Mixed Rumen Microbes Research Articles

A chromatographic method to monitor fructan catabolism in two cool‐season grasses fermented by mixed bovine ruminal microbiota

AbstractBACKGROUNDCool‐season grasses contain fructans (fructose‐based polymers), whose monomers are linked by β‐2,6 or β‐1,2 bonds. Fructans in the rumen are hydrolysed to fructose by bacterial enzymes, but little is known about the kinetics of fructan catabolism in the rumen. A chromatographic method, using high‐performance anion‐exchange chromatography (HPAEC) with pulsed amperometric detection, was developed to monitor changes in fructan profiles during the in vitro fermentation of timothy (Phleum pratense L.) and cocksfoot (Dactylis glomerata L.) by mixed rumen microbes. Sterile suspensions of freeze‐dried and milled tissue in minimal media were inoculated with washed rumen microbial cells under anaerobic conditions, and aliquots of the incubations were profiled 0, 2, and 5 h post‐inoculation.RESULTSTimothy and cocksfoot differed in initial concentrations of glucose and sucrose, and in the amounts remaining 2 h post‐inoculation (p < 0.01). The grasses also differed in initial concentrations of short‐chain fructan, and in concentrations present 2 h post‐inoculation (p = 0.01). Timothy and cocksfoot also differed in amounts of long‐chain fructans remaining 2 and 5 h post‐inoculation (p = 0.03), and in the maximum degree of polymerisation after 2 h (p < 0.01).CONCLUSIONSOver the first 2 h, long‐chain fructans in cocksfoot decreased while short‐chain fructans increased. In timothy, concentrations of short‐chain fructans doubled, and the degree of polymerisation decreased by 15%. The results support a model of degradation of the longest fructan chains and conversion into shorter ones.

Effects of inoculum source, pH, redox potential and headspace di-hydrogen on rumen in vitro fermentation yields

This in vitro study aimed at understanding how abiotic, that is chemical and electrochemical potentials, and biotic factors combine to impact the outputs of rumen volatile fatty acid (VFA). Using a 48-run design optimized by means of an exchange algorithm, the curvilinear effects of pH, Eh and partial pressure of dihydrogen (H2) on fermentation yields were investigated in 6-h batch cultures of mixed rumen microbes, fed on glucose so as to bypass the enzymatic hydrolysis and conversion steps preceding the glycolytic pathway. The role played by rumen microbiota in the expression of these effects was explored by testing three inocula grown on feeds supplying a microflora adapted to fibre, slowly degradable or readily degradable starch as the dominant dietary polysaccharide. Data were fitted to 2nd-order polynomial models. In fibre-adapted cultures, the yields of major VFA were mainly influenced by pH and H2 partial pressure, in opposite ways. In wheat grain-adapted cultures, the VFA yields underwent the opposite influences of pH, in a curvilinear way for propionate, and Eh since acetate production yield was not significantly modified by any factor. In maize grain-adapted cultures, acetate production yield was not modified by any factor but H2 in a quadratic way when the production yields of higher VFA underwent opposite influences of pH and Eh. In conclusion, the effects of environmental factors were dependent on the nature of the inoculum, a major source of variation, and more particularly on its adaptation to high- or low-fibre diets. These effects were loosely interrelated, the pH being the most active factor before the Eh and H2 partial pressure.

Effect of Defaunation on In Vitro Fermentation Characteristics and Methane Emission When Incubated with Forages

An in vitro study was conducted to determine the effects of defaunation (removal of protozoa) and forage sources (rice straw, ryegrass and tall fescue) on ruminal fermentation characteristics, methane (<TEX>$CH_4$</TEX>) production and degradation by rumen microbes. Sodium lauryl sulfate, as a defaunation reagent, was added into the mixed culture solution to remove ruminal protozoa at a concentration of 0.375 mg/ml. Pure cellulose (0.64 g, Sigma, C8002) and three forage sources were incubated in the bottle of culture solution of mixed rumen microbes (faunation) or defaunation for up to 24 h. The concentration of ammonia-N was high under condition of defaunation compared to that from faunation in all incubations (p<0.001). Total VFA concentration was increased at 3, 6 and 12 h (p<0.05~p<0.01) but was decreased at 24 h incubation (p<0.001) under condition of defaunation. Defaunation decreased acetate (p<0.001) and butyrate (p<0.001) proportions at 6, 12 and 24 h incubation times, but increased propionate (p<0.001) proportion at all incubation times for forages. Effective degradability of dry matter was decreased by defaunation (p<0.001). Defaunation not only decreased total gas (p<0.001) and <TEX>$CO_2$</TEX> (p<0.01~0.001) production at 12 and 24 h incubations, but reduced <TEX>$CH_4$</TEX> production (p<0.001) at all incubation times for all forages. The <TEX>$CH_4$</TEX> production, regardless of defaunation, in order of forage sources were rice straw > tall fescue > ryegrass > cellulose (p<0.001) up to 24 h incubation.

The effect of essential oils of Zataria multiflora and Mentha spicata on the in vitro rumen fermentation, and growth and deaminative activity of amino acid-fermenting bacteria isolated from Mehraban sheep

Gas (CO2 and CH4) and ammonia production in the rumen represent major sources of lost carbon and nitrogen, respectively. The essential oils of some plants have been shown to decrease gas and ammonia production by selectively inhibiting rumen microbes. Particularly, those of Zataria multiflora (ZEO; thymol 21%, carvacrol 32%) and Mentha spicata (SEO; carvone 55%) were evaluated in vitro as ruminant-feed additives. The experiments employed mixed rumen microbes and a hyper-ammonia-producing bacterium (HAP) isolated from the rumen of a Mehraban sheep. Both ZEO and SEO decreased in vitro fibre digestibility and also gas production by mixed rumen microbes that were fermenting a typical growing-lamb diet. ZEO decreased ammonia concentration in mixed culture of rumen microbes, but SEO exerted the opposite effect. A bacterial isolate (MT8) was obtained from the rumen of a Mehraban sheep, and the 16S rRNA gene sequence indicated that it was most closely related to Clostridium bifermentans. Isolate MT8 exhibited rapid ammonia production when peptides were the growth substrate, which indicated that MT8 was a HAP. Both oils inhibited the growth and ammonia production of isolate MT8. However, ZEO decreased ammonia production at lower doses, and to a greater degree, than did SEO. These results indicated that both essential oils could potentially be used to modulate rumen fermentation. The detrimental effects on fibre digestion could be problematic in high-forage diets, and this requires further investigation. Isolate MT8 is the first described HAP from the Mehraban sheep rumen. Results on ammonia production by isolate MT8 and mixed rumen microbes indicate differential mode of action of each oil on this parameter.

Quantifying the Responses of Mixed Rumen Microbes to Excess Carbohydrate

The aim of this study was to determine if a mixed microbial community from the bovine rumen would respond to excess carbohydrate by accumulating reserve carbohydrate, energy spilling (dissipating excess ATP energy as heat), or both. Mixed microbes from the rumen were washed with N-free buffer and dosed with glucose. Total heat production was measured by calorimetry. Energy spilling was calculated as heat production not accounted by (i) endogenous metabolism (heat production before dosing glucose) and (ii) synthesis of reserve carbohydrate (heat from synthesis itself and reactions yielding ATP for it). For cells dosed with 5 mM glucose, synthesis of reserve carbohydrate and endogenous metabolism accounted for nearly all heat production (93.7%); no spilling was detected (P = 0.226). For cells dosed with 20 mM glucose, energy spilling was not detected immediately after dosing, but it became significant (P < 0.05) by approximately 30 min after dosing with glucose. Energy spilling accounted for as much as 38.7% of heat production in one incubation. Nearly all energy (97.9%) and carbon (99.9%) in glucose were recovered in reserve carbohydrate, fermentation acids, CO2, CH4, and heat. This full recovery indicates that products were measured completely and that spilling was not a methodological artifact. These results should aid future research aiming to mechanistically account for variation in energetic efficiency of mixed microbial communities.

Evaluation of methods to detect changes in reserve carbohydrate for mixed rumen microbes

Thin-layer chromatography and other analyses revealed that rumen microbes accumulated large amounts of glycogen. The aim of this study was to identify a method that would most accurately quantify accumulation and utilization of this reserve carbohydrate. For whole cells, the anthrone reaction detected more (P<0.001) carbohydrate than did hydrolysis with amyloglucosidase, even after exhaustive extraction by bead beating (45min) or KOH digestion (3h). Less carbohydrate was detected after isolating reserve polysaccharide by ethanol precipitation. Compared to the amyloglucosidase hydrolysis method, the anthrone method detected a larger (P=0.017) increase in cell carbohydrate when glucose (20mM) was dosed in cultures. Additionally, it detected a larger (P=0.049) decrease in cell carbohydrate after glucose was exhausted. This result indicated that the anthrone method detected more carbohydrate that functions as a reserve material, which accumulates during energy excess and is utilized for energy during starvation. For the anthrone method, recoveries for energy (97.5%), carbon (100.2%), and cell components (99.8%) were high, indicating carbohydrate was completely detected. For the amyloglucosidase hydrolysis method, recoveries of energy (88.9%), carbon (91.6%), and cell components (92.8%) were lower. Some authors have inferred from iodine staining that amyloglucosidase hydrolyzes all glycogen in cells. However, iodine did not stain glycogen remaining after cells were incompletely extracted intentionally. The anthrone method appeared to accurately quantify changes in reserve carbohydrate and shows merit for quantitative studies, whereas the amyloglucosidase hydrolysis method detected smaller changes and was less consistent with expected carbon and energy recovery.

In vitro Evaluation of Sheep Rumen Fermentation Pattern After Adding Different Levels of Eugenol – Fumaric acid Combinations

In vitro gas production technique was used to evaluate the effect of three different levels of eugenol + fumaric acid combinations on rumen fermentation. Rumen contents were collected from five rams immediately after slaughtering and used for preparation of inoculums of mixed rumen microbes that were used in generation of five –1 treatment systems, negative control with no additives (T1), fumaric acid 0.5 mg L (T2) and fumaric acid 0.5 mg –1 –1 L in combination with three different doses of eugenol, 100, 200 and 400 mg L (T3, T4 and T5 respectively). Incubations were conducted in triplicates with gas production, pH, ammonia nitrogen (NH -N), total and 3 fractional volatile fatty acids (VFAs) concentrations, cellulase activity, amount of substrate degraded, microbial yield (Y ), fermentation efficiency (FE) and VFAs utilization index (NGGR) were determined after 24 hours of ATP incubation. The results revealed that, different levels of eugenol + fumaric acid combinations were associated with decreased pH value, NH -N concentrations and methane production and increased valeric and isovaleric 3 acids molar proportions. T3 and T4 were associated with increased propionates at the expence of acetates (low A/P), decreased methane production and increased FE, microbial yield (Y ) and VFAs utilization. In contrast, ATP T5 showed decreased total VFAs concentrations, cellulase activity, the amount of substrate degraded, microbial –1 mass generated and VFAs utilization. In conclusion, the authors recommend using 200 mg L eugenol + fumaric acid combination as an alternative for antibiotic feed additives to optimize rumen fermentation pattern. Further investigations are required to apply this work in vivo experiments.

Effect of Exogenous Fibrolytic Enzyme Application on the Microbial Attachment and Digestion of Barley Straw In vitro.

The effects of exogenous fibrolytic enzymes (EFE; a mixture of two preparations from Trichoderma spp., with predominant xylanase and β-glucanase activities, respectively) on colonization and digestion of ground barley straw and alfalfa hay by Fibrobacter succinogenes S85 and Ruminococcus flavefaciens FD1 were studied in vitro. The two levels (28 and 280 μg/ml) of EFE tested and both bacteria were effective at digesting NDF of hay and straw. With both substrates, more NDF hydrolysis (p<0.01) was achieved with EFE alone at 280 than at 28 μg/ml. A synergistic effect (p<0.01) of F. succinogenes S85 and EFE on straw digestion was observed at 28 but not 280 μg/ml of EFE. Strain R. flavefaciens FD1 digested more (p<0.01) hay and straw with higher EFE than with lower or no EFE, but the effect was additive rather than synergistic. Included in the incubation medium, EFE showed potential to improve fibre digestion by cellulolytic ruminal bacteria. In a second batch culture experiment using mixed rumen microbes, DM disappearance (DMD), gas production and incorporation of 15N into particle-associated microbial N (15N-PAMN) were higher (p<0.001) with ammoniated (5% w/w; AS) than with native (S) ground barley straw. Application of EFE to the straws increased (p<0.001) DMD and gas production at 4 and 12 h, but not at 48 h of the incubation. EFE applied onto S increased (p<0.01) 15N-PAMN at 4 h only, but EFE on AS increased (p<0.001) 15N-PAMN at all time points. Prehydrolysis increased (p<0.01) DMD from both S and AS at 4 and 12 h, but reduced (p<0.01) 15N-PAMN in the early stage (4 h) of the incubation, as compared to non-prehydrolyzed samples. Application of EFE to barley straw increased rumen bacterial colonization of the substrate, but excessive hydrolytic action of EFE prior to incubation decreased it.

Effect of Disodium Fumarate on In vitro Rumen Fermentation of Different Substrates and Rumen Bacterial Communities as Revealed by Denaturing Gradient Gel Electrophoresis Analysis of 16S Ribosomal DNA

Two experiments were conducted to investigate the effects of disodium fumarate on the in vitro rumen fermentation profiles of different substrates and microbial communities. In experiment 1, nine diets (high-forage diet (forage:concentrate, e.g. F:C = 7:3, DM basis), medium-forage diet (F:C = 5:5, DM basis), low-forage diet(F:C = 1:9, DM basis), cracked corn, cracked wheat, soluble starch, tall elata (Festuca elata), perennial ryegrass and rice straw) were fermented in vitro by rumen microorganisms from local goats. The results showed that during 24 h incubations, for all substrates, disodium fumarate increased (p<0.05) the gas production, and tended to increase (p<0.10) the acetate, propionate and total VFA concentration and decrease the ratio of acetate to propionate, whereas no treatment effect was observed for the lactate concentration. The apparent DM loss for tall elata, perennial ryegrass and rice straw increased (p<0.05) with the addition of disodium fumarate. With the exception of tall elata, perennial ryegrass and rice straw, disodium fumarate addition increased the final pH (p<0.05) for all substrates. In experiment 2, three substrates (a high-forage diet, a medium-forage diet and a high concentrate diet) were fermented by mixed rumen microbes in vitro. A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique was applied to compare microbial DNA fingerprints between substrates at the end of 24 h incubation. The results showed that when Festuca elata was used as substrate, the control and disodium fumarate treatments had similar DGGE profiles, with their similarities higher than 96%. As the ratio of concentrate increased, however, the similarities in DGGE profiles decreased between the control and disodium fumarate treatment. Overall, these results suggest that disodium fumarate is effective in increasing the pH and gas production for the diets differing in forage: concentrate ratio, grain cereals and soluble starch, and in increasing dry matter loss for the forages (tall elata, perennial ryegrass and rice straw) in vitro, whereas its effect on changes of ruminal microbial community may largely depend on the general nature of the substrate.

Effect of nisin on ruminal methane production and nitrate/nitrite reduction in vitro

The suppressing effects of different concentrations of nitrate (0, 5, 10, 15, and 20 mm) or nisin (0, 5, 10, 15, 20, and 30 μmol/L) on in vitro methane production were examined with mixed rumen microbes using the in vitro continuous incubation system. The effects of different concentrations of nisin (10, 20, and 30 μmol/L) on in vitro nitrate/nitrite reduction were examined for methane suppression without any nitrate toxicity. The culture mixture consisted of 400 mL of strained rumen fluid from 2 non-lactating Holstein cows fed a diet of oaten hay, alfalfa hay cube, and concentrates (35 : 35 : 30) at maintenance level, and 400 mL of autoclaved buffer solution. Methane production was decreased with increasing levels of nitrate. As the concentration of nisin increased from 5 to 30 μmol/L, methane production was decreased by 14–40%. A decrease in acetate to propionate ratio and increase in total volatile fatty acids were observed as the concentration of nisin increased. Toxic nitrite accumulation was unaffected by increasing levels of nisin. In conclusion, nisin improved some of the parameters of ruminal fermentation and inhibited methane production, but did not decrease nitrate toxicity when nitrate was used to inhibit methane production.

Effect of Escherichia coli W3110 on ruminal methanogenesis and nitrate/nitrite reduction in vitro

An in vitro continuous incubation system was used to evaluate effects of nitrate, nitrite and wild-type Escherichia coli W3110 on in vitro methanogenesis by mixed ruminal population, and to determine the reducing ability of wild-type E. coli W3110 on in vitro nitrate/nitrite reduction. The culture mixture consisted of 400 ml of strained rumen fluid from two non-lactating Holstein cows fed a diet of orchardgrass hay at maintenance level, and 400 ml of autoclaved buffer solution. The treatments consisted of nitrate (NaNO 3, 5 or 10 mM), nitrite (NaNO 2, 1 or 2 mM), inoculated E. coli W3110, nitrate (NaNO 3, 5 or 10 mM) plus E. coli W3110, and nitrite (NaNO 2, 1 or 2 mM) plus E. coli W3110. The control culture was incubated without supplements. Methanogenesis was markedly decreased by addition of nitrate and nitrite, and inoculation of E. coli W3110-reduced methanogenesis by 27%. Inoculation of E. coli W3110 to nitrite containing cultures decreased nitrite accumulation, while keeping methanogenesis low. The culture of wild-type E. coli W3110 may decrease methanogenesis in cultures of mixed rumen microbes, suggesting that inoculation of wild-type E. coli W3110 may be a practical way to suppress nitrite toxicity when nitrite is used to abate methanogenesis in the rumen.

Characterisation of proteolytic activity of rumen microbes and commercial proteases

Commercial proteases yield more reproducible estimates of extent of rumen protein degradation than either in situ or rumen in vitro methods. Although extents of protein digestion determined using commercial proteases have been positively correlated to extents of in situ protein degradation, these values have not proven reliable predictors of rates of rumen protein degradation. This is because no protease-based assay has yet been developed that successfully mimics the specificities of the organisms involved in protein degradation in the rumen. If the proteolytic activity of rumen microbes and commercial proteases can be correctly characterised, then it would be possible to elaborate protease blends that match the protein degradative activity of mixed rumen microbes. The research reported here is an extension of earlier work as a prelude to preparing enzyme mixes with “protease fingerprints” similar to rumen microbes.

Mixed Ruminal Microbes of Cattle Produce Isopropanol in the Presence of Acetone But Not 3-d-Hydroxybutyrate

The objective was to evaluate the ability of mixed rumen microbes to synthesize isopropanol from acetone or 3-d-hydroxybutyrate. Rumen fluid from seven mature, nonpregnant, dry Holstein cows was incubated with starch or cellulose and additions of acetone, 3-d-hydroxybutyrate, or saline. Rumen fluid was analyzed for isopropanol after 0, 3, 6, and 9h. No isopropanol was present in any sample at 0h, and none was present in incubations containing saline or 3-d-hydroxybutyrate at any subsequent time. Incubations that included acetone produced small amounts of isopropanol from 0 to 3 and 3 to 6h and significantly larger amounts from 6 to 9h. With starch as the energy substrate, production from 6 to 9h was 3.8μmol/min per liter of rumen fluid and 3.7μmol/min per liter with cellulose as the energy substrate; however, these values did not differ significantly. Mixed rumen microbes could synthesize isopropanol from acetone but not from 3-d-hydroxybutyrate, and rumen microbial metabolism of acetone was the likely source of plasma isopropanol seen in ketotic ruminants.

Optimal mineral composition of artificial saliva for fermentation and methanogenesis in continuous culture of rumen microorganisms

In dual outflow continuous fermenters on a 75 : 25 hay : barley diet, fermentation and gas production by mixed rumen microbes were tested in relation to the concentrations of HPO 4 2−, HCO 3 − and Cl − and the Na +/K + ratio in artificial saliva, by applying a 17-run Franquart design, and by fitting second-order polynomial models. The HPO 4 2−, HCO 3 −, Cl − concentrations and Na +/K + ratio ranged from 0.1 to 4 g l −1, from 0.5 to 7 g l −1, from 0.1 to 0.5 g l −1 and from 0.5 to 15 g g −1, respectively. The major factor was the concentration of HCO 3 −, followed by HPO 4 2− and Na +/K +, while the concentration of Cl − had negligible effects. The volatile fatty acid (VFA) production rate was mostly influenced by changes in pH, from 6.7 to 7.2, mediated by HCO 3 − and HPO 4 2− concentrations. The analysis of the fermentation pattern confirmed the predominant action of HCO 3 −. Butyrate (C4) and branched chain VFA production rates, as well as C4 molar proportion in the fermentation broth were also lowered by high or low values of the Na +/K + ratio. The action of minerals on gas production and protozoa population density was complex and involved to an equal degree several experimental factors with a non-linear behaviour. Protozoa numbers, which varied from 14 to 38 μl −1, were favoured by high Na +/K + ratios. Using response surface analysis, the composition of a mineral base was optimised to simultaneously promote protozoa numbers and methanogenesis in vitro, at 39 μl −1 and 1.20 mmol h −1, respectively. The resulting artificial saliva contained 1.65 g l −1 HPO 4 2−, 4.19 g l −1 HCO 3 −, and 0.22 g l −1 Cl −, and had a Na +/K + ratio of 14.2 g g −1.

Composition and degradability of different fractions of Calliandra leaves, pods and seeds

Calliandra leaves, green pods, and seeds were harvested from the central highlands of Kenya. The leaves were collected from coppice shoots and plants of various ages (4 months, 1.5 and 2.5 years). Samples from the 2.5 year-old plants were further separated into top 30 cm, lower 30 cm and mixed leaves. Dry matter (DM), ash, nitrogen, neutral detergent fibre (NDF), acid detergent fibre (ADF), minerals, amino acids, flavanols and proanthocyanidins (PAs) were determined. Rumen degradability was measured in situ in sheep and in vitro degradability was measured with mixed rumen microbes in a consecutive batch culture (CBC) system. Calliandra leaves and seeds were rich in crude protein (CP) and had a balanced amino acid profile. The pods had higher NDF and ADF contents than the leaves or seeds, and the older leaves had higher calcium content than young leaves. The leaves contained high concentrations of flavanols and PAs whereas the seeds appeared not to have any detectable flavanols. The in situ rumen DM degradability of leaves was surprisingly high. The mean DM degradation asymptote ( A+ B) for all of the leaves was 781 g/kg. The pods were however less degradable in the rumen than the leaves. Except for the mixed leaves with a rather low average CP degradation of 358 g/kg CP after 48 h of incubation, the CP degradation after 48 h of incubation of the other leaves was relatively high. The average CP degradation for the other leaf fractions was 525 g/kg CP. In contrast to the DM and CP degradation in the rumen, NDF in the leaves increased as a proportion of the DM. However, the NDF gain was lower as the period of incubation increased. The DM degradation of the seeds in vitro was higher ( P < 0.05) than those of the other plant parts. The 4 month-old leaves appeared to be the least degradable among the leaves in vitro. The results showed that Calliandra leaves and seeds harvested from Kenya may have potential as dry season feed and protein supplement for ruminants.

Effects of minerals on feed degradation and protein synthesis by rumen micro-organisms in a dual effluent fermenter.

In dual outflow continuous fermenters on a 75:25 hay/barley diet, feed degradation and protein synthesis by mixed rumen microbes were tested in relation to the concentrations of HPO4(2-), HCO3- and Cl- and Na+/K+ ratio in artificial saliva, by applying a 16-run Franquart design, and by fitting second-order polynomial models. The HPO4(2-), HCO3-, Cl- concentrations and Na+/K+ ratio ranged from 0.1 to 4 g.L-1, from 0.5 to 7 g.L-1, from 0.1 to 0.5 g.L-1 and from 0.5 to 15 g.g-1, respectively. Buffer salts, particularly HPO4(2-), were the major factors while Cl- concentration had negligible effects on microbial metabolism. Maximal neutral detergent fibre, acid detergent fibre and organic matter degradabilities occurred at intermediate values of HPO4(2-) and HCO3- concentrations. The outflow of microbial protein and the efficiency of microbial protein synthesis, which varied from 26.2 to 37.1 g.N.kg-1 of organic matter truly degraded, reached minima at the centre of the experimental domain.

Effects of 3 chemical treatments on in vitro fermentation of rice straw by mixed rumen microbes in the presence or absence of anaerobic rumen fungi.

Rice straw (Rs) was treated by a sodium chlorite/acetic acid mixture (Sct), ammoniation (At) and alkaline hydrogen peroxide (Athp) methods. The objective was to compare their degradation and fermentation products in the presence or suppression of anaerobic fungi. Significant differences (P < 0.01) in degradation of straws were observed during all periods of incubation with Sct having the highest digestibilities during the 48-h and 72-h incubations. The degradation of Sct straw was highest in both of the incubations with whole rumen fluid (WRF) and WRF plus cycloheximide. In sacco digestion followed the order of Sct > At > Rs > Athp. Suppressing fungal activity with cycloheximide resulted in a decrease in dry matter degradation, with concomitant decrease in total volatile fatty-acid concentration. While the suppression of fungal activity with cycloheximide depressed acetate and butyrate production, it favored an increase in propionate production.

Effects of sulphate supplementation of untreated and alkali-treated wheat straws on ruminal fermentation and microbial protein synthesis in a semi-continuous fermentor

The effect of sulphate supplementation on the fermentation of untreated or alkali-treated wheat straw by mixed rumen microbes was studied in a semi-continuous fermentor where urea was the main nitrogen source. In addition to the detergent method, chromatography of neutral sugar derivatives was used to estimate cell wall degradation of the treated straw. Microbial protein synthesis was determined using the 15N labelling technique. The most suitable nitrogen supply was determined with untreated straw and estimated to about 40 g N kg −1 of fermented organic matter. With both types of straw, sulphate supplementation improved microbial protein synthesis, all the degradative processes and particularly cellulolysis. The response to sulphate-addition was more intense with treated straw which provided more potential energy. It appears that even after treatment, the availability of sulphur contained in the straw was too low to satisfy rumen microbial sulphate-requirements, though total sulphur was theoretically sufficient for requirements. This study demonstrates the importance of a concomitant and appropriate nitrogen and sulphate supplementation to optimise straw fermentation.

Comparative growth rates of various rumen bacteria in clarified rumen fluid from cows and sheep fed different diets

Pure cultures of strains of different species of rumen bacteria were grown in filter-sterilized rumen fluid supplemented with glucose, bicarbonate, and reducing agent (cysteine and sulfide). Growth rates were determined in a series of experiments. Strains of species most abundant in the rumen grew more rapidly than strains of less abundant bacteria. Ammonia, amino acids, and peptides increased growth rates to some extent, but the greatest stimulatory effect for less abundant bacteria was provided by other factors, present in yeast extract. Factors released from lysates of mixed rumen microbes stimulated growth, but their rate of release was slow. It was concluded that, besides energy and nitrogen sources, growth factors of an as-yet-undetermined nature probably play an important role in determining the predominance of different bacterial species in the rumen.