Ruminal microorganisms hydrogenate polyunsaturated fatty acids (PUFA) present in forages and thereby restrict the availability of health-promoting PUFA in meat and milk. The aim of this study was to investigate PUFA metabolism and the influence of PUFA on members of the ruminal microflora. Eleven of 26 predominant species of ruminal bacteria metabolised linoleic acid (LA; cis-9,cis-12-18:2) substantially. The most common product was vaccenic acid (trans-11-18:1), produced by species related to Butyrivibrio fibrisolvens. alpha-Linolenic acid (LNA; cis-9,cis-12,cis-15-18:3) was metabolised mostly by the same species. The fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n - 3)) and docosahexaenoic acid (DHA; 22:6(n - 3)) were not metabolised. Cellulolytic bacteria did not grow in the presence of any PUFA at 50 microg ml(-1), nor did some butyrate-producing bacteria, including the stearate producer Clostridium proteoclasticum, Butyrivibrio hungatei and Eubacterium ruminantium. Toxicity to growth was ranked EPA > DHA > LNA > LA. Cell integrity, as measured using propidium iodide, was damaged by LA in all 26 bacteria, but to different extents. Correlations between its effects on growth and apparent effects on cell integrity in different bacteria were low. Combined effects of LA and sodium lactate in E. ruminantium and C. proteoclasticum indicated that LA toxicity is linked to metabolism in butyrate-producing bacteria. PUFA also inhibited the growth of the cellulolytic ruminal fungi, with Neocallimastix frontalis producing small amounts of cis-9,trans-11-18:2 (CLA) from LA. Thus, while dietary PUFA might be useful in suppressing the numbers of biohydrogenating ruminal bacteria, particularly C. proteoclasticum, care should be taken to avoid unwanted effects in suppressing cellulolysis.

BackgroundHealth-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.ResultsLinoleic 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%.ConclusionsIt 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.

The Butyrivibrio group comprises Butyrivibrio fibrisolvens and related Gram-positive bacteria isolated mainly from the rumen of cattle and sheep. The aim of this study was to investigate phenotypic characteristics that discriminate between different phylotypes. The phylogenetic position, derived from 16S rDNA sequence data, of 45 isolates from different species and different countries was compared with their fermentation products, mechanism of butyrate formation, lipid metabolism and sensitivity to growth inhibition by linoleic acid (LA). Three clear sub-groups were evident, both phylogenetically and metabolically. Group VA1 typified most Butyrivibrio and Pseudobutyrivibrio isolates, while Groups VA2 and SA comprised Butyrivibrio hungatei and Clostridium proteoclasticum, respectively. All produced butyrate but strains of group VA1 had a butyrate kinase activity <40 U (mg protein)(-1), while strains in groups VA2 and SA all exhibited activities >600 U (mg protein)(-1). The butyrate kinase gene was present in all VA2 and SA bacteria tested but not in strains of group VA1, all of which were positive for the butyryl-CoA CoA-transferase gene. None of the bacteria tested possessed both genes. Lipase activity, measured by tributyrin hydrolysis, was high in group VA2 and SA strains and low in Group VA1 strains. Only the SA group formed stearic acid from LA. Linoleate isomerase activity, on the other hand, did not correspond with phylogenetic position. Group VA1 bacteria all grew in the presence of 200 microg LA ml(-1), while members of Groups VA2 and SA were inhibited by lower concentrations, some as low as 5 microg ml(-1). This information provides strong links between phenotypic and phylogenetic properties of this group of clostridial cluster XIVa Gram-positive bacteria.

The aim of this study was to identify ruminal bacteria that form stearic acid (18 : 0) from linoleic acid (cis-9,cis-12-18 : 2). One 18 : 0-producing isolate, P-18, isolated from the sheep rumen was similar in morphology and metabolic properties to 'Fusocillus' spp. isolated many years ago. Phylogenetic analysis based on nearly full-length 16S rRNA gene sequence (>1300 bp) analysis indicated that the stearate producer was most closely related to Clostridium proteoclasticum B316(T). Clostridium proteoclasticum B316(T) was also found to form 18 : 0, as were other bacteria isolated elsewhere, which occurred in the same family subclass of the low G+C% Gram-positive bacteria, related to Butyrivibrio fibrisolvens. These bacteria are not clostridia, and the ability to form 18 : 0 was present in all strains in contrast to proteolytic activity, which was variable. Production of 18 : 0 occurred in growing, but not in stationary-phase, bacteria, which made detection of biohydrogenating activity difficult, because of the inhibitory effects of linoleic acid on growth.

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Aims: To test various microbial cultures as cattle feed additives. Methods and Results: Four groups of newly born crossbred calves (average body weight 23·5 kg) were reared on green berseem and calf starter which was devoid of cereal grains. Milk was fed up to 8 weeks of age, starting with one tenths and gradually reducing to one twentieths of the body weight. One hundred millilitres of microbial feed additive or 100 g fermented feed was fed to the animals of group 2 (curd containing lactic acid bacteria at 108 cfu ml−1), group 3 (Saccharomyces cerevisiae NCDC‐49 at 106 cfu ml−1) and group 4 (Lactobacillus acidophilus‐15 at 108 cfu ml−1). Group 1 served as control. The incidence and duration of diarrhoea was lower in the animals of probiotic fed groups as compared to control group. Out of three microbial feed additives, yeast feeding showed maximum suppression of diarrhoea followed by Lactobacillus and curd. Conclusions, Significance and Impact of the Study: There was no effect of probiotic feeding on the log number of cells of lactic acid bacteria, yeast and coliform bacteria in the faeces and rumen liquor at any age. The activities of carboxymethylcellulase, xylanase, β‐glucosidase, α‐glucosidase, α‐amylase, protease, urease and pH of the rumen liquor remained unaffected by probiotic feeding at all ages tested in this experiment.

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Two similar Gram-positive rods were isolated from 10 "6 dilutions of ruminal fluid from a sheep receiving a mixed grass hay/concentrate diet, using a medium containing pancreatic casein hydrolysate as sole source of carbon and energy. The isolates did not ferment sugars, but grew on pyruvate or trypticase, forming caproate as the main fermentation product and valerate to a lesser extent. Acetate and propionate were utilized. One of these strains, I-6 T , was selected for further study. Strain I-6 T was a non-motile coccal rod, 1?260?4 mm, with a Gram-positive cell wall ultrastructure and a G+C content of 56?8 mol%. No spores were visible, and strain I-6 T did not survive heating at 80˚C for 10 min. Its rate of NH 3 production was 375 nmol (mg protein) "1 min "1 , placing it in the 'ammonia-hyperproducing' (or HAP) group of ruminal bacteria. 16S rDNA sequence analysis (1296 bases) indicated that it represents a novel species within the 'low-G+C' Grampositive group, for which the name Eubacterium pyruvativorans sp. nov. is proposed. Among cultivated bacteria, strain I-6 T was most closely related (89 % identity) to other asaccharolytic Eubacterium isolates from the mouth and the rumen. It was 98 % identical to uncultured bacterial sequences amplified by others from ruminal digesta.