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Phthalates are endocrine-disrupting chemicals linked to a higher risk of numerous chronic health outcomes. Diet is a primary source of exposure, but prior studies exploring associations between dietary patterns and phthalate exposure are limited.We evaluated the associations between dietary patterns and urinary phthalate biomarkers among a subset of postmenopausal women participating in the Women's Health Initiative (WHI).We included WHI participants selected for a nested case-control study of phthalates and breast cancer (N = 1240). Dietary intake was measured via self-administered food frequency questionnaires at baseline and year-3. We used these data to calculate scores for alignment with the Dietary Approach to Stop Hypertension (DASH), alternative Mediterranean (aMed), and Dietary Inflammatory Index (DII) diets. We measured 13 phthalate metabolites and creatinine in 2-3 urine samples per participant collected over 3-years when all participants were cancer-free. We fit multivariable generalized estimating equation models to estimate the cross-sectional associations.DASH and aMed dietary scores were inversely associated with the sum of di(2-Ethylhexyl) phthalate (-6.48%, 95% CI -9.84, -3.00; -5.23%, 95% CI -8.73, -1.60) and DII score was positively associated (9.00%, 95% CI 5.04, 13.11). DASH and aMed scores were also inversely associated with mono benzyl phthalate and mono-3-carboxypropyl phthalate. DII scores were positively associated with mono benzyl phthalate and the sum of di-n-butyl phthalate.Higher dietary alignment with DASH and aMed dietary patterns were significantly associated with lower concentrations of certain phthalate biomarkers, while an inflammatory diet pattern was associated with higher phthalate biomarker concentrations. These findings suggest that dietary patterns high in fruits, vegetables, and low-fat foods and low in processed foods may be useful in avoiding exposure to phthalates.

Substituted long-chain fatty acids may be useful dietary supplements to suppress ruminal biohydrogenation of unsaturated fatty acids (UFA) and thereby increase the flow of UFA to meat and milk. The aim of this study was to determine if Vernonia galamensis (ironweed, a member of the sunflower family) and vernolic acid (cis-12,13-epoxy-cis-9-octadecenoic acid), the main constituent of the seed oil, affected the biohydrogenation of linoleic acid (LA; cis-9,cis-12-18:2) to rumenic acid (CLA; cis-9, trans-11-18:2), vaccenic acid (VA; trans-11-18:1) and stearic acid (SA; 18:0) by ruminal microorganisms. Ruminal digesta from four sheep receiving a mixed hay-concentrate diet were incubated in vitro with LA (0.2 g/L) ± 0.2 g/L vernolic acid or 5 g/L of dried flowers or leaves of V. galamensis, either alone or combined. Vernolic acid had a substantial effect on LA metabolism, causing decreases in cis-9, trans-11 CLA and VA accumulation as well as SA production (P

Studies on microbial biohydrogenation of fatty acids in the rumen are of importance as this process lowers the availability of nutritionally beneficial unsaturated fatty acids for incorporation into meat and milk but also might result in the accumulation of biologically active intermediates. The impact was studied of adsorption of 22:6n-3 (DHA) to particulate material on its disappearance during 24 h in vitro batch incubations with rumen inoculum. Four adsorbants were used in two doses (1 and 5 mg/ml of mucin, gum arabic, bentonite or silicic acid). In addition, the distribution of 22:6n-3 in the pellet and supernatant of diluted rumen fluid was measured. Bentonite and silicic acid did not alter the distribution of 22:6n-3 between pellet and supernatant nor increased the disappearance of 22:6n-3 during the incubation. Both mucin and gum arabic increased the recovery of 22:6n-3 in the supernatant, indicating that these compounds lowered the adsorption of the fatty acid to ruminal particles. This was associated with an increased disappearance of 22:6n-3, when initial 22:6n-3 was 0.06 or 0.10 mg/ml, and an increased formation of 22:0, when initial 22:6n-3 was 0.02 mg/ml, during the 24 h batch culture experiment. Addition of gum arabic to pure cultures of Butyrivibrio fibrisolvens or Butyrivibrio proteoclasticus did not negate the inhibitory effect of 22:6n-3 on growth. As both mucin and gum arabic provide fermentable substrate for ruminal bacteria, an additional experiment was performed in which mucin and gum arabic were replaced by equal amounts of starch, cellulose or xylan. No differences in disappearance of 22:6n-3 were observed, suggesting that the stimulatory effect of mucin and gum arabic on disappearance of 22:6n-3 most probably is not due to provision of an alternative site of adsorption but related to stimulation of bacterial growth. A relatively high proportion of 22:6n-3 can be reduced to 22:0 provided the initial concentration is low.

Background: Rumen microbes metabolize 22:6n-3. However, pathways of 22:6n-3 biohydrogenation and ruminal microbes involved in this process are not known. In this study, we examine the ability of the well-known rumen biohydrogenating bacteria, Butyrivibrio fibrisolvens D1 and Butyrivibrio proteoclasticus P18, to hydrogenate 22:6n-3. Results: Butyrivibrio fibrisolvens D1 failed to hydrogenate 22:6n-3 (0.5 to 32 mu g/mL) in growth medium containing autoclaved ruminal fluid that either had or had not been centrifuged. Growth of B. fibrisolvens was delayed at the higher 22:6n-3 concentrations; however, total volatile fatty acid production was not affected. Butyrivibrio proteoclasticus P18 hydrogenated 22:6n-3 in growth medium containing autoclaved ruminal fluid that either had or had not been centrifuged. Biohydrogenation only started when volatile fatty acid production or growth of B. proteoclasticus P18 had been initiated, which might suggest that growth or metabolic activity is a prerequisite for the metabolism of 22:6n-3. The amount of 22:6n-3 hydrogenated was quantitatively recovered in several intermediate products eluting on the gas chromatogram between 22:6n-3 and 22:0. Formation of neither 22:0 nor 22:6 conjugated fatty acids was observed during 22:6n-3 metabolism. Extensive metabolism was observed at lower initial concentrations of 22:6n-3 (5, 10 and 20 mu g/mL) whereas increasing concentrations of 22:6n-3 (40 and 80 mu g/mL) inhibited its metabolism. Stearic acid formation (18:0) from 18:2n-6 by B. proteoclasticus P18 was retarded, but not completely inhibited, in the presence of 22:6n-3 and this effect was dependent on 22:6n-3 concentration. Conclusions: For the first time, our study identified ruminal bacteria with the ability to hydrogenate 22:6n-3. The gradual appearance of intermediates indicates that biohydrogenation of 22:6n-3 by B. proteoclasticus P18 occurs by pathways of isomerization and hydrogenation resulting in a variety of unsaturated 22 carbon fatty acids. During the simultaneous presence of 18:2n-6 and 22:6n-3, B. proteoclasticus P18 initiated 22:6n-3 metabolism before converting 18:1 isomers into 18:0.

Concentrations of Na(+), K(+) and Ca(2+) in the growth medium were varied within limits normally found in vivo to determine how cation concentrations affect the sensitivity of ruminal bacteria to the ionophores, monensin (a Na(+)/H(+) and K(+)/H(+) exchanger) and tetronasin (Ca(2+)/H(+)). High [Na(+)] (172 mM cf. 137 mM in control medium) enhanced the efficacy of monensin towards Eubacterium ruminantium 2388, Streptococcus bovis C277, Lactobacillus casei LB17 and Prevotella albensis M384. High [K(+)] (35 mM cf. 19 mM) alone caused a decreased potency of both ionophores, except with L. casei. Added Ca(2+) (7.4 cf. 2.8 mM) increased the potency of tetronasin when [Na(+)] was low. High [Na(+)] alone also potentiated the efficacy of tetronasin. Monensin caused intracellular [Na(+)] and [K(+)] to be decreased in the most sensitive of these organisms, E. ruminantium, whereas only intracellular [Ca(2+)] fell with tetronasin. The changes were small; however, Δp fell by only 20 mV after 2 h when ionophores caused immediate cessation of growth. ATP concentrations fell by 77% and 75% with monensin and tetronasin, respectively. Thus, altering cation concentrations might be used to potentiate the efficacy of ionophores, by increasing the rate of energy expenditure to maintain ionic homoeostasis in sensitive bacteria.

Ricinoleic acid (RA; 12-hydroxy-cis-9-18:1) is the main fatty acid component of castor oil. Although a precursor for CLA synthesis in lactic acid bacteria, RA was found previously not to form CLA in ruminal digesta but to have some inhibitory properties. The present study was undertaken to evaluate the potential of RA to modulate ruminal biohydrogenation and methanogenesis. Ruminal digesta from 4 sheep receiving a mixed hay-concentrate diet was incubated in vitro with 0.167 g/L of linoleic acid (LA; cis-9,cis-12-18:2) or with a combination of LA and RA or LA and castor oil (LA, RA, and castor oil added to a final concentration of 0.167 g/L) in the presence and absence of lipase. The CLA rumenic acid (cis-9,trans-11-18:2) accumulated when either RA or castor oil and lipase was present. Vaccenic acid (VA; trans-11-18:1) also accumulated, and a decrease of the rate of production of stearic acid (SA; 18:0) was observed. When LA was incubated with castor oil in the absence of lipase, no effects on biohydrogenation were observed. Ricinoleic acid at 0.02 g/L did not affect growth of Butyrivibrio fibrisolvens but it inhibited growth of Butyrivibrio proteoclasticus. Butyrivibrio proteoclasticus but not B. fibrisolvens metabolized RA to 12-hydroxystearate. Linoleic acid metabolism by B. proteoclasticus appeared to be unaffected by RA addition whereas rumenic acid accumulation increased (P = 0.015 at 12 h) when RA was added. A 28% decrease (P = 0.004) in methane was obtained in 24 h in vitro incubations of diluted buffered ruminal fluid with added 0.2 g RA/L. There was no effect on the total concentration of VFA after 24 h as a result of RA addition, but the molar proportions of acetate and butyrate were decreased (P = 0.041 and P < 0.001, respectively) whereas that of propionate increased (P < 0.001). It was concluded that, at least in vitro, RA or the combination of castor oil and lipase inhibit biohydrogenation, causing the accumulation of rumenic acid and VA, with potential health benefits for ruminant products. The effect appeared to be mediated via an inhibitory effect on the biohydrogenating activity of B. proteoclasticus. An added environmental benefit could be a concomitant decrease in methane emissions. In vivo studies are now required to confirm the potential of these additives.

Inclusion of a daisy plant, Chrysanthemum coronarium, in a dairy sheep diet has been reported to result in increased concentrations of health-promoting rumenic acid (RA; cis-9,trans-11 CLA) and vaccenic acid (VA; trans-11-18:1) in milk. The aims of the present study were to determine if the reported change in milk fatty acid composition was the result of the effects of C. coronarium on the biohydrogenation of linoleic acid (LA; cis-9,cis-12-18:2) by ruminal microorganisms, and to investigate which constituents of C. coronarium may be responsible for the observed effects. Ruminal digesta from four sheep receiving a mixed hay-concentrate diet were incubated in vitro with LA in the presence or absence of dried whole-plant C. coronarium var. Primrose Gem. Rates of LA disappearance and stearic acid (SA: 18:0) production decreased as a result of C. coronarium addition, and VA accumulation doubled. Chrysanthemum parthenium and Chrysanthemum vulgare had much smaller effects on biohydrogenation. C. coronarium added to cultures of the only known ruminal SA-forming bacterium, Butyrivibrio proteoclasticus, also inhibited LA metabolism by, but not growth of, this species. Lipid analysis indicated that C coronarium var. Primrose Gem had a high content of a-linolenic acid (LNA; cis-9,cis-12,cis-15-18:3; 8.79 mg/g DM) compared to the other samples (

Compounds that mimic vertebrate hormone responses are found throughout the environment, and some are implicated in endocrine disruption. Endocrine disruption has been found in humans, wildlife, and even in the partnership of plants and root symbionts. Most endocrine disruption occurs in estrogenic systems. Estrogens, like other steroid hormones, binds a transcription factor known as a nuclear receptor to regulate gene transcription. Recent research has shown that there are other signaling mechanisms for steroid hormones that involve kinase pathways and G protein-coupled receptors. Mounting evidence suggests estrogen mimics can also act by these pathways which work outside the nucleus. Differential expression of these pathways across cell types, and differential affinity for these pathways by diverse compounds may explain some patterns of endocrine disruption and disease.

Despite the fact that the ruminant diet is rich in polyunsaturated fatty acids (PUFA), ruminant products - meat, milk and dairy - contain mainly saturated fatty acids (SFA) because of bacterial lipolysis and subsequent biohydrogenation of ingested PUFA in the rumen. The link between SFA consumption by man and coronary heart disease is well established. In contrast, ruminant products also contain fatty acids that are known to be beneficial to human health, namely conjugated linoleic acids (CLAs). The aims of research in this field have been to understand the microbial ecology of lipolysis and biohydrogenation and to find ways of manipulating ruminal microbes to increase the flow of PUFA and CLA from the rumen into meat and milk. This review describes our present understanding of the microbial ecology of ruminal lipid metabolism, including some apparently anomalous and paradoxical observations, and the status of how the metabolism may be manipulated and the possible consequential effects on other aspects of ruminal digestion. Intuitively, it may appear that inhibiting the ruminal lipase would cause more dietary PUFA to reach the mammary gland. However, lipolysis releases the non-esterified fatty acids that form the substrates for biohydrogenation, but which can, if they accumulate, inhibit the whole process. Thus, increasing lipase activity could be beneficial if the increased release of non-esterified PUFA inhibited the metabolism of CLA. Rumen ciliate protozoa do not carry out biohydrogenation, yet protozoal lipids are much more highly enriched in CLA than bacterial lipids. How could this happen if protozoa do not metabolise PUFA? The answer seems to lie in the ingestion of plant organelles, particularly chloroplasts, and the partial metabolism of the fatty acids by contaminating bacteria. Bacteria related to Butyrivibrio fibrisolvens are by far the most active and numerous biohydrogenating bacteria isolated from the rumen. But do we misunderstand the role of different bacterial species in biohydrogenation because there are uncultivated species that we need to understand and include in the analysis? Manipulation methods include dietary vegetable and fish oils and plant-derived chemicals. Their usefulness, efficacy and possible effects on fatty acid metabolism and on ruminal microorganisms and other areas of their metabolism are described, and areas of opportunity identified.

Conjugated linoleic acids (CLA) are health-promoting fatty acids found in foods derived from ruminant products that are formed in the rumen during bacterial biohydrogenation of linoleic acid (LA). Although selective antimicrobials might increase CLA production by manipulation of ruminal microflora, feeding of antibiotic growth promoters to livestock is declining due to fears of development of antibiotic resistance in human pathogens. This has initiated a search for alternatives, including plants containing bioactive compounds, which can have similar positive effects on ruminal microflora, but without negative impact on animal and human health. In this study we investigated effects of natural compounds in Australian plants on the bacteria and processes involved in ruminal biohydrogenation. Ethanolic extracts and essential oils were obtained from 91 Australian plants collected during 2004/2005 in Western Australia. Minimal Inhibitory Concentrations for the main bacterial species involved in rumen biohydrogenation were established using an agar dilution method, and effects of selected candidates on LA biohydrogenation was examined using an in vitro system with a mixed rumen bacterial population. A wide range of the plant extracts had selective inhibitory effect towards Clostridium proteoclasticum (which forms stearate from LA) without affecting Butyrivibrio fibrisolvens (which forms CLA and vaccenic acid, but not stearate). However, only a few plants, including Acacia iteaphylla and Kennedia eximia, inhibited LA metabolism or stearate formation in the mixed bacterial community in vitro. Further experiments are needed to investigate effects of selected candidates on CLA production in vivo.

Recent advances in chromatographic identification of CLA isomers, combined with interest in their possible properties in promoting human health (e.g., cancer prevention, decreased atherosclerosis, improved immune response) and animal performance (e.g., body composition, regulation of milk fat synthesis, milk production), has renewed interest in biohydrogenation and its regulation in the rumen. Conventional pathways of biohydrogenation traditionally ignored minor fatty acid intermediates, which led to the persistence of oversimplified pathways over the decades. Recent work is now being directed toward accounting for all possible trans-18:1 and CLA products formed, including the discovery of novel bioactive intermediates. Modern microbial genetics and molecular phylogenetic techniques for identifying and classifying microorganisms by their small-subunit rRNA gene sequences have advanced knowledge of the role and contribution of specific microbial species in the process of biohydrogenation. With new insights into the pathways of biohydrogenation now available, several attempts have been made at modeling the pathway to predict ruminal flows of unsaturated fatty acids and biohydrogenation intermediates across a range of ruminal conditions. After a brief historical account of major past accomplishments documenting biohydrogenation, this review summarizes recent advances in 4 major areas of biohydrogenation: the microorganisms involved, identification of intermediates, the biochemistry of key enzymes, and the development and testing of mathematical models to predict biohydrogenation outcomes.

Aims: All members of the ruminal Butyrivibrio group convert linoleic acid (cis-9,cis-12-18 : 2) via conjugated 18 : 2 metabolites (mainly cis-9,trans-11-18 : 2, conjugated linoleic acid) to vaccenic acid (trans-11-18 : 1), but only members of a small branch, which includes Clostridium proteoclasticum, of this heterogeneous group further reduce vaccenic acid to stearic acid (18 : 0, SA). The aims of this study were to develop a real-time polymerase chain reaction (PCR) assay that would detect and quantify these key SA producers and to use this method to detect diet-associated changes in their populations in ruminal digesta of lactating cows. Methods and Results: The use of primers targeting the 16S rRNA gene of Cl. proteoclasticum was not sufficiently specific when only binding dyes were used for detection in real-time PCR. Their sequences were too similar to some nonproducing strains. A molecular beacon probe was designed specifically to detect and quantify the 16S rRNA genes of the Cl. proteoclasticum subgroup. The probe was characterized by its melting curve and validated using five SA-producing and ten nonproducing Butyrivibrio-like strains and 13 other common ruminal bacteria. Analysis of ruminal digesta collected from dairy cows fed different proportions of starch and fibre indicated a Cl. proteoclasticum population of 2–9% of the eubacterial community. The influence of diet on numbers of these bacteria was less than variations between individual cows. Conclusions: A molecular beacon approach in qPCR enables the detection of Cl. proteoclasticum in ruminal digesta. Their numbers are highly variable between individual animals. Significance and Impact of the Study: SA producers are fundamental to the flow of polyunsaturated fatty acid and vaccenic acid from the rumen. The method described here enabled preliminary information to be obtained about the size of this population. Further application of the method to digesta samples from cows fed diets of more variable composition should enable us to understand how to control these bacteria in order to enhance the nutritional characteristics of ruminant-derived foods, including milk and beef.

The objective of this study was to understand the effects of fumarate addition on methane (CH4) and VFA production in the rumen through a meta-analysis of its effects on ruminal batch cultures. Because the reduction of fumarate to succinate can draw electrons away from ruminal methanogenesis, fumarate has been studied as a potential feed additive to decrease CH4 production in ruminants. The average decrease in CH4 in batch cultures was of 0.037 micromol/micromol of added fumarate, which is considerably lower than 0.25 micromol/micromol, the decrease predicted from the stoichiometry of the reactions involved. One reason that fumarate was not effective at decreasing CH4 in batch cultures was that only an average of 48% of added fumarate appeared to be converted to propionate. Secondly, the incorporation of reducing equivalents in the conversion of fumarate to propionate was almost entirely offset by their release from an average of 20% of added fumarate that appeared to be converted to acetate. Thermodynamic calculations indicated that the conversion of added fumarate to both propionate and acetate was feasible. Fumarate appears to be more effective in decreasing CH4 production and increasing propionate in continuous culture than in batch culture. This suggests that microbial adaptation to fumarate metabolism can be important. Variation in populations of fumarate-reducers, methanogens, and protozoa could all be involved. Fumarate supplementation for an extended period may result in the amplification of otherwise small populations of fumarate-reducers. Addition of some of these organisms may be helpful to improve fumarate conversion to propionate. Strategies based on enhancing the rumen's capacity to convert fumarate to propionate by maintaining a low fumarate concentration have been effective. Thermodynamic considerations should be taken into account when designing strategies for CH4 abatement through the addition of external electron acceptors.

Albumin, as an antifouling agent preventing non-specific adsorption, is widely applied in biosensor construction and in the design of nanocarriers for theranostic applications. However, albumin is not completely inert and it interacts with many endogenous and exogenous compounds. The present work investigates the interactions of bovine serum albumin (BSA) with phenol red dye (PR), a common component of cell-growth media. PR was found to bind to BSA, with the affinity constant KA = (2.5 ± 0.8) × 105 L mol−1, and that it quenches the intrinsic BSA fluorescence through the Forster non-radiative resonance energy transfer (FRET) via static modes, with critical FRET distance of R0 = 2.65 nm. Synchronous fluorescence spectroscopy measurements indicated a conformational transition of BSA in the presence of PR to a conformation with tyrosine residues surrounded by a more hydrophobic environment. Thermodynamic parameters, ΔH°,ΔG° and ΔS°, determined using fluorescence and isothermal titration microcalorimetry, indicated that entropic contribution played a major role at ambient temperature. It was also found that common metal ions increased KA but Ca2+ and Fe3+ decreased it.

Dietarycis-9,trans-11-conjugated linoleic acid (CLA) is generally thought to be beneficial for human health. Fish oil added to ruminant diets increases the CLA concentration of milk and meat, an increase thought to arise from alterations in ruminal biohydrogenation of unsaturated fatty acids. To investigate the mechanism for this effect,in vitroincubations were carried out with ruminal digesta and the main biohydrogenating ruminal bacterium,Butyrivibrio fibrisolvens. Linoleic acid (LA) or α-linolenic acid (LNA) was incubated (1·67g/l) with strained ruminal digesta from sheep receiving a 50:50 grass hay–concentrate ration. Adding fish oil (up to 4·17g/l) tended to decrease the initial rate of LA (P=0·025) and LNA (P=0·137) disappearance, decreased (PPtrans-11-18:1. Concentrations of EPA (20:5n-3) or DHA (22:6n-3), the major fatty acids in fish oil, were low (100mg/l or less) after incubation of fish oil with ruminal digesta. Addition of EPA or DHA (50mg/l) to pure cultures inhibited the growth and isomerase activity ofB. fibrisolvens, while fish oil had no effect. In contrast, similar concentrations of EPA and DHA had no effect on biohydrogenation of LA by mixed digesta, while the addition of LA prevented metabolism of EPA and DHA. Neither EPA nor DHA was metabolised byB. fibrisolvensin pure culture. Thus, fish oil inhibits ruminal biohydrogenation by a mechanism which can be interpreted partly, but not entirely, in terms of its effects onB. fibrisolvens.

Effects of a specific preparation of essential oil compounds on rumen fermentation were determined using four mature sheep, each fitted with a permanent rumen cannula and receiving 110 mg per day of dietary essential oils compounds (EO; CRINA RUMINANTS, AKZO Nobel Surface Chemistry Ltd, Hertfordshire, UK). The basal ration comprised 1 kg DM per day of a 40:60 (DM) concentrate:forage mix fed in two equal meals. The forage was grass silage and the concentrate contained crushed barley grain, rapeseed meal, soyabean meal, molasses and a mineral and vitamin supplement (730, 125, 50, 80 and 15 g/kg fresh weight, respectively). Unsupplemented and EO-supplemented diets were fed in a 2×2 design with 6-week periods. EO inhibited ruminal degradation of soyabean meal N from dacron bags incubated in situ but not heat-treated rapeseed meal or hay. Deamination of amino acids measured in vitro in rumen fluid removed from the sheep decreased by 25% (P

Wallace, R. J., Newbold, C. J., Bequette, B. J., MacRae, J. C., Lobley, G. E. (2001). Increasing the flow of protein from ruminal fermentation - Review. Asian-Australian Journal of Animal Science, 14 (6), 885-893.

International audience; We report for the first time the cloning and characterisation of a protozoal enzyme involved in plant cell wall polysaccharide degradation. A cDNA library was constructed from the ruminal protozoan Polyplastron multivesiculatum and a stable clone expressing xylanase activity was isolated. The encoded enzyme belongs to the glycoside hydrolase family 11, and phylogenetic analysis indicates a closer relationship with catalytic domains from Gram-positive bacteria than the other fibrolytic eukaryotes from the rumen, the anaerobic fungi.

The potential for two complementary fragments of DNA from a clone from the ruminal bacterium Prevotella albensis to encode sequences with homology to at least part of functional proteins is described. One strand contains a sequence with high homology to dnaK, a member of the hsp70 family, and the other strand contains a sequence with some homology to glutamate dehydrogenase genes. Overlapping of these two genes on opposite strands has been reported in eukaryotic species, and is now reported for the first time in a bacterial species. Further investigation of previously described dnaK genes demonstrates that it is more widespread than might be anticipated, with all thirty other dnaK genes investigated also retaining long sequences encoding at least part of a sequence with high homology to a glutamate dehydrogenase gene.

The influence of different N sources on fermentation rate andde novoamino acid synthesis by rumen micro-organisms was investigatedin vitrousing rumen fluid taken from four sheep receiving a mixed diet comprising (g/kg DM): grass hay 500, barley 299·5, molasses 100, fish meal 91, minerals and vitamins 9·5. Pancreatic casein hydrolysate (P; comprising mainly peptides with some free amino acids; 10 g/l), free amino acids (AA; casein acid hydrolysate + added cysteine and tryptophan; 10 g/l), or a mixture of L-proline, glycine, L-valine and L-threonine (M; 0·83 g/l each) were added to diluted (1:3, v/v), strained rumen fluid along with15NH4Cl (A; 1·33 g/l) and 6·7 g/l of a mixture of starch, cellobiose and xylose (1:1:1, by weight). P and AA, but not M, stimulated net gas production after 4 and 8 h incubation (P< 0·05) in comparison with A alone. P increased microbial-protein synthesis (P< 0·05) compared with the other treatments. All of the microbial-N formed after 10 h was synthesizedde novofrom15NH3in treatment A, and the addition of pre-formed amino acids decreased the proportion to 0·37, 0·55, and 0·86 for P, AA, and M respectively.De novosynthesis of amino acids (0·29, 0·42 and 0·69 respectively) was lower than cell-N. Enrichment of alanine, glutamate and aspartate was slightly higher than that of other amino acids, while enrichment in proline was much lower, such that 0·83–0·95 of all proline incorporated into particulate matter was derived from pre-formed proline. Glycine, methionine, lysine, valine and threonine tended to be less enriched than other amino acids. The form in which the amino acids were supplied, as P or AA, had little influence on the pattern ofde novosynthesis. When the concentration of peptides was decreased, the proportion of microbial-N formed from NH3increased, so that at an initial concentration of 1 g peptides/l, similar to the highest reported ruminal peptide concentrations, 0·68 of cell-N was formed from NH3. Decreasing the NH3concentration at 1·0 g peptides/l caused proportionate decreases in the fraction of cell-N derived from NH3, from 0·81 at 0·53 g NH3-N/l to 0·40 at 0·19 g NH3-N/l. It was concluded that different individual amino acids are synthesizedde novoto different extents by mixed rumen micro-organisms when pre-formed amino acids are present, and that the source of N used for synthesis of cell-N and amino acids depends on the respective concentrations of the different N sources available; however, supplementing only with amino acids whose synthesis is lowest when pre-formed amino acids are present does not stimulate fermentation or microbial growth.

Six reductive acetogenic bacteria from a variety of ruminal and non-ruminal environments were investigated for their ability to prevent the accumulation of methane when added to rumen fluid incubated in vitro. Acetitomaculum ruminis, Eubacterium limosum strains ATCC 10825 and ATCC 8486, Ruminococcus productus ATCC 35244, and two acetogenic bacteria, Ser 5 and Ser 8 isolated from 20 h old lambs, were grown in media containing glucose. The bacteria retained the ability to produce acetate from H2 when incubated in the absence of sugar, while no acetate was produced in the absence of H2. When cultures of the acetogens were added to incubations of mixed rumen microorganisms in vitro, providing between 0.05 and 0.13 mg of acetogen protein/ml, methane production decreased by about 5% after 24 h with E. limosum ATCC 8486 and Ser 5, while the other bacteria had no effect on methane production. Increasing the concentration of E. limosum ten-fold did not cause a further decrease in methane production. When E. limosum ATCC 8486 and Ser 5 were added to cultures of mixed ruminal microorganisms in the presence of 2-bromoethanesulfonic acid, which inhibited methane formation and caused H2 to accumulate, both bacteria caused substantial increases in acetate production and decreased H2 formation. It was concluded that although acetogenic bacteria can utilise H2 and CO2 to form acetate in the rumen when methanogenesis is inhibited, even large concentrations of acetogenic bacteria cannot compete for H2 with methanogenic archaea under normal circumstances. # 1999 Elsevier Science B.V. All rights reserved.

Wallace, R. J., Atasoglu, C., Newbold, C. J. (1999). Role of peptides in rumen microbial metabolism - Review. Asian-Australian Journal of Animal Science, 12 (1), 139-147.

The influence of sodium fumarate on rumen fermentation was investigatedin vitrousing batch and semi-continuous cultures of mixed rumen micro-organisms taken from three sheep receiving a basal diet of hay, barley, molasses, fish meal and a mineral–vitamin supplement (500, 299·5, 100, 91 and 9·5 g/kg DM respectively). Batch cultures consisted of 10 ml strained rumen fluid in 40 ml anaerobic buffer containing 200 mg of the same feed given to the sheep. Sodium fumarate was added to achieve a final concentration of 0, 5 or 10 mmol/l, as a result of the addition of 0, 250 or 500 μmol, equivalent to 0, 200 and 400 g/kg feed. CH4production at 24 h (360 μmol in the control cultures) fell (P< 0·05) by 18 and 22 μmol respectively (SED 7·5). Total gas production was increased by the addition of fumarate without significant accumulation of H2. Substantial increases in acetate production (92 and 194 μmol; SED 26·7,P< 0·01) were accompanied by increases in propionate formation (212 and 396 μmol; SED 13·0,P< 0·001). Longer-term effects of fumarate supplementation on ruminal fermentation and CH4production were investigated using the rumen simulation technique (Rusitec). Eight vessels were given 20 g basal diet/d, and half of them received a supplement of fumarate (disodium salt) over a period of 19 d. The response to the daily addition of 6·25 mmol sodium fumarate was a decrease in CH4production of 1·2 mmol (SED 0·39,P< 0·05), equivalent to the consumption of 4·8 mmol H2, and an increase in propionate production of 4·9 mmol (from 10·4 to 15·3 (SED 1·05) mmol/d,P< 0·01). The inhibition of CH4production did not decline during the period of time that fumarate was added to the vessels. Thus, the decrease in CH4corresponded well to the fraction of the fumarate that was converted to propionate. Fumarate had no significant (P> 0·05) effect on total bacterial numbers or on the number of methanogenic archaea, but numbers of cellulolytic bacteria were increased (8·8v. 23·9 (SED 2·49) × 105per ml,P< 0·01). Fumarate also increased DM digestibility of the basal diet after 48 h incubation (0·476v. 0·508 (SED 0·0123),P< 0·05). Thus, it was concluded that sodium fumarate may be a useful dietary additive for ruminants, because it diverts some H2from CH4production and because it is able to stimulate proliferation of cellulolytic bacteria and digestion of fibre.

The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, Prevotella bryantii B 1 4, Selenomonas ruminantium HD4, and Streptococcus bovis ES1, was determined by growing these bacteria in media containing 15 NH 4 Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by P. bryantii , S. ruminantium , and S. bovis , respectively. Concentration-dependent responses were also obtained with amino acids. No individual amino acid was exhausted in any incubation medium. For cultures of P. bryantii , peptides were incorporated and stimulated growth more effectively than amino acids, while cultures of the other species showed no preference for peptides or amino acids. Apparent growth yields increased by between 8 and 57%, depending on the species, when 1 g of peptides or amino acids per liter was added to the medium. Proline synthesis was greatly decreased when peptides or amino acids were added to the medium, while glutamate and aspartate were enriched to a greater extent than other amino acids under all conditions. Thus, the proportion of bacterial protein formed de novo in noncellulolytic ruminal bacteria varies according to species and the form and identity of the amino acid and in a concentration-dependent manner.

The predominant mechanism of peptide breakdown by rumen micro-organisms is aminopeptidase. Thus acetylation of the N-terminus of peptides inhibits their degradation by rumen micro-organisms in short-term incubations with rumen fluid in vitro. An experiment was undertaken to determine if adaptation of the rumen microbial population would take place when acetylated peptides were fed for a prolonged period, which would enable the microbial population to break down the protected peptides and thus decrease their nutritive value. Three adult sheep, fitted with permanent rumen cannulae, received a maintenance hay/concentrate diet to which was added, at each meal, 20 g of casein enzymic hydrolysate ('peptides') or 20 g of peptides previously treated with acetic anhydride. The diets were fed for 28 d in a 3 X 3 latin square and samples were taken during the last 7 d. Fermentation products and NH 3 concentrations indicated that acetylated peptides remained less degradable than untreated peptides. There was a trend towards increased proteolytic activity with acetylated peptides, and dipeptidase activity increased by 18% and 28%, respectively, compared with untreated peptides and control treatments. Activity against N-acetyl-Ala 2 also increased when acetylated peptides were fed, but it remained only 13% of the rate of Ala 2 hydrolysis. No increase was found in the rate of ammonia production from acetylated peptides in animals receiving acetylated peptides-this rate was 26% of that found with untreated peptides-and acetylated peptides continued to persist for longer in the rumen than untreated peptides after feeding. Thus it was concluded that the rumen microbial population did not adapt to utilize acetylated peptides.

Prevotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic aminopeptidase substrates by sonicated extracts and whole cells of different species of rumen bacteria indicated that P. ruminicola had the greatest range and specific activity of dipeptidyl peptidases among the species tested. Streptococcus bovis hydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala2-p-nitroanilide, including Ruminobacter amylophilus, Ruminococcus spp. and Veillonella parvula. Dipeptidyl peptidases, which cleave dipeptides from the amino-terminus of longer peptides, were much more active than aminopeptidases removing single amino acids in P. ruminicola. Ion-exchange chromatography of sonicated extracts of P. ruminicola M384 revealed at least four distinct activities: one hydrolysed Ala2-p-nitroanilide, ValAla-p-nitroanilide, Ala4and Ala5; another was an O2-sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4-methoxynaphthylamide, Gly5 and ValGlySerGlu, similar to dipeptidyl peptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and GlyPro-4-methoxynapthylamide and was similar to dipeptidyl peptidase type IV XDPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. All of the enzymes, and particularly those active against Ala2-p-nitroanilide and GlyPro-p-nitroanilide, were inhibited by serine protease inhibitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and the enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited strongly by iodoacetate. DPP-4 was inhibited completely by diprotin A. Competitive inhibition experiments suggested that DPP-1 was less important than the other enzymes in the breakdown of peptide mixtures.

An experiment with ruminally cannulated sheep was undertaken to determine how the dietary energy source affected the ability of rumen fermentation to respond to pre-formed amino acids compared to non-protein nitrogen in the diet. Four sheep received diets of grass hay (H) or molassed sugar beet pulp (BP) supplemented with 9.3 g N kg−1 DM added either as urea (U) or casein (C) in a 4 × 4 latin square with a factorial design. Measurements of rumen fermentation, microbial numbers and purine excretion were made in the third week of each 21-day period. Rumen total volatile fatty acids concentrations were higher with the BP diets (130 vs. 91 mM; P 0.05). A microbial response to rumen-degradable protein therefore occurred only with the more rapidly degraded BP diet, consistent with the idea that rumen microbial growth responds to pre-formed amino acids only when the energy source is fermented rapidly.

The rate of peptide breakdown in the rumen frequently exceeds the rate at which the amino acids released can be used for microbial growth. The final step in this often wasteful process involves the cleavage of dipeptides. The main rumen bacterial species with high dipeptidase activity, Prevotella ruminicola, Fibrobacter succinogenes, Lachnospira multipara and Megasphaera elsdenii, had activities which were inhibited > 95% by 1,10-phenanthroline, a chelator of divalent metal ions and metalloprotease inhibitor. Dipeptidase activity in digesta taken from the rumen of sheep decreased by 33% in the presence of 1,10-phenanthroline, while mixed bacteria from the same samples were inhibited by 80% and the activity of mixed protozoa decreased by only 15%. Thus a substantial amount of dipeptide breakdown appears to be due to ciliate protozoa in the mixed population. Extensive washing of the protozoa increased the sensitivity of protozoal dipeptidase activity to 1,10-phenanthroline, suggesting that protozoa too have a metallo-dipeptidase activity but that it is normally protected from inhibition by 1,10-phenanthroline. Breakdown of the pentapeptide, Ala5, was also inhibited 27% by 1,10-phenanthroline in the mixed population, and when Trypticase, a pancreatic casein hydrolysate containing a mixture of oligopeptides, dipeptides and amino acids, was incubated with rumen fluid, the production of ammonia and free amino groups was inhibited 71% by 1,10-phenanthroline. It was concluded that metal ion chelation inhibits oligopeptidase and dipeptidase activities of rumen micro-organisms and may be a means of controlling ammonia production from peptides in the rumen.

The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides ) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala 2 and Ala 3 hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala 2 and Ala 3 by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala 2 hydrolysis by EDTA-treated cells was inhibited by the Ca 2+ /H + ionophore, tetronasin. Ala 3 hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola : strains GA33 and B 1 4 had activities which ran at the same R f ; strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same R f for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the M r of the dipeptidases from strains M384 and B 1 4 were 115 000 and 114 500 respectively, and 112 500 and 121 500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.

A ruminal simulation device (Rusitec) was used to compare the effects of Saccharomyces cerevisiae strains NCYC 240, NCYC 694, NCYC 1026, NCYC 1088, and Yea-Sacc (a commercial product containing S. cerevisiae) on ruminal fermentation. S. cerevisiae NCYC 240, NCYC 1088, NCYC 1026, and NCYC 694 were grown on malt extract at 30 degrees C in aerated fed-batch culture and harvested along with spent growth medium by freeze-drying. Each vessel received daily 20 g of a basal diet consisting of hay, barley, molasses, fishmeal, and a minerals/vitamins mixture at 500, 299.5, 100, 91, and 9.5 g/kg of DM, respectively. Yeast preparations (500 mg/d) were added along with the feed. S. cerevisiae NCYC 240, NCYC 1026, and Yea-Sacc stimulated total and cellulolytic bacterial numbers, whereas S. cerevisiae NCYC 694 and NCYC 1088 had no effect on the numbers of bacteria. The effects of S. cerevisiae NCYC 240, NCYC 1026, and Yea-Sacc on ruminal fermentation were further investigated in vivo using ruminally cannulated sheep fed 1.5 kg/d of the diet used in Rusitec, supplemented with 2 g/d of yeast culture. All treatments tended to stimulate total and cellulolytic bacterial numbers. However, the stimulation was only statistically significant for S. cerevisiae NCYC 1026 with total bacterial numbers and S. cerevisiae NCYC 240 with cellulolytic bacteria (P.05). Increased bacterial numbers were associated with an increase in the rate of straw degradation in the rumen and a nonsignificant (P.05) increase in the excretion of purine derivatives in the urine, measured as an index of microbial nitrogen leaving the rumen.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptidase activities against Ala3, Ala4 and Phe3 were increased (P 5 and Phe4 were unchanged and that against Gly-Arg-MNA was decreased (P

Soto, R. C., Muhammed, S. A., Newbold, J., Stewart, C. S., Wallace, R. J. (1994). Influence of peptides, amino acids and urea on microbial activity in the rumen of sheep receiving grass hay and on the growth of rumen bacteria in vitro. Animal Feed Science and Technology, 49 (1-2), 151-161.

SUMMARYMonensin, an ionophore, andAspergillus oryzaefermentation extract (AO), a fungal feed additive, are growth promoters which modify rumen fermentation. The effects of combining these additives were determined in the rumen simulation technique (Rusitec). Sixteen vessels received 20 g/day of a diet of hay, barley, molasses, fishmeal and a mineral/vitamin mixture (500, 299·5, 100, 91 and 9·5 g/kg DM respectively). AO, monensin (M) and AO + monensin (AO/M) were each added to four vessels at 500, 10 and (500 + 10) mg/day respectively. Both M and AO/M increased propionate (14·5 and 138v.91 mmol/day in controls;P< 0·001) and reduced butyrate production (6·6, 5·2 and 9·1 mmol/day respectively;P< 0·01), whereas AO had no effect. AO increased bacterial numbers by 70% (P< 0·055), while M had no effect, and in the presence of M, AO also had no effect. Proteolytic activity in samples from the vessels decreased (P< 0·05) in all treatments (1·06, 0·71, 0·60 and 0·47 mg14C-casein/mg protein/h for control, AO, M and AO/M respectively). In contrast, deamination of amino acids increased (P< 0·001) with AO and AO/M but decreased slightly with M alone (482, 646, 434 and 644 nmol NH3/mg protein/h). Ammonia output was unchanged (47·4, 57·2, 42·7 and 44·8 mg/day). Thus each additive was dominant over the other for different activities, with monensin generally suppressing the effects of AO rather than vice-versa.

Several di-, tri-, and oligopeptides were incubated individually in vitro with rumen fluid from two sheep receiving a mixed grass hay/concentrate diet and with washed cells of Prevotella (formerly Bacteroides) ruminicola M384 and P. ruminicola B(1)4. The rates of breakdown of most peptides were similar in the rumen fluid from the two sheep. Acidic and proline-containing peptides tended to be more slowly degraded than neutral or basic peptides. The dipeptide at the N-terminus of higher peptides was observed as an early product of hydrolysis, confirming that a dipeptidyl aminopeptidase type of activity was present. The relative rates of breakdown of dipeptides by P. ruminicola were different from that of rumen fluid, but the hydrolysis of higher peptides followed a similar pattern, and dipeptides from the N-terminus were detected as early products.

Background Health-promoting polyunsaturated fatty acids (PUFA) are abundant in forages grazed by ruminants and in vegetable and fish oils used as dietary supplements, but only a small proportion of PUFA finds its way into meat and milk, because of biohydrogenation in the rumen. Butyrivibrio fibrisolvens plays a major role in this activity. The aim of this study was to investigate the mechanisms by which PUFA affect the growth of B. fibrisolvens, how PUFA are metabolized and the metabolic response to growth in the presence of PUFA. Results Linoleic acid (LA; cis-9, cis-12-18:2) and α-linolenic acid (LNA; cis-9, cis-12, cis-15-18:3) increased the lag phase of B. fibrisolvens JW11, LNA having the greater effect. Growth was initiated only when the PUFA had been converted to vaccenic acid (VA; trans- 11-18:1). The major fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n- 3)) and docosahexaenoic acid (DHA; 22:6(n- 3)), were not metabolized and prevented growth. Cellular integrity, as determined fluorimetrically by propidium iodide (PI) ingression, was affected as much by 18:1 fatty acids, including VA, as 18:2 fatty acids. The methyl esters of LNA, LA, EPA and DHA had no effect on growth or other measurements. The ATP pool decreased by 2/3 when LA was added to growing bacteria, whereas most acyl CoA pools decreased by >96%. Conclusions It was concluded that biohydrogenation occurs to enable B. fibrisolvens to survive the bacteriostatic effects of PUFA, and that the toxicity of PUFA is probably mediated via a metabolic effect rather than disruption of membrane integrity.

Generally, only a small proportion of dietary fat that is consumed by man reaches the large intestine. Lipids in colonic digesta are derived from this undigested residue of dietary fat and partly from endogenous secretions and shedding of colonocytes. Intestinal bacterial lipases and phospholipases release fatty acids and various glycerides that may then be metabolized further to form products that have implications for intestinal health. Some clinical conditions result in increased fluxes of lipids to the large intestine, and, increasingly, slimming drugs that inhibit pancreatic lipases or adsorbants that enable fat to bypass the small intestine also result in large quantities of dietary lipids reaching the colon. The consequences of increased fat metabolism in the colon are as yet largely unknown.

Short-chain fatty acids (SCFA) are formed from the fermentation of sugars by intestinal bacteria. Acetate is the most abundant SCFA, with lower amounts of propionate and butyrate formed. Propionate and butyrate are also formed from the products of carbohydrate fermentation by other bacteria, for example from lactate and acetate. SCFA play a role in regulating transit of digesta through the intestine, and butyrate formation is thought to be beneficial to health because butyrate decreases the risk of colon cancer. Major butyrate-producing species are among the most abundant present in the colon, including Roseburia and Faecalibacterium spp. Metabolism of longer-chain fatty acids occurs mainly by hydration or hydrogenation of unsaturated fatty acids. Hydroxystearic acids are formed in the intestine, particularly under disease conditions. Metabolism of linoleic acid results in the formation of conjugated linoleic acids (CLA) by several species, including Roseburia hominis and Roseburia inulinovorans. Enhancement of intestinal CLA formation, possibly using probiotics, may be useful in preventing or treating inflammatory bowel disease.

Proteins and peptides were acetylated using acetic anhydride in order to block their N-terminal amino groups and thereby to prevent their hydrolysis by rumen microbial aminopeptidases. The effects of acetylation on peptide breakdown and ammonia production were determined by incubating unmodified and acetylated substrates with sheep rumen micro-organisms in vitro. Ammonia production from casein and lactalbumin was affected little by acetylation, but acetylation of the corresponding enzymic hydrolysates caused ammonia production to be more than halved after 3.6 h incubation. Estimation of peptides remaining in rumen fluid showed that the decreased ammonia production was a consequence of peptides being hydrolysed more slowly. Acetylated Ala-Ala, Ala-Ala-Ala (Ala3), Leu-Gly-Gly, Phe-Gly-Gly and Val-Gly-Ser-Glu survived incubation with rumen fluid in vitro for 6 h, whereas almost none of the corresponding unmodified peptides was present at 6 h. The protection afforded to larger pure peptides was less reliable: for example, 72% of acetylated bradykinin was hydrolysed after 1 h.N-Acetyl Ala3had only a minor inhibitory effect on the breakdown of Ala3and Ala4, suggesting that although acetyl peptides were broken down more slowly than unmodified peptides they did not inhibit peptidase activity.

Decreasing methanogenesis in ruminants would benefit the agricultural industry because it would lead to lower energy losses from the animals as well as being beneficial for the environment in decreasing emissions of a greenhouse gas. Fumaric acid (FA) as a feed supplement has the potential to decrease methane production as well as increase glucogenesis and hence milk yield, but the quantity fed has to be restricted because of a risk of acidosis and a consequent decrease in fibre breakdown and feed intake. The objective of this study was to determine if FA encapsulated in partially hydrogenated vegetable oil (PHVO) could decrease methane formation without the problematic effects on ruminal pH. A commercial sample of encapsulated fumaric acid (EFA) did not affect pH and maintained propionate production when added in vitro to ruminal fluid from sheep receiving 49:51 grass hay:concentrate, and it suppressed methane formation by 19% (P

SUMMARYA rumen simulation device (Rusitec) was used to compare the effects of autoclaved and γ-irradiated Aspergillus oryzae fermentation extract (AO) with those of untreated AO on rumen fermentation. Each vessel received daily 18 g of a basal diet consisting of 500 g hay, 299·5 g rolled barley, 100 g molasses, 91 g fishmeal and 9·5 g of a minerals and vitamins mixture/kg dry matter. AO preparations (0·25 g/day) were added with the feed. Dry matter digestion increased at 24 h, but not at 48 h, after adding AO, suggesting that AO stimulated the rate of digestion but not the extent. AO increased the numbers of total and cellulolytic bacteria by 90 and 50%, respectively. Irradiated AO stimulated numbers of bacteria in the same way as AO, although to a slightly lesser extent. Autoclaved AO had no effect. Thus, the mode of action of AO on rumen fermentation depends on a heat-labile component, possibly a nutrient or an enzyme, or metabolic activity. Viable AO cells are not required to stimulate bacterial growth and activity.

SUMMARYIonophores and inhibitors of bacterial energy metabolism were added to mixed rumen bacteria prepared from sheep receiving grass hay plus concentrate diets, and their influence on the rate of metabolism of alanine (Ala) peptides was determined. Dicyclohexylcarbodiimide (DCCD) had no influence on the rate of breakdown of Ala2, Ala3, Ala4or Ala5, indicating that the metabolism of these peptides did not require ATP. The protonophores tetrachlorosalicylanilide (TCS) and carbonyl cyanidem-chlorophenyl-hydrazone (CCCP) inhibited peptide breakdown to a minor extent (< 15%), whereas the ionophores monensin and tetronasin had greater, but still small (12–31%), inhibitory effects. Toluene stimulated peptide breakdown, consistent with a permeability barrier having been removed. Thus, at least some peptide metabolism depends on transport into bacteria; transport appears not to be ATP-linked, and may well be coupled to the uptake of mineral cations rather than protons. Rumen fluid from sheep receiving a similar diet with added monensin (33 mg/kg) or tetronasin (10 mg/kg) hydrolysed Ala3and Ala4at rates that did not differ significantly from controls. Nevertheless, the peak concentration of free peptides in rumen fluid after feeding was more than doubled in ionophore-fed sheep, and peptides persisted for longer than in control animals.

Aims: Anaerobic fungi are important members of the fibrolytic community of the rumen. The aim of this study was to study their requirement for aromatic amino acids (AA) and related phenyl acids (phenylpropionic and phenylacetic acids) for optimal xylan fermentation. Methods and Results: Neocallimastix frontalis RE1 and Piromyces communis P were grown in a defined medium containing oat spelts xylan as the sole energy source, plus one of the following N sources: ammonia; ammonia plus a complete mixture of 20 AA commonly found in protein; ammonia plus complete AA mixture minus aromatic AA; ammonia plus phenyl acids; ammonia plus complete AA mixture without aromatic AA plus phenyl acids. Both species grew in all the media, indicating no absolute requirement for AA. The complete AA mixture increased (P

One aim of the EC Framework V project, ’Rumen-up’ (QLK5-CT-2001-00 992), was to find plants or plant extracts that would inhibit the nutritionally wasteful degradation of protein in the rumen. A total of 500 samples were screenedin vitrousing14C-labelled casein in a 30-min incubation with ruminal digesta. Eight were selected for further investigation using a batch fermentation system and soya protein and bovine serum albumin as proteolysis substrates; proteolysis was monitored over 12 h by the disappearance of soluble protein and the production of branched SCFA and NH3. Freeze-dried, ground foliage ofPeltiphyllum peltatum,Helianthemum canum,Arbutus unedo,Arctostaphylos uva-ursiandKnautia arvensisinhibited proteolysis (P Daucus carota,Clematis vitalbaandErica arboreahad little effect. Inhibition by the first four samples appeared to be caused by the formation of insoluble tannin–protein complexes. The samples were rich in phenolics and inhibition was reversed by polyethyleneglycol. In contrast,K.arvensiscontained low concentrations of phenolics and no tannins, had no effect in the 30-min assay, yet inhibited the degradation rate of soluble protein (by 14 %,P P 0·05) without precipitating protein in the 12-h batch fermentation. The effects showed some resemblance to those obtained in parallel incubations containing 3 μm-monensin, suggesting thatK.arvensismay be a plant-derived feed additive that can suppress growth and activity of key proteolytic ruminal micro-organisms in a manner similar to that already well known for monensin.

A commercial blend of essential oil (EO) compounds was added to a grass, maize silage, and concentrate diet fed to dairy cattle in order to determine their influence on protein metabolism by ruminal microorganisms. EO inhibited ( P < 0.05) the rate of deamination of amino acids. Pure-culture studies indicated that the species most sensitive to EO were ammonia-hyperproducing bacteria and anaerobic fungi.

This study was conducted to investigate the occurrence of methane oxidation in the rumen, and to identify the electron-sink reaction coupled to the oxidation if it occurred.Mixed ruminal microbes taken from sheep were incubated with 13CH4. Oxidation of methane, estimated from the flux of 13C to CO2 and microbial cells, occurred, but represented only 0.2-0.5% of the methane produced. Methane oxidation was suppressed by the presence of oxygen, and was also inhibited by 2-bromoethane-sulphonate, and molybdate, but not by tungstate.Methane could be oxidized anaerobically in the rumen by reverse methanogenesis in consort with sulphate reduction.

Methods for detecting and measuring the quantity of fibrolytic enzyme preparations added to feeds were investigated by enzymatic and tracer methods. Enzyme preparations added to corn silage, ryegrass silage, and a total mixed ration containing both silages and a concentrate could not be detected using their enzymatic activities. Glycosidase activities of solubles washed from the feed were more than an order of magnitude greater than glycosidases in the added enzymes. Carboxymethylcellulase and xylanase activity determinations, using reducing sugar release as the measurement, were subject to interference from reducing sugars present in the feed. A fluorescent tracer method, using fluorescein added at a rate of 1 g/L of feed enzymes, or 2 g/t of feed, was developed that enabled sensitive detection of liquid enzyme additions to feeds.

Two suggested modes of action of yeast in stimulating rumen fermentation were investigated. The first, that yeast respiratory activity protects anaerobic rumen bacteria from damage by O2, was tested using different strains of yeast that had previously been shown to have differing abilities to increase the viable count of rumen bacteria.Saccharomyces cerevisiaeNCYC 240, NCYC 1026, and the commercial product Yea-Sacc®, added to rumen fluidin vitroat 1·3 mg/ml, increased the rate of O2disappearance by between 46 and 89%. The same three preparations also stimulated bacterial numbers in anin vitrofermenter (Rusitec).S. cerevisiaeNCYC 694 and NCYC 1088, which had no influence on the viable count in Rusitec, also had no effect on O2uptake. Respiration-deficient (RD) mutants ofS. cerevisiaeNCYC 240 and NCYC 1026 were enriched by repeated culturing in the presence of ethidium bromide.S. cerevisiaeNCYC 240 and NCYC 1026 stimulated the total and cellulolytic bacterial populations in Rusitec, while the corresponding RD mutants did not. Rigorous precautions to exclude air from Rusitec resulted i“nS. cevevisiaeNCYC 240 no longer stimulating total bacterial numbers, although it still increased numbers of cellulolytic bacteria. The second hypothesis, that yeast provides malic and other dicarboxylic acids which stimulate the growth of some rumen bacteria, was examined by comparing the effects of yeast and malic acid on rumen fermentation in sheep. Three mature sheep were given 0·85 kg barley/d plus 0·55 kg chopped ryegrass hay/d either unsupplemented, or supplemented with 4 gS. cerevisiaeNCYC 240/d or 100 mgl-malic acid/d either mixed with the diet or in aqueous solution infused continuously into the rumen. Yeast increased the total viable count of bacteria (P< 0·05)whereas malic acid did not, and no other effect of the treatments reached statistical significance. It was concluded, therefore, that the stimulation of rumen bacteria byS.cerevisiaeis at least partly dependent on its respiratory activity, and is not mediated by malic acid.

Prevotella ruminicola plays a prominent role in the breakdown of peptides in the rumen, a process which contributes to excessive ammonia production and inefficient nitrogen retention in ruminants. Various metal ions and chelators were examined to assess how the metal ion-dependent dipeptidase activity of P. ruminicola M384 might be inhibited. Using sonicated extracts, Cu2+, Cr2+ and Hg2+ were most inhibitory, decreasing Ala2 breakdown to 15, 15 and 5% of control activity, whereas Co2+, Mn2+ and Zn2+ stimulated activity by 189, 30 and 26%, respectively. The chelators, EDTA, EGTA, TPEN and 1,10-phenanthroline, were inhibitory, as were several phenanthroline analogues. Among the stereoisomers of 1,10-phenanthroline tested, derivatives methylated on C-2 and C-9 were less effective than the parent molecule, but 3,4,7,8-tetramethyl-1,10-phenanthroline (TMP) was more inhibitory. Titration of the most effective inhibitors showed that EDTA, TPEN and TMP had similar potency and were effective at 0.1 mmol l-1 and above. Thus some metal ions and chelators are potent inhibitors of P. ruminicola dipeptidase, although they are unlikely to be sufficiently specific to peptide metabolism to be useful in vivo.

Treatment of Trypticase peptides with acetic anhydride, succinic anhydride, or maleic anhydride inhibited their breakdown to ammonia by rumen microorganisms by an average of 89% after 12 h of incubation in vitro. All three treatments gave similar protection. Acetylation also protected dipeptides containing lysine and methionine from degradation. However, more effective protection was obtained by linking lysine and methionine as N-epsilon-methionyl lysine.