacteroides, among others.
In the functional metabolic pathway, the main functional pathway-related genes show obvious time and space specificity. For example, genes related to amino acid metabolism, energy metabolism, translation and vitamin cofactor metabolism pathways are mainly found in the rumen, while genes related to carbohydrate metabolism and membrane transport are mainly found in the posterior intestinal mucosa. In the rumen, the gut microbiota may be more involved in the degradation of carbohydrates and the mucosa in energy metabolism, while in the caecum, the gut microbiota may be more involved in amino acid metabolism [28]. KEGG pathway enrichment analysis further revealed that a high-energy diet increased the lipid metabolism of the microbiota. Carbohydrate-active enzyme (CAZy) genes involved in energy metabolism were up-regulated in the high-energy diet group, while genes regulating cell wall degradation were down-regulated in the high-energy diet group. These results suggest that a high-energy diet has little effect on rumen fermentation patterns, but alters the composition of the rumen microbiota, improves lipid metabolism of the microbiota, and limits the metabolism of crude fiber [36]. For example, the potential function of rumen bacteria in Tan sheep was predicted. Especially, the relative abundance of carbohydrate-related genes was higher, and the relative abundance of amino acid metabolism-related genes increased linearly [35]. In the study, 16S functional prediction and comparison between the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway database and the SILVA SSU Ref NR database showed that the functions were concentrated in metabolism, genetic information processing, environmental information processing, cellular processes, etc. And it may contain metabolic enzyme-related genes and a large number of cellulose and lignin-degrading enzyme genes.
To sum up, in the study, 11 Mongolian sheep were selected from a pasture (longitude 116.0, latitude 42.25) in Zhenglan Banner of Xilin Gol League, Inner Mongolia. The 11 adult Mongolia sheep was named as Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10 and Y11. Through 16S rDNA amplicon sequencing, it was found that Firmicutes and Bacteroidetes were dominant in the composition of rumen microorganisms in the Mongolian sheep from Zhenglanqi pasture of Xilin Gol League, Inner Mongolia. Prevotella and other bacteria were also found at the family level, indicating that the rumen of Mongolian sheep was rich in microorganisms degrading lignocellulose. Compared with KEGG metabolic pathway database and Silva SSU ref NR database, it was found that Mongolian sheep rumen was concentrated in metabolism, genetic information processing, environmental information processing, and cellular processes, which may contain metabolic enzyme-related genes and a large number of cellulose and lignin-degrading enzyme genes. The microbial community composition was studied by 16S rDNA amplicon sequencing. The results showed that there were abundant lignocellulose degrading microorganisms and cellulase enzymes in Mongolian sheep rumen. And it guided the screening of cellulolytic enzyme genes with industrial application potential.