Copper Sulfate-induced Fermentation Changes in Continuous Cultures of the Rumen Microbial Ecosystem

The effect of CuSO(4) on fermentation was studied in a continuously cultured rumen ecosystem. CuSO(4), introduced at a level of 50 mg/500 ml of culture volume twice daily, caused a marked inhibition of fermentation of concentrates. Fermentation of alfalfa hay was not inhibited by the same CuSO(4) concentration when the inoculum for the culture was obtained from a cow maintained on a normal concentrate ration. When the inoculum was from a cow on a high concentrate ration, hay fermentation was partially inhibited by CuSO(4). Concentrations of CuSO(4) that did not inhibit the fermentation of alfalfa hay or hay-concentrate mixtures caused preferential production of propionic acid and decreased production of methane.     

Effect of plant oils and organic acids on rumen fermentation in vitro

We determined the effect of plant oils (rapeseed, sunflower, linseed) and organic acids (aspartic and malic) on the fermentation of diet consisting of hay, barley and sugar beet molasses. Rumen fluid was collected from two sheep (Slovak Merino) fed with the same diet twice daily. Mixed rumen microorganisms were incubated in fermentation fluid, which contained rumen fluid and Mc Dougall's buffer. All supplemented diets significantly increased pH, molar proportion of propionate, and numerically decreased methane production. Lactate production was also decreased significantly (except with malate). Incorporation of plant oils into aspartate- and malate-treated incubations negated the decrease of butyrate, lactate and the increase of pH and ammonia with malate treatment, as well asin vitro dry matter digestibility and pH with aspartate treatment. The effect of combined additives on methane production and molar proportion of propionate was lower compared with additives supplemented separately. Combination of additives had no additive effect on rumen fermentation. All additives decreased total protozoan counts in rumen fluid.     

Preliminary in vitro study on the effect of xanthohumol on rumen methanogenesis

There is a great interest in reducing the methane emission from ruminants as one possible cause of global warming. The aim of the presented study was to determine the effects of xanthohumol, one of the hop (Humulus lupulus) phytofactors, on methane production, microbial population and basic parameters of ruminal fermentation. The experiment was carried out in a batch culture system. The basic substrate (400 mg) consisting of meadow hay and barley meal (60:40) was supplemented with 0 (Control), 0.1, 0.2 or 1.0 mg of xanthohumol. The basic parameters of rumen fermentation and composition of microbial population were measured after 24 h of incubation. For the first time, the results of this in vitro study have demonstrated that xanthohumol is capable to reduce the methane production, even at the lowest dose applied (0.1 mg/400 mg). The observed reduction in methane production by 12-13% was not accompanied by altering the basic rumen fermentation parameters. However, the practical utility of this supplement needs further investigation under long-term in vivo conditions.     

Survival of the recombinant Bacteroides thetaiotaomicron strain BTX in in vitro rumen incubations

The survival of Bacteroides thetaiotaomicron strain BTX under rumen-simulating conditions was studied. Strain BTX is a recombinant variant of strain 5482 engineered for the production of high levels of xylanase, an enzyme important in the degradation of hemicellulose. Strain BTX was not inhibited by compounds present in rumen fluid and it grew well in media containing rumen fluid (up to 75%) or high concentrations of volatile fatty acids (total concentration, 100 mmol l⁻¹). The ability of strain BTX to compete with other micro-organisms under rumen-like conditions was studied in in vitro incubations of rumen contents. These experiments employed a consecutive batch culture (CBC) system consisting of alfalfa suspended in a rumen fluid buffer inoculated with blended rumen contents and maintained by transfers (10%, v/v) at 48 h intervals. CBC cultures contained a diversity of microbial morphotypes and accumulated fermentation products in rumen-like proportions. When added alone, the numbers of BTX cells were maintained for only a few hours, and then declined precipitously until undetectable after 48 h. If CBC cultures were also supplemented with chondroitin sulphate (a mucopolysaccharide used by Bact. thetaiotaomicron ), strain BTX grew and the pattern of its population generally followed that of the total population of ruminal bacteria in these cultures. When transferred into fresh CBC cultures containing chondroitin sulphate, BTX was again able to grow and increase in numbers, but to a diminished degree. Although BTX was able to survive and maintain itself in chondroitin sulphate supplemented cultures, this was at a very low level (10¹⁰ ml⁻¹). The potential for manipulation of rumen function by inoculation with recombinant bacteria is discussed.     

In Vitro Lactate Metabolism by Ruminal Ingesta

Ruminal ingesta (300 ml) obtained from a fistulated cow fed alfalfa hay (H), 3.6 kg of grain mixture with corn silage fed ad libitum (S), 2.5:1 grain-alfalfa hay mixture (G), or a 2.5:1 grain-alfalfa hay mixture providing 545 g of sodium and calcium lactate daily (L) were incubated for 8 hr with nonpolymerized sodium lactate or 17% polymerized lactic acid neutralized to pH 6.7. Polymerization had no effect on the rate of lactate utilization. The initial rates of lactate metabolism for the H, G, S, and L ingesta were 0.72, 0.95, 1.8, and 3.4 meq per 100 ml of rumen fluid per hr, respectively. Lactate-2-(14)C was incubated for 4 hr with each type of ruminal ingesta. Of the label recovered in the volatile fatty acids (VFA), 74.1, 61.2, 49.3, and 38.9% was recovered in acetate, and 9.4, 19.8, 23.3, and 51.9% was recovered in propionate with H, G, S, and L ingesta, respectively. The balance of label was distributed between butyrate and valerate. The titratable VFA did not follow this pattern of production. With the hay ingesta, lactate metabolism resulted in a net loss of acetate and a large increase in butyrate. Little propionate was produced. The G, S, and L ingesta metabolized lactate to yield progressively more propionate and less butyrate. Evidence was gathered to suggest that acetate was the primary end product of lactate metabolism but that oxidation of lactate to pyruvate dictated the synthesis of butyrate from acetate to maintain an oxidation-reduction balance. It was noted that acetate and butyrate production from lactate was pH-dependent, with acetate production maximal at pH 7.4 and butyrate at 6.2. Propionate production was largely unaffected within this pH range.     

Fermentation Capacity as a Measure of Net Growth of Rumen Microorganisms

A simple technique for measuring the rate of fermentation of rumen microorganisms is described. It allows quick preparation and handling of the rumen sample immediately after collection. The average rate of fermentation of rumen samples collected from a lactating cow fed on alfalfa hay and concentrate in the ratio of 2:1 was very similar to the rate obtained by other methods. On the assumption that when substrate is in excess, the fermentation rate is proportional to the total microbial cells, the method was used to estimate the net growth of rumen microorganisms. The maximal fermentation rate of subsamples, taken at the beginning and after 1 hr of incubation of a sample, was measured. The results indicate a net average growth of 8% per hr, or 192% per day, in approximate agreement with rumen turnover time. The highest net growth does not necessarily coincide with the highest gas-production rate in the rumen, in part because the bicarbonate concentration in the rumen contents varies. In a cow fed on hay and concentrate, the net growth was lowest before feeding and immediately after feeding.     

Effect of carbon monoxide on fermentation of fiber, starch, and amino acids by mixed rumen microorganisms in vitro.

When 1 atm (101.3 kPa) of carbon monoxide was added to mixed rumen bacterial incubations containing timothy hay, methane production was inhibited by 88% without an increase in hydrogen. The molar ratio of propionate to acetate increased from 0.83 to 1.53, extracellular ammonia declined from 5.2 to 2.4 mM, and hemicellulose and cellulose digestions were inhibited by 40 and 27%, respectively. Even low levels of carbon monoxide (less than 0.1 atm [10.13 kPa]) significantly changed the products of fermentation. With starch, methane production was once again inhibited, but the magnitude of starch fermentation was unaffected. Decrease in acetate was accompanied by an equal molar increase in lactate. Ammonia production from the amino acid source, Trypticase, declined 20% as carbon monoxide was increased to 1.0 atm, and 93% of this decrease was explained by a selective inhibition of branched-chain amino acid fermentation.     

Effect of carbon monoxide on fermentation of fiber, starch, and amino acids by mixed rumen microorganisms in vitro

When 1 atm (101.3 kPa) of carbon monoxide was added to mixed rumen bacterial incubations containing timothy hay, methane production was inhibited by 88% without an increase in hydrogen. The molar ratio of propionate to acetate increased from 0.83 to 1.53, extracellular ammonia declined from 5.2 to 2.4 mM, and hemicellulose and cellulose digestions were inhibited by 40 and 27%, respectively. Even low levels of carbon monoxide (less than 0.1 atm [10.13 kPa]) significantly changed the products of fermentation. With starch, methane production was once again inhibited, but the magnitude of starch fermentation was unaffected. Decrease in acetate was accompanied by an equal molar increase in lactate. Ammonia production from the amino acid source, Trypticase, declined 20% as carbon monoxide was increased to 1.0 atm, and 93% of this decrease was explained by a selective inhibition of branched-chain amino acid fermentation.     

Stoichiometry of carbohydrate fermentation and microbial growth efficiency in a continous culture of mixed rumen bacteria

Summary A population of mixed rumen bacteria was maintained in a chemostat at four different dilution rates, with glocose as the growth limiting carbon and energy substrate. Increasing the dilution rate shifted the proportions of end products: methane decreased and propionate increased. Fermentation and hydrogen balances were calculated from the fermentation end products. Values were similar to earlier ones from batch incubations of rumen contents. This suggests that theoretical overall reaction schemes for carbohydrate fermentation in the rumen, proposed earlier, are also valid in continuous culture.A positive correlation between dilution rate and microbial growth efficiency (gN_inc./kg OM_f was observed, confirming earlier work.Apparently conflicting results of chemostat work and recent in vivo experiments are discussed.     

Effect of tea saponin on methanogenesis,microbial community structure and expression of mcrA gene,in cultures of rumen micro‐organisms

Aims: To determine the in‐vitro effect and mode of action of tea saponin on the rumen microbial community and methane production. Methods and Results: Saponin extracted from tea seeds was added to (1) an in‐vitro fermentation inoculated with rumen fluid and (2) a pure culture of Methanobrevibacter ruminantium. Methane production and expression of the methyl coenzyme‐M reductase subunit A (mcrA) were monitored in both cultures. Abundance of methanogens, protozoa, rumen fungi and cellulolytic bacteria were quantified using real‐time PCR, and bacterial diversity was observed using denaturing gradient gel electrophoresis. Addition of tea saponin significantly reduced methane production and mcrA gene expression in the ruminal fermentation but not with the pure culture of M. ruminantium. The abundance of protozoa and fungi were significantly decreased 50% and 79% respectively but methanogen numbers were not affected, and Fibrobacter succinogenes increased by 41%. Bacterial diversity was similar in cultures with or without tea saponin. Conclusions: Tea saponin appeared to reduce methane production by inhibiting protozoa and presumably lowering methanogenic activity of protozoal‐associated methanogens. Significance and Impact of the Study: Tea saponin may be useful as a supplement to indirectly inhibit methane production in ruminants without a deleterious effect on rumen function.     

Effects of dietary forage particle size and concentrate level on fermentation profile, in vitro degradation characteristics and concentration of liquid- or solid-associated bacterial mass in the rumen of dairy cows

This study investigated effects of dietary forage particle size (PS) and concentrate level (CL) on fermentation profiles of particle-associated rumen liquid (PARL) and free rumen liquid (FRL), in vitro degradation characteristics and concentration of bacterial mass attached to the solid or fluid rumen digesta phase in dairy cows. The experiment was a 4 × 4 Latin square design with four late-lactation dairy cows in four 23 day periods. Cows were restrictively fed (17 kg dry matter (DM)/d) one of four diets varying in the theoretical PS (6 and 30 mm) of grass hay and in the levels (approximately 200 and 550 g/kg, DM basis) of a cereal-based concentrate. Proportion of large particles (>6 mm) and the content of structural fibre in the diet increased by reducing dietary CL and, particularly, by increasing hay PS. This effect was not reflected by changes in mean total volatile fatty acid concentration or pH in the rumen. However, cows fed high concentrate diets had pH of 5.28 and 5.37 in PARL at 3 h after the last meal, when fine or long chopped hay was offered. The low pH may indicate a depression of the capacity of PARL to degrade fibre in vitro. Gas production in vitro of concentrate increased with the high concentrate diet at 12 h, suggesting that amylolytic capacity was affected only in early phases of fermentation. In addition, elevating dietary CL appeared to shift ruminal fermentation outputs from propionate to butyrate and valerate. Inclusion of coarsely chopped hay to a high concentrate diet does not appear to bring advantages due to increased structure in restrictively fed dairy cows. In addition, results suggest that the response of pH in PARL is more sensitive to dietary changes (i.e., forage PS and CL) than the response in FRL, and so PARL might be better to evaluate the risk of ruminal disfunction in dairy cows.     

Characteristics of anaerobic oxalate-degrading enrichment cultures from the rumen.

Enrichment cultures of rumen bacteria degraded oxalate within 3 to 7 days in a medium containing 10% rumen fluid and an initial level of 45 mM sodium oxalate. This capability was maintained in serially transferred cultures. One mole of methane was produced per 3.8 mol of oxalate degraded. Molecular hydrogen and formate inhibited oxalate degradation but not methanogenesis; benzyl viologen and chloroform inhibited both oxalate degradation and methanogenesis. Attempts to isolate oxalate-degrading bacteria from these cultures were not successful. Oxalate degradation was uncoupled from methane production when enrichments were grown in continuous culture at dilution rates greater than or equal to 0.078 h-1. Growth of the uncoupled population (lacking methanogens) in batch culture was accompanied by degradation of 45 mM oxalate within 24 h and production of 0.93 mol of formate per mol of oxalate degraded. Oxalate degradation by the uncoupled population was not inhibited by molecular hydrogen or formate. Cell yields (grams [dry weight]) per mole of oxalate degraded by the primary enrichment and the uncoupled populations were 1.7 and 1.0, respectively.     

Effect of insulin on in vitro fermentation activity of microrganism community of rumen ciliate Entodinium caudatum culture

The influence of insulin (17.4 nmol l-1) on total gas and methane production, the concentration of total and individual fatty acids and dry matter degradability was investigated in the rumen ciliate culture of Entodinium caudatum. The experimental groups consisted of control group (without insulin) and two groups with insulin application--single shot and long-term application (over 30 days). Fermentation activity of each experimental group was observed on two subgroups: whole protozoan culture (protozoa plus bacteria) and bacterial fraction (bacteria without protozoa). Long-term application of insulin significantly increased methane production and DM degradability in the whole protozoan culture. Total VFA concentration was significantly increased by long-term as well as single-dose application of insulin (by 255% and 158%, respectively). The growth of the protozoa was not influenced by insulin treatments. It can be concluded that the fermentation activity of the community of the rumen ciliate Entodinium caudatum culture was marked stimulated by application of insulin.     

Rumen Fungi and Forage Fiber Degradation

The role of anaerobic rumen fungi in in vitro forage fiber degradation was determined in a two forage × two inoculum source × five treatment factorial design. Forages used as substrates for rumen microorganisms were Coastal bermuda grass and alfalfa; inoculum sources were rumen fluid samples from a steer fed Coastal bermuda grass hay or alfalfa hay; treatments were whole rumen fluid (WRF), WRF plus streptomycin (0.2 mg/ml of rumen fluid) and penicillin (1.25 mg/ml of fluid), WRF plus cycloheximide (0.5 mg/ml of fluid), WRF plus streptomycin, penicillin, and cycloheximide, and McDougall buffer. Populations of fungi as shown by sporangial development were greater on bermuda grass leaves than on alfalfa leaflets regardless of inoculum source. However, endogenous fungal populations were greater from the alfalfa hay inoculum. Cycloheximide inhibited the fungi, whereas streptomycin and penicillin, which inhibit bacterial populations, resulted in an increase in numbers of sporangia in the alfalfa inoculum, suggesting an interaction between bacteria and fungi. Bacteria (i.e., WRF plus cycloheximide) were equal to the total population in degrading dry matter, neutral-detergent fiber (NDF), acid-detergent fiber (ADF), and cellulose for both inocula and both forages. Degradation of dry matter, NDF, ADF, and cellulose by anaerobic fungi (i.e., WRF plus streptomycin and penicillin) was less than that due to the total population or bacteria alone. However, NDF, ADF, and cellulose digestion was 1.3, 2.4, and 7.9 percentage units higher, respectively, for bermuda grass substrate with the alfalfa versus bermuda grass inoculum, suggesting a slight benefit by rumen fungi. No substantial loss of lignin (72% H₂SO₄ method) occurred due to fungal degradation. The most active fiber-digesting population in the rumen was the bacteria, even when streptomycin and penicillin treatment resulted in an increase in rumen fungi over untreated WRF. The development of large numbers of sporangia on fiber may not indicate a substantial role as digesters of forage.     

Methanogenesis in rumen ciliate cultures ofEntodinium caudatum andEpidinium ecaudatum after long-term cultivation in a chemically defined medium

The methanogenic activity in the presence of Entodinium caudatum and Epidinium ecaudatum was well preserved after long-term cultivation. Microscopic observation revealed that methane production in the presence of E. caudatum was probably caused by their intracellular methanogenic activity, while methane production in the presence of E. ecaudatum f caudatum et ecaudatum could be attributed to both the methanogenic bacterial fraction of their external surface and their intracellular activity. Methane production per protozoan cell of E. caudatum and E. ecaudatum was 2.1 nmol per cell per d and 6.0 nmol per cell per d, respectively. E. caudatum was responsible for almost the entire methane production in the culture. The activity of free methanogens constituted approximately 50% of the total methane production in the E. ecaudatum culture. Decrease of digestibility of substrates and differences in the fermentation end products accompanied the inhibition of methanogenesis in both cultures by penicillin G, streptomycin, chloramphenicol, 2-bromoethanesulfonate, and pyromellitic diimide. E. caudatum appeared to be more sensitive than E. ecaudatum to the compounds tested. Hydrogen recoveries based on both volatile fatty acids and methane production suggested that the methanogenic population appeared not to be fully able to consume hydrogen produced in the protozoan cultures. The culture conditions tested were found to be suitable for experiments on the relationship between rumen ciliates and rumen bacteria.     

Evaluation of a Method of Measuring Fermentation Rates and Net Growth of Rumen Microorganisms

The method of el-Shazly and Hungate for measuring gas production in rumen contents was slightly modified and used throughout this investigation. The variation in the fermentation rates due to samples collected separately from a sheep fed on hay was less than 2%. When samples obtained through a stomach tube were compared with samples collected through the rumen fistula, the variation was about 3%. The rates of gas and acid production were approximately similar in samples obtained from the rumen at the same time when no sodium bicarbonate was added. During in vitro incubation of whole ruminal contents, there was a highly significant correlation between the net growth rate values (obtained by using fermentation capacity as an index) and the change in concentration of viable rumen bacteria or total ciliate protozoa.     

Rumen microbial changes in cattle fed diets with or without salinomycin.

Four rumen-fistulated steers, randomly assigned to two groups (control and salinomycin fed) were used to monitor the changes in rumen microbial populations and volatile fatty acids (VFA) concentrations associated with feeding salinomycin (0.22 mg X kg-1 X day-1). Steers were adapted to an alfalfa hay and grain (80:20) diet before supplementing the diet with salinomycin, and then the diet was changed to 50:50 and 20:80 ratios of alfalfa hay to grain at 2-week intervals. Rumen samples for total and selective enumeration of anaerobic bacteria. VFA analysis, and enumeration of protozoa were collected during the 80:20 alfalfa hay-to-grain diet before salinomycin feeding, and during the 80:20, 50:50, and 20:80 hay-to-grain diets with salinomycin. At each sampling period, rumen samples were collected at 3 h after feeding on three consecutive days. Salinomycin feeding had no effect on rumen pH and total VFA concentration. The acetate-to-propionate ratio was significantly lower in salinomycin-fed steers than in the control. The molar proportion of butyrate increased in both control and salinomycin-fed steers. Total anaerobic bacterial counts were lower in salinomycin-fed steers than in the control steers after 8 weeks of salinomycin feeding. Salinomycin-resistant bacteria increased from 7.6 to 15.6% in salinomycin-fed steers but remained unchanged in control steers. Salinomycin had no effect on cellulolytic and lactate-utilizing bacteria, but the proportion of amylolytic bacteria was higher in salinomycin-fed steers than in control steers. The total number of protozoa decreased initially in salinomycin-fed steers. The initial reduction was due to reduced numbers of Entodinium species. Holotrichs were unaffected by salinomycin feeding.     

Rumen microbial changes in cattle fed diets with or without salinomycin

Four rumen-fistulated steers, randomly assigned to two groups (control and salinomycin fed) were used to monitor the changes in rumen microbial populations and volatile fatty acids (VFA) concentrations associated with feeding salinomycin (0.22 mg X kg-1 X day-1). Steers were adapted to an alfalfa hay and grain (80:20) diet before supplementing the diet with salinomycin, and then the diet was changed to 50:50 and 20:80 ratios of alfalfa hay to grain at 2-week intervals. Rumen samples for total and selective enumeration of anaerobic bacteria. VFA analysis, and enumeration of protozoa were collected during the 80:20 alfalfa hay-to-grain diet before salinomycin feeding, and during the 80:20, 50:50, and 20:80 hay-to-grain diets with salinomycin. At each sampling period, rumen samples were collected at 3 h after feeding on three consecutive days. Salinomycin feeding had no effect on rumen pH and total VFA concentration. The acetate-to-propionate ratio was significantly lower in salinomycin-fed steers than in the control. The molar proportion of butyrate increased in both control and salinomycin-fed steers. Total anaerobic bacterial counts were lower in salinomycin-fed steers than in the control steers after 8 weeks of salinomycin feeding. Salinomycin-resistant bacteria increased from 7.6 to 15.6% in salinomycin-fed steers but remained unchanged in control steers. Salinomycin had no effect on cellulolytic and lactate-utilizing bacteria, but the proportion of amylolytic bacteria was higher in salinomycin-fed steers than in control steers. The total number of protozoa decreased initially in salinomycin-fed steers. The initial reduction was due to reduced numbers of Entodinium species. Holotrichs were unaffected by salinomycin feeding.     

Effect of glyphosate contaminated feed on rumen fermentation parameters and in sacco degradation of grass hay and corn grain

Abstract Four rumen fistulated wethers were used to investigate the effect of glyphosate contaminated feed on rumen fermentation. The rations were based on corn silage, urea and a vitamin-mineral premix, either in the absence or presence of 0.77 g glyphosate per kg DM. Furthermore, rations were fed either with or without aromatic amino acid supplementation. During four periods of 28 days, sheep received each of the four dietary treatments according to a Latin square. After 14 days of adaptation rumen fermentation parameters (pH, ammonia, volatile fatty acids) were measured on day 15 over a five-hour period after the morning feeding. The remaining 13 days served for in sacco degradation studies with grass hay and corn grain. Ammonia (NH3) and pH of rumen fluid were within the normal range for all dietary treatments (NH3: 9.1-32.3 mmol x l(- l), pH: 6.2-6.7). Neither rumen fermentation parameters nor in sacco DM and NDF degradation of incubated feedstuffs were significantly affected by glyphosate, with or without aromatic amino acid supplementation. Kinetic profiles of the in sacco dry matter and NDF degradation of grass hay were almost identical for the dietary treatments.     

Effects of monolaurin on ruminal methanogens and selected bacterial species from cattle, as determined with the rumen simulation technique

Before being able to implement effective ruminal methane mitigation strategies via feed supplementation, the assessment of side effects on ruminal fermentation and rumen microbial populations is indispensable. In this respect we investigated the effects of monolaurin, a methane-mitigating lipid, on methanogens and important carbohydrate-degrading bacteria present in ruminal fluid of dairy cattle in continuous culture employing the rumen simulation technique. In six experimental runs, each lasting for 10 days, four diets with different carbohydrate composition, based on hay, maize, wheat and a maize-wheat mixture, either remained non-supplemented or were supplemented with monolaurin and incubated in a ruminal-fluid buffer mixture. Incubation liquid samples from days 6 to 10 of incubation were analyzed with relative quantitative polymerase chain reaction (qPCR) of 16S rRNA genes to assess monolaurin-induced shifts in specific rumen microbial populations in relation to the corresponding non-supplemented diets. Monolaurin completely inhibited Fibrobacter succinogenes in all diets while the response of the other cellulolytic bacteria varied in dependence of the diet. Megasphaera elsdenii remained unaffected by monolaurin in the two diets containing maize, but was slightly stimulated by monolaurin with the wheat and largely with the hay diet. The supply of monolaurin suppressed Methanomicrobiales below the detection limit with all diets, whereas relative 16S rRNA gene copy numbers of Methanobacteriales increased by 7-fold with monolaurin in case of the hay diet. Total Archaea were decreased by up to over 90%, but this was significant only for the wheat containing diets. Thus, monolaurin exerted variable effects mediated by unknown mechanisms on important ruminal microbes involved in carbohydrate degradation, along with its suppression of methane formation. The applicability of monolaurin for methane mitigation in ruminants thus depends on the extent to which adverse effects on carbohydrate-degrading bacteria actually impair the supply of digested carbohydrates to the animal.