Genomic architecture of three newly isolated unclassified Butyrivibrio species elucidate their potential role in the rumen ecosystem.

One cellulose-degrading strain CB08 and two xylan-degrading strains XB500-5 and X503 were isolated from buffalo rumen. All the strains were designated as putative novel species of Butyrivibrio based on phylogeny, phylogenomy, digital DNA-DNA hybridization, and average nucleotide identity with their closest type strains. The draft genome length of CB08 was ~3.54 Mb, while X503 and XB500-5 genome sizes were ~3.24 Mb and ~3.27 Mb, respectively. Only 68.28% of total orthologous clusters were shared among three genomes, and 40-44% of genes were identified as hypothetical proteins. The presence of genes encoding diverse carbohydrate-active enzymes (CAZymes) exhibited the lignocellulolytic potential of these strains. Further, the genome annotations revealed the metabolic pathways for monosaccharide fermentation to acetate, butyrate, lactate, ethanol, and hydrogen. The presence of genes for chemotaxis, antibiotic resistance, antimicrobial activity, synthesis of vitamins, and essential fatty acid suggested the versatile metabolic nature of these Butyrivibrio strains in the rumen environment.

Crystal Structures of Bacterial Pectin Methylesterases Pme8A and PmeC2 from Rumen Butyrivibrio.

Pectin is a complex polysaccharide that forms a substantial proportion of the plant's middle lamella of forage ingested by grazing ruminants. Methanol in the rumen is derived mainly from methoxy groups released from pectin by the action of pectin methylesterase (PME) and is subsequently used by rumen methylotrophic methanogens that reduce methanol to produce methane (CH4). Members of the genus Butyrivibrio are key pectin-degrading rumen bacteria that contribute to methanol formation and have important roles in fibre breakdown, protein digestion, and the biohydrogenation of fatty acids. Therefore, methanol release from pectin degradation in the rumen is a potential target for CH4 mitigation technologies. Here, we present the crystal structures of PMEs belonging to the carbohydrate esterase family 8 (CE8) from Butyrivibrio proteoclasticus and Butyrivibrio fibrisolvens, determined to a resolution of 2.30 Å. These enzymes, like other PMEs, are right-handed ß-helical proteins with a well-defined catalytic site and reaction mechanisms previously defined in insect, plant, and other bacterial pectin methylesterases. Potential substrate binding domains are also defined for the enzymes.

Sharpea azabuensis: a ruminal bacterium that produces trans-11 intermediates from linoleic and linolenic acid.

To investigate the metabolism of 18:2n-6 and 18:3n-3 by pure cultures of Sharpea azabuensis, two different strains (RL 1 and ST18) were each incubated in the presence of 40 µg ml-1 18:2n-6 or 18:3n-3. Pure cultures of Butyrivibriofibrisolvens D1 and Butyrivibrio proteoclasticus P18 were included as control treatments. Similar to the metabolism of B. fibrisolvens, both S. azabuensis strains converted 18:2n-6 or 18:3n-3 to cis-9, trans-11 CLA or cis-9, trans-11, cis-15 CLnA, after which it was further reduced to trans-11 18:1 or trans-11, cis-15 18:2, respectively. B. proteoclasticus additionally reduced trans-11 18:1 to 18:0. Trans-11, cis-15 18:2 was also further metabolized by B. proteoclasticus, although trans-11 18:1 did not accumulate, and only minor amounts of 18:0 were formed. The time frame of 18:2n-6 and 18:3n-3 biohydrogenation by S. azabuensis was comparable with B. fibrisolvens, indicating that S. azabuensis and B. fibrisolvens might be alternative biohydrogenators of 18:2n-6 and 18:3n-3 in the rumen.

Butyrivibrio hungatei MB2003 Competes Effectively for Soluble Sugars Released by Butyrivibrio proteoclasticus B316T during Growth on Xylan or Pectin.

Rumen bacterial species belonging to the genus Butyrivibrio are important degraders of plant polysaccharides, particularly hemicelluloses (arabinoxylans) and pectin. Currently, four species are recognized; they have very similar substrate utilization profiles, but little is known about how these microorganisms are able to coexist in the rumen. To investigate this question, Butyrivibrio hungatei MB2003 and Butyrivibrio proteoclasticus B316T were grown alone or in coculture on xylan or pectin, and their growth, release of sugars, fermentation end products, and transcriptomes were examined. In monocultures, B316T was able to grow well on xylan and pectin, while MB2003 was unable to utilize either of these insoluble substrates to support significant growth. Cocultures of B316T grown with MB2003 revealed that MB2003 showed growth almost equivalent to that of B316T when either xylan or pectin was supplied as the substrate. The effect of coculture on the transcriptomes of B316T and MB2003 was assessed; B316T transcription was largely unaffected by the presence of MB2003, but MB2003 expressed a wide range of genes encoding proteins for carbohydrate degradation, central metabolism, oligosaccharide transport, and substrate assimilation, in order to compete with B316T for the released sugars. These results suggest that B316T has a role as an initiator of primary solubilization of xylan and pectin, while MB2003 competes effectively for the released soluble sugars to enable its growth and maintenance in the rumen.IMPORTANCE Feeding a future global population of 9 billion people and climate change are the primary challenges facing agriculture today. Ruminant livestock are important food-producing animals, and maximizing their productivity requires an understanding of their digestive systems and the roles played by rumen microbes in plant polysaccharide degradation. Butyrivibrio species are a phylogenetically diverse group of bacteria and are commonly found in the rumen, where they are a substantial source of polysaccharide-degrading enzymes for the depolymerization of lignocellulosic material. Our findings suggest that closely related species of Butyrivibrio have developed unique strategies for the degradation of plant fiber and the subsequent assimilation of carbohydrates in order to coexist in the competitive rumen environment. The identification of genes expressed during these competitive interactions gives further insight into the enzymatic machinery used by these bacteria as they degrade the xylan and pectin components of plant fiber.

Comparative Genomics of Rumen Butyrivibrio spp. Uncovers a Continuum of Polysaccharide-Degrading Capabilities.

Plant polysaccharide breakdown by microbes in the rumen is fundamental to digestion in ruminant livestock. Bacterial species belonging to the rumen genera Butyrivibrio and Pseudobutyrivibrio are important degraders and utilizers of lignocellulosic plant material. These bacteria degrade polysaccharides and ferment the released monosaccharides to yield short-chain fatty acids that are used by the ruminant for growth and the production of meat, milk, and fiber products. Although rumen Butyrivibrio and Pseudobutyrivibrio species are regarded as common rumen inhabitants, their polysaccharide-degrading and carbohydrate-utilizing enzymes are not well understood. In this study, we analyzed the genomes of 40 Butyrivibrio and 6 Pseudobutyrivibrio strains isolated from the plant-adherent fraction of New Zealand dairy cows to explore the polysaccharide-degrading potential of these important rumen bacteria. Comparative genome analyses combined with phylogenetic analysis of their 16S rRNA genes and short-chain fatty acid production patterns provide insight into the genomic diversity and physiology of these bacteria and divide Butyrivibrio into 3 species clusters. Rumen Butyrivibrio bacteria were found to encode a large and diverse spectrum of degradative carbohydrate-active enzymes (CAZymes) and binding proteins. In total, 4,421 glycoside hydrolases (GHs), 1,283 carbohydrate esterases (CEs), 110 polysaccharide lyases (PLs), 3,605 glycosyltransferases (GTs), and 1,706 carbohydrate-binding protein modules (CBM) with predicted activities involved in the depolymerization and transport of the insoluble plant polysaccharides were identified. Butyrivibrio genomes had similar patterns of CAZyme families but varied greatly in the number of genes within each category in the Carbohydrate-Active Enzymes database (CAZy), suggesting some level of functional redundancy. These results suggest that rumen Butyrivibrio species occupy similar niches but apply different degradation strategies to be able to coexist in the rumen.IMPORTANCE Feeding a global population of 8 billion people and climate change are the primary challenges facing agriculture today. Ruminant livestock are important food-producing animals, and maximizing their productivity requires an understanding of their digestive systems and the roles played by rumen microbes in plant polysaccharide degradation. Members of the genera Butyrivibrio and Pseudobutyrivibrio are a phylogenetically diverse group of bacteria and are commonly found in the rumen, where they are a substantial source of polysaccharide-degrading enzymes for the depolymerization of lignocellulosic material. Our findings have highlighted the immense enzymatic machinery of Butyrivibrio and Pseudobutyrivibrio species for the degradation of plant fiber, suggesting that these bacteria occupy similar niches but apply different degradation strategies in order to coexist in the competitive rumen environment.

Effects of co-inoculating rice straw with ruminal microbiota and anaerobic sludge: digestion performance and spatial distribution of microbial communities.

Ruminal microbiota (RM) were co-inoculated with anaerobic sludge (AS) at different ratios to study the digestion of rice straw in batch experiments. The CH4 yield reached 273.64 mL/g volatile solid (VS) at a co-inoculum ratio of 1:1. The xylanase and cellulase activities were 198.88-212.88 and 24.51-29.08 U/mL in co-inoculated samples, respectively, and were significantly different compared to the results for single inoculum (p < 0.05). Higher ratios of AS enhanced acetoclastic methanogenesis, and propionate accumulation could be the main reason for the longer lag phase observed in samples with a higher RM ratio. The microbial compositions were clearly altered after digestion. Fibrobacter, Ruminococcus and Butyrivibrio from the rumen did not settle in the co-inoculated system, whereas Clostridiales members became the main polysaccharide degraders. Microbial interactions involving hydrolytic bacteria and acetoclastic methanogens in the residue were considered to be significant for hydrolysis activities and methane production. Syntrophy involving propionate oxidizers with associated methanogens occurred in the liquid phase. Our findings provide a better understanding of the anaerobic digestion of rice straw that is driven by specific microbial populations.

Biohydrogenation of 22:6n-3 by Butyrivibrio proteoclasticus P18.

BACKGROUND: Rumen microbes metabolize 22:6n-3. However, pathways of 22:6n-3 biohydrogenation and ruminal microbes involved in this process are not known. In this study, we examine the ability of the well-known rumen biohydrogenating bacteria, Butyrivibrio fibrisolvens D1 and Butyrivibrio proteoclasticus P18, to hydrogenate 22:6n-3. RESULTS: Butyrivibrio fibrisolvens D1 failed to hydrogenate 22:6n-3 (0.5 to 32 µg/mL) in growth medium containing autoclaved ruminal fluid that either had or had not been centrifuged. Growth of B. fibrisolvens was delayed at the higher 22:6n-3 concentrations; however, total volatile fatty acid production was not affected. Butyrivibrio proteoclasticus P18 hydrogenated 22:6n-3 in growth medium containing autoclaved ruminal fluid that either had or had not been centrifuged. Biohydrogenation only started when volatile fatty acid production or growth of B. proteoclasticus P18 had been initiated, which might suggest that growth or metabolic activity is a prerequisite for the metabolism of 22:6n-3. The amount of 22:6n-3 hydrogenated was quantitatively recovered in several intermediate products eluting on the gas chromatogram between 22:6n-3 and 22:0. Formation of neither 22:0 nor 22:6 conjugated fatty acids was observed during 22:6n-3 metabolism. Extensive metabolism was observed at lower initial concentrations of 22:6n-3 (5, 10 and 20 µg/mL) whereas increasing concentrations of 22:6n-3 (40 and 80 µg/mL) inhibited its metabolism. Stearic acid formation (18:0) from 18:2n-6 by B. proteoclasticus P18 was retarded, but not completely inhibited, in the presence of 22:6n-3 and this effect was dependent on 22:6n-3 concentration. CONCLUSIONS: For the first time, our study identified ruminal bacteria with the ability to hydrogenate 22:6n-3. The gradual appearance of intermediates indicates that biohydrogenation of 22:6n-3 by B. proteoclasticus P18 occurs by pathways of isomerization and hydrogenation resulting in a variety of unsaturated 22 carbon fatty acids. During the simultaneous presence of 18:2n-6 and 22:6n-3, B. proteoclasticus P18 initiated 22:6n-3 metabolism before converting 18:1 isomers into 18:0.

The fructanolytic abilities of the rumen bacterium Butyrivibrio fibrisolvens strain 3071.

AIMS: To determine if Butyrivibrio fibrisolvens strain 3071 is able to use fructose polymers for growth and to identify the enzymes involved in their digestion. METHODS AND RESULTS: Strain 3071 utilized 97, 89, 85 and 60% of sucrose, timothy grass fructan, inulin oligosaccharides and inulin, respectively, in the growth medium. A cell extract from timothy grass fructan-grown bacteria was used for identification of fructanolytic enzymes by anion exchange chromatography, gel filtration, zymography and thin-layer chromatography. The bacterium synthesizes a specific endolevanase and a nonspecific ß-fructofuranosidase. Both enzymes occurred in two forms differing in molecular weight. The ß-fructofuranosidase was not able to digest long-chain inulin or timothy grass fructan, but degraded inulin oligosaccharides and sucrose. Addition of 1,4-dithioerythritol to an enzyme solution did not affect the activity of endolevanase(s), but increased the ability of ß-fructofuranosidase to digest sucrose. The digestion of timothy grass fructan by endolevanase(s) was described by Michaelis-Menten kinetics in which Km  = 2·82 g l(-1) and Vmax  = 4·01 µmoles reducing sugar equivalents × mg(-1)  × min(-1) . CONCLUSION: Strain 3071 synthesizes enzymes enabling it to use grass fructans for growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Butyrivibrio fibrisolvens strain 3071 can be considered a member of the rumen fructanolytic guild.

Analysis of the rumen bacterial diversity of goats during shift from forage to concentrate diet.

High-grain feeding used in the animal production is known to affect the host rumen bacterial community, but our understanding of consequent changes in goats is limited. This study was therefore aimed to evaluate bacterial population dynamics during 20 days adaptation of 4 ruminally cannulated goats to the high-grain diet (grain: hay - ratio of 40:60). The dietary transition of goats from the forage to the high-grain-diet resulted in the significant decrease of rumen fluid pH, which was however still higher than value established for acute or subacute ruminal acidosis was not diagnosed in studied animals. DGGE analysis demonstrated distinct ruminal microbial populations in hay-fed and grain-fed animals, but the substantial animal-to-animal variation were detected. Quantitative PCR showed for grain-fed animals significantly higher number of bacteria belonging to Clostridium leptum group at 10 days after the incorporation of corn into the diet and significantly lower concentration of bacteria belonging to Actinobacteria phylum at the day 20 after dietary change. Taxonomic distribution analysed by NGS at day 20 revealed the similar prevalence of the phyla Firmicutes and Bacteroidetes in all goats, significantly higher presence of the unclassified genus of groups of Bacteroidales and Ruminococcaceae in grain-fed animals and significantly higher presence the genus Prevotella and Butyrivibrio in the forage-fed animals. The three different culture-independent methods used in this study show that high proportion of concentrate in goat diet does not induce any serious disturbance of their rumen ecosystem and indicate the good adaptive response of caprine ruminal bacteria to incorporation of corn into the diet.

Impairment of rumen biohydrogenation and bacteria of the Butyrivibrio group in the rumen of goats through a 20:5 n-3 (EPA) rich supplement.

BACKGROUND: Marine products can inhibit biohydrogenation in the rumen, but the mechanism is not clear. This study investigated a 20:5 n-3 rich supplement effects on rumen biohydrogenation, microbial change and fermentation characteristics in goats. RESULTS: The supplementation decreased 18:0 proportions in rumen fatty acids (P < 0.001), while it increased cis-9, trans-11 conjugated linoleic acid (CLA) (P < 0.001) and trans-10, cis-12 CLA proportions (P < 0.001). The supplement reduced the number of Butyrivibrio spp. and B. proteoclasticus (P < 0.01). Denaturing gradient gel electrophoresis redundancy analysis indicated that some species, mainly from the rumen of goats receiving the 2.5 and 5.0 g d(-1) supplement, were positively correlated with cis-9, trans-11 CLA proportions; some species, mainly from the rumen of control goats, were positively correlated with 18:0 proportions. The supplement reduced the NH3 -N concentrations and acetate molar proportions in the rumen (P < 0.05), but increased propionate and butyrate molar proportions (P < 0.01), and had no effect on total volatile fatty acid concentration. CONCLUSION: The supplement rich in 20:5 n-3 reduced the biohydrogenation of 18-carbon unsaturated fatty acids with a significant reduction of the 18:0 proportion and this was coupled with the suppression of the abundance of biohydrogenating bacteria and unknown bacteria.

Rapid changes in key ruminal microbial populations during the induction of and recovery from diet-induced milk fat depression in dairy cows.

The ruminant provides a powerful model for understanding the temporal dynamics of gastrointestinal microbial communities. Diet-induced milk fat depression (MFD) in the dairy cow is caused by rumen-derived bioactive fatty acids, and is commonly attributed to the changes in the microbial population. The aim of the present study was to determine the changes occurring in nine ruminal bacterial taxa with well-characterised functions, and abundance of total fungi, ciliate protozoa and bacteria during the induction of and recovery from MFD. Interactions between treatment and time were observed for ten of the twelve populations. The total number of both fungi and ciliate protozoa decreased rapidly (days 4 and 8, respectively) by more than 90% during the induction period and increased during the recovery period. The abundance of Streptococcus bovis (amylolytic) peaked at 350% of control levels on day 4 of induction and rapidly decreased during the recovery period. The abundance of Prevotella bryantii (amylolytic) decreased by 66% from day 8 to 20 of the induction period and increased to the control levels on day 12 of the recovery period. The abundance of Megasphaera elsdenii and Selenomonas ruminantium (lactate-utilising bacteria) increased progressively until day 12 of induction (>170%) and decreased during the recovery period. The abundance of Fibrobacter succinogenes (fibrolytic) decreased by 97% on day 4 of induction and increased progressively to an equal extent during the recovery period, although smaller changes were observed for other fibrolytic bacteria. The abundance of the Butyrivibrio fibrisolvens/Pseudobutyrivibrio group decreased progressively during the induction period and increased during the recovery period, whereas the abundance of Butyrivibrio hungatei was not affected by treatment. Responsive taxa were modified rapidly, with the majority of changes occurring within 8 d and their time course was similar to the time course of the induction of MFD, demonstrating a strong correlation between changes in ruminal microbial populations and MFD.

Milk fatty acid composition, rumen microbial population, and animal performances in response to diets rich in linoleic acid supplemented with chestnut or quebracho tannins in dairy ewes.

The aim of the study was to evaluate milk fatty acid (FA) profile, animal performance, and rumen microbial population in response to diets containing soybean oil supplemented or not with chestnut and quebracho tannins in dairy ewes. Eighteen Comisana ewes at 122±6 d in milking were allotted into 3 experimental groups. Diets were characterized by chopped grass hay administered ad libitum and by 800 g/head and day of 3 experimental concentrates containing 84.5 g of soybean oil/kg of dry matter (DM) and 52.8 g/kg of DM of bentonite (control diet), chestnut tannin extract (CHT diet), or quebracho tannin extract (QUE diet). The trial lasted 4 wk. Milk yield was recorded daily, and milk composition and blood parameters were analyzed weekly. At the end of the experiment, samples of rumen fluid were collected to analyze pH, volatile fatty acid profile, and the relative proportions of Butyrivibrio fibrisolvens and Butyrivibrio proteoclasticus in the rumen microbial population. Hepatic functionality, milk yield, and gross composition were not affected by tannin extracts, whereas milk FA composition was characterized by significant changes in the concentration of linoleic acid (CHT +2.77% and QUE +9.23%), vaccenic acid (CHT +7.07% and QUE +13.88%), rumenic acid (CHT -1.88% and QUE +24.24%), stearic acid (CHT + 8.71% and QUE -11.45%), and saturated fatty acids (CHT -0.47% and QUE -3.38%). These differences were probably due to the ability of condensed versus hydrolyzable tannins to interfere with rumen microbial metabolism, as indirectly confirmed by changes in the relative proportions of B. fibrisolvens and B. proteoclasticus populations and by changes in the molar proportions of volatile fatty acids. The effect of the CHT diet on the milk FA profile and microbial species considered in this trial was intermediate between that of QUE and the control diet, suggesting a differential effect of condensed and hydrolyzable tannins on rumen microbes. Compared with control animals, the presence of B. fibrisolvens increased about 3 times in ewes fed CHT and about 5 times in animals fed QUE. In contrast, the abundance of B. proteoclasticus decreased about 5- and 15-fold in rumen liquor of ewes fed CHT and QUE diets, respectively. The use of soybean oil and a practical dose of QUE or CHT extract in the diet of dairy ewes can be an efficient strategy to improve the nutritional quality of milk.

Effects of dietary Capsicum oleoresin on productivity and immune responses in lactating dairy cows.

This study investigated the effect of Capsicum oleoresin in granular form (CAP) on nutrient digestibility, immune responses, oxidative stress markers, blood chemistry, rumen fermentation, rumen bacterial populations, and productivity of lactating dairy cows. Eight multiparous Holstein cows, including 3 ruminally cannulated, were used in a replicated 4×4 Latin square design experiment. Experimental periods were 25 d in duration, including a 14-d adaptation and an 11-d data collection and sampling period. Treatments included control (no CAP) and daily supplementation of 250, 500, or 1,000 mg of CAP/cow. Dry matter intake was not affected by CAP (average 27.0±0.64 kg/d), but milk yield tended to quadratically increase with CAP supplementation (50.3 to 51.9±0.86 kg/d). Capsicum oleoresin quadratically increased energy-corrected milk yield, but had no effect on milk fat concentration. Rumen fermentation variables, apparent total-tract digestibility of nutrients, and N excretion in feces and urine were not affected by CAP. Blood serum ß-hydroxybutyrate was quadratically increased by CAP, whereas the concentration of nonesterified fatty acids was similar among treatments. Rumen populations of Bacteroidales, Prevotella, and Roseburia decreased and Butyrivibrio increased quadratically with CAP supplementation. T cell phenotypes were not affected by treatment. Mean fluorescence intensity for phagocytic activity of neutrophils tended to be quadratically increased by CAP. Numbers of neutrophils and eosinophils and the ratio of neutrophils to lymphocytes in peripheral blood linearly increased with increasing CAP. Oxidative stress markers were not affected by CAP. Overall, in the conditions of this experiment, CAP did not affect feed intake, rumen fermentation, nutrient digestibility, T cell phenotypes, and oxidative stress markers. However, energy-corrected milk yield was quadratically increased by CAP, possibly as a result of enhanced mobilization of body fat reserves. In addition, CAP increased neutrophil activity and immune cells related to acute phase immune response.

Abundance of ruminal bacteria, epithelial gene expression, and systemic biomarkers of metabolism and inflammation are altered during the peripartal period in dairy cows.

Seven multiparous Holstein cows with a ruminal fistula were used to investigate the changes in rumen microbiota, gene expression of the ruminal epithelium, and blood biomarkers of metabolism and inflammation during the transition period. Samples of ruminal digesta, biopsies of ruminal epithelium, and blood were obtained during -14 through 28d in milk (DIM). A total of 35 genes associated with metabolism, transport, inflammation, and signaling were evaluated by quantitative reverse transcription-PCR. Among metabolic-related genes, expression of HMGCS2 increased gradually from -14 to a peak at 28 DIM, underscoring its central role in epithelial ketogenesis. The decrease of glucose and the increase of nonesterified fatty acids and ß-hydroxybutyrate in the blood after calving confirmed the state of negative energy balance. Similarly, increases in bilirubin and decreases in albumin concentrations after calving were indicative of alterations in liver function and inflammation. Despite those systemic signs, lower postpartal expression of TLR2, TLR4, CD45, and NFKB1 indicated the absence of inflammation within the epithelium. Alternatively, these could reflect an adaptation to react against inducers of the immune system arising in the rumen (e.g., bacterial endotoxins). The downregulation of RXRA, INSR, and RPS6KB1 between -14 and 10 DIM indicated a possible increase in insulin resistance. However, the upregulation of IRS1 during the same time frame could serve to restore sensitivity to insulin of the epithelium as a way to preserve its proliferative capacity. The upregulation of TGFB1 from -14 and 10 DIM coupled with upregulation of both EGFR and EREG from 10 to 28 DIM indicated the existence of 2 waves of epithelial proliferation. However, the downregulation of TGFBR1 from -14 through 28 DIM indicated some degree of cell proliferation arrest. The downregulation of OCLN and TJP1 from -14 to 10 DIM indicated a loss of tight-junction integrity. The gradual upregulation of membrane transporters MCT1 and UTB to peak levels at 28 DIM reflected the higher intake and fermentability of the lactation diet. In addition, those changes in the diet after calving resulted in an increase of butyrate and a decrease of ruminal pH and acetate, which partly explain the increase of Anaerovibrio lipolytica, Prevotella bryantii, and Megasphaera elsdenii and the decrease of fibrolytic bacteria (Fibrobacter succinogenes, Butyrivibrio proteoclasticus). Overall, these multitier changes revealed important features associated with the transition into lactation. Alterations in ruminal epithelium gene expression could be driven by nutrient intake-induced changes in microbes; microbial metabolism; and the systemic metabolic, hormonal, and immune changes. Understanding causes and mechanisms driving the interaction among ruminal bacteria and host immunometabolic responses merits further study.

Effect of ethanol and methanol on growth of ruminal bacteria Selenomonas ruminantium and Butyrivibrio fibrisolvens.

The effect of ethanol and methanol on growth of several ruminal bacterial strains was examined. Ethanol concentrations as low as 0.2% had a significant, but moderate, inhibitory effect on lag time or growth over time and 3.3% ethanol significantly inhibited maximum optical density obtained by both Selenomonas ruminantium and Butyrivibrio fibrisolvens. Little growth of either strain occurred at 10% ethanol concentrations. Methanol concentrations below 0.5% had little effect on either growth or maximum optical density of Selenomonas ruminantium whereas methanol concentrations below 3.3% had little effect on growth or maximum optical density of Butyrivibrio fibrisolvens. Higher methanol concentrations increasingly inhibited growth of both strains and no growth occurred at a 10% methanol concentration. Concentrations of ethanol or methanol used to add hydrophobic compounds to culture media should be kept below 1%.

Effect of stoned olive pomace on rumen microbial communities and polyunsaturated fatty acid biohydrogenation: an in vitro study.

BACKGROUND: Stoned olive pomace (SOP), which represents approximately 50% of the conversion process of olives to olive oil, is largely not utilised and creates costs for its disposal and has negative environmental impacts. In vitro trial experiments were employed to study the effect of feeds integrated with this bio-waste, which is rich in polyphenols, on rumen biohydrogenation, using sheep rumen liquor as inoculum. RESULTS: Fatty acid (FA) analysis and a polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) approach aimed at characterising the microbial community indicated that including SOP in feeds at the level of 50 g/kg and 90 g/kg induced changes in the FA profile and microbial populations. The simultaneous decrease of Butyrivibrio proteoclasticus and accumulation of vaccenic acid was observed. A depression in the populations of Neisseria weaveri, Ruminobacter amylophilus and other unclassified bacteria related to members of the Lachnospiraceae and Pasteurellaceae families was detected, suggesting that these microbial groups may be involved in rumen biohydrogenation. CONCLUSIONS: Supplementation of feeds with SOP alters the rumen bacterial community, including bacteria responsible for the hydrogenation of vaccenic acid to stearic acid, thereby modifying the FA profile of the rumen liquor. Hence, a use of SOP aimed to produce meat or dairy products enriched in functional lipids can be hypothesised.

Effects of partial mixed rations and supplement amounts on milk production and composition, ruminal fermentation, bacterial communities, and ruminal acidosis.

Late-lactation Holstein cows (n=144) that were offered 15kg dry matter (DM)/cow per day of perennial ryegrass to graze were randomized into 24 groups of 6. Each group contained a fistulated cow and groups were allocated to 1 of 3 feeding strategies: (1) control (10 groups): cows were fed crushed wheat grain twice daily in the milking parlor and ryegrass silage at pasture; (2) partial mixed ration (PMR; 10 groups): PMR that was isoenergetic to the control diet and fed twice daily on a feed pad; (3) PMR+canola (4 groups): a proportion of wheat in the PMR was replaced with canola meal to produce more estimated metabolizable protein than other groups. Supplements were fed to the control and PMR cows at 8, 10, 12, 14, or 16kg of DM/d, and to the PMR+canola cows at 14 or 16kg of DM/d. The PMR-fed cows had a lower incidence of ruminal acidosis compared with controls, and ruminal acidosis increased linearly and quadratically with supplement fed. Yield of milk fat was highest in the PMR+canola cows fed 14 or 16kg of total supplement DM/d, followed by the PMR-fed cows, and was lowest in controls fed at these amounts; a similar trend was observed for milk fat percentage. Milk protein yield was higher in the PMR+canola cows fed 14 or 16kg of total supplement DM/d. Milk yield and milk protein percentage were not affected by feeding strategy. Milk, energy-corrected milk, and milk protein yields increased linearly with supplement fed, whereas milk fat percentage decreased. Ruminal butyrate and d-lactate concentrations, acetate-to-propionate ratio, (acetate + butyrate)/propionate, and pH increased in PMR-fed cows compared with controls for all supplement amounts, whereas propionate and valerate concentrations decreased. Ruminal acetate, butyrate, and ammonia concentrations, acetate-to-propionate ratio, (acetate + butyrate)/propionate, and pH linearly decreased with amounts of supplement fed. Ruminal propionate concentration linearly increased and valerate concentration linearly and quadratically increased with supplement feeding amount. The Bacteroidetes and Firmicutes were the dominant bacterial phyla identified. The Prevotellaceae, Ruminococcaceae, and Lachnospiraceae were the dominant bacterial families, regardless of feeding group, and were influenced by feeding strategy, supplement feeding amount, or both. The Veillonellaceae family decreased in relative abundance in PMR-fed cows compared with controls, and the Streptococcaeae and Lactobacillaceae families were present in only minor relative abundances, regardless of feeding group. Despite large among- and within-group variation in bacterial community composition, distinct bacterial communities occurred among feeding strategies, supplement amounts, and sample times and were associated with ruminal fermentation measures. Control cows fed 16kg of DM of total supplement per day had the most distinct ruminal bacterial community composition. Bacterial community composition was most significantly associated with supplement feeding amount and ammonia, butyrate, valerate, and propionate concentrations. Feeding supplements in a PMR reduced the incidence of ruminal acidosis and altered ruminal bacterial communities, regardless of supplement feeding amount, but did not result in increased milk measures compared with isoenergetic control diets component-fed to late-lactation cows.

Effects of incremental amounts of fish oil on trans fatty acids and Butyrivibrio bacteria in continuous culture fermenters.

Previous studies have shown that adding fish oil (FO) to ruminant animal diets increased vaccenic acid (VA; t11 C18:1) accumulation in the rumen. Therefore, the objective of this study was to evaluate the effect of dietary FO amounts on selected strains of rumen bacteria involved in biohydrogenation. A single-flow continuous culture system consisting of four fermenters was used in a 4 × 4 Latin square design with four 9 days consecutive periods. Treatment diets were as follows: (i) control diet (53:47 forage to concentrate; CON), (ii) control plus FO at 0.5% (DM basis; FOL), (iii) control plus FO at 2% (DM basis; FOM) and (iv) control plus FO at 3.5% (DM basis; FOH). Fermenters were fed treatment diets three times daily at 120 g/day. Samples were collected from each fermenter on day 9 of each period at 1.5, 3 and 6 h post-morning feeding and then composited into one sample per fermenter. Increasing dietary FO amounts resulted in a linear decrease in acetate and isobutyrate concentrations and a linear decrease in acetate-to-propionate ratio. Propionate, butyrate, valerate and isovalerate concentrations were not affected by FO supplementation. Concentrations of C18:0 in fermenters linearly decreased, while concentrations of t10 C18:1 and VA linearly increased as dietary FO amounts increased. The concentrations of c9t11 and t10c12 conjugated linoleic acid were not affected by FO supplementation. The DNA abundance for Butyrivibrio fibrisolvens, Butyrivibrio vaccenic acid subgroup, Butyrivibrio stearic acid subgroup and Butyrivibrio proteoclasticus linearly decreased as dietary FO amounts increased. In conclusion, FO effects on trans fatty acid accumulation in the rumen may be explained in part by FO influence on Butyrivibrio group.

Structure and function of an acetyl xylan esterase (Est2A) from the rumen bacterium Butyrivibrio proteoclasticus.

Butyrivibrio proteoclasticus is a significant component of the microbial population of the rumen of dairy cattle. It is a xylan-degrading organism whose genome encodes a large number of open reading frames annotated as fiber-degrading enzymes. We have determined the three-dimensional structure of Est2A, an acetyl xylan esterase from B. proteoclasticus, at 2.1 Å resolution, along with the structure of an inactive mutant (H351A) at 2.0 Å resolution. The structure reveals two domains-a C-terminal SGNH domain and an N-terminal jelly-roll domain typical of CE2 family structures. The structures are accompanied by experimentally determined enzymatic parameters against two model substrates, para-nitrophenyl acetate and para-nitrophenyl butyrate. The suite of fiber-degrading enzymes produced by B. proteoclasticus provides a rich source of new enzymes of potential use in industrial settings.

Characterisation of caecal microbial diversity of lactating does and their offspring given diets with different neutral detergent soluble to insoluble fibre ratios.

The effect of neutral detergent soluble fibre (NDSF) to neutral detergent fibre (NDF) dietary ratio (0.29, LR and 0.43, HR) on the caecal ecosystem of lactating does and their offspring was studied. From the 17th day of lactation, each diet was given to four does, allowing for free access to their litters. Does were sampled at 17 and 28 days of lactation, and also two pups per litter at 17 (milk-fed only), 28 (milk and solid fed) and 49 days of age. DGGE was used to study bacterial caecal biodiversity, and total bacterial concentration and relative proportions of Ruminococcus albus and Butyrivibrio fibrisolvens were quantified by real time PCR. In does, diet did not affect (P > 0.10) diversity indexes, total bacterial concentration or relative abundance of B. fibrisolvens, but at 28 days of lactation the proportion of R. albus was higher with LR (interaction Diet × Time, P = 0.037). Caecal communities of pups of 17 days were grouped by litter, but the influence of the mother was reduced at 28 days with solid feed intake, and at 49 days rabbits clustered by diet. Caecal biodiversity increased from 17 to 28 days, and was reduced at 49 days (Shannon index of 3.60, 3.71 and 3.57, respectively; P = 0.049). Total bacterial concentration and relative abundance of R. albus and B. fibrisolvens increased with solid feed intake from 17 to 28 days (P < 0.01), remaining unaffected thereafter. Access of pups to solid feed from 17 days of age modulates the development and composition of the caecal microbiota at weaning.