Effects of Different Formulations of Glyphosate on Rumen Microbial Metabolism and Bacterial Community Composition in the Rumen Simulation Technique System.

The use of the herbicide glyphosate and its formulations on protein-rich feedstuff for cattle leads to a considerable intake of glyphosate into the rumen of the animals, where glyphosate may potentially impair the 5-enolpyruvylshikimate-3-phosphate pathway of the commensal microbiota, which could cause dysbiosis or proliferation of pathogenic microorganisms. Here, we evaluated the effects of pure glyphosate and the formulations Durano TF and Roundup® LB plus in different concentrations on the fermentation pattern, community composition and metabolic activity of the rumen microbiota using the Rumen Simulation Technique (RUSITEC). Application of the compounds in three concentrations (0.1 mg/l, 1.0 mg/l or 10 mg/l, n = 4 each) for 9 days did not affect fermentation parameters such as pH, redox potential, NH3-N concentration and production of short-chain fatty acids compared to a control group. Microbial protein synthesis and the degradation of different feed fractions did not vary among the treatments. None of the used compounds or concentrations did affect the microbial diversity or abundance of microbial taxa. Metaproteomics revealed that the present metabolic pathways including the shikimate pathway were not affected by addition of glyphosate, Durano TF or Roundup® LB plus. In conclusion, neither pure glyphosate, nor its formulations Durano TF and Roundup® LB plus did affect the bacterial communities of the rumen.

Effect of a Glyphosate-Containing Herbicide on Escherichia coli and Salmonella Ser. Typhimurium in an In Vitro Rumen Simulation System.

Glyphosate (N-(phosphonomethyl)glycine) is the most-used herbicide worldwide. Many studies in the past have shown that residues of the herbicide can be found in many cultivated plants, including those used as livestock feed. Sensitivity to glyphosate varies with bacteria, particularly those residing in the intestine, where microbiota is exposed to glyphosate residues. Therefore, less susceptible pathogenic isolates could have a distinct advantage compared to more sensitive commensal isolates, probably leading to dysbiosis. To determine whether the ruminal growth and survival of pathogenic Escherichia coli or Salmonella serovar Typhimurium are higher when glyphosate residues are present in the feed, an in vitro fermentation trial with a "Rumen Simulation System" (RUSITEC) and a glyphosate-containing commercial formulation was performed. Colony forming units of E. coli and Salmonella ser. Typhimurium decreased steadily in all fermenters, regardless of the herbicide application. Minimum inhibitory concentrations of the studied Salmonella and E. coli strains did not change, and antibiotic susceptibility varied only slightly but independent of the glyphosate application. Overall, application of the glyphosate-containing formulation in a worst-case concentration of 10 mg/L neither increased the abundance for the tested E. coli and Salmonella strain in the in vitro fermentation system, nor promoted resistance to glyphosate or antibiotics.

Evaluation of enterotoxin gene expression and enterotoxin production capacity of the probiotic strain Bacillus toyonensis BCT-7112T.

The aim of the present study was to evaluate the safety of the probiotic strain Bacillus toyonensis BCT-7112T (active ingredient of Toyocerin) in relation to the enterotoxins haemolysin BL (Hbl) and the non-haemolytic enterotoxin (Nhe) by performing a quantitative reverse transcription (RT) real-time polymerase chain reaction (PCR) and a Western blot assay. The expression levels of the enterotoxin genes hblA, hblD, nheA, nheB and nheC, determined by means of RT real-time PCR in B. toyonensis, were lower than those in B. cereus reference strains. No expression of hblC was detected. The Western blot assays of native and 25-fold concentrated supernatants from B. toyonensis, using monoclonal antibodies directed against the Hbl component L1 and the Nhe component NheB, showed weak bands. The NheC component was not detected in the native supernatant, but weakly in the 25-fold concentrated supernatant. According to the results of the present study, the enterotoxin expression and protein levels of B. toyonensis BCT-7112T were absent or clearly lower compared to the B. cereus reference strains. Thus, their ability to form functional enterotoxins can also be considered to be lower or unlikely compared to the B. cereus reference strains. This experimental approach can be implemented when studying the health and safety as well as harmlessness of probiotic microorganisms.

Effects of drought-stressed temperate forage legumes on the degradation and the rumen microbial community in vitro.

According to climate change scenarios, central Europe may expect extending drought periods during summer. Lower water availability may influence the ruminal digestion of individual forage legume species differently. To test this hypothesis, Lotus corniculatus L. (var. Bull), Medicago lupulina L. (var. Ekola), Medicago falcata L. (wild seeds) and Trifolium repens L. (var. Rivendel) were each grown in parallel lots of control and drought-stressed monocultures. Rainout shelters (installed in May 2011 on a regrowth after first cut until harvest in mid of June) withheld rainfall of 40 mm in the drought stress treatment. Samples of dried (60°C) and milled (5 mm screen) forage legumes were incubated in a simulation experiment using Rusitec to assess drought effects on parameters for microbial metabolism. Degradability of dry matter and organic matter as well as methane production decreased in incubations with drought-stressed compared to control variants of legume species. Degradability of crude protein, neutral detergent fibre, acid detergent fibre and residual organic matter including non-fibre carbohydrates and lipids were affected by interactions between drought stress and species. Significant interactions were also found for ammonia concentrations, molar SCFA proportions and the microbial communities. It is concluded that drought stress for growing forage legumes influences their ruminal degradation and fermentation as well as the ruminal microbial communities of Bacteria and Archaea differently in a legume species-dependent manner.

Application of MootralTM Reduces Methane Production by Altering the Archaea Community in the Rumen Simulation Technique.

The reduction of methane emissions by ruminants is a highly desirable goal to mitigate greenhouse gas emissions. Various feed additives have already been tested for their ability to decrease methane production; however, practical use is often limited due to negative effects on rumen fermentation or high costs. Organosulphur compounds from garlic (Allium sativum) and flavonoids have been identified as promising plant-derived compounds which are able to reduce methane production. Here, we evaluated the effects of a combination of garlic powder and bitter orange (Citrus aurantium) extracts, Mootral, on ruminal methane production, ruminal fermentation and the community of methanogenic Archaea by using the rumen simulation technique as ex vivo model. The experiment consisted of an equilibration period of 7 days, an experimental period of 8 days and a withdrawal period of 4 days. During the experimental period three fermenters each were either treated as controls (CON), received a low dose of Mootral (LD), a high dose of Mootral (HD), or monensin (MON) as positive control. Application of Mootral strongly reduced the proportion of methane in the fermentation gas and the production rate of methane. Moreover, the experimental mixture induced a dose-dependent increase in the production rate of short chain fatty acids and in the molar proportion of butyrate. Some effects persisted during the withdrawal period. Both, single strand conformation polymorphism and Illumina MiSeq 16S rRNA amplicon sequencing indicated an archaeal community distinct from CON and MON samples in the LD and HD samples. Among archaeal families the percentage of Methanobacteriaceae was reduced during application of both doses of Mootral. Moreover, several significant differences were observed on OTU level among treatment groups and after withdrawal of the additives for LD and HD group. At day 14, 4 OTUs were positively correlated with methane production. In conclusion this mixture of garlic and citrus compounds appears to effectively reduce methane production by alteration of the archaeal community without exhibiting negative side effects on rumen fermentation.

Alterations in the Rumen Liquid-, Particle- and Epithelium-Associated Microbiota of Dairy Cows during the Transition from a Silage- and Concentrate-Based Ration to Pasture in Spring.

In spring dairy cows are often gradually transitioned from a silage- and concentrate-based ration (total mixed ration, TMR) to pasture. Rumen microbiota adaptability is a key feature of ruminant survival strategy. However, only little is known on the temporal and spatial microbial alterations involved. This study aims to investigate how the rumen liquid (LAAB), particle (PAAB), and epithelium (EAAB) associated archaea and bacteria are influenced by this nutritional change. A 10-wk trial was performed, including 10 rumen-fistulated dairy cows, equally divided into a pasture- and a confinement- group (PG and CG). The CG stayed on a TMR-based ration, while the PG was gradually transitioned from TMR to pasture (wk 1: TMR-only, wk 2: 3 h/day on pasture, wk 3 & 4: 12 h/day on pasture, wk 5-10: pasture-only). In wk 1, wk 5, and wk 10 samples of solid and liquid rumen contents, and papillae biopsies were collected. The DNA was isolated, and PCR-SSCP and 16S rRNA gene amplicon sequencing analysis were performed. Cluster analysis revealed a higher similarity between LAAB and PAAB, compared to the EAAB, characterized by higher species diversity. At all three locations the microbiota was significantly influenced by the ration change, opposite the generally acknowledged hypothesis that the EAAB remain more consistent throughout dietary changes. Even though the animals in the PG were already on a full-grazing ration for 4-6 days in wk 5, the microbiota at all three locations was significantly different compared to wk 10, suggesting an adaptation period of several days to weeks. This is in line with observations made on animal level, showing a required time for adaptation of 2-3 weeks for production and metabolic variables. A large part of the rumen prokaryote species remained unaltered upon transition to pasture and exhibited a strong host influence, supporting the hypothesis that the rumen microbiota consists of a core and a variable microbiota. For the effect of the location as well as the ration change either very similar or opposite trends among member species of common taxa were observed, demonstrating that microbes that are phylogenetically close may still exhibit substantially different phenotypes and functions.

Studies on potential effects of fumaric acid on rumen microbial fermentation, methane production and microbial community.

The greenhouse gas methane (CH4) contributes substantially to global climate change. As a potential approach to decrease ruminal methanogenesis, the effects of different dosages of fumaric acid (FA) on ruminal microbial metabolism and on the microbial community (archaea, bacteria) were studied using a rumen simulation technique (RUSITEC). FA acts as alternative hydrogen acceptor diverting 2H from methanogenesis of archaea towards propionate formation of bacteria. Three identical trials were conducted with 12 fermentation vessels over a period of 14 days. In each trial, four fermentation vessels were assigned to one of the three treatment groups differing in FA dosage: low fumaric acid (LFA), high fumaric acid (HFA) and without FA (control). FA was continuously infused with the buffer. Grass silage and concentrate served as substrate. FA led to decreases in pH and to higher production rates of total short chain fatty acids (SCFA) mediated by increases in propionate for LFA of 1.69 mmol d(-1) and in propionate and acetate production for HFA of 4.49 and 1.10 mmol d(-1), respectively. Concentrations of NH3-N, microbial crude protein synthesis, their efficiency, degradation of crude nutrients and detergent fibre fraction were unchanged. Total gas and CH4 production were not affected by FA. Effects of FA on structure of microbial community by means of single strand conformation polymorphism (SSCP) analyses could not be detected. Given the observed increase in propionate production and the unaffected CH4 production it can be supposed that the availability of reduction equivalents like 2H was not limited by the addition of FA in this study. It has to be concluded from the present study that the application of FA is not an appropriate approach to decrease the ruminal CH4 production.

Elevated CO(2) and drought stress effects on the chemical composition of maize plants, their ruminal fermentation and microbial diversity in vitro.

Climate changes are supposed to influence productivity and chemical composition of plants. In the present experiments, it was hypothesised that the incubation of plants exposed to elevated atmospheric carbon dioxide concentrations ([CO2]) and drought stress will result in different ruminal fermentation pattern and microbial diversity compared to unaffected plants. Maize plants were grown, well-watered under ambient (380 ppm CO2, Variant A) and elevated [CO2] (550 ppm CO2, Variant B). Furthermore, each CO2 treatment was also exposed to drought stress (380 ppm and 550 ppm CO2,Variants C and D, respectively), which received only half as much water as the well-watered plants. Plant material from these treatments was incubated in a semi-continuous in vitro fermentation experiment using the rumen simulation technique. Single strand conformation polymorphism (SSCP) analysis was conducted for Bacteria and Archaea specific profiles. The analysis of crude nutrients showed higher contents of fibre fraction in drought stress Variants C and D. Crude protein content was increased by drought stress under ambient but not under elevated [CO2]. Fermentation of drought stress variants resulted in significantly increased pH values, decreased digestibilities of organic matter and increased ammonia-N (NH3-N) concentrations compared with well-watered variants. Additionally, the 550 ppm CO2 Variants B and D showed significantly lower NH3-N concentrations than Variants A and C. The Bacteria- and Archaea-specific SSCP profiles as well as the production rates of short-chain fatty acids and their molar percentages were not affected by treatments. During the first four days of equilibration period, a decrease of molar percentage of acetate and increased molar percentages of propionate were observed for all treatments. These alterations might have been induced by adaptation of the in vitro system to the new substrate. The rumen microflora appeared to be highly adaptive and could cope with altered contents of crude nutrients in plants as induced by elevated [CO2] and drought stress.