Assessment of different enrichment methods revealed the optimal approach to identify bovine circRnas.

Although circular RNAs (circRNAs) play important roles in regulating gene expression, the understanding of circRNAs in livestock animals is scarce due to the significant challenge to characterize them from a biological sample. In this study, we assessed the outcomes of bovine circRNA identification using six enrichment approaches with the combination of ribosomal RNAs removal (Ribo); linear RNAs degradation (R); linear RNAs and RNAs with structured 3' ends degradation (RTP); ribosomal RNAs coupled with linear RNAs elimination (Ribo-R); ribosomal RNA, linear RNAs and RNAs with poly (A) tailing elimination (Ribo-RP); and ribosomal RNA, linear RNAs and RNAs with structured 3' ends elimination (Ribo-RTP), respectively. RNA-sequencing analysis revealed that different approaches led to varied ratio of uniquely mapped reads, false-positive rate of identifying circRNAs, and the number of circRNAs per million clean reads (Padj <0.05). Out of 2,285 and 2,939 highly confident circRNAs identified in liver and rumen tissues, respectively, 308 and 260 were commonly identified from five methods, with Ribo-RTP method identified the highest number of circRNAs. Besides, 507 of 4,051 identified bovine highly confident circRNAs had shared splicing sites with human circRNAs. The findings from this work provide optimized methods to identify bovine circRNAs from cattle tissues for downstream research of their biological roles in cattle.

Methylation-GC-MS/FID-Based Glycosidic Linkage Analysis of Unfractionated Polysaccharides in Red Seaweeds.

Glycosidic linkage analysis was conducted on the unfractionated polysaccharides in alcohol-insoluble residues (AIRs) prepared from six red seaweeds (Gracilariopsis sp., Prionitis sp., Mastocarpus papillatus, Callophyllis sp., Mazzaella splendens, and Palmaria palmata) using GC-MS/FID analysis of partially methylated alditol acetates (PMAAs). The cell walls of P. palmata primarily contained mixed-linkage xylans and small amounts of sulfated galactans and cellulose. In contrast, the unfractionated polysaccharides of the other five species were rich in galactans displaying diverse 3,6-anhydro-galactose and galactose linkages with varied sulfation patterns. Different levels of cellulose were also observed. This glycosidic linkage method offers advantages for cellulose analysis over traditional monosaccharide analysis that is known for underrepresenting glucose in crystalline cellulose. Relative linkage compositions calculated from GC-MS and GC-FID measurements showed that anhydro sugar linkages generated more responses in the latter detection method. This improved linkage workflow presents a useful tool for studying polysaccharide structural variations across red seaweed species. Furthermore, for the first time, relative linkage compositions from GC-MS and GC-FID measurements, along with normalized FID and total ion current (TIC) chromatograms without peak assignments, were analyzed using principal component analysis (PCA) as a proof-of-concept demonstration of the technique's potential to differentiate various red seaweed species.

3-Nitrooxypropanol supplementation had little effect on fiber degradation and microbial colonization of forage particles when evaluated using the in situ ruminal incubation technique.

3-Nitrooxypropanol (3-NOP) is an investigational compound that acts as an enzyme inhibitor to decrease ruminal methanogenesis. We hypothesized that when feeding 3-NOP to cattle fed a high-forage diet, H2 would accumulate in the rumen, which could suppress microbial colonization of feed particles and fiber degradation. Therefore, the study investigated the effects of supplementing a high-forage diet with 3-NOP on ruminal fiber degradability and microbial colonization of feed particles using the in situ technique. Eight ruminally cannulated beef cattle were allocated to 2 groups (4 cattle/group) in a crossover design with 2 periods and 2 dietary treatments. The treatments were control (basal diet) and 3-NOP (basal diet supplemented with 3-NOP, 150 mg/kg of dry matter). The basal diet consisted of 45% barley silage, 45% chopped grass hay, and 10% concentrate (dry matter basis). Samples of dried, ground barley silage and grass hay were incubated in the rumen of each animal for 0, 4, 12, 24, 36, 48, 96, 120, 216, and 288 h to determine neutral detergent fiber (NDF) degradation kinetics. An additional 2 bags were incubated for 4 and 48 h to evaluate the bacterial community attached to the incubated forages. Dietary supplementation of 3-NOP decreased (-53%) the dissolved methane concentration and increased (+780%) the dissolved H2 concentration in ruminal fluid, but did not substantially alter in situ NDF degradation. The addition of 3-NOP resulted in a decrease in the α-diversity of the microbial community with colonizing communities showing reduced numbers of amplicon sequence variants and phylogenetic diversity compared with control diets. Principal coordinate analysis plots indicated that forages incubated in animals fed 3-NOP resulted in highly specific changes to targeted microbes compared with control diets based on unweighted analysis (considering only absence and presence of taxa), but did not alter the overall composition of the colonizing community based on weighted UniFrac distances; unchanged relative abundances of major taxa included phyla Bacteroidetes, Firmicutes, and Fibrobacteres. The effect of 3-NOP on colonizing methanogenic microbes differed depending upon the forage incubated, as abundance of genus Methanobrevibacter was decreased for barley silage but not for grass hay. In conclusion, 3-NOP supplementation of a high-forage diet decreased ruminal methanogenesis and increased dissolved H2 concentration, but had no negative effects on ruminal fiber degradation and only minor effects on relative abundances of the major taxa of bacteria adhered to forage substrates incubated in the rumen.

Bacterial PhyA protein-tyrosine phosphatase-like myo-inositol phosphatases in complex with the Ins(1,3,4,5)P4 and Ins(1,4,5)P3 second messengers.

myo-Inositol phosphates (IPs) are important bioactive molecules that have multiple activities within eukaryotic cells, including well-known roles as second messengers and cofactors that help regulate diverse biochemical processes such as transcription and hormone receptor activity. Despite the typical absence of IPs in prokaryotes, many of these organisms express IPases (or phytases) that dephosphorylate IPs. Functionally, these enzymes participate in phosphate-scavenging pathways and in plant pathogenesis. Here, we determined the X-ray crystallographic structures of two catalytically inactive mutants of protein-tyrosine phosphatase-like myo-inositol phosphatases (PTPLPs) from the non-pathogenic bacteria Selenomonas ruminantium (PhyAsr) and Mitsuokella multacida (PhyAmm) in complex with the known eukaryotic second messengers Ins(1,3,4,5)P4 and Ins(1,4,5)P3 Both enzymes bound these less-phosphorylated IPs in a catalytically competent manner, suggesting that IP hydrolysis has a role in plant pathogenesis. The less-phosphorylated IP binding differed in both the myo-inositol ring position and orientation when compared with a previously determined complex structure in the presence of myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP6 or phytate). Further, we have demonstrated that PhyAsr and PhyAmm have different specificities for Ins(1,2,4,5,6)P5, have identified structural features that account for this difference, and have shown that the absence of these features results in a broad specificity toward Ins(1,2,4,5,6)P5 These features are main-chain conformational differences in loops adjacent to the active site that include the extended loop prior to the penultimate helix, the extended Ω-loop, and a ß-hairpin turn of the Phy-specific domain.

Discovery and characterization of family 39 glycoside hydrolases from rumen anaerobic fungi with polyspecific activity on rare arabinosyl substrates.

Enzyme activities that improve digestion of recalcitrant plant cell wall polysaccharides may offer solutions for sustainable industries. To this end, anaerobic fungi in the rumen have been identified as a promising source of novel carbohydrate active enzymes (CAZymes) that modify plant cell wall polysaccharides and other complex glycans. Many CAZymes share insufficient sequence identity to characterized proteins from other microbial ecosystems to infer their function; thus presenting challenges to their identification. In this study, four rumen fungal genes (nf2152, nf2215, nf2523, and pr2455) were identified that encode family 39 glycoside hydrolases (GH39s), and have conserved structural features with GH51s. Two recombinant proteins, NF2152 and NF2523, were characterized using a variety of biochemical and structural techniques, and were determined to have distinct catalytic activities. NF2152 releases a single product, ß1,2-arabinobiose (Ara2) from sugar beet arabinan (SBA), and ß1,2-Ara2 and α-1,2-galactoarabinose (Gal-Ara) from rye arabinoxylan (RAX). NF2523 exclusively releases α-1,2-Gal-Ara from RAX, which represents the first description of a galacto-(α-1,2)-arabinosidase. Both ß-1,2-Ara2 and α-1,2-Gal-Ara are disaccharides not previously described within SBA and RAX. In this regard, the enzymes studied here may represent valuable new biocatalytic tools for investigating the structures of rare arabinosyl-containing glycans, and potentially for facilitating their modification in industrial applications.

Structure and mechanism of Staphylococcus aureus TarM, the wall teichoic acid α-glycosyltransferase.

Unique to Gram-positive bacteria, wall teichoic acids are anionic glycopolymers cross-stitched to a thick layer of peptidoglycan. The polyol phosphate subunits of these glycopolymers are decorated with GlcNAc sugars that are involved in phage binding, genetic exchange, host antibody response, resistance, and virulence. The search for the enzymes responsible for GlcNAcylation in Staphylococcus aureus has recently identified TarM and TarS with respective α- and ß-(1-4) glycosyltransferase activities. The stereochemistry of the GlcNAc attachment is important in balancing biological processes, such that the interplay of TarM and TarS is likely important for bacterial pathogenicity and survival. Here we present the crystal structure of TarM in an unusual ternary-like complex consisting of a polymeric acceptor substrate analog, UDP from a hydrolyzed donor, and an α-glyceryl-GlcNAc product formed in situ. These structures support an internal nucleophilic substitution-like mechanism, lend new mechanistic insight into the glycosylation of glycopolymers, and reveal a trimerization domain with a likely role in acceptor substrate scaffolding.

Contributions of a unique ß-clamp to substrate recognition illuminates the molecular basis of exolysis in ferulic acid esterases.

Lignocellulosic biomass is a promising renewable resource; however, deconstruction of this material is still the rate-limiting step. Major obstacles in the biocatalytic turnover of lignocellulose are ester-linked decorations that prevent access to primary structural polysaccharides. Enzymes targeting these esters represent promising biotools for increasing bioconversion efficiency. Ruminant livestock are unique in their ability to degrade lignocellulose through the action of their gut microbiome. The anaerobic fungi (phylum Neocallimastigomycota) are key members of this ecosystem that express a large repertoire of carbohydrate-active enzymes (CAZymes) with little sequence identity with characterized CAZymes [Lombard, Golaconda, Drula, Coutinho and Henrissat (2014) Nucleic Acids Res. 42: , D490-D495]. We have identified a carbohydrate esterase family 1 (CE1) ferulic acid esterase (FAE) belonging to Anaeromyces mucronatus(AmCE1/Fae1a), and determined its X-ray structure in both the presence [1.55 Å (1 Å=0.1 nm)] and absence (1.60 Å) of ferulic acid. AmCE1 adopts an α/ß-hydrolase fold that is structurally conserved with bacterial FAEs, and possesses a unique loop, termed the ß-clamp, that encloses the ligand. Isothermal titration calorimetry reveals that substrate binding is driven by enthalpic contributions, which overcomes a large entropic penalty. A comparative analysis of AmCE1 with related enzymes has uncovered the apparent structural basis for differential FAE activities targeting cross-linking ferulic acid conjugates compared with terminal decorations. Based on comparisons to structurally characterized FAEs, we propose that the ß-clamp may define the structural basis of exolytic activities in FAEs. This provides a structure-based tool for predicting exolysis and endolysis in CE1. These insights hold promise for rationally identifying enzymes tailored for bioconversion of biomass with variations in cell wall composition.

Structure of the mycosin-1 protease from the mycobacterial ESX-1 protein type VII secretion system.

Mycobacteria use specialized type VII (ESX) secretion systems to export proteins across their complex cell walls. Mycobacterium tuberculosis encodes five nonredundant ESX secretion systems, with ESX-1 being particularly important to disease progression. All ESX loci encode extracellular membrane-bound proteases called mycosins (MycP) that are essential to secretion and have been shown to be involved in processing of type VII-exported proteins. Here, we report the first x-ray crystallographic structure of MycP1(24-407) to 1.86 Å, defining a subtilisin-like fold with a unique N-terminal extension previously proposed to function as a propeptide for regulation of enzyme activity. The structure reveals that this N-terminal extension shows no structural similarity to previously characterized protease propeptides and instead wraps intimately around the catalytic domain where, tethered by a disulfide bond, it forms additional interactions with a unique extended loop that protrudes from the catalytic core. We also show MycP1 cleaves the ESX-1 secreted protein EspB from both M. tuberculosis and Mycobacterium smegmatis at a homologous cut site in vitro.

Evaluating the liver abscess microbiota of beef cattle during a reduction in tylosin supplementation shows differences according to abscess size and fraction.

Liver abscesses (LA) resulting from bacterial infection in cattle pose a significant global challenge to the beef and dairy industries. Economic losses from liver discounts at slaughter and reduced animal performance drive the need for effective mitigation strategies. Tylosin phosphate supplementation is widely used to reduce LA occurrence, but concerns over antimicrobial overuse emphasize the urgency to explore alternative approaches. Understanding the microbial ecology of LA is crucial to this, and we hypothesized that a reduced timeframe of tylosin delivery would alter LA microbiomes. We conducted 16S rRNA sequencing to assess severe liver abscess bacteriomes in beef cattle supplemented with in-feed tylosin. Our findings revealed that shortening tylosin supplementation did not notably alter microbial communities. Additionally, our findings highlighted the significance of sample processing methods, showing differing communities in bulk purulent material and the capsule-adhered material. Fusobacterium or Bacteroides ASVs dominated LA, alongside probable opportunistic gut pathogens and other microbes. Moreover, we suggest that liver abscess size correlates with microbial community composition. These insights contribute to our understanding of factors impacting liver abscess microbial ecology and will be valuable in identifying antibiotic alternatives. They underscore the importance of exploring varied approaches to address LA while reducing reliance on in-feed antibiotics.

Substrate binding in protein-tyrosine phosphatase-like inositol polyphosphatases.

Protein-tyrosine phosphatase-like inositol polyphosphatases are microbial enzymes that catalyze the stepwise removal of one or more phosphates from highly phosphorylated myo-inositols via a relatively ordered pathway. To understand the substrate specificity and kinetic mechanism of these enzymes we have determined high resolution, single crystal, x-ray crystallographic structures of inactive Selenomonas ruminantium PhyA in complex with myo-inositol hexa- and pentakisphosphate. These structures provide the first glimpse of a myo-inositol polyphosphatase-ligand complex consistent with its known specificity and reveal novel features of the kinetic mechanism. To complement the structural studies, fluorescent binding assays have been developed and demonstrate that the K(d) for this enzyme is several orders of magnitude lower than the K(m). Together with rapid kinetics data, these results suggest that the protein tyrosine phosphatase-like inositol polyphosphatases have a two-step, substrate-binding mechanism that facilitates catalysis.

Identification of Genes Involved in the Degradation of Lignocellulose Using Comparative Transcriptomics.

Lignocellulosic biomass represents an abundant, renewable resource that can be used to produce biofuels, low-cost livestock feed, and high-value chemicals. The potential of this bioresource has led to intensive research efforts to develop cost-effective methods to break down lignocellulose. The efficiency with which the anaerobic fungi (phylum Neocallimastigomycota) degrade plant biomass is well recognized and in recent years has received renewed interest. Transcriptomics has been used to identify enzymes that are expressed by these fungi and are involved in the degradation of a range of lignocellulose feedstocks. The transcriptome is the entire complement of coding and non-coding RNA transcripts that are expressed by a cell under a particular set of conditions. Monitoring changes in gene expression can provide fundamental information about the biology of an organism. Here we outline a general methodology that will enable researchers to conduct comparative transcriptomic studies with the goal of identifying enzymes involved in the degradation of the plant cell wall. The method described will include growth of fungal cultures, isolation and sequencing of RNA, and a basic description of data analysis for bioinformatic identification of differentially expressed transcripts.

Isolation and Preparation of Extracellular Proteins from Lignocellulose-Degrading Fungi for Comparative Proteomic Studies Using Mass Spectrometry.

Fungi utilize a unique mechanism of nutrient acquisition involving extracellular digestion. To understand the biology of these microbes, it is important to identify and characterize the function of proteins that are secreted and involved in nutrient acquisition. Mass spectrometry-based proteomics is a powerful tool to study complex mixtures of proteins and understand how the proteins produced by an organism change in response to different conditions. Many fungi are efficient decomposers of plant cell walls, and anaerobic fungi are well recognized for their ability to digest lignocellulose. Here we outline a protocol for the enrichment and isolation of proteins secreted by anaerobic fungi after growth on simple (glucose) and complex (straw and alfalfa hay) carbon sources. We provide detailed instruction on generating protein fragments and preparing these for proteomic analysis using reversed-phase chromatography and mass spectrometry. The interpretation of results and their relevance to a particular biological system is study-dependent and beyond the scope of this protocol.

3-Nitrooxypropanol supplementation of a forage diet decreased enteric methane emissions from beef cattle without affecting feed intake and apparent total-tract digestibility.

Supplementation of ruminant diets with the methane (CH4) inhibitor 3-nitrooxypropanol (3-NOP; DSM Nutritional Products, Switzerland) is a promising greenhouse gas mitigation strategy. However, most studies have used high grain or mixed forage-concentrate diets. The objective of this study was to evaluate the effects of supplementing a high-forage diet (90% forage DM basis) with 3-NOP on dry matter (DM) intake, rumen fermentation and microbial community, salivary secretion, enteric gas emissions, and apparent total-tract nutrient digestibility. Eight ruminally cannulated beef heifers (average initial body weight (BW) ±â€…SD, 515 ±â€…40.5 kg) were randomly allocated to two treatments in a crossover design with 49-d periods. Dietary treatments were: 1) control (no 3-NOP supplementation); and 2) 3-NOP (control + 150 mg 3-NOP/kg DM). After a 16-d diet adaption, DM intake was recorded daily. Rumen contents were collected on days 17 and 28 for volatile fatty acid (VFA) analysis, whereas ruminal pH was continuously monitored from days 20 to 28. Eating and resting saliva production were measured on days 20 and 31, respectively. Diet digestibility was measured on days 38-42 by the total collection of feces, while enteric gas emissions were measured in chambers on days 46-49. Data were analyzed using the mixed procedure of SAS. Dry matter intake and apparent total-tract digestibility of nutrients (DM, neutral and acid detergent fiber, starch, and crude protein) were similar between treatments (P ≥ 0.15). No effect was observed on eating and resting saliva production. Relative abundance of the predominant bacterial taxa and rumen methanogen community was not affected by 3-NOP supplementation but rather by rumen digesta phase and sampling hour (P ≤ 0.01). Total VFA concentration was lower (P = 0.004) following 3-NOP supplementation. Furthermore, the reduction in acetate and increase in propionate molar proportions for 3-NOP lowered (P < 0.001) the acetate to propionate ratio by 18.9% as compared with control (4.1). Mean pH was 0.21 units lower (P < 0.001) for control than 3-NOP (6.43). Furthermore, CH4 emission (g/d) and yield (g/kg DMI) were 22.4 and 22.0% smaller (P < 0.001), respectively, for 3-NOP relative to control. Overall, the results indicate that enteric CH4 emissions were decreased by more than 20% with 3-NOP supplementation of a forage diet without affecting DM intake, predominant rumen microbial community, and apparent total-tract nutrients digestibility.

This study evaluated the effects of supplementing forage fed cattle with 3-nitrooxypropanol (150 mg/kg dry matter) on feed intake, rumen fermentation and microbial community composition, methane emissions, and nutrient digestibility. Eight ruminally cannulated beef heifers were used for the experiment. The results indicated that 3-nitrooxypropanol supplementation substantially reduced methane emissions without affecting feed intake and total-tract digestibility of nutrients.

Comparative analysis of macroalgae supplementation on the rumen microbial community: Asparagopsis taxiformis inhibits major ruminal methanogenic, fibrolytic, and volatile fatty acid-producing microbes in vitro.

Seaweeds have received a great deal of attention recently for their potential as methane-suppressing feed additives in ruminants. To date, Asparagopsis taxiformis has proven a potent enteric methane inhibitor, but it is a priority to identify local seaweed varieties that hold similar properties. It is essential that any methane inhibitor does not compromise the function of the rumen microbiome. In this study, we conducted an in vitro experiment using the RUSITEC system to evaluate the impact of three red seaweeds, A. taxiformis, Palmaria mollis, and Mazzaella japonica, on rumen prokaryotic communities. 16S rRNA sequencing showed that A. taxiformis had a profound effect on the microbiome, particularly on methanogens. Weighted Unifrac distances showed significant separation of A. taxiformis samples from the control and other seaweeds (p < 0.05). Neither P. mollis nor M. japonica had a substantial effect on the microbiome (p > 0.05). A. taxiformis reduced the abundance of all major archaeal species (p < 0.05), leading to an almost total disappearance of the methanogens. Prominent fiber-degrading and volatile fatty acid (VFA)-producing bacteria including Fibrobacter and Ruminococcus were also inhibited by A. taxiformis (p < 0.05), as were other genera involved in propionate production. The relative abundance of several other bacteria including Prevotella, Bifidobacterium, Succinivibrio, Ruminobacter, and unclassified Lachnospiraceae were increased by A. taxiformis suggesting that the rumen microbiome adapted to an initial perturbation. Our study provides baseline knowledge of microbial dynamics in response to seaweed feeding over an extended period and suggests that feeding A. taxiformis to cattle to reduce methane may directly, or indirectly, inhibit important fiber-degrading and VFA-producing bacteria.

Evaluation of Rumen Fermentation and Microbial Adaptation to Three Red Seaweeds Using the Rumen Simulation Technique.

Several red seaweeds have been shown to inhibit enteric CH4 production; however, the adaptation of fermentation parameters to their presence is not well understood. The objective of this study was to examine the effect of three red seaweeds (Asparargopsis taxiformis, Mazzaella japonica, and Palmaria mollis) on in vitro fermentation, CH4 production, and adaptation using the rumen simulation technique (RUSITEC). The experiment was conducted as a completely randomized design with four treatments, duplicated in two identical RUSITEC apparatus equipped with eight fermenter vessels each. The four treatments included the control and the three red seaweeds added to the control diet at 2% diet DM. The experimental period was divided into four phases including a baseline phase (d 0-7; no seaweed included), an adaptation phase (d 8-11; seaweed included in treatment vessels), an intermediate phase (d 12-16), and a stable phase (d 17-21). The degradability of organic matter (p = 0.04) and neutral detergent fibre (p = 0.05) was decreased by A. taxiformis during the adaptation phase, but returned to control levels in the stable phase. A. taxiformis supplementation resulted in a decrease (p < 0.001) in the molar proportions of acetate, propionate, and total volatile fatty acid (VFA) production, with an increase in the molar proportions of butyrate, caproate, and valerate; the other seaweeds had no effect (p > 0.05) on the molar proportions or production of individual VFA. A. taxiformis was the only seaweed to suppress CH4 production (p < 0.001), with the suppressive effect increasing (p < 0.001) across phases. Similarly, A. taxiformis increased (p < 0.001) the production of hydrogen (H2, %, mL/d) across the adaptation, intermediate, and stable phases, with the intermediate and stable phases having greater H2 production than the adaptation phase. In conclusion, M. japonica and P. mollis did not impact rumen fermentation or inhibit CH4 production within the RUSITEC. In contrast, we conclude that A. taxiformis is an effective CH4 inhibitor and its introduction to the ruminal environment requires a period of adaptation; however, the large magnitude of CH4 suppression by A. taxiformis inhibits VFA synthesis, which may restrict the production performance in vivo.

Application of 3-nitrooxypropanol and canola oil to mitigate enteric methane emissions of beef cattle results in distinctly different effects on the rumen microbial community.

BACKGROUND: The major greenhouse gas from ruminants is enteric methane (CH4) which in 2010, was estimated at 2.1 Gt of CO2 equivalent, accounting for 4.3% of global anthropogenic greenhouse gas emissions. There are extensive efforts being made around the world to develop CH4 mitigating inhibitors that specifically target rumen methanogens with the ultimate goal of reducing the environmental footprint of ruminant livestock production. This study examined the individual and combined effects of supplementing a high-forage diet (90% barley silage) fed to beef cattle with the investigational CH4 inhibitor 3-nitrooxypropanol (3-NOP) and canola oil (OIL) on the rumen microbial community in relation to enteric CH4 emissions and ruminal fermentation. RESULTS: 3-NOP and OIL individually reduced enteric CH4 yield (g/kg dry matter intake) by 28.2% and 24.0%, respectively, and the effects were additive when used in combination (51.3% reduction). 3-NOP increased H2 emissions 37-fold, while co-administering 3-NOP and OIL increased H2 in the rumen 20-fold relative to the control diet. The inclusion of 3-NOP or OIL significantly reduced the diversity of the rumen microbiome. 3-NOP resulted in targeted changes in the microbiome decreasing the relative abundance of Methanobrevibacter and increasing the relative abundance of Bacteroidetes. The inclusion of OIL resulted in substantial changes to the microbial community that were associated with changes in ruminal volatile fatty acid concentration and gas production. OIL significantly reduced the abundance of protozoa and fiber-degrading microbes in the rumen but it did not selectively alter the abundance of rumen methanogens. CONCLUSIONS: Our data provide a mechanistic understanding of CH4 inhibition by 3-NOP and OIL when offered alone and in combination to cattle fed a high forage diet. 3-NOP specifically targeted rumen methanogens and partly inhibited the hydrogenotrophic methanogenesis pathway, which increased H2 emissions and propionate molar proportion in rumen fluid. In contrast, OIL caused substantial changes in the rumen microbial community by indiscriminately altering the abundance of a range of rumen microbes, reducing the abundance of fibrolytic bacteria and protozoa, resulting in altered rumen fermentation. Importantly, our data suggest that co-administering CH4 inhibitors with distinct mechanisms of action can both enhance CH4 inhibition and provide alternative sinks to prevent excessive accumulation of ruminal H2.

Effect of ergot alkaloids and a mycotoxin deactivating product on in vitro ruminal fermentation using the Rumen simulation technique (RUSITEC).

The rumen simulation technique (RUSITEC) was used to investigate the effect of ergot alkaloids (EA) and a mycotoxin deactivating product (Biomin AA; MDP) on nutrient digestion, ruminal fermentation parameters, total gas, methane, and microbial nitrogen production. Ruminal fermentation vessels received a feedlot finishing diet of 90:10 concentrate:barley silage (DM basis). Using a randomized complete block design, treatments were assigned (n = 4 vessels/treatment) within two RUSITEC apparatuses in a 2 × 2 factorial arrangement. Treatments included: (1) control (CON) diet (no EA and no MDP); (2) CON diet + 1 g/d MDP; (3) CON diet + 20 mg/kg EA; and (4) CON diet + 20 mg/kg EA + 1 g/d MDP. The study was conducted over 14 d with 7 d of adaptation and 7 d of sample collection. Data were analyzed in SAS using PROC MIXED including fixed effects of EA, MDP, and the EA×MDP interaction. Random effects included RUSITEC apparatus and cow rumen inoculum (n = 4). Ergot alkaloids decreased dry matter (DMD) (P = 0.01; 87.9 vs. 87.2%) and organic matter disappearance (OMD) (P = 0.02; 88.8 vs. 88.4%). Inclusion of MDP increased OMD (P = 0.01; 88.3 vs. 88.9%). Neutral detergent fiber disappearance (NDFD) was improved with MDP; however, an EA×MDP interaction was observed with MDP increasing (P < 0.001) NDFD more with EA diet compared to CON. Acetate proportion decreased (P = 0.01) and isovalerate increased (P = 0.03) with EA. Consequently, acetate:propionate was reduced (P = 0.03) with EA. Inclusion of MDP increased total volatile fatty acid (VFA) production (P < 0.001), and proportions of acetate (P = 0.03) and propionate (P = 0.03), and decreased valerate (P < 0.001), isovalerate (P = 0.04), and caproate (P = 0.002). Treatments did not affect (P ≥ 0.17) ammonia, total gas, or methane production (mg/d or mg/g of organic matter fermented). The inclusion of MDP reduced (P < 0.001) microbial nitrogen (MN) production in the effluent and increased (P = 0.01) feed particle-bound MN. Consequently, total MN decreased (P = 0.001) with MDP. In all treatments, the dominant microbial phyla were Firmicutes, Bacteroidota, and Proteobacteria, and the major microbial genus was Prevotella. Inclusion of MDP further increased the abundance of Bacteroidota (P = 0.04) as it increased both Prevotella (P = 0.04) and Prevotellaceae_UCG-003 (P = 0.001). In conclusion, EA reduced OMD and acetate production due to impaired rumen function, these responses were successfully reversed by the addition of MDP.

Ergot formed from a parasitic fungus (Claviceps purpurea) affects various types of grains (rye, wheat, or oats) and may contain several toxic ergot alkaloids (EA). Individual EA may impact the rumen microorganisms, and cattle feed intake, digestibility, health, and overall performance. A common method to alleviate toxicity in mycotoxin-contaminated feed is through the addition of mycotoxin binders (MDP); however, their efficacy against EA is unknown. To better understand the effect of EA in cattle, we performed an in vitro experiment to examine the impact of EA on the ruminal microbial populations and fermentation of a finishing feedlot diet using an artificial rumen (RUSITEC). Additionally, an MDP was added to test if it could reduce the detrimental effects of EA on rumen fermentation. MDP increased total volatile fatty acids (VFA) and reduced total microbial protein synthesis. Furthermore, EA reduced microbial diversity and the acetate:propionate ratio. Although EA reduced organic matter digestibility and acetate production, these negative effects were reversed by the addition of the MDP.

Combined effects of 3-nitrooxypropanol and canola oil supplementation on methane emissions, rumen fermentation and biohydrogenation, and total tract digestibility in beef cattle.

The individual and combined effects of 3-nitrooxypropanol (3-NOP) and canola oil (OIL) supplementation on enteric methane (CH4) and hydrogen (H2) emissions, rumen fermentation and biohydrogenation, and total tract nutrient digestibility were investigated in beef cattle. Eight beef heifers (mean body weight ± SD, 732 ± 43 kg) with ruminal fistulas were used in a replicated 4 × 4 Latin square with a 2 (with and without 3-NOP) × 2 (with and without OIL) arrangement of treatments and 28-d periods (13 d adaption and 15 d measurements). The four treatments were: control (no 3-NOP, no OIL), 3-NOP (200 mg/kg dry matter [DM]), OIL (50 g/kg DM), and 3-NOP (200 mg/kg DM) plus OIL (50 g/kg DM). Animals were fed restrictively (7.6 kg DM/d) a basal diet of 900 g/kg DM barley silage and 100 g/kg DM supplement. 3-NOP and OIL decreased (P < 0.01) CH4 yield (g/kg DM intake) by 31.6% and 27.4%, respectively, with no 3-NOP × OIL interaction (P = 0.85). Feeding 3-NOP plus OIL decreased CH4 yield by 51% compared with control. There was a 3-NOP × OIL interaction (P = 0.02) for H2 yield (g/kg DM intake); the increase in H2 yield (P < 0.01) due to 3-NOP was less when it was combined with OIL. There were 3-NOP × OIL interactions for molar percentages of acetate and propionate (P < 0.01); individually, 3-NOP and OIL decreased acetate and increased propionate percentages with no further effect when supplemented together. 3-NOP slightly increased crude protein (P = 0.02) and starch (P = 0.01) digestibilities, while OIL decreased the digestibilities of DM (P < 0.01) and neutral detergent fiber (P < 0.01) with no interactions (P = 0.15 and 0.10, respectively). 3-NOP and OIL increased (P = 0.04 and P < 0.01, respectively) saturated fatty acid concentration in rumen fluid, with no interaction effect. Interactions for ruminal trans-monounsaturated fatty acids (t-MUFA) concentration and percentage were observed (P = 0.02 and P < 0.01); 3-NOP had no effect on t-MUFA concentration and percentage, while OIL increased the concentration (P < 0.01) and percentage (P < 0.01) of t-MUFA but to a lesser extent when combined with 3-NOP. In conclusion, the CH4-mitigating effects of 3-NOP and OIL were independent and incremental. Supplementing ruminant diets with a combination of 3-NOP and OIL may help mitigate CH4 emissions, but the decrease in total tract digestibility due to OIL may decrease animal performance and needs further investigation.

Ecology and molecular targets of hypermutation in the global microbiome.

Changes in the sequence of an organism's genome, i.e., mutations, are the raw material of evolution. The frequency and location of mutations can be constrained by specific molecular mechanisms, such as diversity-generating retroelements (DGRs). DGRs have been characterized from cultivated bacteria and bacteriophages, and perform error-prone reverse transcription leading to mutations being introduced in specific target genes. DGR loci were also identified in several metagenomes, but the ecological roles and evolutionary drivers of these DGRs remain poorly understood. Here, we analyze a dataset of >30,000 DGRs from public metagenomes, establish six major lineages of DGRs including three primarily encoded by phages and seemingly used to diversify host attachment proteins, and demonstrate that DGRs are broadly active and responsible for >10% of all amino acid changes in some organisms. Overall, these results highlight the constraints under which DGRs evolve, and elucidate several distinct roles these elements play in natural communities.

Pretreatment of crop residues by ammonia fiber expansion (AFEX) alters the temporal colonization of feed in the rumen by rumen microbes.

This study examines the colonization of barley straw (BS) and corn stover (CS) by rumen bacteria and how this is impacted by ammonia fiber expansion (AFEX) pre-treatment. A total of four ruminally cannulated beef heifers were used to investigate in situ microbial colonization in a factorial design with two crop residues, pre-treated with or without AFEX. Crop residues were incubated in the rumen for 0, 2, 4, 8 and 48 h and the colonizing profile was determined using 16 s rRNA gene sequencing. The surface colonizing community clustered based on incubation time and pre-treatment. Fibrobacter, unclassified Bacteroidales, and unclassified Ruminococcaceae were enriched during late stages of colonization. Prevotella and unclassified Lachnospiraceae were enriched in the early stages of colonization. The microbial community colonizing BS-AFEX and CS was less diverse than the community colonizing BS and CS-AFEX. Prevotella, Coprococcus and Clostridium were enriched in both AFEX crop residues, while untreated crop residues were enriched with Methanobrevibacter. Several pathways associated with simple carbohydrate metabolism were enriched in the primary colonizing community of AFEX crop residues. This study suggests that AFEX improves the degradability of crop residues by increasing the accessibility of polysaccharides that can be metabolized by the dominant taxa responsible for primary colonization.