Characterizing the Alteration in Rumen Microbiome and Carbohydrate-Active Enzymes Profile with Forage of Muskoxen Rumen through Comparative Metatranscriptomics.

Muskox (Ovibos moschatus), as the biggest herbivore in the High Arctic, has been enduring the austere arctic nutritional conditions and has evolved to ingest and digest scarce and high lignified forages to support the growth and reproduce, implying probably harbor a distinct microbial reservoir for the deconstruction of plant biomass. Therefore, metagenomics approach was applied to characterize the rumen microbial community and understand the alteration in rumen microbiome of muskoxen fed either triticale straw or brome hay. The difference in the structure of microbial communities including bacteria, archaea, fungi, and protozoa between the two forages was observed at the taxonomic level of genus. Further, although the highly abundant phylotypes in muskoxen rumen fed either triticale straw or brome hay were almost the same, the selective enrichment different phylotypes for fiber degrading, soluble substrates fermenting, electron and hydrogen scavenging through methanogenesis, acetogenesis, propionogenesis, and sulfur-reducing was also noticed. Specifically, triticale straw with higher content of fiber, cellulose selectively enriched more lignocellulolytic taxa and electron transferring taxa, while brome hay with higher nitrogen content selectively enriched more families and genera for degradable substrates-digesting. Intriguingly, the carbohydrate-active enzyme profile suggested an over representation and diversity of putative glycoside hydrolases (GHs) in the animals fed on triticale straw. The majority of the cellulases belonged to fiver GH families (i.e., GH5, GH6, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus, Piromyces, Neocallimastix, and Fibrobacter. Abundance of major genes coding for hemicellulose digestion was higher than cellulose mainly including GH8, GH10, GH16, GH26, and GH30, and these enzymes were produced by members of the genera Fibrobacter, Ruminococcus, and Clostridium. Oligosaccharides were mainly of the GH1, GH2, GH3, and GH31 types and were associated with the genera Prevotella and Piromyces. Our results strengthen metatranscriptomic evidence in support of the understanding of the microbial community and plant polysaccharide response to changes in the feed type and host animal. The study also establishes these specific microbial consortia procured from triticale straw group can be used further for efficient plant biomass hydrolysis.

Composition of bacterial and archaeal communities in the rumen of dromedary camel using cDNA-amplicon sequencing.

The camel is known to survive in harsh environmental conditions, due to its higher digestive efficiency of high-fiber diets compared with other ruminants. However, limited data are available on the microbial community in the rumen of a camel. In this study, the Illumina sequencing of V4 region of 16S rRNA genes based on RNA isolation was employed to get insight into the bacterial and archaeal communities associated with liquid and solid rumen fractions in eight camels under different feeding systems. Camels in group C1 were fed Egyptian clover hay plus concentrates mixture and camels of group C2 were fed fresh Egyptian clover. The results showed that liquid fraction has higher operational taxonomic units (OTUs) than solid fraction, and camel group C1 showed a higher microbial diversity than C2. The UniFrac analysis indicated that the microbial communities in camel groups are distinct. Moreover, phylum Firmicutes and Bacteroidetes dominated the bacterial community and Candidatus Methanomethylophilus dominated the archaeal community with a significant difference in the relative abundance between camel groups. Dominant bacterial genera were Prevotella, Fibrobacteres, Ruminococcus, and Butyrivibrio. There were many negative and positive correlations between and within bacterial and archaeal genera. The composition of microbial community in the rumen of a camel is similar to other ruminants with differences in the abundance.

Community structure and fibrolytic activities of anaerobic rumen fungi in dromedary camels.

Anaerobic fungi colonize the rumen and degrade cellulose and hemicellulose, which enable them to be key players in the lignocellulose fermentation. Consequently, an expansion of knowledge about rumen fungi could increase animal productivity, utilization of lignified forages like alfalfa hay, and enhance fibrolytic enzymes production. Here, we used an Internal Transcribed Spacer 1 (ITS1) clone library to investigate the anaerobic rumen fungi in camel and to investigate their ability to produce cellulase and xylanase in vitro. Rumen fluid was collected from camels fed Egyptian clover (n = 14), and wheat straw (n = 7) and fecal samples were collected from camels fed wheat straw and concentrates (n = 5), or natural grazing plants (n = 10). Neocallimastix and Cyllamyces were the most abundant anaerobic fungi in all camel groups. An anaerobic rumen fungi media containing alfalfa hay as a carbon source was inoculated by rumen and fecal samples to assess the ability of anaerobic rumen fungi in camel gut to produce cellulase and xylanase. The anaerobic gut fungi in the camel is diverse and has cellulolytic and xylanolytic activities, fungal culture from rumen samples of camel fed wheat straw (R2) exhibited highest cellulase production. In addition, many of the sequences in the current study have no equivalent cultured representative, indicating a novel diversity within the camel gut.

Total rRNA-Seq Analysis Gives Insight into Bacterial, Fungal, Protozoal and Archaeal Communities in the Rumen Using an Optimized RNA Isolation Method.

Advances in high throughput, next generation sequencing technologies have allowed an in-depth examination of biological environments and phenomena, and are particularly useful for culture-independent microbial community studies. Recently the use of RNA for metatranscriptomic studies has been used to elucidate the role of active microbes in the environment. Extraction of RNA of appropriate quality is critical in these experiments and TRIzol reagent is often used for maintaining stability of RNA molecules during extraction. However, for studies using rumen content there is no consensus on (1) the amount of rumen digesta to use or (2) the amount of TRIzol reagent to be used in RNA extraction procedures. This study evaluated the effect of using various quantities of ground rumen digesta and of TRIzol reagent on the yield and quality of extracted RNA. It also investigated the possibility of using lower masses of solid-phase rumen digesta and lower amounts of TRIzol reagent than is used currently, for extraction of RNA for metatranscriptomic studies. We found that high quality RNA could be isolated from 2 g of ground rumen digesta sample, whilst using 0.6 g of ground matter for RNA extraction and using 3 mL (a 5:1 TRIzol : extraction mass ratio) of TRIzol reagent. This represents a significant savings in the cost of RNA isolation. These lower masses and volumes were then applied in the RNA-Seq analysis of solid-phase rumen samples obtained from 6 Angus X Hereford beef heifers which had been fed a high forage diet (comprised of barley straw in a forage-to-concentrate ratio of 70:30) for 102 days. A bioinformatics analysis pipeline was developed in-house that generated relative abundance values of archaea, protozoa, fungi and bacteria in the rumen and also allowed the extraction of individual rRNA variable regions that could be analyzed in downstream molecular ecology programs. The average relative abundances of rRNA transcripts of archaea, bacteria, protozoa and fungi in our samples were 1.4 ± 0.06, 44.16 ± 1.55, 35.38 ± 1.64, and 16.37 ± 0.65% respectively. This represents the first study to define the relative active contributions of these populations to the rumen ecosystem and is especially important in defining the role of the anaerobic fungi and protozoa.

Investigation and manipulation of metabolically active methanogen community composition during rumen development in black goats.

This study was performed to investigate the initial colonization of metabolically active methanogens and subsequent changes in four fractions: the rumen solid-phase (RS), liquid-phase (RL), protozoa-associated (RP), and epithelium-associated (RE) from 1 to 60 d after birth, and manipulate methanogen community by early weaning on 40 d and supplementing rhubarb from 40 to 60 d in black goats. The RNA-based real-time quantitative PCR and 16S rRNA amplicon sequencing were employed to indicate the metabolically active methanogens. Results showed that active methanogens colonized in RL and RE on 1 d after birth. RP and RE contained the highest and lowest density of methanogens, respectively. Methanobrevibacter, Candidatus Methanomethylophilus, and Methanosphaera were the top three genera. The methanogen communities before weaning differed from those post weaning and the structure of the methanogen community in RE was distinct from those in the other three fractions. The discrepancies in the distribution of methanogens across four fractions, and various fluctuations in abundances among four fractions according to age were observed. The addition of rhubarb significantly (P < 0.05) reduced the abundances of Methanimicrococcus spp. in four fractions on 50 d, but did not change the methanogen community composition on 60 d.