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.

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.

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.

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.

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.

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.

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.

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.

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.

Effect of ammonia fiber expansion-treated wheat straw and a recombinant fibrolytic enzyme on rumen microbiota and fermentation parameters, total tract digestibility, and performance of lambs.

The objective of this study was to evaluate the effect of ammonia fiber expansion (AFEX)-treated wheat straw pellets and a recombinant fibrolytic enzyme on the rumen microbiome, rumen fermentation parameters, total tract diet digestibility, and performance of lambs. Eight rumen cannulated wethers and 60 lambs (n = 15 per diet, 8 rams and 7 ewes) were used in a replicated 4 × 4 Latin square design digestibility study and a complete randomized growth performance study, respectively. Four treatment diets were arranged in a 2 × 2 factorial structure with AFEX wheat straw (0% or 30% AFEX straw pellets on a dietary DM basis replacing alfalfa hay pellets) and fibrolytic enzyme (with or without XYL10C, a ß-1,4-xylanase, from Aspergillus niger) as main factors. Enzyme was applied at 100 mg/kg of diet DM, 22 h before feeding. Rumen bacteria diversity Pielou evenness decreased (P = 0.05) with AFEX compared with the control diet and increased (P < 0.01) with enzyme. Enzyme increased (P ≤ 0.02) the relative abundancies of Prevotellaceae UCG-004, Christensenellaceae R-7 group, Saccharofermentans, and uncultured Kiritimatiellaeota. Total protozoa counts were greater (P ≤ 0.04) in the rumen of lambs fed AFEX compared with control, with enzyme reducing (P ≤ 0.05) protozoa counts for both diets. Digestibility of DM did not differ (P > 0.10) among diets, but digestibility of CP was reduced (P = 0.001), and digestibility of NDF and ADF increased (P < 0.05) as AFEX replaced alfalfa. Compared with control, AFEX promoted greater DMI (P = 0.003) and improved ADG up to 42 d on feed (P = 0.03), but not (P = 0.51) over the full ~94-d experiment. Consequently, overall G:F was reduced (P = 0.04) for AFEX when compared with control (0.188 vs. 0.199), but days on feed were lower (P = 0.04) for AFEX (97 vs. 91 d). Enzyme improved DMI of AFEX up to day 70 (P = 0.01), but did not affect DMI of the control diet. Enzyme addition improved ADG of lambs fed both diets in the first 28 d (P = 0.02), but not over the entire feeding period (P ≥ 10). As a result, G:F was improved with enzyme for the first 28 d (P = 0.04), but not overall (P = 0.45). This study shows that AFEX-treated wheat straw can replace alfalfa hay with no loss in lamb growth performance. Additionally, the enzyme XYL10C altered the rumen microbiome and improved G:F in the first month of the feeding.

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.

A Pine Enhanced Biochar Does Not Decrease Enteric CH4 Emissions, but Alters the Rumen Microbiota.

The objective of this study was to examine the effect of a pine enhanced biochar (EB) on rumen fermentation, apparent total tract digestibility, methane (CH4) emissions, and the rumen and fecal microbiome of Angus × Hereford heifers fed a barley silage-based diet. The experiment was a replicated 4 × 4 Latin square using 8 ruminally cannulated heifers (565 ± 35 kg initial BW). The basal diet contained 60% barley silage, 35% barley grain and 5% mineral supplement with EB added at 0% (control), 0.5, 1.0, or 2.0% (DM basis). Each period lasted 28 days, consisting of 14 days adaptation and 14 days of measurements. Samples for profiling of the microbiome in rumen liquid, solids and feces were collected on d 15 before feeding. Rumen samples for fermentation characterization were taken at 0, 3, 6, and 12 h post feeding. Total collection of urine and feces was conducted from days 18 to 22. Heifers were housed in open-circuit respiratory chambers on days 26-28 to estimate CH4 emissions. Ruminal pH was recorded at 1-min intervals during CH4 measurements using indwelling pH loggers. Data were analyzed with the fixed effects of dietary treatment and random effects of square, heifer within square and period. Dry matter intake was similar across treatments (P = 0.21). Ammonia N concentration and protozoa counts responded quadratically (P = 0.01) to EB in which both were decreased by EB included at 0.5 and 1.0%, compared to the control and 2.0% EB. Minimum pH was increased (P = 0.04), and variation of pH was decreased (P = 0.03) by 2.0% EB. Total tract digestibility, N balance and CH4 production were not affected (P ≥ 0.17) by EB. Enhanced biochar decreased the relative abundance of Fibrobacter (P = 0.05) and Tenericutes (P = 0.01), and increased the relative abundance of Spirochaetaes (P = 0.01), Verrucomicrobia (P = 0.02), and Elusimicrobia (P = 0.02). Results suggest that at the examined concentrations, EB was ineffective at decreasing enteric CH4 emissions, but did alter specific rumen microbiota.

Addressing Global Ruminant Agricultural Challenges Through Understanding the Rumen Microbiome: Past, Present, and Future.

The rumen is a complex ecosystem composed of anaerobic bacteria, protozoa, fungi, methanogenic archaea and phages. These microbes interact closely to breakdown plant material that cannot be digested by humans, whilst providing metabolic energy to the host and, in the case of archaea, producing methane. Consequently, ruminants produce meat and milk, which are rich in high-quality protein, vitamins and minerals, and therefore contribute to food security. As the world population is predicted to reach approximately 9.7 billion by 2050, an increase in ruminant production to satisfy global protein demand is necessary, despite limited land availability, and whilst ensuring environmental impact is minimized. Although challenging, these goals can be met, but depend on our understanding of the rumen microbiome. Attempts to manipulate the rumen microbiome to benefit global agricultural challenges have been ongoing for decades with limited success, mostly due to the lack of a detailed understanding of this microbiome and our limited ability to culture most of these microbes outside the rumen. The potential to manipulate the rumen microbiome and meet global livestock challenges through animal breeding and introduction of dietary interventions during early life have recently emerged as promising new technologies. Our inability to phenotype ruminants in a high-throughput manner has also hampered progress, although the recent increase in "omic" data may allow further development of mathematical models and rumen microbial gene biomarkers as proxies. Advances in computational tools, high-throughput sequencing technologies and cultivation-independent "omics" approaches continue to revolutionize our understanding of the rumen microbiome. This will ultimately provide the knowledge framework needed to solve current and future ruminant livestock challenges.

Effect of humic substances on rumen fermentation, nutrient digestibility, methane emissions, and rumen microbiota in beef heifers1.

Ruminants play an important role in food security, but there is a growing concern about the impact of cattle on the environment, particularly regarding greenhouse gas emissions. The objective of this study was to examine the effect of humic substances (HS) on rumen fermentation, nutrient digestibility, methane (CH4) emissions, and the rumen microbiome of beef heifers fed a barley silage-based diet. The experiment was designed as a replicated 4 × 4 Latin square using 8 ruminally cannulated Angus × Hereford heifers (758 ± 40.7 kg initial BW). Heifers were offered a basal diet consisting of 60% barley silage and 40% concentrate (DM basis) with either 0- (control), 100-, 200- or 300-mg granulated HS/kg BW. Each period was 28 d with 14 d of adaptation. Rumen samples were taken on day 15 at 0, 3, 6, and 12 h postfeeding. Total urine and feces were collected from days 18 to 22. Blood samples were taken on day 22 at 0 and 6 h postfeeding. Between days 26 and 28, heifers were placed in open-circuit respiratory chambers to measure CH4. Ruminal pH was recorded continuously during the periods of CH4 measurement using indwelling pH loggers. Intake was similar (P = 0.47) across treatments. Concentration of ammonia-N and counts of rumen protozoa responded quadratically (P = 0.03), where both increased at H100 and then decreased for the H300 treatments. Apparent total tract digestibility of CP (P = 0.04) was linearly increased by HS and total N retention (g/d, % N intake, g/kg BW0.75) was improved (P = 0.04) for HS when compared with the control. There was no effect of HS on CH4 production (g/d; P = 0.83); however, HS decreased the relative abundance of Proteobacteria (P = 0.04) and increased the relative abundance of Synergistetes (P = 0.01) and Euryarchaeota (P = 0.04). Results suggest that HS included at up to 300 mg/kg BW may improve N retention and CP digestibility, but there was no impact on CH4 production.

Application of Transcriptomics to Compare the Carbohydrate Active Enzymes That Are Expressed by Diverse Genera of Anaerobic Fungi to Degrade Plant Cell Wall Carbohydrates.

The efficiency with which the anaerobic fungi (phylum Neocallimastigomycota) degrade plant biomass is well-recognized and in recent years has received renewed interest. To further understand the biological mechanisms that are utilized by the rumen anaerobic fungi to break down lignocellulose, we have used a transcriptomic approach to examine carbohydrate digestion by Neocallimastix frontalis, Piromyces rhizinflata, Orpinomyces joyonii, and Anaeromyces mucronatus cultured on several carbon sources. The number of predicted unique transcripts ranged from 6,633 to 12,751. Pfam domains were identified in 62-70% of the fungal proteins and were linked to gene ontology terms to infer the biological function of the transcripts. Most of the predicted functions are consistent across species suggesting a similar overall strategy evolved for successful colonization of the rumen. However, the presence of differential profiles in enzyme classes suggests that there may be also be niche specialization. All fungal species were found to express an extensive array of transcripts encoding carbohydrate active enzymes (CAZymes) ranging from 8.3 to 11.3% of the transcriptome. CAZyme families involved in hemicellulose digestion were the most abundant across all four fungi. This study provides additional insight into how anaerobic fungi have evolved to become specialists at breaking down the plant cell wall in the complex and, strictly anaerobic rumen ecosystem.

A Novel aadA Aminoglycoside Resistance Gene in Bovine and Porcine Pathogens.

A novel variant of the AAD(3″) class of aminoglycoside-modifying enzymes was discovered in fatal bovine respiratory disease-associated pathogens Pasteurella multocida and Histophilus somni. The aadA31 gene encodes a spectinomycin/streptomycin adenylyltransferase and was located in a variant of the integrative and conjugative element ICEMh1, a mobile genetic element transmissible among members of the family Pasteurellaceae. The gene was also detected in Mannheimia haemolytica from a case of porcine pneumonia and in Moraxella bovoculi from a case of keratoconjunctivitis. IMPORTANCE Aminoglycosides are important antimicrobials used worldwide for prophylaxis and/or therapy in multiple production animal species. The emergence of new resistance genes jeopardizes current pathogen detection and treatment methods. The risk of resistance gene transfer to other animal and human pathogens is elevated when resistance genes are carried by mobile genetic elements. This study identified a new variant of a spectinomycin/streptomycin resistance gene harbored in a self-transmissible mobile element. The gene was also present in four different bovine pathogen species.

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.