Biohydrogenation of C18 unsaturated fatty acids to stearic acid by a strain of Butyrivibrio hungatei from the bovine rumen

AIMS: To identify a ruminal isolate which transforms oleic, linoleic and linolenic acids to stearic acid and to identify transient intermediates formed during biohydrogenation. METHODS AND RESULTS: The stearic acid-forming bacterium, isolated from the rumen of a grazing cow, was a Gram-negative motile rod which utilized a range of growth substrates including starch and pectin but not cellulose or xylan. From its 16S rRNA gene sequence, the isolate was identified as a strain of Butyrivibrio hungatei. During conversion of linoleic acid, 9,11-conjugated linoleic acid formed as a transient intermediate before trans-vaccenic acid accumulated together with stearic acid. Unlike previously studied ruminal biohydrogenating bacteria, B. hungatei Su6 was able to convert alpha-linolenic acid to stearic acid. Linolenic acid was converted to stearic via conjugated linolenic acid, linoleic acid and trans-vaccenic acid as intermediates. Oleic acid and cis-vaccenic acid were converted to a series of trans monounsaturated isomers as well as stearic acid. An investigation of these isomers indicated that mixed trans positional isomers are intermediate in the biohydrogenation of cis monounsaturated fatty acids to stearic acid. CONCLUSION: This, the first rigorous identification and characterization of a ruminal bacterium which forms stearic acid, shows that B. hungatei plays an important role in unsaturated fatty acid transformations in the rumen. SIGNIFICANCE AND IMPACT OF THE STUDY: Biohydrogenating bacteria which convert C18 unsaturated fatty acids to stearic acid have not been available for study for many years. Access to B. hungatei Su6 now provides a fresh opportunity for understanding biohydrogenation mechanisms and rumen processes which lead to saturated fat in ruminant products.     

Rumen microbes and fermentation of wild sika deer on the Shiretoko peninsula of Hokkaido Island,Japan

A total of 32 wild Hokkaido sika deer (Cervus nippon yesoensis) were shot (13 in summer, nine in autumn and 10 in winter) in the Syari district, Shiretoko Peninsula of Hokkaido Island, Japan. The ingested foods, rumen fermentation parameters and microbes were determined to evaluate digestive strategy and food availability in each season. Ingested foods and ruminal characteristics greatly varied by season. Rumen digesta mainly comprised of graminoids in summer, graminoids and agricultural products in autumn, and bark and twigs in winter. Rumen pH showed seasonal differences (P<0.05) and was lowest in summer, highest in winter, and intermediate in autumn, reflecting the seasonal differences in ruminal concentration of total volatile fatty acids which were significantly lower (P<0.05) in winter than in summer and autumn. Acetate proportions were significantly higher in winter than in other seasons (P<0.05), while the opposite trend was seen in proportions of propionate and butyrate. Rumen ammonia levels showed significant seasonal differences (P<0.05), decreasing from summer to autumn to winter. Rumen protozoa levels in autumn and winter decreased to 28 and 10% of the levels observed in summer, respectively (P<0.05 for both). The rumen bacteria level in winter was lower (P<0.05) than that in autumn, but no difference was seen for the other seasonal comparisons. Gram negative cocci were present in significantly higher proportions in winter than in other seasons (P<0.05), while Gram negative curved rods were less frequently observed in winter (P<0.05). Based on these results, wild sika deer in this area are shown to survive with rumen microbial populations altered with the dietary conditions that vary greatly by season.     

Establishment of a spinated type of Diplodinium rangiferi by transfaunation of the rumen ciliates of Japanese sika deer (Cervus nippon centralis) to the rumen of two Japanese shorthorn calves (Bos taurus taurus)

One liter of rumen fluid containing 4.7 x 10(4) ciliates/ml, representing four genera including nine species of ciliates from a Japanese sika deer was inoculated into two unfaunated Japanese shorthorn calves. Two weeks after inoculation, all species originally present in the inoculum were subsequently detected in the rumen fluid of one or both calves. Ciliate densities ranged from 10(5)-10(6) cells/ml over the remainder of the 33-wk experiment. The inoculum contained Diplodinium rangiferi. which lacks caudal appendages, as is characteristic for the species. However, three weeks later, the rumen fluid of both calves contained D. rangiferi, which possesses caudal appendages varying from a single spine to multiple spines with a complicated furcate appearance. The caudal spines of D. rangiferi did not disappear during the experiment, even when the diet of the calves was switched to the ration of sika deer from which the inoculum was obtained.     

Influence of methylamine on anaerobic rumen bacterial growth and plant fiber digestion

Anaerobic rumen bacteria were screened for growth inhibition by methylamine. Under nitrogen-limited growth conditions only Ruminobacter amylophilus, Prevotella ruminicola and Megasphaera elsdenii were significantly inhibited. Ruminobacter amylophilus was inhibited more than any other species. Inhibition of R. amylophilus growth occured at all increments in methylamine for all NH₄Cl concentrations tested (0·2–1·8 m ). Dimethylamine was less inhibitory, while trimethyl-, ethyl- and diethylamine were not inhibitory. When methylamine was added to in vitro mixed culture, ruminal fermentations containing alfalfa, neutral detergent fiber disappearance and ammonia production were not affected. This suggests that although methylamine may inhibit specific rumen bacteria, it does not alter overall mixed ruminal microorganism activities with alfalfa as a substrate.     

Exposure of sheep scrapie brain homogenate to rumen-simulating conditions does not result in a reduction of PrP(Sc) levels

AIMS: Experiments were designed to evaluate the potential of rumen-simulating conditions to reduce PrP(Sc) levels. METHODS AND RESULTS: Scrapie-positive brain material was incubated under rumen-simulating conditions. Time points were taken over a 24-h period and PrP(Sc) levels were analysed by Western blot. No loss of PrP(Sc) was observed over a 24-h time period. CONCLUSIONS: Our results indicate that a fully developed rumen fermentation does not provide significant protection against prion infection via the oral route. Developmental changes including senescence of immune system function or other developmental changes in the gastrointestinal tract are potential mechanisms by which relative bovine spongiform encephalopathy (BSE) susceptibility might vary with age. SIGNIFICANCE AND IMPACT OF THE STUDY: Epidemiology of the BSE outbreak in the United Kingdom indicates that younger animals were at higher risk of infection. The rumen undergoes pronounced developmental changes early in life, coinciding with the introduction of fibre into the diet. The timeframe of highest risk of infection overlaps the time in life prior to full rumen development. This work indicates that a fully developed rumen does not provide significant protection against prion infection via the oral route of infection. This result implicates other developmental changes that are responsible for the age-dependent susceptibility of cattle to BSE.     

Short-chain fatty acids and CO2 as regulators of Na+ and Cl− absorption in isolated sheep rumen mucosa

Summary Unidirectional ²²Na⁺ and ³⁶Cl^– fluxes were determined in short-circuited, stripped rumen mucosa from sheep by using the Ussing chamber technique. In both CO₂/HCO _– ³ -containing and CO₂/HCO _– ³ -free solutions, replacement of gluconate by short-chain fatty acids (SCFA, 39 mM) significantly enhanced mucosal-toserosal Na⁺ absorption without affecting the Cl^– transport in the same direction. Short-chain fatty acid stimulation of Na⁺ transport was at least partly independent of Cl^– and could almost completely be abolished by 1 mM mucosal amiloride, while stimulation of Na⁺ transport was enhanced by lowering the mucosal pH from 7.3 to 6.5. Similar to the SCFA action, raising the PCO₂ in the mucosal bathing solution led to an increase in the amiloride-sensitive mucosal-to-serosal Na⁺ flux. Along with its effect on sodium transport, raising the PCO₂ also stimulated chloride transport. The results are best explained by a model in which undissociated SCFA and/or CO₂ permeate the cell membrane and produce a raise in intracellular H⁺ concentration. This stimulates an apical Na⁺/H⁺ exchange, leading to increased Na⁺ transport. The stimulatory effect of CO₂ on Cl^– transport is probably mediated by a Cl^–/HCO _– ³ exchange mechanism in the apical membrane. Binding of SCFA anions to that exchange as described for the rat distal colon (Binder and Mehta 1989) probably does not play a major role in the rumen.Abbreviations DIDS 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid - G _t transepithelial conductance (mS·cm^-2) - HSCFA undissociated short-chain fatty acids - J _ms mucosal-to-serosal flux (Eq · cm^-2 · h^-1) - J _net net flux (Eq · cm^-2 · h^-1) - J _sm serosal-to-mucosal flux (Eq · cm^-2 · h^-1) - PD transepithelial potential difference (mV) - SCFA dissociated short-chain fatty acids - SCFA short-chain fatty acids