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

Nährstoffverfügbarkeit und Nährstoffnutzung von klee- und kräuterreichen Aufwüchsen ökologisch bewirtschafteten Grünlandes entlang der Produktionskette Erzeugung - Konservierung - Verdauung

Ziel des Vorhabens war es, kräuterreiche Grünlandaufwüchse, die im Ökologischen Landbau eine große Bedeutung haben, im Hinblick auf die Futterqualität und die Tierernährung zu bewerten. Für Frühjahrs- und Sommeraufwüchse wurden die Futterqualitäts- und Konservierungseigenschaften eines ungedüngten kräuterreichen Grünlandes und eines ungedüngten Weißklee-Weidelgrasbestandes mit dem eines mit mineralischem Stickstoff gedüngten reinen Weidelgrasbestandes (konventionelle Vergleichsvariante) verglichen. Beim kräuterreichen Grünland bestand der Kräuteranteil aus den feinblättrigen Arten Löwenzahn und Spitzwegerich. Futter wurde im Labormaßstab einsiliert und die Gärfähigkeit sowie die Silagequalität ermittelt. Die Verdauungsphysiologie wurde anhand der Pansensimulationstechnik ‚Rusitec’ studiert. Die kräuterreichen Aufwüchse wiesen eine im Vergleich zum reinen Grasbestand gute Siliereignung und Silagequalität auf mit geringen Ammoniakgehalten und niedrigem Proteinabbau. Kräuterreiches Futter enthielt weniger Rohprotein und Gerüstsubstanzen. Bei der Enzymlöslichkeit der organischen Substanz (ELOS) und der energetischen Bewertung (HFT) traten keine signifikanten Unterschiede zwischen den Varianten auf. Dagegen erwiesen sich die Silagen aus kräuterreichen Aufwüchsen in der Pansensimulation als geringer abbaubar, die mikrobielle Zell- bzw. Proteinsynthese war im Vergleich zur Gras-Klee- und insbesondere gegenüber der Grasvariante vermindert. Die Ergebnisse weisen auf ein hohes, N-effizientes Verwertungspotential kräuterreicher Grünlandaufwüchse in der Wiederkäuerfütterung hin. Es bleibt zu klären, ob die nicht mikrobiell im Pansen abbaubaren Futterbestandteile postruminal zur Energie- und Nährstoffversorgung des Wiederkäuers beizutragen vermögen

Effet de l’huile d’olive, de tournesol ou de graines de lin sur le patron de fermentation et la production de méthane dans le système de simulation ruminale RUSITEC

6 páginas, 2 tablas.-- Contributed to: 13th Seminar of the FAO-CIHEAM Sub-Network on Sheep and Goat Nutrition: Challenging strategies to promote sheep and goat sector in the current global (León, Spain, oct 14-16, 2009).The objective of this study was to investigate the effects of the addition of different vegetable oils to the diet on the fermentation pattern and methane production in Rusitec fermenters. For this purpose four treatments were randomly allocated to sixteen fermenters in a completely random design. Inoculum was obtained from four ruminally fistulated Merino sheep fed lucerne hay and concentrate. Treatments were defined by the diet supplied to the fermenters, so that the control treatment (C) diet was a total mixed ration for ewes on lactation. In the other experimental treatments the control diet was supplemented with olive oil (OO), sunflower oil (SO) or linseed oil (LO) to reach a final concentration of 50 g oil/kg diet. After 7 days of adaptation, samples of effluent, fermenters’ fluid content and digesta of each fermenter were collected during 10 days. There were no significant differences (P>0.05) between treatments in pH values in the fermenters’ fluid content. The LO diet showed (P0.05). All three oils reduced methane production compared with control cultures (P0.05) of oil supplementation on Llactate concentration. Ammonia N concentration was higher (P<0.05) in the control treatment, but there were no significant differences between treatments in microbial protein output. In conclusion, these results indicate that fermentation pattern is affected by oil supplementation mainly by decreasing the acetate to propionate ratio, as well as methane production.Funding received from Spanish MICINN (Projects AGL2008-04805 and AGL2005-04760) and from Junta de Castilla y León (Project GR158) is gratefully acknowledged.Peer Reviewe

Rumen simulation technique study on the interactions of dietary lauric and myristic acid supplementation in suppressing ruminal methanogenesis

The interactions of lauric (C12) and myristic acid (C14) in suppressing ruminal methanogenesis and methanogens were investigated with the rumen simulation technique (Rusitec) using bovine ruminal fluid. The fatty acids were added to basal substrates (grass hay:concentrate, 1:1.5) at a level of 48 g/kg DM, provided in C12:C14 ratios of 5:0, 4:1, 3:2, 2·5:2.5, 2:3, 1:4 and 0:5. Additionally, an unsupplemented control consisting of the basal substrates only was employed. Incubation periods lasted for 15 (n 4) and 25 (n 2) d. CH4 formation was depressed by any fatty acid mixture containing at least 40 % C12, and effects persisted over the complete incubation periods. The greatest depression (70 % relative to control) occurred with a C12:C14 ratio of 4:1, whereas the second most effective treatment in suppressing CH4 production (60 % relative to control) was found with a ratio of 3:2. Total methanogenic counts were decreased by those mixtures of C12 and C14 also successful in suppressing methanogenesis, the 4:1 treatment being most efficient (60 % decline). With this treatment in particular, the composition of the methanogenic population was altered in such a way that the proportion of Methanococcales increased and Methanobacteriales decreased. Initially, CH4 suppression was associated with a decreased fibre degradation, which, however, was reversed after 10 d of incubation. The present study demonstrated a clear synergistic effect of mixtures of C12 and C14 in suppressing methanogenesis, mediated probably by direct inhibitory effects of the fatty acids on the methanogen

Efficiency of monolaurin in mitigating ruminal methanogenesis and modifying C-isotope fractionation when incubating diets composed of either C3 or C4 plants in a rumen simulation technique (Rusitec) system

Mitigation of methanogenesis in ruminants has been an important goal for several decades. Free lauric acid, known to suppress ruminal methanogenesis, has a low palatability; therefore, in the present study the aim was to evaluate the mitigation efficacy of its esterified form (monolaurin). Further, 13C-isotope abundance (δ13C) and 13C-12C fractionation during methanogenesis and fermentation were determined to evaluate possible microbial C-isotope preferences. Using the rumen simulation technique, four basal diets, characterised either by the C3 plants grass (hay) and wheat (straw and grain), or the C4 plant (13C excess compared with C3 plants) maize (straw and grain), and a mixture of the latter two, were incubated with and without monolaurin (50g/kg dietary DM). Added to hay, monolaurin did not significantly affect methanogenesis. When added to the other diets (P<0·05 for the wheat-based diet) methane formation was lowered. Monolaurin decreased fibre disappearance (least effect with the hay diet), acetate:propionate ratio, and protozoal counts. Feed residues and SCFA showed the same δ13C as the diets. Methane was depleted in 13C while CO2 was enriched in 13C compared with the diets. Monolaurin addition resulted in 13C depletion of CO2 and enrichment in CH4 (the latter only in the hay diet). In conclusion, monolaurin proved to effectively decrease methanogenesis in the straw-grain diets although this effect might partly be explained by the concomitantly reduced fibre disappearance. The influence on 13C-isotope abundance and fractionation supports the hypothesis that ruminal microbes seem to differentiate to some extent between C-isotopes during methanogenesis and fermentatio

Nutritive Value of Barley Fodder Grown in a Hydroponics System

A series of experiments were conducted to evaluate barley fodder (BF) as a feed for ruminants. Barley seeds were grown in a specially designed and controlled environment cabin at 15 - 18°C. The first experiment was conducted to germinate barley seeds from day 0 (dO) to day 10 (dIO, n=ll) and samples were collected daily for chemical composition analysis. In the second experiment, the rumen simulation technique (RUSITEC) was used to measure the in vitro dry matter (DM) and crude protein (CP) digestibility and the volatile fatty acids (VF A) produced by BF at different times. The nylon bag technique was also used to measure the DM degradability of BF in the rumen of cattle at 2, 4, 8, 12, 24 and 48 hours. The third experiment on the intake and digestibility of eight-day old BF was measured by an in vivo technique, using four bucks. The fourth experiment involved the measurement of rumen fluid parameters (pH, ammonia and VFA) in three rumen fistulated bucks that were fed on eight-day old BF ad libitum. The CP, organic matter (OM), ether extract (EE), neutral detergent fibre (NDF), acid detergent fibre (ADF) and crude fibre (CF) contents of BF from d1 to d10 increased, but the DM content decreased with the age of BF. DM content at d7 and d8 were 16 and 18% while CP content were 12 and 13% respectively. The amount of calcium (Ca), phosphorus (P) and Nitrate (N03) did not change significantly throughout sprouting time. The in vivo, in vitro and in sacco DM degradability studies showed that 80 to 100% DM loss for BF harvested on d7 to dW. DM intake was lower in the third experiment although digestion coefficient (DC) of BF was very high (98%). This condition was probably due to the abnormal growth of BF during the experimental period. Rumen fluid pH was 5.9-6.0 and ammonia-N (NH3-N) content was 7.0 to 8.8 mg/mL. Total fatty acids production was 70 to 1 00 mmoVL between two and eight hour of collection. The acetic:propionic:butyric ratios were 58:36:1.3, 68:25:2, 61:35:1.2, 64:33:0.7 and 65:32:1 at 0, 2, 4, 6 and 8 hours, respectively. Overall results showed that VFA and ammonia-nitrogen (NH3-N) were produced at a minimum level when BF was given ad libitum although the digestibility was higher in both in sacco and in vitro studies. BF should be given at different levels in order to get optimum pH, VFA and NH3-N production

Synergism of Cattle and Bison Inoculum on Ruminal Fermentation and Select Bacterial Communities in an Artificial Rumen (Rusitec) Fed a Barley Straw Based Diet

This study evaluated the effect of increasing the proportion of bison relative to cattle inoculum on fermentation and microbial populations within an artificial rumen (Rusitec). The experiment was a completely randomized design with a factorial treatment structure (proportion cattle:bison inoculum; 0:100, 33:67, 67:33 and 100:0) replicated in two Rusitec apparatuses (n=8 fermenters). The experiment was 15 d with 8 d of adaptation and 7 d of sampling. Fermenters were fed a diet of 70:30 barley straw:concentrate (DM basis). True digestibility of DM was determined after 48 h of incubation from d 13-15, and daily ammonia (NH3) and volatile fatty acid (VFA) production were measured on d 9-12. Protozoa counts were determined at d 9, 11, 13 and 15 and particle-associated bacteria (PAB) from d 13-15. Select bacterial populations in the PAB were measured using RT-qPCR. Fermenter was considered the experimental unit and day of sampling as a repeated measure. Increasing the proportion of bison inoculum resulted in a quadratic effect (P0.05). Increasing bison inoculum linearly increased (P<0.05) concentrate aNDF disappearance, total and concentrate N disappearance as well as total daily VFA and acetate production. A positive quadratic response (P<0.05) was observed for daily NH3-N, propionate, butyrate, valerate, isovalerate and isobutyrate production, as well as the acetate:propionate ratio. Increasing the proportion of bison inoculum linearly increased (P<0.05) total protozoa numbers. No effects were observed on pH, total gas and methane production, microbial N synthesis, or copies of 16S rRNA associated with total bacteria, Selenomonas ruminantium or Prevotella bryantii. Increasing bison inoculum had a quadratic effect (P<0.05) on Fibrobacter succinogenes, and tended to linearly (P<0.10) increase Ruminococcus flavefaciens and decrease (P<0.05) Ruminococcus albus copy numbers. In conclusion, bison inoculum increased the degradation of feed protein and fibre. A mixture of cattle and bison rumen inoculum acted synergistically, increasing the DM and aNDF disappearance of barley straw

In vitro rumen simulations show a reduced disappearance of deoxynivalenol, nivalenol and enniatin B at conditions of rumen acidosis and lower microbial activity

Ruminants are generally considered to be less susceptible to the effects of mycotoxins than monogastric animals as the rumen microbiota are capable of detoxifying some of these toxins. Despite this potential degradation, mycotoxin-associated subclinical health problems are seen in dairy cows. In this research, the disappearance of several mycotoxins was determined in an in vitro rumen model and the effect of realistic concentrations of those mycotoxins on fermentation was assessed by volatile fatty acid production. In addition, two hypotheses were tested: (1) a lower rumen pH leads to a decreased degradation of mycotoxins and (2) rumen fluid of lactating cows degrade mycotoxins better than rumen fluid of non-lactating cows. Maize silage was spiked with a mixture of deoxynivalenol (DON), nivalenol (NIV), enniatin B (ENN B), mycophenolic acid (MPA), roquefortine C (ROQ-C) and zearalenone (ZEN). Fresh rumen fluid of two lactating cows (L) and two non-lactating cows (N) was added to a buffer of normal pH (6.8) and low pH (5.8), leading to four combinations (L6.8, L5.8, N6.8, N5.8), which were added to the spiked maize substrate. In this study, mycotoxins had no effect on volatile fatty acid production. However, not all mycotoxins fully disappeared during incubation. ENN B and ROQ-C disappeared only partially, whereas MPA showed almost no disappearance. The disappearance of DON, NIV, and ENN B was hampered when pH was low, especially when the inoculum of non-lactating cows was used. For ZEN, a limited transformation of ZEN to alpha-ZEL and beta-ZEL was observed, but only at pH 6.8. In conclusion, based on the type of mycotoxin and the ruminal conditions, mycotoxins can stay intact in the rumen

A review

Urea in diets of ruminants has been investigated to substitute expensive animal and vegetable protein sources for more than a century, and has been widely incorporated in diets of ruminants for many years. Urea is also recycled to the fermentative parts of the gastrointestinal (GI) tracts through saliva or direct secretory flux from blood depending upon the dietary situations. Within the GI tracts, urea is hydrolyzed to ammonia by urease enzymes produced by GI microorganisms and subsequent ammonia utilization serves the synthesis of microbial protein. In ruminants, excessive urease activity in the rumen may lead to urea/ammonia toxicity when high amounts of urea are fed to animals; and in non-ruminants, ammonia concentrations in the GI content and milieu may cause damage to the GI mucosa, resulting in impaired nutrient absorption, futile energy and protein spillage and decreased growth performance. Relatively little attention has been directed to this area by researchers. Therefore, the present review intends to discuss current knowledge in ureolytic bacterial populations, urease activities and factors affecting them, urea metabolism by microorganisms, and the application of inhibitors of urease activity in livestock animals. The information related to the ureolytic bacteria and urease activity could be useful for improving protein utilization efficiency in ruminants and for the reduction of the ammonia concentration in GI tracts of monogastric animals. Application of recent molecular methods can be expected to provide rationales for improved strategies to modulate urease and urea dynamics in the GI tract. This would lead to improved GI health, production performance and environmental compatibility of livestock production

Microbial rDNA sequences as markers to determine microbial synthesis in Rusitec fermenters: A comparison with 15N

Microbial rDNA sequences have been proposed as potential internal markers to determine microbial synthesis in the rumen. The objective of this experiment was to compare values of microbial growth determined using 15N as external marker with concentrations of microbial DNA in fermenters, and to assess if both procedures detected similar differences between diets and solid (SOL) and liquid (LIQ) digesta phases