Ruminococcus Albus Research Articles

Effect of a Blend of Essential Oils on Buffalo Rumen Microbial and Enzyme Profiles and In Vitro Feed Fermentation

Three rumen-fistulated adult Murrah buffaloes (BW 426±45 kg) were arranged in a 3×3 LSD. The treatments involved supplementation of a blend (BEO-CL) of clove bud oleoresin and lemongrass oil (in 1:1 ratio) at the rate of 0, 0.75 and 1.5% of BW. After 20d of feeding, the rumen liquor and rumen contents were sampled before (0h) and 4h after feeding and analyzed for various parameters. There was neither any effect of feed additive nor sampling time on the concentration of VFAs, NH3-N, lactic acid and activities of select enzymes (carboxymethylcellulase, avicelase, xylanase, amylase, β-glucosidase, α-glucosidase and acetyl esterase). The population of rumen bacteria, fungi, ciliate protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus and methanogens were not affected by BEO-CL supplementation. In vitro gas production test to evaluate various feedstuffs namely hays (berseem, oat and maize); dry roughages (wheat straw, paddy straw and sugarcane bagasse) complete feeds with 20:80, 35:65 and 50:50 concentrate: roughage ratio) as substrates was conducted using rumen liquor of these buffaloes as inocula. There was an increase in gas production with rumen liquor of BEO-CL supplemented buffaloes irrespective of substrate. Methane production was decreased with 0.75% BEO-CL supplemented inoculum but not with paddy straw and sugarcane bagasse. The in vitro true digestibility (IVTD) of feed was increased with BEO-CL inoculum only with complete diet having 20:80 concentrate:roughage ratio as substrate. It is concluded that BEO-CL at the level of 0.75% of BW did not affect rumen characteristics and major microbial populations but inhibited in vitro methane production; hence, it can be further explored for its use as a feed additive to mitigate methane emission in buffaloes.

Synergism of Cattle and Bison Inoculum on Ruminal Fermentation and Select Bacterial Communities in an Artificial Rumen (Rusitec) Fed a Barley Straw Based Diet.

This study evaluated the effect of increasing the proportion of bison relative to cattle inoculum on fermentation and microbial populations within an artificial rumen (Rusitec). The experiment was a completely randomized design with a factorial treatment structure (proportion cattle:bison inoculum; 0:100, 33:67, 67:33, and 100:0) replicated in two Rusitec apparatuses (n = 8 fermenters). The experiment was 15 d with 8 d of adaptation and 7 d of sampling. Fermenters were fed a diet of 70:30 barley straw:concentrate (DM basis). True digestibility of DM was determined after 48 h of incubation from d 13 to 15, and daily ammonia (NH3) and volatile fatty acid (VFA) production were measured on d 9–12. Protozoa counts were determined at d 9, 11, 13, and 15 and particle-associated bacteria (PAB) from d 13 to 15. Select bacterial populations in the PAB were measured using RT-qPCR. Fermenter was considered the experimental unit and day of sampling as a repeated measure. Increasing the proportion of bison inoculum resulted in a quadratic effect (P < 0.05) on straw, concentrate and total true DM disappearance and on straw and total neutral detergent fiber (aNDF) disappearance, with greater disappearances observed with mixed inoculum. There were no effect of source or proportion of inoculum on ADF disappearance (P > 0.05). Increasing bison inoculum linearly increased (P < 0.05) concentrate aNDF disappearance, total and concentrate N disappearance as well as total daily VFA and acetate production. A positive quadratic response (P < 0.05) was observed for daily NH3-N, propionate, butyrate, valerate, isovalerate and isobutyrate production, as well as the acetate:propionate ratio. Increasing the proportion of bison inoculum linearly increased (P < 0.05) total protozoa numbers. No effects were observed on pH, total gas and methane production, microbial N synthesis, or copies of 16S rRNA associated with total bacteria, Selenomonas ruminantium or Prevotella bryantii. Increasing bison inoculum had a quadratic effect (P < 0.05) on Fibrobacter succinogenes, and tended to linearly (P < 0.10) increase Ruminococcus flavefaciens and decrease (P < 0.05) Ruminococcus albus copy numbers. In conclusion, bison inoculum increased the degradation of feed protein and fiber. A mixture of cattle and bison rumen inoculum acted synergistically, increasing the DM and aNDF disappearance of barley straw.

Fermentation of Ammonia Fiber Expansion Treated and Untreated Barley Straw in a Rumen Simulation Technique Using Rumen Inoculum from Cattle with Slow versus Fast Rate of Fiber Disappearance.

The purpose of this study was to determine the effect of rumen inoculum from heifers with fast vs. slow rate of in situ fiber digestion on the fermentation of complex versus easily digested fiber sources in the forms of untreated and Ammonia Fiber Expansion (AFEX) treated barley straw, respectively, using an artificial rumen simulation technique (Rusitec). In situ fiber digestion was measured in a previous study by incubating untreated barley straw in the rumen of 16 heifers fed a diet consisting of 700 g/kg barley straw and 300 g/kg concentrate. The two heifers with fastest rate of digestion (Fast ≥ 4.18% h-1) and the two heifers with the slowest rate of digestion (Slow ≤ 3.17% h-1) were chosen as inoculum donors for this study. Two Rusitec apparatuses each equipped with eight fermenters were used in a completely randomized block design with two blocks (apparatus) and four treatments in a 2 × 2 factorial arrangement of treatments (Fast or Slow rumen inoculum and untreated or AFEX treated straw). Fast rumen inoculum and AFEX straw both increased (P < 0.05) disappearance of dry matter (DMD), organic matter, true DMD, neutral detergent fiber, acid detergent fiber, and nitrogen (N) with an interactive effect between the two (P < 0.05). Fast rumen inoculum increased (P > 0.05) methane production per gram of digested material for both untreated and AFEX straw, and reduced (interaction, P < 0.05) acetate: propionate ratio for untreated straw. Greater relative populations of Ruminococcus albus (P < 0.05) and increased microbial N production (P = 0.045) were observed in Fast rumen inoculum. AFEX straw in Fast inoculum had greater total bacterial populations than Slow, but for untreated straw this result was reversed (interaction, P = 0.013). These findings indicate that differences in microbial populations in rumen fluid contribute to differences in the capacity of rumen inoculum to digest fiber.

Milk fatty acid profile in cows fed red clover- or alfalfa-silage based diets differing in rumen-degradable protein supply

Polyphenol oxidase in red clover (RC) silage reduces lipolysis and consequently protects its constituent fatty acids (FA) against biohydrogenation by ruminal microorganisms. Fatty acid biohydrogenation could be further inhibited by reducing the nitrogen (N) supply to ruminal bacteria. To compare the effects of RC and alfalfa (AL) silage fed in diets differing in rumen-degradable protein supply on the transfer efficiency of polyunsaturated FA from diet to milk, and on the resulting FA profile of milk fat, 8 multiparous Holstein dairy cows (72±17days in milk) were used in a replicated 4×4 Latin square design (21-day periods including 14days of adaptation). Four treatments were compared in a 2×2 factorial arrangement with AL or RC fed in diets formulated to provide 100% (RDP‐100) or 85% (RDP‐85) of calculated rumen-degradable protein requirements. Rumen-degradable protein concentrations were adjusted by varying the supplies of ground vs. steam-flaked corn grains, and untreated vs. heat-treated soybean meals. Feeding RC, as compared with AL diets, decreased ruminal ammonia-N concentration as well as urea-N in plasma and milk. Intakes of c9c12 18:2 and c9c12c15 18:3 were lower for cows fed RC, but their secretion in milk and their apparent transfer efficiency from diet were greater as compared to cows fed AL. As a result, feeding RC increased the concentrations of c9c12 18:2 and c9c12c15 18:3, but decreased the proportions of t11 18:1, c9t11 18:2, and t11c15 18:2 in milk fat. These results are consistent with the lower effective ruminal disappearance of c9c12 18:2 and c9c12c15 18:3 in RC as determined by a 96h in sacco incubation, and the lower abundance of ruminal bacteria capable of hydrolyzing dietary lipids and hydrogenating polyunsaturated FA (Ruminococcus albus and Ruminococcus flavefaciens) in the rumen content. The apparent transfer efficiency of c9c12 18:2 from diet to milk was further increased by feeding RDP‐85 as compared with RDP‐100 diet. Feeding RC decreased concentrations of linear odd-chain FA (11:0, 13:0, 15:0, 17:0, and c9 17:1) and increased proportions of branched-chain FA (iso13:0, iso14:0, iso15:0, iso16:0, iso17:0, anteiso15:0, and anteiso17:0) in milk fat as compared with AL. In conclusion, as compared with AL, cows fed RC produced milk with greater concentrations of major forage FA (c9c12 18:2 and c9c12c15 18:3) and lower proportions of intermediates in ruminal biohydrogenation of these FA. Variations in milk fat concentrations of odd- and branched-chain FA, which are synthesized in the rumen by various microbial populations, may be reflecting the effects of forage source on ruminal fermentation.

Illumina Miseq platform analysis caecum bacterial communities of rex rabbits fed with different antibiotics.

Antibiotics have been widely used for the prevention and the treatment of diseases to humans and animals, and they have fed additives for agricultural animals to promote growth. However, there is a growing concern over the practice due to its side effects on intestinal microbial communities which plays a vital role in animals’ health. To investigate the effect of antibiotics on the bacterial population of the caecum in rex rabbits, 80 rex rabbits were randomly divided into four groups: control group (B, basal diet), chlortetracycline group (C, 50 mg/kg), colistin sulfate group (S, 20 mg/kg) and zinc bacitracin group (Z, 40 mg/kg). Caecum microbial communities of rex rabbits from the four groups were analyzed through Illumina Miseq platform after being fed 28 days. The results showed that most obtained sequences belongs to Firmicutes followed by Bacteroidetes, and the ratio of Bacteroidetes/Firmicutes in C group (42.31 %) was higher than that in Z group (21.84 %). Zinc bacitracin supplementation caused a significant decreased of the Proteobacteria phylum and Lactobacillus spp. (P < 0.05), while the Lactobacillus spp. significantly increased in S group (P < 0.05). In addition, Ruminococcus spp., especially Ruminococcus albus were the predominant bacterial species found in both S and Z groups. The proportion of Coprococcus spp. significantly increased in Z group (P < 0.05). These findings suggested that the antibiotics used may cause significant changes in the caecum microbiota of rex rabbits, and we also found C group had a similarity caecum bacteria structure with B group which was probably due to the high levels of chlortetracycline resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0273-1) contains supplementary material, which is available to authorized users.

Effects of Dietary Forage and Calf Starter Diet on Ruminal pH and Bacteria in Holstein Calves during Weaning Transition.

We investigated the relationship between ruminal pH and bacteria in calves fed calf starter with and without forage during weaning transition. First, 16 Holstein bull calves were obtained from dairy farms and equipped with rumen cannulas by cannulation surgery. Then, calves (73.5 ± 4.2 kg; mean ± SE) were assigned to groups fed calf starter either with forage (HAY, n = 8) or without forage (CON, n = 8), and all calves were weaned at 8 weeks of age. Ruminal pH was measured continuously, and rumen fluid samples were collected at 7, 8, 9, and 11 weeks of age, namely −1, 0, 1, and 3 weeks after weaning, respectively, to assess volatile fatty acid concentrations and bacterial DNA. The 24-h mean ruminal pH was significantly (P < 0.05) different between the two groups. Diurnal changes in the 1-h mean ruminal pH were observed throughout the study in the HAY group; however, they were not observed at 0 and 1 weeks after weaning in the CON group. Moreover, the HAY group had significantly (P < 0.05) higher proportions of acetate and butyrate and lower proportion of propionate, and significantly (P < 0.05) lower ruminal acetate-to-propionate ratios were observed in the CON group. The ruminal bacterial diversity indices decreased after −1 week in both groups and increased at 0 and 1 weeks after weaning in the HAY and CON groups, respectively. From the 454 pyrosequencing analysis, significant differences (P < 0.05) were observed in the relative abundance of several phyla (Bacteroidetes, Actinobacteria, and Tenericutes) and one genus (Prevotella) between the two groups. From quantitative real-time PCR analysis, the HAY group had the higher copy numbers of cellulolytic bacteria (Ruminococcus flavefaciens and Ruminococcus albus) compared with the CON group. This study demonstrated that feeding of dietary forage alleviates subacute ruminal acidosis due to diurnal changes in ruminal pH. Furthermore, changes in ruminal pH affect the ruminal bacterial diversity and relative abundance, and these changes might have influenced the establishment of fermentative ruminal functions during weaning transition.

Multiple cellobiohydrolases and cellobiose phosphorylases cooperate in the ruminal bacterium Ruminococcus albus 8 to degrade cellooligosaccharides.

Digestion of plant cell wall polysaccharides is important in energy capture in the gastrointestinal tract of many herbivorous and omnivorous mammals, including humans and ruminants. The members of the genus Ruminococcus are found in both the ruminant and human gastrointestinal tract, where they show versatility in degrading both hemicellulose and cellulose. The available genome sequence of Ruminococcus albus 8, a common inhabitant of the cow rumen, alludes to a bacterium well-endowed with genes that target degradation of various plant cell wall components. The mechanisms by which R. albus 8 employs to degrade these recalcitrant materials are, however, not clearly understood. In this report, we demonstrate that R. albus 8 elaborates multiple cellobiohydrolases with multi-modular architectures that overall enhance the catalytic activity and versatility of the enzymes. Furthermore, our analyses show that two cellobiose phosphorylases encoded by R. albus 8 can function synergistically with a cognate cellobiohydrolase and endoglucanase to completely release, from a cellulosic substrate, glucose which can then be fermented by the bacterium for production of energy and cellular building blocks. We further use transcriptomic analysis to confirm the over-expression of the biochemically characterized enzymes during growth of the bacterium on cellulosic substrates compared to cellobiose.

Effect of dietary interventions on the intestinal microbiota of Mongolian hosts

The gut microbiota of Mongolian hosts has distinctive characteristics due to their meat- and dairy-oriented daily diets and unique genotype. The aim of the present study was to investigate the effect of switching from the typical high protein and fat Mongolian diets to carbohydrate-rich meals composed principally of wheat, rice and naked oats on the host gut microbiota within 3 weeks. Our study took the advantage of the long sequence reads produced by the PacBio single molecule real-time sequencing technology to enable the profiling of subjects’ gut microbiota communities along the diet intervention to the species precision. We found that the bacterial richness and diversity decreased apparently along the diet intervention. During the diet intervention, the gut microbiota composition displayed no significant difference at phylum level (with major phyla of Firmicutes, Bacteroidetes, Tenericutes and Proteobacteria). The relative abundances of some genera such as Bacteroidetes, Faecalibacterium, Roseburia, Alistipes, Streptococcus, and Oscillospira were significantly altered after the diet switching started. Notably, significant changes were also observed in the proportions of the species Bacteroides dorei, Bacteroides fragilis, Bacteroides thetaiotaomicron, Ruminococcus albus, Ruminococcus faecis, Roseburia faecis and Eubacterium ventriosum. These results have demonstrated that diet and host gut microbiota is closely linked.

Rumen microbial community and nitrogen metabolism in goats fed blend of palm oil and canola oil

This study assessed the influence of dietary blend of 20% palm oil and 80% canola oil on the population of rumen microbiota and nitrogen economy in goats. Twenty-four Boer bucks (4–5 months old; initial BW, 20.54 ± 0.474 kg) were randomly allotted to diets containing on a dry matter basis, 0, 4 or 8% oil blend, fed daily for 100 days and slaughtered. The rumen microbiota was examined by quantitative real-time polymerase chain reaction (PCR) using the 16S rRNA gene. The population of total protozoa and methanogens was lower (p < .05) while the population of Fibrobacter succinogenes was greater (p < .05) in goats fed oil blend compared with the control goats. The population of total bacteria was greater (p < .05) in goats fed 8% oil blend compared with those fed other diets. The population of Ruminococcus albus and Ruminococcus flavefaciens was greater (p < .05) in goats fed 4% oil blend compared with those fed other diets. Diet had no effect (p> 0.05) on microbial N yield, microbial protein synthesis, efficiency of microbial protein synthesis and the intake, retention and excretion of N. Dietary supplementation of blend of palm oil and canola oil altered rumen microbial profile but did not affect nitrogen metabolism in goats.

Characterization of a Novel α-l-Arabinofuranosidase from Ruminococcus albus 7 and Rational Design for Its Thermostability.

An α-l-arabinofuranosidase (Abf) encoding gene was obtained via genomic mining from a Ruminococcus albus strain. The specific activity of this GH 51 Abf was 73.3 U/mg at pH 6.0 and 50 °C. The modification of Abf, aimed at improving thermostability, was performed through different strategies. Structure-based rational design using the PoPMuSiC and the Enzyme Thermal Stability System (ETSS) predicted thermal stability of Abf and enhanced the half-life of thermal inactivation (t1/2) at 50 °C for K208W more than 11.1 times versus the wild-type (WT). Sequence-based rational design was also conducted by substituting histidine with lysine at various sites. Among eight mutants, the t1/2 at 50 °C of H337K was prolonged by 5.0-fold, and the specific activity of this mutant was increased to 121.8 U/mg. In addition, the mutant H337K was utilized with some enzymes to extract pectin from apple pomace. The enzymatically produced pectin got less moisture and ash, milder pH, and higher viscosity than its acid-extracted counterpart, indicating that Abf has an application prospect in pectin production.

Effects of dietary supplementation of rumen-protected folic acid on rumen fermentation, degradability and excretion of urinary purine derivatives in growing steers

ABSTRACTThe present experiment was undertaken to determine the effects of dietary addition of rumen-protected folic acid (RPFA) on ruminal fermentation, nutrient degradability, enzyme activity and the relative quantity of ruminal cellulolytic bacteria in growing beef steers. Eight rumen-cannulated Jinnan beef steers averaging 2.5 years of age and 419 ± 1.9 kg body weight were used in a replicated 4 × 4 Latin square design. The four treatments comprised supplementation levels of 0 (Control), 70, 140 and 210 mg RPFA/kg dietary dry matter (DM). On DM basis, the ration consisted of 50% corn silage, 47% concentrate and 3% soybean oil. The DM intake (averaged 8.5 kg/d) was restricted to 95% of ad libitum intake. The intake of DM, crude protein (CP) and net energy for growth was not affected by treatments. In contrast, increasing RPFA supplementation increased average daily gain and the concentration of total volatile fatty acid and reduced ruminal pH linearly. Furthermore, increasing RPFA supplementation enhanced the acetate to propionate ratio and reduced the ruminal ammonia N content linearly. The ruminal effective degradability of neutral detergent fibre from corn silage and CP from concentrate improved linearly and was highest for the highest supplementation levels. The activities of cellobiase, xylanase, pectinase and α-amylase linearly increased, but carboxymethyl-cellulase and protease were not affected by the addition of RPFA. The relative quantities of Butyrivibrio fibrisolvens, Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes increased linearly. With increasing RPFA supplementation levels, the excretion of urinary purine derivatives was also increased linearly. The present results indicated that the supplementation of RPFA improved ruminal fermentation, nutrient degradability, activities of microbial enzymes and the relative quantity of the ruminal cellulolytic bacteria in a dose-dependent manner. According to the conditions of this experiment, the optimum supplementation level of RPFA was 140 mg/kg DM.

Oral administration of Lactobacillus plantarum and Bacillus subtilis on rumen fermentation and the bacterial community in calves.

The objective of this study was to assess the effect of dietary probiotics on rumen fermentation and the bacterial community in dairy calves. Twelve Holstein calves were randomly allocated to three treatments: a basal diet, the basal diet supplemented with Lactobacillus plantarum GF103 (LB) or basal diet supplemented with a mixture of Lactobacillus plantarum GF103 and Bacillus subtilis B27 (LBS). A milk replacer was fed to calves from 8 days of age. A starter and alfalfa hay was offered ad libitum from 21 and 28 days of age, respectively, and the orts were weighted daily. The ruminal fluid was sampled at 56 and 83 days of age to determine the rumen fermentation characteristics. The bacterial community was analyzed by denaturing gradient gel electrophoresis (DGGE) and the number of certain bacteria was quantified by real-time polymerase chain reaction. The ratio of total dry matter intake to average body wieght was higher in the control (P < 0.05). The DGGE fingerprint of the 16S ribosomal RNA gene was affected by the blended probiotics at 83 days of age. The number of Ruminococcus albus was lower in the LB and LBS treatment (P < 0.05). Oral administration of the probiotics affected the rumen bacterial community and the numbers of cellulolytic bacteria decreased.

Effects of isovalerate supplementation on growth performance and ruminal fermentation in pre- and post-weaning dairy calves

SUMMARYThe objective of the present study was to evaluate the effects of isovalerate supplementation on growth performance and ruminal fermentation in pre- and post-weaning dairy calves. Forty-eight Chinese Holstein male calves at 15 days of age and 45·1 ± 0·36 kg body weight (BW) were assigned randomly to four groups. The treatments were: control, low-isovalerate, moderate-isovalerate (MIV) and high-isovalerate (HIV) with 0, 3, 6 and 9 g isovalerate per calf per day, respectively. Isovalerate was hand-mixed into milk in pre-weaning calves and the concentrate portion in post-weaning calves. The study lasted 75 days, including a 15-day adaptation period followed by a 60-day sampling period. Weaning was conducted when calves were 60 days old. Six calves were chosen from each treatment at random and slaughtered at 30 and 90 days of age. Average daily weight gain increased linearly whether during pre-weaning or post-weaning period with increasing isovalerate supplementation. Dry matter intake linearly increased at 90 days of age with increasing isovalerate supplementation. During weaning, ruminal pH and ammonia nitrogen (N) decreased linearly, whereas total ruminal volatile fatty acid concentration increased linearly with increasing isovalerate supplementation. The ratio of acetate to propionate increased linearly with increasing isovalerate supplementation due to increased acetate concentration and the unchanged propionate concentration. Activities of caboxymethyl-cellulase, cellobiase, xylanase and pectinase linearly increased at 90 days of age, α-amylase and β-amylase activities linearly increased at 30 and 90 days of age. Relative quantities of Butyrivibrio fibrisolvens, Ruminococcus albus, Fibrobacter succinogenes and Ruminococcus flavefaciens increased linearly with increasing isovalerate supplementation. Ruminal fermentation, enzyme activities and cellulolytic bacteria were higher for HIV and MIV than for the control. The present results indicate that isovalerate accelerated growth of calves by improving ruminal fermentation, microbial enzyme activities and cellulolytic bacteria growth during weaning. In the experimental conditions of the current trial, the optimum isovalerate dose was about 6·0 g isovalerate per calf per day.

Effect of crude glycerine in supplement on the intake, rumen fermentation, and microbial profile of Nellore steers grazing tropical grass

The objective of this study was to evaluate the effect of crude glycerine (CG) inclusion in feed supplement (0, 70, 140, 210 and 280g/kg DM basis of supplement) on the forage intake and digestibility, ruminal fermentation parameters, kinetics of fibre degradation, and rumen microbial profile of Nellore steers grazing on tropical grass. Ten ruminally cannulated Nellore steers [490kg±47 body weight (BW)] were used in a replicated 5×5 Latin square design with 14-d periods. Steers were individually supplemented at the rate of 300g/100kg of BW. Inclusion of CG in the supplement did not affect (P>0.05) dry matter intake, apparent total tract digestibility, ruminal pH (P=0.784) or total ruminal VFA (P=0.291), but linearly decreased the NH3-N concentration (P=0.021). The inclusion of CG in the supplement linearly increased (P <0.001) the molar proportions of butyrate and valerate; linearly decreased the acetate (P=0.007) concentration, thus reducing the acetate to propionate ratio (P<0.001); and did not affect the molar proportions of propionate and isobutyrate. Inclusion of GC had a quadratic effect (P=0.010) on the in situ potential degradable fraction of NDF and rate of fibre degradation (P=0.006). Addition of CG linearly decreased the number of protozoa of the genera Entodinium (P=0.015) and Isotricha (P=0.058) and the relative proportions of Ruminococcus albus (P=0.047) and Ruminococcus flavefaciens in the rumen (P=0.036), but did not affect Fibrobacter succinogenes (P=0.420) or the methanogens (P=0.150). The inclusion of CG in the supplement up to 280g/kg DM altered ruminal fermentation and negatively affected in situ fibre degradation and the gram-positive cellulolytic bacterial population, but did not affect intake and apparent total tract digestibility for Nellore steers grazing tropical grass.

Ruminal methanogens and bacteria populations in sheep are modified by a tropical environment

Microbial fermentation of carbohydrates in the rumen is largely responsible for the emission of methane by ruminants. Ruminants fed tropical forages usually produce more enteric methane than ruminants fed temperate forages. The relative influence of forage type, breed and temperate vs tropical environment on rumen microbial populations is not known. This experiment aimed to separate these effects. We designed two parallel experiments in sheep in two sites: temperate (France) and tropical (French West Indies), using in each site two breeds, Texel (temperate origin), and Blackbelly (tropical origin) fed the same temperate forages (C3 carbon fixation, permanent grasslands of high and low quality) and tropical forages (C4 carbon fixation, permanent grasslands of high and low quality). We determined diet digestibility, ruminal end-products of fermentation and microbial groups: total protozoa, methanogens and bacteria, and selected fibrolytic bacteria. Dry matter digestibility coefficient was higher in tropical site (612 vs 580g/kg on average, P=0.004) but no difference was observed between C3 and C4 forages. There was no effect of site on total VFA concentration, but the acetate:propionate ratio was higher for the tropical site (4.30 vs 3.93 on average, P=0.007). The acetate:propionate ratio was also affected by forage type with higher values for C3 than C4 forage (4.24 vs 3.99 on average, P=0.03). Concentration of total rumen bacteria and methanogens was determined by qPCR targeting, respectively, the rrs (16S ribosomal RNA subunit) and mcrA (methyl coenzyme-M reductase) genes. For both groups, the number of gene copies per gram of DM rumen content was higher in the tropical site (P<0.001). For cellulolytic bacteria, higher number of rrs copies per gram of DM of rumen content were detected for Fibrobacter succinogenes in the temperate site (P<0.001), whereas no differences were observed for Ruminococcus flavefaciens or Ruminococcus albus numbers between sites, breeds and forage type. Protozoa numbers determined by counting did not vary between sites, forages or breeds, but a site×forage interaction was observed (P=0.01): there were more protozoa and R. albus in tropical sites for tropical forages. Our results suggest that rumen microbiota was mainly influenced by environment (temperate vs tropical) and that forage type (C3 vs C4) and breed had minor effects. However, an interaction between environment and forage type was observed for some variables.

Influence of rumen contents’ processing method on microbial populations in the fluid and subsequent in vitro fermentation of substrates of variable composition

The in vitro batch culture technique is being increasingly used to study rumen fermentation, but the results are affected by several factors, being the source of the inoculum one of the most relevant. This work was conducted to assess the effects of different processing methods of ruminal contents on microbial populations in the obtained fluid, and its influence on fermentation parameters when the fluid was used as inoculum for in vitro incubations. Rumen contents were obtained from four rumen-fistulated sheep fed a 2:1 alfalfa hay:concentrate diet and subjected to the following treatments: SQ: squeezed through four layers of cheesecloth; FL: SQ treatment and further filtration through a 100-μm nylon cloth; STO: blended for three min at 230revmin−1 in a Stomacher® and further filtrations as in SQ. Microbial populations’ abundance and bacterial diversity in the ruminal fluids were analysed by quantitative PCR (qPCR) and automated ribosomal intergenic spacer analysis (ARISA), respectively. Three forages (alfalfa hay, grass hay and barley straw) were incubated in vitro, either alone or mixed with concentrate (1:1), using each of the ruminal fluids as inoculum. There were no differences between SQ and FL methods in any of the microbial populations analysed, but STO increased the relative abundance of Fibrobacter succinogenes and Ruminococcus albus (P<0.05) and decreased (P<0.05) the concentration of protozoal DNA compared with SQ. Bacterial diversity was not affected (P>0.05) by the processing methods. There were no interactions (P>0.05) between the processing method and the characteristics of the substrates for any of the fermentation parameters analysed. Compared with SQ, the STO method resulted in greater (P<0.05) methane production and ammonia-N concentrations in 8h incubations. After 24h of incubation, the use of STO inoculum increased (P<0.05) methane production and dry matter degradability compared with SQ, with no differences in the rest of the parameters. No differences between SQ and FL methods were detected in any parameter. The results show that stomaching the rumen contents prior to inoculation of in vitro cultures modified some microbial populations, but had only subtle effects on fermentation parameters.

Functional and modular analyses of diverse endoglucanases from Ruminococcus albus 8, a specialist plant cell wall degrading bacterium

Ruminococcus albus 8 is a specialist plant cell wall degrading ruminal bacterium capable of utilizing hemicellulose and cellulose. Cellulose degradation requires a suite of enzymes including endoglucanases, exoglucanases, and β-glucosidases. The enzymes employed by R. albus 8 in degrading cellulose are yet to be completely elucidated. Through bioinformatic analysis of a draft genome sequence of R. albus 8, seventeen putatively cellulolytic genes were identified. The genes were heterologously expressed in E. coli, and purified to near homogeneity. On biochemical analysis with cellulosic substrates, seven of the gene products (Ra0185, Ra0259, Ra0325, Ra0903, Ra1831, Ra2461, and Ra2535) were identified as endoglucanases, releasing predominantly cellobiose and cellotriose. Each of the R. albus 8 endoglucanases, except for Ra0259 and Ra0325, bound to the model crystalline cellulose Avicel, confirming functional carbohydrate binding modules (CBMs). The polypeptides for Ra1831 and Ra2535 were found to contain distantly related homologs of CBM65. Mutational analysis of residues within the CBM65 of Ra1831 identified key residues required for binding. Phylogenetic analysis of the endoglucanases revealed three distinct subfamilies of glycoside hydrolase family 5 (GH5). Our results demonstrate that this fibrolytic bacterium uses diverse GH5 catalytic domains appended with different CBMs, including novel forms of CBM65, to degrade cellulose.

Effects of feeding goats with Leucaena leucocephala and Manihot esculenta leaves supplemented diets on rumen fermentation profiles, urinary purine derivatives and rumen microbial population

ABSTRACTThe effects of different levels of Leucaena leucocephala and Manihot esculenta leaves’ supplementation on in vitro gas production, rumen fermentation and microbial populations and urinary purine derivatives (PD) were investigated. Seven treatment groups – T1:C/rice straw (RS) (40:60) (Control); T2:C/RS/leucaena leaves (40:45:15); T3:C/RS/leucaena leaves (40:30:30); T4: C/RS/leucaena leaves (40:15:45); T5:C/RS/cassava leaves (40:45:15); T6:C/RS/cassava leaves (40:30:30) and T7:C/RS/cassava leaves (40:15:45) – were used in this experiment. In the in vitro study, acetate, propionate, butyrate and total VFA were found to increase significantly in T7. No significant difference was observed in in vitro gas production except the control diet although in vitro dry matter digestibility (IVDMD) was recorded significantly decreased. Determination of urinary PD, rumen fermentation and microbial population were done using 21 local Boer goats. Rumen NH₃-N, acetic and total VFA production were found to improve (P < .05) in all supplemented group. Significant (P < .05) reduction was noted in the urinary allantoin production and total PD at T7. The populations of total protozoa and Ruminococcus flavefacien had significantly increased (P < .05) while Ruminococcus albus, and Fibrobacter succinogenes were significantly reduced (P < .05) in supplemented group. In conclusion, the result from the present study suggested that 25% of L. leucocephala leaves diet and 50% of M. esculenta leaves diet can be incorporated in the goat diet so as to improve the nutritive value of poor quality diet.

Rumen fermentation and bacterial communities in weaned Chahaer lambs on diets with different protein levels

We evaluated the effects of diets with different crude protein (CP) levels on growth performance, rumen fermentation and bacterial communities in weaned Chahaer lambs. 128 weaned Chahaer lambs ((20.56±1.43) kg body weight; ram:ewe 1:1) aged (61±1.85) d were randomly allotted to one of four diets with CP content of 11.17% (T1), 12.06% (T2), 13.40% (T3) or 14.36% (T4). Ruminal fermentation parameters were measured and bacterial communities were analysed using PCR-dena-turing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR. The average daily gain and feed utilization efficiency in T3 were higher than those in the other groups (P<0.05), although the dry matter intake and metabolizable energy intake were similar. Total volatile fatty acid concentration in the ruminal fluid of T3 was lower than that of T1 (P=0.011), T2 (P=0.008) or T4 (P=0.309). The ammonia nitrogen concentration and acetate/propionate ratio of ruminal fluid were significantly higher in lambs fed the higher CP diets, whereas the molar concentrations of propionate and butyrate of ruminal fluid were lower. The rumen bacterial community was similar in T2 and T3 which shown more stable and diverse rumen microbes ecosystem compared with the other groups. The DGGE profiles and phylogenetic tree indicated that Bacteroides uniformis, Clostridium alkalicellulosi, Alkalibaculum bacchi and Saccharofermentans sp. were common bacterium of Chahaer lamb rumen. B. uniformis, C. alkalicellulosi, Saccharofermentans sp. and Gracilibacter thermotolerans, which belong to the Bacteroidetes and Firmicutes phyla, were the dominant species in the rumen of lambs fed 13.40% CP. However, Ruminococcus albus, Ruminococcus flavefaciens and Butyrivibrio fibrisolvens were not different in lambs fed different CP diets. Therefore, it could be concluded that B. uniformis, C. alkalicellulosi, A. bacchi and Saccharofermentans sp. were common bacteria of Chahaer lamb rumen. Furthermore, the dietary CP of 13.04% could improve performance and change rumen fermentation model by increasing the dominant species' peak intensities of B. uniformis, C. alkalicellulosi, Saccharofermentans sp. and Gracilibacter thermotolerans and stabilizing rumen microbial ecosystem.

Effects of dietary supplementing tannic acid in the ration of beef cattle on rumen fermentation, methane emission, microbial flora and nutrient digestibility.

Four adult Simmental male cattle (376±9.0kg initial BW), fitted with permanent rumen cannulas, were used in a 4×4 Latin square design to investigate the effects of dietary supplementing tannic acid (TA) on rumen fermentation, methane (CH4 ) production, rumen microbes, nutrient digestibility and plasma biochemical parameters. Four levels of TA, that is 0, 6.5, 13.0 or 26.0g/kg dry matter (DM), were added to the basal ration (composed of corn silage and concentrate mixture) as experimental treatments respectively. Each experimental period consisted of a 12-day adaptation phase followed by a 3-day sampling phase. The results showed that supplementing TA at 26.0 g/kg DM decreased the relative abundance of protozoa, methanogens and Ruminococcus albus to the total ruminal bacterial 16S rDNA in beef cattle (p<0.05). The results also showed that supplementing TA at 6.5, 13.0 or 26.0g/kg DM decreased (p<0.01) the CH4 production (l/kg DM intake) by 11.1%, 14.7% and 33.6% respectively. Supplementing TA at 13.0 or 26.0g/kg DM decreased the ratio of acetate to propionate and ammonia nitrogen (NH3 -N) (p<0.05) and tended to decrease the total volatile fatty acid (VFA) concentration of rumen fluid (p=0.07). Supplementing TA at 26.0g/kg DM decreased DM and organic matter (OM) digestibility (p<0.05), supplementing TA at 6.5, 13.0 or 26.0g/kg DM decreased (p<0.01) crude protein (CP) digestibility by 5.0%, 8.6% and 15.7%, respectively, and supplementing TA at 6.5, 13.0 or 26.0g/kg DM increased (p<0.05) the plasma total antioxidant capability. It was concluded that supplementing TA in the ration of beef cattle decreased the CH4 production and digestibility of CP of beef cattle. Supplementing TA could be an effective option to mitigate CH4 emission form cattle, further research is necessary to study the effects of TA on the performance of cattle.