Dynamic genome-based metabolic modeling of the predominant cellulolytic rumen bacterium Fibrobacter succinogenes S85.

Fibrobacter succinogenes is a cellulolytic bacterium that plays an essential role in the degradation of plant fibers in the rumen ecosystem. It converts cellulose polymers into intracellular glycogen and the fermentation metabolites succinate, acetate, and formate. We developed dynamic models of F. succinogenes S85 metabolism on glucose, cellobiose, and cellulose on the basis of a network reconstruction done with the automatic reconstruction of metabolic model workspace. The reconstruction was based on genome annotation, five template-based orthology methods, gap filling, and manual curation. The metabolic network of F. succinogenes S85 comprises 1,565 reactions with 77% linked to 1,317 genes, 1,586 unique metabolites, and 931 pathways. The network was reduced using the NetRed algorithm and analyzed for the computation of elementary flux modes. A yield analysis was further performed to select a minimal set of macroscopic reactions for each substrate. The accuracy of the models was acceptable in simulating F. succinogenes carbohydrate metabolism with an average coefficient of variation of the root mean squared error of 19%. The resulting models are useful resources for investigating the metabolic capabilities of F. succinogenes S85, including the dynamics of metabolite production. Such an approach is a key step toward the integration of omics microbial information into predictive models of rumen metabolism. IMPORTANCE F. succinogenes S85 is a cellulose-degrading and succinate-producing bacterium. Such functions are central for the rumen ecosystem and are of special interest for several industrial applications. This work illustrates how information of the genome of F. succinogenes can be translated to develop predictive dynamic models of rumen fermentation processes. We expect this approach can be applied to other rumen microbes for producing a model of rumen microbiome that can be used for studying microbial manipulation strategies aimed at enhancing feed utilization and mitigating enteric emissions.

Effect of Hydrogen-Consuming Compounds on In Vitro Ruminal Fermentation, Fatty Acids Profile, and Microbial Community in Water Buffalo.

The present study aimed to investigate the effect of hydrogen-consuming compounds on ruminal methane (CH4) production, in vitro fermentation parameters, fatty acids profile, and microbial community in water buffalo. Different sodium nitrate to disodium fumarate ratios [2:1 (F), 1:1 (S), 1:2 (T)] were studied in vitro by batch culture technique in the presence of linoleic acid. Results revealed that the dominant bacterial communities were not affected with sodium nitrate and disodium fumarate, whereas CH4 production and Verrucomicrobia, Succiniclasticum, norank_f__Muribaculaceae, and Prevotellaceae_UCG-003 were reduced (P < 0.05). However, ruminal pH, unsaturated fatty acids/saturated fatty acids (UFA/SFA) and Campilobacterota, Selenomonas, Succinivibrio, Oribacterium, Christensenellaceae_R-7_group, Campylobacter, Shuttleworthia, Schwartzia, and Prevotellaceae_YAB2003_group were increased (P < 0.05). Total volatile fatty acids (TVFA) and Spirochaetae, Fibrobacterota, Verrucomicrobia, Fibrobacter, Treponema, and Prevotellaceae were decreased in F (P < 0.05), but cis-9, trans-11CLA, acetate/propionate and Proteobacteria, Campilobacterota, Selenomonas, Succinivibrio, and Campylobacter were increased in F (P < 0.05). The highly selected bacterial genera in F were Campylobacter and Succinivibrio. The disodium fumarate, enhanced (P < 0.05) the TVFA, propionate, total bacteria, Butyrivibrio proteoclasticus, and Atypical butyrivibrio. The concentrations of C18:3n3, C20:3n6, C21:0, C22:2n6, and C22:1n9, as well as the populations of total fungi, protozoa, methanogens, Butyrivibrio hungatei in T were higher (P < 0.05). The highly selected bacterial genera in T were Fibrobacter and Treponema. Conclusively, the addition of sodium nitrate and disodium fumarate can reduce the CH4 production and optimize ruminal fatty acid composition. Furthermore, disodium fumarate can alleviate the adverse effect of sodium nitrate on the rumen fermentation.

Characterization of microbial intolerances and ruminal dysbiosis towards different dietary carbohydrate sources using an in vitro model.

AIM: This study aimed to characterize the critical points for determining the development of dysbiosis associated with feed intolerances and ruminal acidosis. METHODS AND RESULTS: A metabologenomics approach was used to characterize dynamic microbial and metabolomics shifts using the rumen simulation technique (RUSITEC) by feeding native cornstarch (ST), chemically modified cornstarch (CMS), or sucrose (SU). SU and CMS elicited the most drastic changes as rapidly as 4 h after feeding. This was accompanied by a swift accumulation of d-lactate, and the decline of benzoic and malonic acid. A consistent increase in Bifidobacterium and Lactobacillus as well as a decrease in fibrolytic bacteria was observed for both CMS and ST after 24 h, indicating intolerances within the fibre degrading populations. However, an increase in Lactobacillus was already evident in SU after 8 h. An inverse relationship between Fibrobacter and Bifidobacterium was observed in ST. In fact, Fibrobacter was positively correlated with several short-chain fatty acids, while Lactobacillus was positively correlated with lactic acid, hexoses, hexose-phosphates, pentose phosphate pathway (PENTOSE-P-PWY), and heterolactic fermentation (P122-PWY). CONCLUSIONS: The feeding of sucrose and modified starches, followed by native cornstarch, had a strong disruptive effect in the ruminal microbial community. Feed intolerances were shown to develop at different rates based on the availability of glucose for ruminal microorganisms. SIGNIFICANCE AND IMPACT OF THE STUDY: These results can be used to establish patterns of early dysbiosis (biomarkers) and develop strategies for preventing undesirable shifts in the ruminal microbial ecosystem.

Effects of yeast culture supplementation on lactation performance and rumen fermentation profile and microbial abundance in mid-lactation Holstein dairy cows.

The continuous trend for a narrowing margin between feed cost and milk prices across dairy farms in the United States highlights the need to improve and maintain feed efficiency. Yeast culture products are alternative supplements that have been evaluated in terms of milk performance and feed efficiency; however, less is known about their potential effects on altering rumen microbial populations and consequently rumen fermentation. Therefore, the objective of this study was to evaluate the effect of yeast culture supplementation on lactation performance, rumen fermentation profile, and abundance of major species of ruminal bacteria in lactating dairy cows. Forty mid-lactation Holstein dairy cows (121 ± 43 days in milk; mean ± standard deviation; 32 multiparous and 8 primiparous) were used in a randomized complete block design with a 7-d adaptation period followed by a 60-d treatment period. Cows were blocked by parity, days in milk, and previous lactation milk yield and assigned to a basal total mixed ration (TMR; 1.6 Mcal/kg of dry matter, 14.6% crude protein, 21.5% starch, and 38.4% neutral detergent fiber) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.). Treatments were top-dressed over the TMR once a day. Cows were individually fed 1 × /d throughout the trial. Blood and rumen fluid samples were collected in a subset of cows (n = 10/treatment) at 0, 30, and 60 d of the treatment period. Rumen fluid sampled via esophageal tubing was analyzed for ammonia-N, volatile fatty acids (VFA), and ruminal bacteria populations via quantitative PCR amplification of 16S ribosomal DNA genes. Milk yield was not affected by treatment effects. Energy balance was lower in YC cows than CON, which was partially explain by the trend for lower dry matter intake as % body weight in YC cows than CON. Cows fed YC had greater overall ruminal pH and greater total VFA (mM) at 60 d of treatment period. There was a contrasting greater molar proportion of isovalerate and lower acetate proportion in YC-fed cows compared with CON cows. Although the ruminal abundance of specific fiber-digesting bacteria, including Eubacterium ruminantium and Ruminococcus flavefaciens, was increased in YC cows, others such as Fibrobacter succinogenes were decreased. The abundance of amylolytic bacteria such as Ruminobacter amylophilus and Succinimonas amylolytica were decreased in YC cows than CON. Our results indicate that the yeast culture supplementation seems to promote some specific fiber-digesting bacteria while decreasing amylolytic bacteria, which might have partially promoted more neutral rumen pH, greater total VFA, and isovalerate.

Effects of peripartal yeast culture supplementation on lactation performance, blood biomarkers, rumen fermentation, and rumen bacteria species in dairy cows.

Feeding yeast culture fermentation products has been associated with improved feed intake and milk yield in transition dairy cows. These improvements in performance have been further described in terms of rumen characteristics, metabolic profile, and immune response. The objective of this study was to evaluate the effects of a commercial yeast culture product (YC; Culture Classic HD, Phibro Animal Health) on performance, blood biomarkers, rumen fermentation, and rumen bacterial population in dairy cows from -30 to 50 d in milk (DIM). Forty Holstein dairy cows were enrolled in a randomized complete block design from -30 to 50 DIM and blocked according to expected calving day, parity, previous milk yield, and genetic merit. At -30 DIM, cows were assigned to either a basal diet plus 114 g/d of ground corn (control; n = 20) or a basal diet plus 100 g/d of ground corn and 14 g/d of YC (n = 20), fed as a top-dress. Cows received the same close-up diet from 30 d prepartum until calving [1.39 Mcal/kg of dry matter (DM) and 12.3% crude protein (CP)] and lactation diet from calving to 50 DIM (1.60 Mcal/kg of DM and 15.6% CP). Blood samples and rumen fluid were collected at various time points from -30 to 50 d relative to calving. Cows fed YC compared with control showed a trend for increased energy-corrected milk (+3.2 kg/d). Lower somatic cell counts were observed in YC cows than in control. We detected a treatment × time interaction in nonesterified fatty acids (NEFA) that could be attributed to a trend for greater NEFA in YC cows than control at 7 DIM, followed by lower NEFA in YC cows than control at 14 and 30 DIM. In the rumen, YC contributed to mild changes in rumen fermentation, mainly increasing postpartal valerate while decreasing prepartal isovalerate. This was accompanied by alterations in rumen microbiota, including a greater abundance of cellulolytic (Fibrobacter succinogenes) and lactate-utilizing bacteria (Megasphaera elsdenii). These results describe the potential benefits of supplementing yeast culture during the late pregnancy through early lactation, at least in terms of rumen environment and performance.

In Vivo Competitions between Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminoccus albus in a Gnotobiotic Sheep Model Revealed by Multi-Omic Analyses.

Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens are the three predominant cellulolytic bacterial species found in the rumen. In vitro studies have shown that these species compete for adherence to, and growth upon, cellulosic biomass. Yet their molecular interactions in vivo have not heretofore been examined. Gnotobiotically raised lambs harboring a 17-h-old immature microbiota devoid of culturable cellulolytic bacteria and methanogens were inoculated first with F. succinogenes S85 and Methanobrevibacter sp. strain 87.7, and 5 months later, the lambs were inoculated with R. albus 8 and R. flavefaciens FD-1. Longitudinal samples were collected and profiled for population dynamics, gene expression, fibrolytic enzyme activity, in sacco fibrolysis, and metabolite profiling. Quantitative PCR, metagenome and metatranscriptome data show that F. succinogenes establishes at high levels initially but is gradually outcompeted following the introduction of the ruminococci. This shift resulted in an increase in carboxymethyl cellulase (CMCase) and xylanase activities but not in greater fibrolysis, suggesting that F. succinogenes and ruminococci deploy different but equally effective means to degrade plant cell walls. Expression profiles showed that F. succinogenes relied upon outer membrane vesicles and a diverse repertoire of CAZymes, while R. albus and R. flavefaciens preferred type IV pili and either CBM37-harboring or cellulosomal carbohydrate-active enzymes (CAZymes), respectively. The changes in cellulolytics also affected the rumen metabolome, including an increase in acetate and butyrate at the expense of propionate. In conclusion, this study provides the first demonstration of in vivo competition between the three predominant cellulolytic bacteria and provides insight on the influence of these ecological interactions on rumen fibrolytic function and metabolomic response.IMPORTANCE Ruminant animals, including cattle and sheep, depend on their rumen microbiota to digest plant biomass and convert it into absorbable energy. Considering that the extent of meat and milk production depends on the efficiency of the microbiota to deconstruct plant cell walls, the functionality of predominant rumen cellulolytic bacteria, Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens, has been extensively studied in vitro to obtain a better knowledge of how they operate to hydrolyze polysaccharides and ultimately find ways to enhance animal production. This study provides the first evidence of in vivo competitions between F. succinogenes and the two Ruminococcus species. It shows that a simple disequilibrium within the cellulolytic community has repercussions on the rumen metabolome and fermentation end products. This finding will have to be considered in the future when determining strategies aiming at directing rumen fermentations for animal production.

Association of ionophores, yeast, and bacterial probiotics alters the abundance of ruminal microbial species of pasture intensively finished beef cattle.

The effect of the association of non-protein nitrogen, yeast, and bacterial probiotics on the ruminal microbiome of beef cattle intensively finished on pasture was evaluated. The experiment was carried out in a completely randomized design with five treatments and four replications. The treatments consisted of a group of animals kept on pasture that received low consumption supplementation (LS) and four groups that received for 98 days, 17.5 g concentrate kg-1 body weight. The supplements were composed of the association of additives: urea (U), slow-release non-protein nitrogen (U+SRN), yeast (Saccharomyces cerevisiae; U+SRN+Y), and bacterial probiotics (live strains of bacteria; U+SRN+Y+BP). All supplements also contained salinomycin and virginiamycin. After slaughtering the animals, samples of ruminal content were collected to quantify groups of fibrolytic bacteria (Ruminococcus albus and Fibrobacter succinogenes), non-fibrolytic (Prevotella ruminicola, Selenomonas ruminantium, and Streptococcus bovis), Archaea, and ciliate protozoa, using the qPCR technique. The abundance of F. succinogenes was the same for the LS animals and those that received the supplement U+SRN+Y (1.42×108 copies mL-1) but higher than the other treatments. Supplementation reduced by 90% the abundance of S. bovis compared to the LS. The inclusion of yeast increased the abundance of fibrolytic bacteria by 2.2-fold. For animals that received the supplement U+SRN+Y+BP and the LS, there was no difference for non-fibrolytic bacteria (3.07×109 copies mL-1). The use of yeasts and sources of non-protein nitrogen in high-concentrate diets for beef cattle stimulates the growth of fibrolytic bacteria, which can contribute to the reduction of digestive disorders and metabolic diseases in animals that receive diets with high concentrate in pasture intensive termination systems.

The Horse Gut Microbiome Responds in a Highly Individualized Manner to Forage Lignification.

Although contributions of the equine gut microbiome to forage utilization are well recognized, the impact of alfalfa (Medicago sativa L.) lignification on the equine gut microbiome remains unknown; thus, we characterized microbial communities in the equine gut when feeding reduced lignin (RL) and conventional (CON) alfalfa hays to adult stock-type horses. Dietary treatments were fed to six horses in a crossover study. Experimental periods consisted of a 9-day dietary adaptation phase followed by a 5-day total fecal collection phase, during which horses were housed in individual box stalls and manure was removed on a continuous 24-hour basis. At 12-hour intervals, manure was mixed, frozen, and processed for V4, 16S rRNA amplicon MiSeq sequencing. Reduced lignin alfalfa did not shift microbiome composition equally across all horses; however, each subject's microbiome responded to hay lignin content in an individualized manner, mostly, in terms of beta diversity. Amplicon sequence variants affiliated to Akkermansia, Fibrobacter succinogenes, Treponema, and Paludibacter fluctuated significantly when RL alfalfa was fed, with abundance patterns unique to each horse. Horse-specific associations between individual gut microbiome traits and characteristics of the digested CON or RL alfalfa were also observed, mainly in regards to dry matter digestibility and mean fecal particle size. These results indicate that the horse gut microbiome responds in an individualized manner to changes in the amount of acid detergent lignin in alfalfa hay, potentially impacting several feed digestibility characteristics. The implications of these horse-specific responses to hay lignification, for metabolic health and performance, remain to be elucidated.

Modulation of rumen fermentation and microbial community through increasing dietary cation-anion difference in Chinese Holstein dairy cows under heat stress conditions.

AIMS: The effect of increasing dietary cation-anion difference (DCAD) on rumen fermentation and ruminal microbial community in dairy cows under heat stress (HS) conditions were evaluated. METHODS AND RESULTS: This study was performed as a two-period cross-over design during the summer season, with eight lactating dairy cows randomly distributed to either a control DCAD diet (CON: 33·5 mEq/100 g DM) or high DCAD diet (HDCAD: 50·8 mEq/100 g DM). Throughout the present study, the temperature and humidity index (THI; 80·2 ± 4·29) was generally elevated above the threshold (THI = 72) that is reported to cause HS in lactating dairy cows. Rumen liquid samples were collected on 15 and 21 d during each 21 d-period. The absolute concentration of ruminal total volatile fatty acid (TVFA) in HDCAD treatment was significantly (P < 0·05) higher than those in the control, whilst the ruminal pH, NH3 -N, and VFA molar percentages were unaffected through increasing DCAD. Furthermore, the copy numbers of the cellulolytic bacteria Ruminococcus albus and Ruminococcus flavefaciens in rumen fluid significantly (P < 0·05) rose along with the increment of DCAD. Although the Alpha diversity indexes and the bacterial microbiota structure were unaffected, increasing DCAD significantly (P < 0·05) enriched the phylum Fibrobacteres and genus Fibrobacter in the microflora of rumen fluid, whilst the genera Flexilinea and Dubosiella were the most differentially abundant taxa in the control. CONCLUSIONS: Increasing DCAD under HS conditions resulted in a greater concentration of total VFA without affecting rumen bacteria diversity or structure, although the enrichment of some cellulolytic/hemicellulolytic bacteria was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides information on the modulation of rumen fermentation and microbial community through the increment of DCAD in Holstein dairy cows under HS conditions.

Bacterial community analysis on the different mucosal immune inductive sites of gastrointestinal tract in Bactrian camels.

The microbial communities colonize the mucosal immune inductive sites could be captured by hosts, which could initiate the mucosal immune responses. The aggregated lymphoid nodule area (ALNA) and the ileal Payer's patches (PPs) in Bactrian camels are both the mucosal immune inductive sites of the gastrointestinal tract. Here, the bacteria community associated with the ALNA and ileal PPs were analyzed using of 16S rDNA-Illumina Miseq sequencing. The mutual dominant bacterial phyla at the two sites were the Bacteroidetes, Firmicutes, Verrucomicrobia and Proteobacteria, and the mutual dominant genus in both sits was Prevotella. The abundances of the Fibrobacter, Campylobacter and RFP12 were all higher in ALNA than in ileal PPs. While, the abundances of the 5-7N15, Clostridium, and Escherichia were all higher in ileal PPs than in ALNA. The results suggested that the host's intestinal microenvironment is selective for the symbiotic bacteria colonizing the corresponding sites, on the contrary, the symbiotic bacteria could impact on the physiological functions of this local site. In ALNA and ileal PPs of Bactrian camel, the bacteria which colonized different immune inductive sites have the potential to stimulate different immune responses, which is the result of the mutual selection and adaptation between microbial communities and their host.

Effects of sodium selenite and coated sodium selenite on lactation performance, total tract nutrient digestion and rumen fermentation in Holstein dairy cows.

Se can enhance lactation performance by improving nutrient utilization and antioxidant status. However, sodium selenite (SS) can be reduced to non-absorbable elemental Se in the rumen, thereby reducing the intestinal availability of Se. The study investigated the impacts of SS and coated SS (CSS) supplementation on lactation performance, nutrient digestibility, ruminal fermentation and microbiota in dairy cows. Sixty multiparous Holstein dairy cows were blocked by parity, daily milk yield and days in milk and randomly assigned to five treatments: control, SS addition (0.3 mg Se/kg DM as SS addition) or CSS addition (0.1, 0.2 and 0.3 mg Se/kg DM as CSS addition for low CSS (LCSS), medium CSS (MCSS) and high CSS (HCSS), respectively). Experiment period was 110 days with 20 days of adaptation and 90 days of sample collection. Dry matter intake was higher for MCSS and HCSS compared with control. Yields of milk, milk fat and milk protein and feed efficiency were higher for MCSS and HCSS than for control, SS and LCSS. Digestibility of DM and organic matter was highest for CSS addition, followed by SS addition and then control. Digestibility of CP was higher for MCSS and HCSS than for control, SS and LCSS. Higher digestibility of ether extract, NDF and ADF was observed for SS or CSS addition. Ruminal pH decreased with dietary Se addition. Acetate to propionate ratio and ammonia N were lower, and total volatile fatty acids (VFAs) concentration was greater for SS, MCSS and HCSS than control. Ruminal H ion concentration was highest for MCSS and HCSS and lowest for control. Activities of cellobiase, carboxymethyl-cellulase, xylanase and protease and copies of total bacteria, fungi, Ruminococcus flavefaciens, Fibrobacter succinogenes and Ruminococcus amylophilus increased with SS or CSS addition. Activity of α-amylase, copies of protozoa, Ruminococcus albus and Butyrivibrio fibrisolvens and serum glucose, total protein, albumin and glutathione peroxidase were higher for SS, MCSS and HCSS than for control and LCSS. Dietary SS or CSS supplementation elevated blood Se concentration and total antioxidant capacity activity. The data implied that milk yield was elevated due to the increase in total tract nutrient digestibility, total VFA concentration and microorganism population with 0.2 or 0.3 mg Se/kg DM from CSS supplementation in dairy cows. Compared with SS, HCSS addition was more efficient in promoting lactation performance of dairy cows.

Effects of different sources of nitrogen on performance, relative population of rumen microorganisms, ruminal fermentation and blood parameters in male feedlotting lambs.

Slow-release urea (SRU) can substitute dietary protein sources in the diet of feedlotting ruminant species . However, different SRU structures show varying results of productive performance. This study was conducted to investigate the effect of different sources of nitrogen on performance, blood parameter, ruminal fermentation and relative population of rumen microorganisms in male Mehraban lambs. Thirty-five male lambs with an average initial BW of 34.7 ± 1.8 kg were assigned randomly to five treatments. Diets consisted of concentrate mixture and mineral and vitamin supplements plus (1) alfalfa and soybean meal, (2) wheat straw and soybean meal, (3) wheat straw and urea, (4) wheat straw and Optigen® (a commercial SRU supplement) and (5) wheat straw and SRU produced in the laboratory. No statistical difference was observed in animal performance and DM intake among treatments. The mean value of ruminal pH and ammonia was higher (P < 0.05) for the SRU diet compared with WU diet. The difference in pH is likely to be due to the higher ammonia level as VFAs concentrations were unchanged. The level of blood urea nitrogen (BUN) was different among treatments (P = 0.065). The highest concentration of BUN was recorded in Optigen diet (183.1 mg/l), whereas the lowest value was recorded in wheat straw-soybean meal diet (147 mg/l). The amount of albumin and total protein was not affected by the treatments. The relative population of total protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminococcus albus in the SRU treatment was higher (P < 0.01) than that in urea treatment at 3 h post-feeding. During the period of lack of high-quality forage and in order to reduce dietary costs, low-quality forage with urea sources can be used in the diet. Results of microbial populations revealed that SRU can be used as a nitrogen source which can sustainably provide nitrogen for rumen microorganism without negative effects on the performance of feedlotting lambs.

Supplementation of live yeast based feed additive in early life promotes rumen microbial colonization and fibrolytic potential in lambs.

Rumen microbiota is of paramount importance for ruminant digestion efficiency as the microbial fermentations supply the host animal with essential sources of energy and nitrogen. Early separation of newborns from the dam and distribution of artificial milk (Artificial Milking System or AMS) could impair rumen microbial colonization, which would not only affect rumen function but also have possible negative effects on hindgut homeostasis, and impact animal health and performance. In this study, we monitored microbial communities in the rumen and the feces of 16 lambs separated from their dams from 12 h of age and artificially fed with milk replacer and starter feed from d8, in absence or presence of a combination of the live yeast Saccharomyces cerevisiae CNCM I-1077 and selected yeast metabolites. Microbial groups and targeted bacterial species were quantified by qPCR and microbial diversity and composition were assessed by 16S rDNA amplicon sequencing in samples collected from birth to 2 months of age. The fibrolytic potential of the rumen microbiota was analyzed with a DNA microarray targeting genes coding for 8 glycoside hydrolase (GH) families. In Control lambs, poor establishment of fibrolytic communities was observed. Microbial composition shifted as the lambs aged. The live yeast supplement induced significant changes in relative abundances of a few bacterial OTUs across time in the rumen samples, among which some involved in crucial rumen function, and favored establishment of Trichostomatia and Neocallimastigaceae eukaryotic families. The supplemented lambs also harbored greater abundances in Fibrobacter succinogenes after weaning. Microarray data indicated that key cellulase and hemicellulase encoding-genes were present from early age in the rumen and that in the Supplemented lambs, a greater proportion of hemicellulase genes was present. Moreover, a higher proportion of GH genes from ciliate protozoa and fungi was found in the rumen of those animals. This yeast combination improved microbial colonization in the maturing rumen, with a potentially more specialized ecosystem towards efficient fiber degradation, which suggests a possible positive impact on lamb gut development and digestive efficiency.

Deciphering the unique cellulose degradation mechanism of the ruminal bacterium Fibrobacter succinogenes S85.

Fibrobacter succinogenes S85, isolated from the rumen of herbivores, is capable of robust lignocellulose degradation. However, the mechanism by which it achieves this is not fully elucidated. In this study, we have undertaken the most comprehensive quantitative proteomic analysis, to date, of the changes in the cell envelope protein profile of F. succinogenes S85 in response to growth on cellulose. Our results indicate that the cell envelope proteome undergoes extensive rearrangements to accommodate the cellulolytic degradation machinery, as well as associated proteins involved in adhesion to cellulose and transport and metabolism of cellulolytic products. Molecular features of the lignocellulolytic enzymes suggest that the Type IX secretion system is involved in the translocation of these enzymes to the cell envelope. Finally, we demonstrate, for the first time, that cyclic-di-GMP may play a role in mediating catabolite repression, thereby facilitating the expression of proteins involved in the adhesion to lignocellulose and subsequent lignocellulose degradation and utilisation. Understanding the fundamental aspects of lignocellulose degradation in F. succinogenes will aid the development of advanced lignocellulosic biofuels.

Corn oil supplementation enhances hydrogen use for biohydrogenation, inhibits methanogenesis, and alters fermentation pathways and the microbial community in the rumen of goats.

Enteric methane (CH4) emissions are not only an important source of greenhouse gases but also a loss of dietary energy in livestock. Corn oil (CO) is rich in unsaturated fatty acid with >50% PUFA, which may enhance ruminal biohydrogenation of unsaturated fatty acids, leading to changes in ruminal H2 metabolism and methanogenesis. The objective of this study was to investigate the effect of CO supplementation of a diet on CH4 emissions, nutrient digestibility, ruminal dissolved gases, fermentation, and microbiota in goats. Six female goats were used in a crossover design with two dietary treatments, which included control and CO supplementation (30 g/kg DM basis). CO supplementation did not alter total-tract organic matter digestibility or populations of predominant ruminal fibrolytic microorganisms (protozoa, fungi, Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes), but reduced enteric CH4 emissions (g/kg DMI, -15.1%, P = 0.003). CO supplementation decreased ruminal dissolved hydrogen (dH2, P < 0.001) and dissolved CH4 (P < 0.001) concentrations, proportions of total unsaturated fatty acids (P < 0.001) and propionate (P = 0.015), and increased proportions of total SFAs (P < 0.001) and acetate (P < 0.001), and acetate to propionate ratio (P = 0.038) in rumen fluid. CO supplementation decreased relative abundance of family Bacteroidales_BS11_gut_group (P = 0.032), increased relative abundance of family Rikenellaceae (P = 0.021) and Lachnospiraceae (P = 0.025), and tended to increase relative abundance of genus Butyrivibrio_2 (P = 0.06). Relative abundance (P = 0.09) and 16S rRNA gene copies (P = 0.043) of order Methanomicrobiales, and relative abundance of genus Methanomicrobium (P = 0.09) also decreased with CO supplementation, but relative abundance (P = 0.012) and 16S rRNA gene copies (P = 0.08) of genus Methanobrevibacter increased. In summary, CO supplementation increased rumen biohydrogenatation by facilitating growth of biohydrogenating bacteria of family Lachnospiraceae and genus Butyrivibrio_2 and may have enhanced reductive acetogenesis by facilitating growth of family Lachnospiraceae. In conclusion, dietary supplementation of CO led to a shift of fermentation pathways that enhanced acetate production and decreased rumen dH2 concentration and CH4 emissions.

The Phylogenomic Diversity of Herbivore-Associated Fibrobacter spp. Is Correlated to Lignocellulose-Degrading Potential.

Members of the genus Fibrobacter are cellulose-degrading bacteria and common constituents of the gastrointestinal microbiota of herbivores. Although considerable phylogenetic diversity is observed among members of this group, few functional differences explaining the distinct ecological distributions of specific phylotypes have been described. In this study, we sequenced and performed a comparative analysis of whole genomes from 38 novel Fibrobacter strains against the type strains for the two formally described Fibrobacter species F. succinogenes strain S85 and F. intestinalis strain NR9. Significant differences in the number of genes encoding carbohydrate-active enzyme families involved in plant cell wall polysaccharide degradation were observed among Fibrobacter phylotypes. F. succinogenes genomes were consistently enriched in genes encoding carbohydrate-active enzymes compared to those of F. intestinalis strains. Moreover, genomes of F. succinogenes phylotypes that are dominant in the rumen had significantly more genes annotated to major families involved in hemicellulose degradation (e.g., CE6, GH10, and GH43) than did the genomes of F. succinogenes phylotypes typically observed in the lower gut of large hindgut-fermenting herbivores such as horses. Genes encoding a putative urease were also identified in 12 of the Fibrobacter genomes, which were primarily isolated from hindgut-fermenting hosts. Screening for growth on urea as the sole source of nitrogen provided strong evidence that the urease was active in these strains. These results represent the strongest evidence reported to date for specific functional differences contributing to the ecology of Fibrobacter spp. in the herbivore gut.IMPORTANCE The herbivore gut microbiome is incredibly diverse, and a functional understanding of this diversity is needed to more reliably manipulate this community for specific gain, such as increased production in ruminant livestock. Microbial degraders of plant cell wall polysaccharides in the herbivore gut, particularly Fibrobacter spp., are of fundamental importance to their hosts for digestion of a diet consisting primarily of recalcitrant plant fibers. Considerable phylogenetic diversity exists among members of the genus Fibrobacter, but much of this diversity remains cryptic. Here, we used comparative genomics, applied to a diverse collection of recently isolated Fibrobacter strains, to identify a robust association between carbohydrate-active enzyme gene content and the Fibrobacter phylogeny. Our results provide the strongest evidence reported to date for functional differences among Fibrobacter phylotypes associated with either the rumen or the hindgut and emphasize the general significance of carbohydrate-active enzymes in the evolution of fiber-degrading bacteria.

Quantitative qPCR Analysis of Ruminal Microorganisms in Beef Cattle Grazing in Pastures in the Rainy Season and Supplemented with Different Protein Levels.

We tested the hypothesis that supplementation with three protein levels improves fermentation parameters without changing the rumen microbial population of grazing beef cattle in the rainy season. Four rumen-cannulated Nellore bulls (432 ± 21 kg of body weight) were used in a 4 × 4 Latin square design with four supplements and four experimental periods of 21 days each. The treatments were mineral supplement (ad libitum) and supplements with low, medium (MPS), and high protein supplement (HPS), supplying 106, 408, and 601 g/day of CP, respectively. The abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and domain bacteria primers. Supplemented animals showed lower (P < 0.05) proportions of Ruminococcus flavefaciens and greater (P < 0.05) proportions of Ruminococcus albus and Butyrivibrio fibrisolvens than animals that received only the mineral supplement. The HPS supplement resulted in higher (P < 0.05) proportions of Fibrobacter succinogenes, R. flavefaciens, and B. fibrisolvens and lower (P < 0.05) proportions of R. albus than the MPS supplement. Based on our results, high protein supplementation improves the ruminal conditions and facilitates the growth of cellulolytic bacteria in the rumen of bulls on pastures during the rainy season.

Biochanin A improves fibre fermentation by cellulolytic bacteria.

AIMS: The objective was to determine the effect of the isoflavone biochanin A (BCA) on rumen cellulolytic bacteria and consequent fermentative activity. METHODS AND RESULTS: When bovine microbial rumen cell suspensions (n = 3) were incubated (24 h, 39°C) with ground hay, cellulolytic bacteria proliferated, short-chain fatty acids were produced and pH declined. BCA (30 µg ml-1 ) had no effect on the number of cellulolytic bacteria or pH, but increased acetate, propionate and total SCFA production. Addition of BCA improved total digestibility when cell suspensions (n = 3) were incubated (48 h, 39°C) with ground hay, Avicel, or filter paper. Fibrobacter succinogenes S85, Ruminococcus flavefaciens 8 and Ruminococcus albus 8 were directly inhibited by BCA. Synergistic antimicrobial activity was observed with BCA and heat killed cultures of cellulolytic bacteria, but the effects were species dependent. CONCLUSIONS: These results indicate that BCA improves fibre degradation by influencing cellulolytic bacteria competition and guild composition. SIGNIFICANCE AND IMPACT OF THE STUDY: BCA could serve as a feed additive to improve cellulosis when cattle are consuming high-fibre diets. Future research is needed to evaluate the effect of BCA on fibre degradation and utilization in vivo.

Benefits of procyanidins on gut microbiota in Bama minipigs and implications in replacing antibiotics

Several studies have reported the effect of absorption of procyanidins and their contribution to the small intestine. However, differences between dietary interventions of procyanidins and interventions via antibiotic feeding in pigs are rarely reported. Following 16S rRNA gene Illumina MiSeq sequencing, we observed that both procyanidin administration for 2 months (procyanidin-1 group) and continuous antibiotic feeding for 1 month followed by procyanidin for 1 month (procyanidin-2 group) increased the number of operational taxonomic units, as well as the Chao 1 and ACE indices, compared to those in pigs undergoing antibiotic administration for 2 months (antibiotic group). The genera Fibrobacter and Spirochaete were more abundant in the antibiotic group than in the procyanidin-1 and procyanidin-2 groups. Principal component analysis revealed clear separations among the three groups. Additionally, using the online Molecular Ecological Network Analyses pipeline, three co-occurrence networks were constructed; Lactobacillus was in a co-occurrence relationship with Trichococcus and Desulfovibrio and a co-exclusion relationship with Bacillus and Spharerochaeta. Furthermore, metabolic function analysis by phylogenetic investigation of communities by reconstruction of unobserved states demonstrated modulation of pathways involved in the metabolism of carbohydrates, amino acids, energy, and nucleotides. These data suggest that procyanidin influences the gut microbiota and the intestinal metabolic function to produce beneficial effects on metabolic homeostasis.

Changes in fibre-adherent and fluid-associated microbial communities and fermentation profiles in the rumen of cattle fed diets differing in hay quality and concentrate amount.

The rumen microbiota enable important metabolic functions to the host cattle. Feeding of starch-rich concentrate feedstuffs to cattle has been demonstrated to increase the risk of metabolic disorders and to significantly alter the rumen microbiome. Thus, alternative feeding strategies like the use of high-quality hay, rich in sugars, as an alternative energy source need to be explored. The aim of this study was to investigate changes in rumen microbial abundances in the liquid and solid-associated fraction of cattle fed two hay qualities differing in sugar content with graded amounts of starchy concentrate feeds using Illumina MiSeq sequencing and quantitative polymerase chain reaction. Operational taxonomic units clustered separately between the liquid and the solid-associated fraction. Phyla in the liquid fraction were identified as mainly Firmicutes, Proteobacteria and Bacteroidetes, whereas main phyla of the fibre-associated fraction were Bacteroidetes, Fibrobacteres and Firmicutes. Significant alterations in the rumen bacterial communities at all taxonomic levels as a result of changing the hay quality and concentrate proportions were observed. Several intermicrobial correlations were found. Genera Ruminobacter and Fibrobacter were significantly suppressed by feeding sugar-rich hay, whereas others such as Selenomonas and Prevotella proliferated. This study extends the knowledge about diet-induced changes in ruminal microbiome of cattle.