Estimating ruminal microbial efficiencies in silage-fed cattle: comparison of an in vitro method with a combination of in situ and in vivo measurements

The achievement of maximum ruminal feed conversion into microbial biomass is a widely accepted concept of ruminant nutrition because high microbial efficiency improves microbial protein supply to the small intestine and, proportionally, reduces fermentative gaseous carbon losses (Beever 1993; Leng 1993). It has recently been demonstrated that differences between forages in in vitro microbial efficiencies, i.e. differences in the proportion of fermented substrate incorporated into microbial biomass, could be determined by a combination of in vitro gas volume measurements with a concomitant evaluation of the amount of substrate truly degraded during 24 h of incubation (for review see Blümmel et al. 1997a). It has been pointed out that these two measurements are not synonymous but complementary. The measurement of degradability is a modification by Goering and van Soest (1970) of the Tilley and Terry (1963) method to remove any residual microbial biomass from the undegraded substrate, thus allowing the calculation of the total amount of substrate dissimilated into all fermentation products, i.e. microbial biomass, short chain fatty acids (SCFA) and gases. In contrast, the gas volume measurement indicates how much of the fermented substrate was used for the formation of SCFA and gases since these two fermentation products are stoichiometrically very closely associated (Blümmel et al. 1997a). The measurements described above were performed after 24 h of incubation because the analytical approach used required all substrate solubles to already be fermented at the time of the residue determination; they would otherwise be removed by the treatment, leading to an overestimation of substrate degradability. On the other hand the determination should not be conducted too far beyond the microbial peak yield in order to minimize the possible contribution of microbial lysis to gas production. The ratio of truly degraded substrate to the gas volume thereby produced in 24 h was termed partitioning factor (PF). This factor denotes the substrate specific variation of in vitro microbial efficiency. The concept of the PF value was derived from, and applied to, crop residues of temperate and tropical origin (Blümmel et al. 1997b) and to Mediterranean grass and legume hays (Blümmel and Bullerdieck 1997). Forages with a high PF value, i.e. proportionally low gas production per unit of substrate degraded, were related to higher voluntary feed intakes than those with a low PF value. High in vitro PF values were also associated with high excretion of renal purine derivatives in Malawian goats fed maize stover leaves with different PF values but similar digestibilities (Mgomezulu and Blümmel 1996). For 61 straws and hays examined, the production of 1 ml of gas was associated with the concomitant in vitro true degradability of 2.74 to 4.65 mg of substrate i.e. PF values ranged from 2.74 to 4.65 mg/ml of which a minimum of 2.20 mg (Blümmel et al. 1997a) were required for the formation of acetate, propionate, butyrate and fermentative CO₂, CH₄ and H₂O, the latter produced upon the reduction of CO₂ to CH₄ as follows: CO₂+8H > CH₄+2H₂O. Carbon dioxide also arises from buffering the SCFA but this CO₂ is, contrary to the fermentative CO₂, not derived from the incubated substrate but from the buffer medium. Substrate degraded on top of these requirements (2.20 mg/ml) was available for microbial cell synthesis; proportionally more microbial biomass has been synthesized in fermentations having high PF values (Blümmel et al. 1997a). The objective of this work was to further investigate the relationship between in vitro microbial efficiencies as estimated by the PF value and in vivo microbial efficiencies using four perennial rye-grass silages harvested at different stages of maturity. These silages were fed to steers and the in vivo microbial protein synthesis was estimated from renal allantoin excretion, thus providing the opportunity for the comparison of in vitro and in vivo microbial efficiencies. Some aspects of the in vivo work have been reported by Philipczyk et al. (1996) and Schröder et al. (1997).     

Gas and short‐chain fatty acid production from feeds commonly fed to red deer (Cervus elaphus L.) and incubated with rumen inoculum from red deer and sheep

Efficient red deer supplementary feeding depends on estimations of the nutritive value of offered feeds, frequently estimated with the use of equations derived from domestic ruminants. The aim of this study was to compare the 24‐hour in vitro true dry matter degradability (ivTD₂₄), in vitro gas production (GP) kinetic parameters, GP in 24 hr of incubation (GAS₂₄) and short‐chain fatty acid (SCFA) and microbial biomass (MBS) produced after 24‐hour incubation of feeds in inoculum prepared from sheep and red deer rumen fluid. Eleven feeds, frequently consumed by red deer in Slovenia, which occur either naturally (two fresh grasses, chestnut fruits and common and sessile oak acorns) or are fed as winter supplemental feeds (two grass hays, two grass silages, apple pomace, fresh sugar beetroot), were investigated. The in vitro GP kinetic parameters, GAS₂₄ and ivTD₂₄, did not differ between animal species. Amounts of SCFAs were greater (p < 0.05) when feeds were incubated in sheep inoculum, while molar proportions of acetic and propionic acids did not differ. Molar proportions of butyric acid produced during incubation of high fibre feeds did not differ between animal species, but were higher (p < 0.05) when feeds high in starch or sugar were incubated in red deer inoculum. Greater production of SCFA by sheep rumen microbes suggests better coverage of host animal with energy precursors, while greater production of MBS by red deer rumen microbes suggests better coverage of host animal with protein. Results also suggest that rumens of sheep and red deer are inhabited by different microbial communities, which did not affect the extent of in vitro GP and degradation of feeds used in the present experiment. However, the possibility exists that the divergent nutrient use could be a consequence of different priming by different feeds of the donor animal diets.     

Micronized fibres affect in vitro fermentation under normal buffered and osmotic stress conditions using porcine inocula

In this in vitro study, the modified Hohenheim gas test was used to determine fermentation activity and bacterial composition of pig's faecal microbial inoculum, when fermenting a standard pig diet with varying levels of crude protein (CP; 20, 24 and 28% CP), and supplemented with one of three fibre sources manufactured by micronization treatment. These were wheat envelopes (MWE), pea fibre (MPF) and lupine fibre (MLF). For comparison, inulin was used. As intestinal bacteria have to cope with varying osmotic conditions in their ecosystem, fermentation was performed under normal buffered and osmotic stress conditions. After 24 h of fermentation, total gas production and ammonia production were measured. In addition, the effect of MWE and inulin on short‐chain fatty acid (SCFA) production and numbers of total eubacteria, Lactobacillus spp., Bifidobacterium spp., Enterobacteriaceae, Enterococcus spp., Clostridium cluster XIVa and Clostridium cluster IV, were determined using quantitative real‐time PCR. Under normal buffered conditions, supplementation of MWE resulted in increased (p < 0.05) SCFA, acetic, propionic and valerianic acid production at CP levels of 20 and 28%. There was an increase (p < 0.05) in ammonia production for the micronized supplements, and for MWE an increased (p < 0.05) branched‐chain proportion was observed, possibly due to higher availability of protein for fermentation which was released during the micronization process. Osmotic stress conditions reduced (p < 0.05) total gas as well as total SCFA, acetic and propionic acid production for all treatments, while cell counts were increased (p < 0.05) for Bifidobacterium spp., Enterococcus spp. and Lactobacillus spp. Under normal buffered conditions in combination with 24 and 28% CP levels, lactobacilli were increased for MWE, compared to inulin (p < 0.05). In conclusion, micronized supplements such as MWE may beneficially modulate pigs' intestinal microbiota by increasing SCFA production in addition to a selective proliferation of lactobacilli.     

Characterization of the nutritive value of tropical legume grains as alternative ingredients for small‐scale pork producers using in vitro enzymatic hydrolysis and fermentation*

In the tropic, the small‐scale pork production is negatively influenced by the low availability of high protein ingredients. The study aimed to compare the protein and starch hydrolysis as well as fibre fermentation of five tropical legume grains (Canavalia brasiliensis, CB; Lablab purpureus, LP; Vigna unguiculata, white WVU; pink PVU and red RVU) and a control (extruded full‐fat soybean (SB)), using an in vitro model that simulated digestion in the gastrointestinal tract of pigs. A sequential in vitro hydrolysis was carried out with pepsin (120 min) and pancreatin (240 min) to determine the degree of hydrolysis (DH) of protein and starch. The indigestible residue was fermented in vitro with pig faecal inoculum to compare the modelled kinetics of gas production over 72 h and the production of short‐chain fatty acids (SCFA). After 360 min of pepsin–pancreatin hydrolysis, SB and WVU had the highest protein hydrolysis (76% and 66%) and PVU and WVU the highest starch hydrolysis (70% and 64%) (p < 0.01). The in vitro fermentation of the indigestible residue of WVU resulted in the highest (482 ml/g DM; p < 0.001) and CB the lowest (335 ml/g DM) gas production. These data were consistent with the SCFA production. Butyrate, propionate and total SCFA were higher (or tended) for RVU and WVU when compared with CB and SB (p = 0.015–0.085). In conclusion, the high DH of protein and starch as well as the high gas and SCFA production obtained with raw WVU makes it an interesting alternative to SB as a feedstuff for swine nutrition in the tropic. Other legume grains (LP and CB) cannot be used by pigs in their raw form.     

Effect of supplementation of rice bran and fumarate alone or in combination on in vitro rumen fermentation,methanogenesis and methanogens

This study investigated the effect of fumarate (FUM) and rice bran (RB), alone and together, on in vitro rumen fermentation, methanogenesis and methanogens. In vitro incubation was performed with six media that were either unsupplemented (control) or supplemented with 10% RB, 5 mmol/L FUM, 10% RB + 5 mmol/L FUM, 10 mmol/L FUM, or 10% RB + 10 mmol/L FUM. Methane (CH₄) production, dry matter digestibility, CH₄ per digested dry matter, total short‐chain fatty acid (SCFA) production, proportion of SCFA, acetate : proprionate ratio, production of NH₃‐N, and population density of rumen microbes were determined. Supplementation with 10% RB + 10 mmol/L FUM yielded a 36% decrease in CH₄ production compared to the control. Supplementation of FUM, in the presence or absence of RB, provided increases in total SCFA production and propionate proportion up to 61% and 31%, respectively. Total bacteria, methanogens and protozoa populations were significantly (P < 0.05) decreased with the 10% RB + 10 mmol/L FUM supplementation. The effect of anti‐methanogenesis of FUM was enhanced by the addition of RB. Notably, the CH₄ production attenuation was achieved by 10% RB + 10 mmol/L FUM without reduction of digestibility or of ruminal fermentation.     

Contribution of different rumen microbial groups to gas,short‐chain fatty acid and ammonium production from different diets—an approach in an in vitro fermentation system

In this study, the relative contribution of different microbial groups to ruminal metabolism was investigated for different diets. The rumen microbial cultures included whole rumen fluid, fungi + protozoa, bacteria + protozoa, protozoa and bacteria + fungi and were established by physical and chemical methods. Gas production, short‐chain fatty acid (SCFA) and ammonium production were measured at 24 hr in in vitro incubations using the Hohenheim gas test (HGT) procedure. Seven donor animal diets with different concentrate‐to‐roughage ratios (C:R: 10:90, 30:70, 50:50, 70:30, 70:30BC (BC = NaHCO₃), 90:10 and 90:10BC) and five HGT diets (C:R: 10:90, 30:70, 50:50, 70:30 and 90:10) were formulated. Incubations in the HGT were always based on inoculum from sheep diets with the respective C:R ratio. Gas and ammonium production increased (p < 0.001) as a result of a gradual increase in concentrate proportion of the diets. In general, SCFA production followed the same trend. Whole rumen fluid and bacteria + fungi produced approximately 50% higher gas volume than protozoa and fungi + protozoa fractions, whereas gas production with bacteria + protozoa was at an intermediate level. Coculture of protozoa either with bacteria or with fungi produced more ammonium. Populations without bacteria were characterized by a particularly high acetate/propionate ratio. Although an interaction between microbial group and diet was observed for several variables, no clear direction could be established. Manipulating rumen fluid by selectively suppressing specific rumen microbial groups may be a helpful tool in elucidating their role in nutrient degradation and turnover in vitro.     

Effect of monensin withdrawal on rumen fermentation,methanogenesis and microbial populations in cattle

This study was designed to obtain information on the residual influence of dietary monensin on ruminant fermentation, methanogenesis and bacterial population. Three ruminally cannulated crossbreed heifers (14 months old, 363 ± 11 kg) were fed Italian ryegrass straw and concentrate supplemented with monensin for 21 days before sampling. Rumen fluid samples were collected for analysis of short chain fatty acid (SCFA) profiles, monensin concentration, methanogens and rumen bacterial density. Post‐feeding rumen fluid was also collected to determine in vitro gas production. Monensin was eliminated from the rumen fluid within 3 days. The composition of SCFA varied after elimination of monensin, while total production of SCFA was 1.78 times higher than on the first day. Methane production increased 7 days after monensin administration ceased, whereas hydrogen production decreased. The methanogens and rumen bacterial copy numbers were unaffected by the withdrawal of monensin.     

Effects of heat treatment on protein feeds evaluated in vitro by the method of estimating utilisable crude protein at the duodenum

The effect of heat treatment on the protein value of field beans, lupins and peas was studied using an in vitro method. Protein feeds were subjected to heat treatment for 30, 60 and 90 min in forced air oven at 120, 140 and 160 °C and in autoclave at 105, 120 and 135 °C. The heat‐treated protein feeds were incubated in buffered rumen fluid together with grass silage and barley in complete isonitrogenous diets. The gas production (GP) was recorded continuously, and ammonia‐N (NH₃‐N) concentrations were determined during the in vitro incubation at 8, 24 and 48 h and used to determine the utilisable crude protein (uCP) at the duodenum at 16 h of incubation (uCP₁₆). Heat treatments decreased the concentration of soluble crude protein and increased neutral detergent insoluble CP (NDICP) in all protein feeds compared to untreated. Inclusion of protein feeds to basal diet showed no increase in the uCP₁₆ in untreated field bean diet and only a small numerical increase in the uCP₁₆ concentrations from 160 g/kg dry matter (DM) to 166 and 172 g/kg DM in untreated lupine and pea diets, respectively, indicating high degradability of untreated feeds. Increasing the time and temperature of the heat treatment linearly increased the uCP₁₆ concentrations in field bean and pea diets, but not in lupin diets. Autoclave treatment was more effective in decreasing uCP₁₆ than oven treatment despite the lower temperatures used. However, the combination of highest temperatures and treatment time in autoclave increased acid detergent insoluble CP (ADICP) concentrations in protein feeds, indicating protein damage and decreased intestinal digestibility. Determining in vitro uCP and ADICP shows to be a promising method for evaluating protein value in heat‐treated animal feeds.     

Influence of Aguamiel (Agave atrovirens) as a Natural Feed Additive on Cecal Fermentation Kinetics of Some Forage Species in Horse Feeding

This study aimed to evaluate the effect of different dose levels of aguamiel (Agave atrovirens) on in vitro cecal gas, methane (CH₄), and carbon dioxide (CO₂) productions of five forage species (Avena sativa [hay]), Moringa oleifera, Caesalpinia coriacea, Salix babylonica, and Eichhornia crassipes using inocula from the horse. The forage samples were incubated with three doses of aguamiel: 0, 34, and 68 μg of aguamiel/g dry matter (DM) of substrate. Cecal inocula were collected from four adult female Criolla horses (3–4 years of age and weighing 300 ± 15.0 kg) grazed on native grasses for about 8 hours without supplementation. Forage type affected (P < .001) cecal asymptotic, rate and lag time of gas, CH₄ and CO₂ productions (mL/g DM), pH and DM degradability. Aguamiel dose had linear and quadratic effects (P < .05) on the asymptotic and rate of CH₄ productions and rate and lag time of CO₂ productions (mL/g DM). Forage type × aguamiel dose interactions were significant (P < .05) for asymptotic, rate and lag time of gas, and CH₄ and CO₂ productions (mL/g DM). Forage species effects were pronounced (P < .05) on CH₄ and CO₂ productions (mL/g incubated and degraded DM) and proportional CH₄ production at all hours of incubation, except for CO₂ production (mL/g incubated DM). Aguamiel dose affected (P < .05) CO₂ production (mL/g incubated DM) and proportional CO₂ production at the incubated hours. Forage type × aguamiel dose interactions were observed (P < .05) for CO₂ production (mL/g incubated DM) and proportional CO₂ production at the incubated hours but had no impact on CH₄ production. It is concluded that addition of aguamiel to five forage species affected fermentation kinetics of gas production resulting in different in vitro cecal gas, CH₄ and CO₂ productions from these substrates.     

Effects of dissolved carbon dioxide on the integrity of the rumen epithelium: An agent in the development of ruminal acidosis

The carbon dioxide released and dissolved in rumen fluid may easily permeate across the epithelial cell membrane. Thus, we hypothesized that CO₂ may act as proton carrier and induce epithelial damage under acidotic conditions. Ovine ruminal epithelia were mounted in Ussing chambers under short‐circuit conditions. The serosal buffer solution had a constant pH of 7.4 and was gassed either with 100% oxygen or with carbogen (95% O₂/5% CO₂). The mucosal solution was gassed with either 100% oxygen or 100% carbon dioxide. The mucosal pH was lowered stepwise from 6.6 to 5.0 in the presence or absence of short‐chain fatty acids (SCFA). The transepithelial conductance (G_t) as an indicator of epithelial integrity and the short‐circuit current (I_sc) as an indicator of active electrogenic ion transfer were continuously monitored. At an initial mucosal pH of 6.6, there was no significant difference in G_t between the treatment groups. In the absence of both SCFA and CO₂, G_t remained constant when the mucosal solution was acidified to pH 5.0. In the presence of SCFA, mucosal acidification induced a significant rise in G_t when the solutions were gassed with oxygen. A small increase in G_t was observed in the mucosal presence of CO₂. However, no difference in final G_t was observed between SCFA‐containing and SCFA‐free conditions under carbon dioxide gassing during stepwise mucosal acidification. The SCFA+proton‐induced increase in G_t could also be minimized by serosal gassing with carbogen. Because of the SCFA+proton‐induced changes in G_t and their attenuation by CO₂, a protective role for mucosally available carbon dioxide may be assumed. We suggest that this effect may be due to the intraepithelial conversion of carbon dioxide to bicarbonate. However, the serosal presence of CO₂ at a physiological concentration may be sufficient to protect the epithelia from SCFA+proton‐induced damage for a certain period of time.     

Microbial protein formation of different carbohydrates in vitro

The aim of this study was to investigate the microbial protein yield of different pure carbohydrates to contribute to a more precise prediction of the microbial protein formed in the rumen. In a first experiment, sucrose, wheat starch, microcrystalline cellulose and citrus pectin were incubated for 8 and 24 hr in the modified Hohenheim gas test (HGT) system (3 runs × 2 syringes) including gas production, ammonia and short‐chain fatty acid concentration measurements. Ammonia values were used for estimation of the microbial protein formation. In a second experiment, the same substrates were incubated for 96 hr in the HGT system (2 runs × 3 syringes) and gas production was measured after 2, 4, 6, 8, 12, 16, 24, 30, 36, 48, 60, 72 and 96 hr of incubation to obtain the fermentation kinetics and the time of half‐maximal gas production (t_1/2) of the substrates. The substrates differed considerably in their fermentation kinetics, and therefore, comparison on the basis of t_1/2 was chosen as the most meaningful. At t_1/2, microbial protein yield [g/kg dry matter] was higher for cellulose than for sucrose and pectin and higher for starch than for sucrose. The microbial protein expressed in g/L gas production was higher for starch and cellulose than for sucrose and pectin at t_1/2. Effects of carbohydrates related to ruminal pH may remain undetected in in vitro trials.     

Effect of medicinal and aromatic plants on rumen fermentation,protozoa population and methanogenesis in vitro

The potential of tannins from 21 medicinal and aromatic plant leaves as antimethanogenic additives in ruminant feeds was investigated. The effect of tannin from these leaves on rumen fermentation parameters, protozoa population and methanogenesis was studied by incubating the samples [200 mg dry matter (DM)] without and with polyethylene glycol (PEG)‐6000 (400 mg DM) as a tannin binder during 24‐h incubation in the in vitro Hohenheim gas method. Based on the methane percentage estimated in the total gas produced, methane production in millilitre was calculated [methane volume (ml) = methane % × total gas produced (ml) in 24 h]. In the samples, crude protein and neutral detergent fibre (g/kg DM) ranged from 113 to 172 and from 352 to 444 respectively. The total phenol (TP; g/kg DM) content was highest in Terminalia chebula (274) followed by Hemigraphis colorata (71) and Sapindus laurifolia (51) respectively. In the remaining samples, it was <43 g/kg DM. Activity of tannins, as represented by the increase in gas volume on addition of PEG, ranged from 0 to 133%, with the highest being recorded in T. chebula. The per cent increase in methane on PEG addition was 0 for Ammi majus, Aristolochia indica, Cascabela thevetia, Ipomea nil and Lantana camara, illustrating that tannins present in these samples had no effect on methane concentration. The PEG addition increased the total protozoa count by >50% in A. indica and C. thevetica. One of the important findings of our study was that of the 21 samples screened, Entodinia population increased in 12 with PEG as compared to 7 where Holotricha increased, indicating higher susceptibility of Entodinia to tannin. There was no increase in the protozoa population with PEG when incubating Cardiospermum halicacabum, Clerodendrum inerme, Dioscorea floribunda, Nerium oleander and Selastras paniculatus, which strongly suggested that methane suppression recorded in these samples was not because of a defaunating effect of their tannins per se. The fermentation pattern reflected increased total volatile fatty acid (TVFA) concentration from 0 to 28.3% with PEG addition among the leaves. Our results confirmed further observations that methanogenesis in vitro is not essentially related to density of protozoa population. Secondly, medicinal and aromatic plants such as C. inerme, Gymnema sylvestre and Sapindus laurifolia containing tannins appear to have a potential to suppress in vitro methanogenesis.     

Effect of cellobiose supplementation on in vitro fermentation activity and bacterial numbers of porcine inocula

In this in vitro study, the modified Hohenheim gas test (HGT) was applied to determine fermentation activity and bacterial composition of pig's faecal microbial inoculum using different concentrations of cellobiose. Incubation procedures included normal buffered and osmotic stress conditions (elevated medium salinity). After 24 hr of fermentation, production of gas, ammonia and short‐chain fatty acid (SCFA) was measured, and the gene copy numbers of total bacteria, Lactobacillus spp., Bifidobacterium spp., Roseburia spp., Clostridium Cluster IV spp. and Enterobacteriaceae were analysed using real‐time polymerase chain reaction. There was a significant reduction in gas production after 24 hr when comparing osmotic stress conditions with normal buffered conditions. Under osmotic stress, increasing cellobiose concentrations linearly increased gas production (p < .001), while ammonia, acetic acid and isobutyric acid concentrations decreased (p < .001, p = .012, p = .035 respectively). Under normal buffered conditions, Roseburia spp. gene copies linearly increased with increasing cellobiose concentrations (p = .048). Lactobacillus spp. and Bifidobacterium spp. numbers were higher under osmotic stress (p < .001) compared to normal conditions. Results might point towards a positive impact of cellobiose supplementation on gut health especially under osmotic stress conditions.     

In vitro microbial protein synthesis,ruminal degradation and post‐ruminal digestibility of crude protein of dairy rations containing Quebracho tannin extract

This study evaluated the effects of Quebracho tannin extract (QTE) on in vitro ruminal fermentation, chemical composition of rumen microbes, ruminal degradation and intestinal digestibility of crude protein (iCPd). Three treatments were tested, the control (basal diet without QTE), the basal diet with 15 g QTE/kg dry matter (DM) and the basal diet with 30 g QTE/kg DM. The basal diet contained (g/kg DM): 339 grass silage, 317 maize silage and 344 concentrate. In vitro gas production kinetic was determined using the Hohenheim gas test (Experiment 1). The Ankom RF technique, a batch system with automatic gas pressure recordings, was used to determine in vitro production of short‐chain fatty acids (SCFA) and ammonia–nitrogen concentration (NH₃‐N), as well as nitrogen and purine bases content in liquid‐associated microbes (LAM) and in a residue of undegraded feed and solid‐associated microbes (Feed+SAM) (Experiment 2). Ruminal degradation and iCPd were determined using the nylon bag technique and the mobile nylon bag technique, respectively (Experiment 3). Gas production (Experiment 1), total SCFA and NH₃‐N (Experiment 2) decreased with increasing QTE levels. Microbial mass and composition of LAM were not affected by QTE, but total mass of Feed+SAM linearly increased, likely due to decreased substrate degradation with increasing QTE levels. The total amount of N in microbial mass and undegraded feed after the in vitro incubation increased with increasing QTE levels, suggesting a potential greater N flow from the rumen to the duodenum. In contrast to in vivo studies with the same QTE, no effects were detected on ruminal effective degradability and iCPd, when using the nylon bag techniques. Based on the in vitro procedures, QTE increased the supply of N post‐rumen; however, some evidence of a decreased fibre degradation were also observed. Therefore, the benefit of adding QTE to diets of cattle is still questionable.     

Effect of methanol on methanogenesis and fermentation in the rumen simulation technique (RUSITEC)

Introduction Major methanol sources in ruminant feeds are pectins esterified with methoxyl groups. Methanol can be released by pectin esterase activity of rumen bacteria (R exova -B enkova and M arkovic 1976). It has been detected in rumen fluid of cows in vivo and in vitro (V antcheva et al. 1970, 1972). It is, however, not likely to accumulate in the rumen fluid since it can be readily used by methylotrophic organisms. Methanogenic archaebacteria such as Methanosarcina barkeri (H utten et al. 1980; M& uuml ; ller et al. 1986) are able to use methanol as energy and carbon source. Two equations have been reported for this conversion: 4CH₄ → 3CH₄ + CO₂ + 2 H₂O (1) CH₃OH + H₂ → CH₄ + H₂O (2) Methanogenesis from methanol has been shown to occur during in vitro fermentations with rumen fluid as inoculum (C zerkawski and B reckenridge 1972; P ol and D emeyer 1988). However, acidogenic micro-organisms, such as Eubacterium limosum or Butyribacterium methylotrophicum, may also utilize methanol. In rumen fluid of sheep fed a molasses-rich diet, Eubacterium limosum was the predominant methanol-utilizing bacterium (G enthner et al. 1981). Major products of these acidogenic methylotrophs are acetate and butyrate. These different fermentation end-products, methane versus fatty acids, will affect the metabolizable energy content of feedstuffs that are rich in highly methoxylated pectins or other methyl group-containing compounds. The purpose of the present study was to investigate whether the rumen simulation technique (RUSITEC) would be a suitable model to evaluate the methanogenic or acidogenic potential of methanol during ruminal fermentation. The study focused on methane production, fermentation characteristics and methanol turnover. Fermentation traits included pH, redox potential, ammonia and volatile fatty acid production, and degradation of feed constituents.     

Reducing methane production by supplementation of Terminalia chebula RETZ. containing tannins and saponins

This study investigates the effects of Terminalia chebula Retz. meal supplementation on rumen fermentation and methane (CH₄) production by using an in vitro gas technique. The experimental design was a completely randomized design (CRD) and the dietary treatments were T. chebula supplementation at 0, 4, 8, 12, 16 and 20 mg with 0.5 g of roughage and concentrate ratio at 60:40. The results revealed that cumulative gas production (96 h of incubation) were higher (P < 0.01) with T. chebula supplementation at 12, 16 and 20 mg than other treatments. However, in vitro dry matter degradability (IVDMD) and in vitro organic matter digestibility (IVOMD) were not significantly different among treatments (P > 0.05). The NH₃‐N concentrations tended to quadratically increase with increasing levels of T. chebula in the diet. In addition, total volatile fatty acids (VFA) and propionate concentrations were increased (P < 0.01), while acetate concentration, acetate‐to‐propionate ratio, CH₄ production and protozoal populations were decreased (P < 0.01) when supplemented with T. chebula at 8, 12 and 16 mg, respectively. Based on this study, it could be concluded that supplementation of T. chebula at 12 mg could improve rumen fermentation by reducing CH₄ production and protozoa populations, thus improving in vitro gas production and VFA profiles.     

In vitro fermentation characteristics of different carbohydrate sources in two dog breeds (German shepherd and Neapolitan mastiff)

Few studies have been published on the normal intestinal biota of canines unlike the wealth of information regarding livestock animal species. The in vitro gas production technique (IVGPT) including measurements of accumulating gas during fermentation and end‐product determinations allows obtaining a complete picture of microbial activity kinetics. The aim of this study was to study the in vitro fermentation characteristics of different carbohydrate sources using inocula from two dog breeds (German Shepherd and Neapolitan mastiff). Faeces sampled from rectum of two GS and NM adult dogs, fed the same dry food, were used as inocula. The samples, diluted and filtered, were incubated at 39 °C under anaerobic condition with nine substrates different for carbohydrate composition (rice, corn, potato, spelt, pure cellulose, beet pulp, wheat bran, inulin and fructo‐oligosaccharide). Gas production was recorded 17 times using a manual pressure transducer. After 48 h, the fermentation was stopped and fermenting liquor was analysed for pH and volatile fatty acids (VFA). Organic matter digestibility (OMD) was calculated as difference after burning the residuals. OMD, gas production and end‐products were significantly correlated with chemical composition of substrates, in particular carbohydrate fractions (total dietary fibre and starch), confirming the effectiveness of the IVGPT in evaluating dog feeds. Concerning the comparison between breeds significant differences (p < 0.01) were found for OMD, gas production, fermentation kinetic parameters and end‐products, suggesting a different pathway of fermentation and consequently, a different anaerobic population.     

Dose–response effect of a pine bark extract on in vitro ruminal ammonia and methane formation kinetics

ABSTRACT

This study assessed the potential of a pine bark extract (PBE) to decrease methane (CH₄) and ammonia nitrogen (NH₃-N) production in vitro. Dietary substrates, mixed hay, soybean meal and corn grain, were supplemented 0, 2, 4 and 6% of PBE and incubated in an in vitro batch culture for 24-h. Incubations were run three times. Total gas production (GP) was determined at 6, 12 and 24-h and gas samples were analysed for CH₄. Samples were collected for volatile fatty acids (VFA) and NH₃-N analysis. Treatments were compared by polynomial contrasts for PBE concentration. Increasing PBE caused linear decreases in NH₃-N, microbial biomass production and digestibility, whereas the degradation rate was quadratically reduced. Total VFA were decreased but total GP and CH₄ production and kinetics were unaffected. The inclusion of 2% PBE in ruminant feed has the capability to reduce NH₃-N concentration by 50%, without affecting diet digestibility or CH₄ production.     

Effect of cashew nut shell liquid on metabolic hydrogen flow on bovine rumen fermentation

Effect of cashew nut shell liquid (CNSL), a methane inhibitor, on bovine rumen fermentation was investigated through analysis of the metabolic hydrogen flow estimated from concentrations of short‐chain fatty acids (SCFA) and methane. Three cows were fed a concentrate and hay diet without or with a CNSL‐containing pellet. Two trials were conducted using CNSL pellets blended with only silica (trial 1) or with several other ingredients (trial 2). Methane production was measured in a respiration chamber system, and energy balance and nutrient digestibility were monitored. The estimated flow of metabolic hydrogen demonstrated that a part of metabolic hydrogen was used for hydrogen gas production, and a large amount of it flowed into production of methane and SCFA in both trial 1 and 2, when CNSL was administered to the bovine rumen. The results obtained by regression analyses showed that the effect of CNSL supply on methane reduction was coupled with a significant (P < 0.01) decrease of acetate and a significant (P < 0.01) increase of propionate and hydrogen gas. These findings reveal that CNSL is able to reduce methane and acetate production, and to increase hydrogen gas and propionate production in vivo.     

The effect of various anionic salts on ruminal pH and short-chain fatty acids in non-pregnant and non-lactating cows

AIM: To evaluate whether the different anionic salts used in the prevention of parturient paresis have an impact on the ruminal pH and the production of short-chain fatty acids (SCFA) in mature non-pregnant, non-lactating cows.

METHODS: Eleven Holstein-Friesian crossbred cows were administered 2,000 mEq of either one of three chloride salts, viz CaCl₂, MgCl₂, or NH₄Cl; four sulphate salts, viz CaSO₄, CaSO₄ with a grain size of 10 µm, MgSO₄, or (NH₄)₂SO₄; two combinations of anionic salts, viz CaCl₂+MgSO₄, or CaSO₄+NH₄Cl; NaCl; or water, via a ruminal cannula over a 14-day treatment period. The salts and controls were assigned in an 11 × 11 Latin square, and the cows were distributed randomly. Ruminal fluid was collected four times in each treatment period for monitoring the ruminal pH, and four times a day at Days 7 and 14 for monitoring any changes in the concentrations of SCFA.

RESULTS: Feeding anionic salts did not change the ruminal pH, total concentration of SCFA, or distribution pattern of the main SCFA acetic acid, propionic acid, butyric acid or valeric acid (p>0.05).

CONCLUSIONS AND CLINICAL RELEVANCE: Feeding anionic salts has no negative side effect on the ruminal pH and concentrations of SCFA in mature non-pregnant, non-lactating cattle. Impaired function of the rumen due to the feeding of anionic salts is not likely.