Browsing by Subject "Monensin"

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    Adaptations of Prevotella bryantii B14 to short-chain fatty acids and monensin exposure
    (2023) Trautmann, Andrej; Seifert, Jana
    The rumen microbiome constitutes a complex ecosystem including a vast diversity of organisms that produce and consume short-chain fatty acids (SCFAs). It is of great interest to analyze these activities as they are of benefit for both, the microbiome and the host. This dissertation aims to display the proteome and metabolome of the predominant ruminal representative Prevotella bryantii B14 in presence of various SCFA and under exposure of the antibiotic monensin in pure and mixed culture (in vitro). Due to the strong contributing abundance of Prevotellaceae in the rumen microbiome, the representative P. bryantii B14 (DSM 11371) was chosen to investigate biochemical factors for the success of withstanding monensin and the impact of SCFA on their growth. The current work is composed of two effective publications. The formatting was aligned to the dissertation. The first publication, studying the supplementation of various SCFAs, showed SCFAs as growth promoting but not essential for P. bryantii B14. Pure cultures of P. bryantii B14 were grown in Hungate tubes under anaerobic conditions. Gas chromatography time of flight mass spectrometry (GC-ToF MS) was used to quantify long-chain fatty acid (LCFA) profiles of P. bryantii B14. Proteins of P. bryantii B14 were identified and quantified by using a mass spectrometry-based, label-free approach. Different growth behavior was observed depending on the supplemented SCFA. An implementation of SCFAs on LCFAs and the composition on membrane proteins became evident. Supplementing P. bryantii B14 with branched-chain fatty acids (BCFAs), in particular isovaleric acid, showed an increase of the 3-IPM pathway, which is part of the branched-chain amino acid (BCAA) metabolism. Findings point out that the structure similarity of isovaleric acid and valine is most likely enhancing the conversion of BCFA into BCAA. The required set of enzymes of the BCAA metabolism supported this perspective. The ionophore monensin has antibiotic properties which are used in cattle fattening but also for treating ketosis and acidosis in ruminants. In the second publication, P. bryantii B14 was exposed to different concentrations of monensin (0, 10, 20 and 50 uM) and to different exposure times (9, 24, 48 and 72 h) with and without monensin. Growth behavior, glucose and intracellular sodium concentration were determined. Proteins were analyzed by label-free quantification method using the same method as in the previous mentioned experiment. Fluorescence microscopy was used to observe extracellular polysaccharides (EPS) of P. bryantii B14. A progressing monensin exposure triggered disconnection between P. bryantii B14 cells to the sacrificial EPS layer by increasing its number and amount of carbohydrate active enzymes (CAZymes). Simultaneously, an increase of extracellular glucose was monitored. Reduction of intracellular sodium was likely to be performed by increasing the abundance of ion-transporters and an increased activity of Na+-translocating NADH:quinone oxidoreductase under monensin supplementation. The role of monensin supplemented Prevotella in a mixed culture of the rumen microbiome was described. Extracted rumen fluid from cows was incubated anaerobically by using the rumen simulation technique (Rusitec). Proteomics of the solid phase was applied by using a similar approach as in the previous related studies. Metabolomics of the liquid phase from the Rusitec content was performed by using 1H-nuclear magnetic resonance (NMR) spectroscopy. Further parameters such as pH, gas and methane production were monitored over time. The experiment was constituted out of three phases starting with an adaptation phase of 7 days. A subsequent treatment phase followed, where monensin was supplemented via the daily introduced total mixed ration (TMR) for further 7 days. The elution phase was the final phase when monensin supplementation was stopped and monitoring was continued for further 3 days. Metabolomics and proteomics showed that members of the genus Prevotella remained most abundant under monensin supplementation. Furthermore, shifting the ruminal metabolism to an increased production of propionate by shifting the metabolism of Prevotella sp. to an enhanced succinate production. The current work shows the impact of SCFAs on various metabolic functions of P. bryantii B14. Diverse defence mechanisms of Prevotella sp., in particular P. bryantii B14, were shown to avoid the antibiotic effects of monensin.
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    Effects of monensin and tannin extract supplementation on methane production and other criteria of rumen fermentation in vitro and in long-term studies with sheep
    (2013) Wischer, Gerald; Rodehutscord, Markus
    Ruminants increasingly attract public concern due to their methane release and contribution to the greenhouse effect. One strategy to reduce the release of methane is to modify microbial fermentation in the rumen by the use of feed additives such as monensin and tannin extracts. However, other characteristics of fermentation including the synthesis of microbial protein may also be affected. The aim of the present studies was to provide a comprehensive evaluation of the effects of monensin and tannin extracts on ruminal fermentation and methane production. The ionophore monensin is known to increase feed efficiency in ruminants. Although the use of silages is common practice in cattle feeding, the effects of monensin on the fermentation of silages in the rumen and microbial protein synthesis are lacking. Monensin has often been described to have indirect effects on methane production resulting from its effects on feed intake, protozoa and Gram-positive bacteria. It has rarely been studied whether monensin can reduce methane production without adverse effects on other criteria of rumen fermentation. The first objective therefore was to investigate the effects of different dosages of monensin on methane production and microbial protein synthesis when supplemented to different silages in two in vitro systems (Study 1). In Experiment 1 of Study 1, 15 g of oven-dried grass silage alone or combined with a concentrate was incubated in a rumen simulation (Rusitec) over a period of 13 d to examine the effects of monensin supplementation (2 or 4 mg/d, n = 4) on the production of total gas, methane, volatile fatty acids (VFA), degradation of nutrients and microbial protein synthesis. In Experiment 2 of Study 1, different dosages of monensin (0.5, 1, 2, 6 and 10 µg) were supplemented to syringes containing 120 mg of grass silage alone, grass silage combined with concentrates, or maize silage alone. After 24 h of incubation the effects of monensin on total gas, methane and VFA production were determined. In Experiment 1 monensin inclusion to grass silage and grass silage combined with concentrate resulted in a decreased total gas, methane and acetate production, while propionate production was increased. Along with a decreased degradation of crude protein, ammonia concentration in the system was reduced. While microbial protein originating from solid associated microbes decreased with monensin inclusion, microbial protein from liquid associated microbes was increased, resulting in an increase in total microbial protein synthesis. In Experiment 2, different dosages of monensin reduced methane production in grass silage (17%), grass silage combined with concentrate (10%) and maize silage (13%) without adverse effects on total gas production. Based on these two in vitro experiments it was concluded that monensin is able to reduce methane production without a major decrease in total gas and VFA production and degradation of organic matter. Although microbial fractions were differently affected, the total microbial protein synthesis was increased upon monensin supplementation. Tannins are secondary plant compounds that are known to complex with feed and microbial proteins. Several products from this heterogeneous group have shown potential to affect rumen fermentation in vivo and, even more, in vitro, but are often accompanied by negative effects on digestibility, feed intake and microbial protein synthesis. In Study 2 of the present work, ten tannin extracts (chestnut, mimosa, myrabolan, quebracho, sumach, tara, valonea, oak, cocoa and grape seed) and four monomers of rapeseed tannin (pelargonidin, catechin, cyanidin and sinapinic acid) were screened in grass silage based diets in successive runs using the Hohenheim Gas Test. The objective was to determine the optimal dosage of each tannin extract to cause a maximal methane reduction without negative effects on total gas production. Whereas the supplementation of pelargonidin and cyanidin to grass silage did not reduce methane production; catechin and sinapinic acid reduced methane production without affecting total gas production. Except tara extract, all tannin extracts reduced methane production by 8 to 28% without adverse effects on total gas production. Based on these results, chestnut, grape seed, myrabolan, sumach and valonea extract were investigated in a second step in a Rusitec to determine their effects on degradation of nutrients, VFA and ammonia production, and particularly on microbial protein synthesis. All tannin extracts were supplemented at similar dosages of 1.5 g to 15 g of grass silage. The supplementation of chestnut resulted in the greatest decrease in methane production (63%), followed by valonea (35%), grape seed (23%), sumach (18%), and myrabolan (7%; not significantly different from the control). While chestnut extract reduced acetate production by 19%, supplementation with grape seed or myrabolan extract increased acetate production; however, degradation of fibre fractions was reduced in all tannin treatments. Degradation of dry and organic matter was reduced by all tannin extracts, but there were no differences between tannin treatments. Crude protein degradation and ammonia production were also reduced by tannin extract supplementation. Microbial protein synthesis and its efficiency were not affected by tannin supplementation, which indicates that a reduction in methane production due to tannin extract supplementation is possible without negatively affecting microbial protein synthesis. Chestnut and valonea extract had the greatest potential in reducing methane production without negative effects on rumen fermentation of grass silage and microbial protein synthesis. Therefore, these tannin extracts were investigated for their long-term effects in sheep (Study 3). In Experiment 1 of Study 3, sheep receiving the control, chestnut or valonea treatment (each n = 4) were fed 842 g/d of hay (fresh weight). The animals on the control treatment also received 464 g/d of concentrate, and animals on the tannin treatments received the same amount of concentrate but were also fed 20 g of the respective tannin extract. Following initiation of tannin feeding, methane release from sheep was measured in 23.5 h intervals in respiration chambers on day 1, 8, 15, 29, 57, 85, 113, 148, and 190. In three balances periods faeces and urine were collected for 6 and 3 days, respectively. Effects on nutrient digestibility, nitrogen and energy metabolism were evaluated, with microbial protein synthesis estimated from the urinary excretion of purine derivatives. Based on the results of Experiment 1, a second experiment was conducted four month after the start of Experiment 1. Experiment 2 had the same study design and data collected, but the dosage of tannin extracts was doubled compared to Experiment 1 (0.9 vs. 1.7 g tannin extract/kg body weight) and the duration was shorter (85 days). Hay and concentrates used in both experiments were also evaluated using the Hohenheim Gas for their effects on total gas and methane production. In both experiments, methane release was not significantly reduced by tannin extract supplementation when analysed over the whole experimental period. In Experiment 1 the supplementation of chestnut extract on day 190 resulted in a reduced methane release. In both experiments, on day 1 a numeric reduction in methane release for the tannin treatments was observed, with a greater reduction recorded for the higher dosage used in Experiment 2. This trend disappeared by day 57. In the third balance period of Experiment 1, digestibility of dry and organic matter was reduced by tannin supplementation. The digestibility of crude protein was reduced in both experiments, whereas the digestibility of fibre fractions was not influenced. In both experiments a long-lasting shift in nitrogen excretion from urine to faeces was observed, which occurred to a greater extent in Experiment 2. The urinary excretion of purine derivatives was not significantly affected by tannin supplementation, indicating that the microbial protein synthesis was not altered in either experiment. The in vitro methane production was reduced for concentrates containing tannin extracts, but it was not significantly affected when concentrates were incubated with hay. It is concluded that monensin added to different silages caused a decrease in methane production without affecting total gas production but with an increased microbial protein synthesis. Nine of the ten considered tannin extracts and two tannin monomers decreased methane production without affecting total gas production. The Rusitec study confirmed the great potential of chestnut and valonea extract to reduce methane production without negative effects on microbial protein synthesis. However, neither chestnut nor valonea extract reduced the methane release in sheep when fed over a longer period of time. It is assumed, that rumen microbes adapted to the tannin dosages in terms of methane release but not nitrogen metabolism, as there were long-lasting effects on nitrogen excretion. The shift in nitrogen excretion can have a positive effect on the environment due to the reduced potential of ammonia emission from the urine. Both in vitro systems used in the present studies showed effects of tannin extracts that were considerably different from those observed in sheep. The monomers investigated in the present study are the basic units of condensed tannins, whereas the tannin extracts selected in vitro only contain hydrolysable tannins. It is possible that monomers of chestnut and valonea extract may reduce methane production, whereas higher dosages of these tannin extracts cause negative effects on feed intake, digestibility and microbial protein synthesis. Further investigations should focus systematically on the transfer of in vitro studies to estimate in vivo responses. Therefore, a parallel implementation of different in vitro and respiration studies would be of great value.
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    Na+-coupled respiration and reshaping of extracellular polysaccharide layer counteract monensin-induced cation permeability in Prevotella bryantii B14
    (2021) Trautmann, Andrej; Schleicher, Lena; Pfirrmann, Jana; Boldt, Christin; Steuber, Julia; Seifert, Jana
    Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B14 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P. bryantii, as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P. bryantii B14 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na+-translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P. bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.
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    A shift towards succinate‐producing Prevotella in the ruminal microbiome challenged with monensin
    (2022) Trautmann, Andrej; Schleicher, Lena; Koch, Ariane; Günther, Johannes; Steuber, Julia; Seifert, Jana
    The time‐resolved impact of monensin on the active rumen microbiome was studied in a rumen‐simulating technique (Rusitec) with metaproteomic and metabolomic approaches. Monensin treatment caused a decreased fibre degradation potential that was observed by the reduced abundance of proteins assigned to fibrolytic bacteria and glycoside hydrolases, sugar transporters and carbohydrate metabolism. Decreased proteolytic activities resulted in reduced amounts of ammonium as well as branched‐chain fatty acids. The family Prevotellaceae exhibited increased resilience in the presence of monensin, with a switch of the metabolism from acetate to succinate production. Prevotella species harbour a membrane‐bound electron transfer complex, which drives the reduction of fumarate to succinate, which is the substrate for propionate production in the rumen habitat. Besides the increased succinate production, a concomitant depletion of methane concentration was observed upon monensin exposure. Our study demonstrates that Prevotella sp. shifts its metabolism successfully in response to monensin exposure and Prevotellaceae represents the key bacterial family stabilizing the rumen microbiota during exposure to monensin.