A Proteomic Analysis of Fibre Degradation and Assimilation by Butyrivibrio Proteoclasticus

<p>Butyrivibrio proteoclasticus B316T is a Gram-positive, lignocellulose degrading bacterium that is prevalent in the rumen of animals grazing pasture, and is one of only a few rumen microbes known to degrade and utilise xylan in vitro. Xylan is a hemicellulose that comprises up to 45% of the polysaccharide component of ruminant forages. Often as little as 30% of the total energy content of forages is utilised by the ruminant due to poor hemicellulose degradation by the fibrolytic rumen microbes. An opportunity exists to improve forage degradation in the rumen, which is predicted to improve the productivity of forage fed ruminants. A clearer understanding of the strategies employed by fibrolytic rumen microbes to degrade and utilise lignocellulose is important in realising this goal. Almost 10% of the B. proteoclasticus genome encodes proteins involved in polysaccharide metabolism and transport, which includes 134 fibrolytic enzymes that are active upon plant fibre. Many of these are clustered into one of 36 polysaccharide utilisation loci that also contain transmembrane transporters, transcriptional regulators, environmental sensors and genes involved in further polysaccharide metabolism. Gel-based and gel-free proteomic analyses of the cytosolic, cell-associated, and secreted fractions of cells grown on xylan were used to identify proteins involved in the degradation, assimilation, and metabolism of hemicellulose. A set of 416 non-redundant proteins were identified, which included 12 extracellular and 24 cytosolic polysaccharidases, and 59 proteins involved in the uptake and further metabolism of polysaccharide degradation products, many of which were substrate-binding protein components of ATP-driven transporter systems. In cells grown on xylan, several of these proteins displayed significant protein abundance changes relative to cells grown on the monomeric sugar xylose, in a pattern that reflected the growth substrates used. A model of xylan degradation by B. proteoclasticus based on these results hypothesises that B. proteoclasticus attacks the xylan backbone and main substituent groups of hemicellulose in the extracellular space, assimilates the xylooligosaccharides and performs the final stages of degradation within the cell. These results provide insight into a xylan degrading enzyme system that has evolved to efficiently degrade and utilise hemicellulose, extend our understanding of the enzymes that are likely to play important roles in hemicellulose degradation, and support the notion that Butyrivibrio species are important contributors to rumen fibre degradation.</p>

A Proteomic Analysis of Fibre Degradation and Assimilation by Butyrivibrio Proteoclasticus

<p>Butyrivibrio proteoclasticus B316T is a Gram-positive, lignocellulose degrading bacterium that is prevalent in the rumen of animals grazing pasture, and is one of only a few rumen microbes known to degrade and utilise xylan in vitro. Xylan is a hemicellulose that comprises up to 45% of the polysaccharide component of ruminant forages. Often as little as 30% of the total energy content of forages is utilised by the ruminant due to poor hemicellulose degradation by the fibrolytic rumen microbes. An opportunity exists to improve forage degradation in the rumen, which is predicted to improve the productivity of forage fed ruminants. A clearer understanding of the strategies employed by fibrolytic rumen microbes to degrade and utilise lignocellulose is important in realising this goal. Almost 10% of the B. proteoclasticus genome encodes proteins involved in polysaccharide metabolism and transport, which includes 134 fibrolytic enzymes that are active upon plant fibre. Many of these are clustered into one of 36 polysaccharide utilisation loci that also contain transmembrane transporters, transcriptional regulators, environmental sensors and genes involved in further polysaccharide metabolism. Gel-based and gel-free proteomic analyses of the cytosolic, cell-associated, and secreted fractions of cells grown on xylan were used to identify proteins involved in the degradation, assimilation, and metabolism of hemicellulose. A set of 416 non-redundant proteins were identified, which included 12 extracellular and 24 cytosolic polysaccharidases, and 59 proteins involved in the uptake and further metabolism of polysaccharide degradation products, many of which were substrate-binding protein components of ATP-driven transporter systems. In cells grown on xylan, several of these proteins displayed significant protein abundance changes relative to cells grown on the monomeric sugar xylose, in a pattern that reflected the growth substrates used. A model of xylan degradation by B. proteoclasticus based on these results hypothesises that B. proteoclasticus attacks the xylan backbone and main substituent groups of hemicellulose in the extracellular space, assimilates the xylooligosaccharides and performs the final stages of degradation within the cell. These results provide insight into a xylan degrading enzyme system that has evolved to efficiently degrade and utilise hemicellulose, extend our understanding of the enzymes that are likely to play important roles in hemicellulose degradation, and support the notion that Butyrivibrio species are important contributors to rumen fibre degradation.</p>

An Overview: The Toxicity of Ageratina adenophora on Animals and Its Possible Interventions

Ageratina adenophora is one of the major invasive weeds that causes instability of the ecosystem. Research has reported that A. adenophora produces allelochemicals that inhibit the growth and development of food crops, and also contain some toxic compounds that cause toxicity to animals that consume it. Over the past decades, studies on the identification of major toxic compounds of A. adenophora and their toxic molecular mechanisms have been reported. In addition, weed control interventions, such as herbicides application, was employed to reduce the spread of A. adenophora. However, the development of therapeutic and prophylactic measures to treat the various A. adenophora—induced toxicities, such as hepatotoxicity, splenotoxicity and other related disorders, have not been established to date. The main toxic pathogenesis of A. adenophora is oxidative stress and inflammation. However, numerous studies have verified that some extracts and secondary metabolites isolated from A. adenophora possess anti-oxidation and anti-inflammation activities, which implies that these extracts can relieve toxicity and aid in the development of drug or feed supplements to treat poisoning-related disorders caused by A. adenophora. Furthermore, beneficial bacteria isolated from rumen microbes and A. adenophora can degrade major toxic compounds in A. adenophora so as to be developed into microbial feed additives to help ameliorate toxicity mediated by A. adenophora. This review presents an overview of the toxic mechanisms of A. adenophora, provides possible therapeutic strategies that are available to mitigate the toxicity of A. adenophora and introduces relevant information on identifying novel prophylactic and therapeutic measures against A. adenophora—induced toxicity.

Development of Protocol for Lyophilization of Goat Rumen Fluid

Background: Rumen fluid has been used as microbial inoculum to treat indigestion in ruminant animals and to conduct in vitro rumen fermentation experiments. Lyophilization of the goat rumen fluid will provide continuous supply of rumen inoculums either for laboratory studies or for transfaunation in treating digestive disorders sequelae to high grain rations. However, no standard protocol is available for lyophilizing goat rumen fluid. Hence, this study was designed to develop a protocol to lyophilize goat rumen fluid as an alternate source for fresh goat rumen fluid. Methods: The study was conducted using 5 × 3 × 3 factorial design with four different cryoprotectants viz., 10% skim milk powder, 10% skim milk powder + 5% sodium glutamate, 5% glycerol, 5% DMSO and no cryoprotectant, at three pre freezing (2, 24 and 48 hours) and three freeze drying (8, 24 and 32 hours) time intervals to standardize protocol for lyophilizing goat rumen fluid. The viability of rumen microbes in the “lyophilized goat ruminal inoculum”, was determined via in vitro gas production study. Result: Pre freezing (-80°C deep freezer) duration of 48 hours with 32 hours of time duration in lyophilizer (-45°C) was ideal for lyophilizing goat rumen fluid with or without the addition of various cryoprotectants. Glycerol used at 5% as cryoprotectant resulted in significantly (P less than 0.05) highest gas production at all (12, 24 and 48) incubation hours studied indicating better viability of rumen microbes.

Characterizing Effects of Ingredients Differing in Ruminally Degradable Protein and Fiber Supplies on the Ovine Rumen Microbiome Using Next-Generation Sequencing

The ratio of concentrate to forage within diets is known to alter rumen microbial profiles, but comparatively less information is available on the effect of differing sources of individual nutrients on the microbiome. The objective of this study was to investigate rumen microbial responses to diets composed of protein and fiber sources expected to vary in nutrient degradability. The responses of interest included relative abundances of bacterial taxa as well as estimations of community richness and diversity. Ten ruminally cannulated wethers (Suffolk, Dorset, or Suffolk × Dorset) received four diet treatments consisting of either beet pulp or timothy hay and soybean meal (SBM) or heat-treated soybean meal (HSBM) in a partially replicated 4 × 4 Latin square experiment for 21 days. Timothy hay and beet pulp were expected to provide differing rumen degradabilities of neutral detergent fiber (NDF) while the soybean meals were expected to provide differing rumen degradabilities of crude protein (CP). Solid and liquid samples of rumen contents were collected for microbial DNA isolation and Next-Generation sequencing. Numerous rumen bacterial population shifts were observed due to change in fiber source, with increased abundances (P &lt; 0.05) of fibrolytic populations associated with timothy hay diets compared with beet pulp diets. Conversely, populations of the pectin-degrading genera, Treponema and Lachnospira, increased on the beet pulp treatment (P = 0.015 and P = 0.0049, respectively). Limited impact on bacterial taxa was observed between diets differing in protein source. The Paraprevotellaceae genus YRC22 was observed to increase in abundance on HSBM diets (P = 0.023) and the phylum Spirochaetes tended to be more abundant on SBM than HSBM diets (P = 0.071). Beet pulp decreased rumen bacterial diversity (P = 0.0027) and tended to decrease bacterial species richness (P = 0.051) compared to timothy hay. Our results serve to further underscore the sensitivity of rumen microbes to changes in their preferred substrates, particularly of those associated with fiber degradation.

PSX-A-22 Late-Breaking: Rumen microbiome composition and microbe network dynamics in beef cattle predict finishing performance under different backgrounding systems

Backgrounding (BKG) impacts growth and finishing performance in beef cattle. However, specific microbiome contributions to growth performance during this period, considering different BKG systems, remain unknown. A longitudinal study was designed to characterize the rumen microbiome and average daily gain (ADG) of Angus and Angus x Simmental calves (n = 38) placed under different BKG systems for 55 d after weaning: DL: a high roughage diet within a dry lot and CC: annual cover crop within a strip and PP, while a third group, PP: remained on perennial pasture vegetation within rotational paddocks, just as before weaning. After BKG, calves went to a feedlot for 142 d and finished with a high energy ration. Rumen bacterial communities were profiled by collecting fluid samples via oral probe, and sequencing the V4 region of the 16S rRNA bacterial gene, at weaning, during backgrounding and finishing. For calves moved to CC and DL BKG, bacterial composition diverged drastically, including sharp decreases in bacterial diversity (P &lt; 0.001), while PP claves conserved more stable diversity patterns. During BKG, DL calves also showed the greatest ADG (P &lt; 0.05), which coincided with increased abundance of taxa affiliated to the Aeromondales (Succinivibrio, Succcinimonas and Ruminobacter) (P &lt; 0.001). However, once in the finishing phase, PP calves showed compensatory ADG, with significantly higher values, particularly compared with calves on DL BKG (v = 0.02). Network theory analyses were concordant with these patterns, highlighting the importance of understanding microbe-microbe interactions at early developmental stages to predict finishing performance. These results indicate that rumen microbes and their network interactions during backgrounding successfully predict finishing performance in beef cattle, underscoring the importance of early post weaning stages as potential targets for feeding interventions that can modulate the rumen microbiome to enhance lifelong animal performance.

329 Supplementing Native Rumen Microorganisms Improves Feedlot Steer Digestive Health and Performance

The rumen microbiome functions as a synchronized entity that digests feed in order to provide nutrients for its host. High-concentrate diets destabilize the rumen microbiome by biasing the community towards microorganisms that readily ferment simple carbohydrates resulting in decreased pH, increased CO2, and an increased solvent concentration in the rumen content. This chemical shift interrupts rumen fermentation and can lead to the development of metabolic diseases that negatively impact animal performance. This study evaluated the benefit of a daily, in-feed microbial feed supplement (MFS; Magnius, Native Microbials Inc, San Diego, CA) containing three native rumen microbes (Chordicoccus ruminifurens ASCUSBF65, Prevotella albensis ASCUSBF41, and Succinivibrio dextrinosolvens ASCUSBF53) on commercial feedlot steer performance and rumen microbiome composition. The trial was conducted by HMS Veterinary Development in Reedley, CA using 200 steers in 20 pens over 109 days with a 2x2 factorial design (with and without step-up period x with and without MFS). The without step-up group was directly fed the finisher ration (94% concentrate, 0.95 Mcal/lb NEm), while the step-up group was adapted to the finisher ration over a period of 21 days. Rumen microbiome samples were collected via stomach tubing periodically throughout the trial. At the end of 109 days, the FCR of the animals receiving MFS were significantly lower than the control animals (7.67% improvement, P = 0.013) in the step-up group, although no significant differences were observed in ADG and DMI. No significant performance differences were observed in the group without a step-up (Table 1). The relationship between rumen pH, dissolved CO2, and the observed changes in the microbiome suggest a potential interplay between acetogenesis and methanogenesis where CO2 consuming bacteria may be important in improving rumen digestive health. Collectively, these results suggest that feeding native rumen microorganisms can improve rumen resilience and health of high-grain consuming cattle.

Spent Craft Brewer's Yeast Reduces Production of Methane and Ammonia by Bovine Rumen Microbes

Methane and ammonia are byproducts of rumen fermentation that do not promote animal growth, and methane is a key contributor to anthropogenic climate disruption. Cows eructate every few breaths and typically emit 250–500 L of methane gas daily. Significant research is focused on finding diets and additives that lower the production of methane and ammonia. Emerging research has shown that humulones and lupulones, molecules that are found in the cones of hops (Humulus lupulus), have potential in this regard. These molecules, which are also key flavor components in beer, are biologically active: they are known inhibitors of Gram-positive bacteria. Ruminants' sophisticated digestive systems host billions of microorganisms, and these systems' outputs will likely be affected in the presence of brewer's yeast (Saccharomyces cerevisiae). So-called spent yeast is produced during the beer-brewing process and contains humulones and lupulones in concentrations that vary by beer style, but it is generally discarded as waste. Our research suggests that adding spent craft brewer's yeast to rumen microbes by single time-point 24-h in vitro incubations suppresses production of methane and ammonia. This project examines the correlation between the quantities of hop acids in spent yeast and the production of methane and ammonia by bovine rumen microbes in vitro. We determined, by HPLC, the hop acid concentrations in spent yeast obtained from six beer styles produced at a local brewery. We performed anaerobic incubation studies on bovine rumen microbes, comparing the effects of these materials to a baker's yeast control and to the industry-standard antibiotic monensin. Results include promising decreases in both methane (measured by GC–FID) and ammonia (measured by colorimetric assay) in the presence of craft brewer's yeast, and a strong correlation between the quantities of hop acids in the spent yeast and the reduction of methane and ammonia. Notably, two of the yeast samples inhibited methane production to a greater degree than the industry-standard antibiotic monensin. Our results suggest that spent brewer's yeast has potential to improve ruminant growth while reducing anthropogenic methane emission.

Rumen Sampling Methods Bias Bacterial Communities Observed

The rumen is a complex ecosystem that plays a critical role in our efforts to improve feed efficiency of cattle and reduce their environmental impacts. Sequencing of the 16S rRNA gene provides a powerful tool to survey shifts in the microbial community in response to feed additives and dietary changes. Oral stomach tubing a cow for a rumen sample is a rapid, cost-effective alternative to rumen cannulation for acquiring rumen samples. In this study, we determined how sampling method, as well as type of sample collected (liquid vs solid), bias the microbial populations observed. The abundance of major archaeal populations was not different at the family level in samples acquired via rumen cannula or stomach tube. Liquid samples were enriched for the order WCHB1-41 (phylum Kiritimatiellaeota) as well as the family Prevotellaceae and had significantly lower abundance of Lachnospiraceae compared with grab samples from the rumen cannula. Solid samples most closely resembled the grab samples; therefore, inclusion of particulate matter is important for an accurate representation of the rumen microbes. Stomach tube samples were the most variable and were most representative of the liquid phase. In comparison with a grab sample, stomach tube samples had significantly lower abundance of Lachnospiraceae, Fibrobacter and Treponema. Fecal samples did not reflect the community composition of the rumen, as fecal samples had significantly higher relative abundance of Ruminococcaceae and significantly lower relative abundance of Lachnospiraceae compared with samples from the rumen.

The rumen microbiome of yak co-evolves with its host probably adding the adaptation to its harsh environments

Background Rumen microbes play an important role in ruminant energy supply and animal performance. Previous studies showed that yak (Bos grunniens) rumen microbiome and fermentation differ from other ruminants. However, little is understood on the features of the rumen microbiome that make yak adapted to its unique environmental and dietary conditions. Here we investigated the rumen microbiome and metabolome to understand how yak adapts to the coarse forage and harsh environment in the high Qinghai-Tibetan plateau. Result Metataxonomic analysis of the rumen microbiota revealed that yak (Bos grunniens), domesticated cattle (Bos taurus), and dzo (a hybrid between the yak and domestic cattle) have distinct rumen microbiota. Metagenomic analysis displayed a larger gene pool encoding a richer repertoire of carbohydrate-active enzymes (CAZymes) in the rumen microbiome of yak and dzo than cattle. Some of the genes encoding glycoside hydrolases (GH) that mediate the digestion of cellulose and hemicellulose were significantly enriched in the rumen of yak than cattle, but the cattle rumen microbiome had more genes assigned to GH57 that primarily includes amylases. The rumen fermentation profile differed also, with cattle having a higher molar proportion of acetate but a lower molar proportion of propionate than dzo and yak. Metabolomic analysis showed differences in both rumen microbial metabolic pathways and metabolites, mainly amino acids, carboxylic acids, sugars, and bile acids. Notably, styrene degradation, primary bile acid biosynthesis, glyoxylate, and dicarboxylate metabolism significantly differed between cattle and dzo; streptomycin biosynthesis was significantly different between cattle and yak; and the pathways for biotin metabolism and styrene degradation significantly differed between dzo and yak. Correlation analysis revealed certain microbial species correlated with differential rumen metabolites. Nine differential metabolites showed a positive correlation with seven species belonging to Bacteroides and Alistipes but a negative correlation with ten species belonging to Prevotella and Ruminococcus. Conclusion The present study showed that the rumen microbiome of yak and its host had probably co-evolved aiding in the adaptation of yak to the harsh dietary environment of the Qinghai-Tibetan plateau. In particular, the yak rumen microbiome has more enzymes involved in the degradation of rough forage than that of cattle, providing sufficient energy for its host.