Microbial biohydrogenation of oleic acid to trans isomers in vitro

Ruminant products are significant sources of dietary trans fatty acids. Trans fatty acids, including various conjugated linoleic acid isomers, have been shown to act as metabolic modifiers of lipid metabolism. Trans fatty acids originate from biohydrogenation of dietary unsaturated fatty acids by gut microbes; however, the exact synthetic pathways are unclear. It was our goal to examine the biohydrogenation pathway for oleic acid, where oleic acid is hydrogenated directly to stearic acid. Our objective in this study was to trace the time course of appearance of 13C in labeled oleic acid to determine if trans monoenes are formed from the 13C-labeled oleic acid or if the 13C appears only in stearic acid as described in reviews of earlier work. Enrichments were calculated from the mass abundance of 13C in major fatty acid fragments and expressed as a percentage of total carbon isotopomers. Significant 13C enrichment was found in stearic acid, oleic acid, trans-6, trans-7, and in all trans C18:1 in positions 9-16. We concluded that the biohydrogenation of oleic acid by mixed ruminal microbes involves the formation of several positional isomers of trans monoenes rather than only direct biohydrogenation to form stearic acid as previously described.     

The hydrogenation of unsaturated fatty acids by five bacterial isolates from the sheep rumen, including a new species.

Five strictly anaerobic bacteria able to hydrogenate unsaturated fatty acids were isolated from sheep rumen. One was characterized as Ruminococcus albus, two as Eubacterium spp. and two as Fusocillus spp., one of which is named as a new species. The Fusocillus organisms were able to hydrogenate oleic acid and linoleic acid to stearic acid, and linolenic acid to cis-octadec-15-enoic acid. The R. albus and the two Eubacteria did not hydrogenate oleic acid but converted linoleic and linolenic acids to a mixture of octadecenoic acids; trans-octadec-II-enoic acid predominated but several isomeric cis and trans octadecenoic acids were produced together with isomers of non-conjugated octadecadienoic acids. The intermediate and final products of hydrogenation by each organism were compatible with the results from mixed rumen bacteria.     

Biohydrogenation of unsaturated fatty acids by a mixed culture of rumen microorganisms

The biohydrogenation of C-18 unsaturated fatty acids was examined in a mixed culture of microorganisms prepared by inoculating a proper growth medium with a sample of rumen fluid. Some major factors influencing the hydrogenation capacity have been investigated. The age of the mixed culture, the type of inoculum used, the concentration of substrates as well as the presence of sterile rumen fluid in the growth medium were found to be important factors determining biohydrogenation behavior. It could be shown that the mixed microbial culture, which had been grown for about 24 h on a medium similar to that of Bryant and Robinson, contained sterile rumen fluid (10% v/v), and had been inoculated with a sample of the whole untreated rumen content, had the best biohydrogenation capacity. The culture was able to carry out the complete conversion of linoleic and linolenic acid to stearic acid.     

Microbial fatty acid conversion within the rumen and the subsequent utilization of these fatty acids to improve the healthfulness of ruminant food products

Consumers are aware of foods containing microcomponents that may have positive effects on health maintenance and disease prevention. In ruminant milk, meat, and milk products; these functional food components include eicosapentaenoic acid (20:5n3), docosahexaenoic acid (22:6n3), 9c11t-conjugated linoleic acid, and vaccenic acid (11t-18:1). Modifying ruminal microbial metabolism of fatty acid in rumen through animal diet formulation is an effective way to enhance these functional fatty acids in ruminant-derived food products. However, it requires an understanding of the interrelationship between supply of lipid through the diet and rumen fermentation. Lipids in ruminant diets undergo extensive hydrolysis and biohydrogenation in the rumen. Apparent transfer efficiency of eicosapentaenoic acid and docosahexaenoic acid from feed to milk is very low (1.9 to 3.3%), which is, to a large extent, related to their extensive biohydrogenation in the rumen. Therefore, feeding a rumen-protected supplement containing eicosapentaenoic acid and docosahexaenoic acid, can be used to bypass the rumen. Ruminant-derived foods also contain different types of conjugated linoleic acid isomers, which are intermediates of rumen biohydrogenation of linoleic acid (9c12c-18:2). The predominant isomer of conjugated linoleic acid is 9c11t, which has numerous health benefits in animal models. The concentration of conjugated linoleic acid in ruminant-derived food products can be significantly enhanced through animal diet modification. We conclude that most current functional food products from ruminants have potential for their health-supporting properties, and for this market to succeed, an evidence-based approach should be developed in humans.     

Biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria

AIMS: To investigate biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria, and to identify the fungus with the fastest biohydrogenation rate. METHODS AND RESULTS: Biohydrogenation of linoleic acid by mixed rumen fungi and mixed rumen bacteria were compared in vitro. With mixed rumen bacteria, all biohydrogenation reactions were finished within 100 min of incubation and the end product of biohydrogenation was stearic acid. With mixed rumen fungi, biohydrogenation proceeded more slowly over a 24-h period. Conjugated linoleic acid (CLA; cis-9, trans-11 C18 : 2) was an intermediate product, and vaccenic acid (VA; trans-11 C18 : 1) was the end product of biohydrogenation. Fourteen pure fungal isolates were tested for biohydrogenation rate. DNA sequencing showed that the isolate with the fastest rate belonged to the Orpinomyces genus. CONCLUSIONS: It is concluded that rumen fungi have the ability to biohydrogenate linoleic acid, but biohydrogenation is slower in rumen fungi than in rumen bacteria. The end product of fungal biohydrogenation is VA, as for group A rumen bacteria. Orpinomyces is the most active biohydrogenating fungus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate that rumen fungi can biohydrogenate fatty acids. Fungi could influence CLA content of ruminant products.     

Hydrogenation of polyunsaturated fatty acids by human colonic bacteria

Emulsions of the fatty acids linoleic (C18:2 n-6), alpha-linolenic (C18:3 n-3) and arachidonic acid (C20:4 n-6) were incubated for 4 h under anaerobic conditions with human faecal suspensions. Linoleic acid was significantly decreased (P < 0.001) and there was a significant rise (P < 0.05) in its hydrogenation product, stearic acid. Linolenic acid was also significantly decreased (P < 0.01), and significant increases in C18:3 cis-trans isomers (P < 0.01) and linoleic acid (P < 0.05) were seen. With each acid, there were non-significant increases in acids considered to be intermediates in biohydrogenation. The study provides evidence that bacteria from the human colon can hydrogenate C18 essential polyunsaturated fatty acids. However, with arachidonic acid there was no evidence of hydrogenation.     

Isolation of a new phospholipid, phosphatidyl-N-(2-hydroxyethyl)alanine, from rumen protozoa

1. Phosphatidyl-N-(2-hydroxyethyl)alanine was isolated from the mixed protozoal fraction of rumen and characterized. 2. Of the fatty acids 31% was octadecenoic acid, 91% of which was the trans-Delta(11)-isomer, an intermediary in the ruminal hydrogenation of linolenic acid and linoleic acid.     

Isomerization of stable isotopically labeled elaidic acid to cis and trans monoenes by ruminal microbes

A previous study showed that oleic acid was converted by mixed ruminal microbes to stearic acid and also converted to a multitude of trans octadecenoic acid isomers. This study traced the metabolism of one of these trans C18:1 isomers upon its incubation with mixed ruminal microbes. Unlabeled and labeled (18-[13C]trans-9 C18:1) elaidic acid were each added to four in vitro batch cultures with three cultures inoculated with mixed ruminal bacteria and one uninoculated culture. Samples were taken at 0, 12, 24, and 48 h and analyzed for 13C enrichment in component fatty acids by gas chromatography-mass spectrometry. At 0 h of incubation, enrichment was detected only in elaidic acid. By 48 h of incubation, 13C enrichment was 18% (P < 0.01) for stearic acid, 7% to 30% (P < 0.01) for all trans C18:1 isomers having double bonds between carbons six through 16, and 5% to 10% for cis-9 and cis-11 monoenes. After 48 h, 13C enrichment in the uninoculated cultures was only detected in the added elaidic acid. This study shows trans fatty acids exposed to active ruminal cultures are converted to stearic acid but also undergo enzymic isomerization yielding a multitude of positional and geometric isomers.     

Increased expression of a molecular chaperone GroEL in response to unsaturated fatty acids by the biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens

Butyrivibrio fibrisolvens is the most active bacterial species in the biohydrogenation of polyunsaturated fatty acids (PUFA) in the rumen. It needs to remove the unsaturated bonds in order to detoxify the PUFA to enable the growth of the bacterium. Here, we investigated the response of cell membrane-associated proteins in B. fibrisolvens to growth in the presence of PUFA. Numerous changes were observed in the cell membrane-associated proteome. One of the main modifications occurring when the 18:2 fatty acids, linoleic acid and conjugated linoleic acid, were added, was an increased expression of the molecular chaperone GroEL.     

The role of holotrichs in the metabolism of dietary linoleic acid in the rumen

The uptake and metabolism of linoleic acid by rumen holotrichs (mainly Isotricha prostoma and I. intestinalis) has been examined in in vitro infusion experiments. Maximum absorption and metabolism of [1-14C]linoleate by 2 . 10(6) Isotricha suspended in 100 ml buffer was obtained using an infusion rate of 1.6 mg linoleate/h. After 90 min, 84% of the added substrate was recovered within the cells, mainly as free fatty acid or phospholipid. There was a rapid incorporation of radioactivity into phospholipid, mainly phosphatidylcholine, at the commencement of linoleate infusion but no further incorporation after about 40 min. The presence of bacteria during incubations, in approximately the same Isotricha/bacteria ratio as found in the rumen, reduced the uptake of linoleate and the accumulation of free fatty acid by holotrichs but the incorporation into phospholipid remained similar to that obtained in the absence of bacteria. Very little biohydrogenation of linoleic acid occurred in incubations with holotrichs alone. Bacterial suspensions converted linoleic acid to mainly trans monoene and a small amount of stearic acid, but in incubations containing both bacteria and holotrichs, both stearic acid and trans monoene were major products. Using the latter mixed culture, about 20% of the added [1-14C]linoleic acid was present in holotrich phospholipid of which 62% remained as octadecadienoic acid. The Isotricha population was 3 . 10(3)--2 . 10(4)/ml rumen fluid and it contributed about 23% of the linoleic acid in the rumen of a cow on a hay diet.     

The role of microbes in rumen lipolysis and biohydrogenation and their manipulation

Despite the fact that the ruminant diet is rich in polyunsaturated fatty acids (PUFA), ruminant products – meat, milk and dairy – contain mainly saturated fatty acids (SFA) because of bacterial lipolysis and subsequent biohydrogenation of ingested PUFA in the rumen. The link between SFA consumption by man and coronary heart disease is well established. In contrast, ruminant products also contain fatty acids that are known to be beneficial to human health, namely conjugated linoleic acids (CLAs). The aims of research in this field have been to understand the microbial ecology of lipolysis and biohydrogenation and to find ways of manipulating ruminal microbes to increase the flow of PUFA and CLA from the rumen into meat and milk. This review describes our present understanding of the microbial ecology of ruminal lipid metabolism, including some apparently anomalous and paradoxical observations, and the status of how the metabolism may be manipulated and the possible consequential effects on other aspects of ruminal digestion. Intuitively, it may appear that inhibiting the ruminal lipase would cause more dietary PUFA to reach the mammary gland. However, lipolysis releases the non-esterified fatty acids that form the substrates for biohydrogenation, but which can, if they accumulate, inhibit the whole process. Thus, increasing lipase activity could be beneficial if the increased release of non-esterified PUFA inhibited the metabolism of CLA. Rumen ciliate protozoa do not carry out biohydrogenation, yet protozoal lipids are much more highly enriched in CLA than bacterial lipids. How could this happen if protozoa do not metabolise PUFA? The answer seems to lie in the ingestion of plant organelles, particularly chloroplasts, and the partial metabolism of the fatty acids by contaminating bacteria. Bacteria related to Butyrivibrio fibrisolvens are by far the most active and numerous biohydrogenating bacteria isolated from the rumen. But do we misunderstand the role of different bacterial species in biohydrogenation because there are uncultivated species that we need to understand and include in the analysis? Manipulation methods include dietary vegetable and fish oils and plant-derived chemicals. Their usefulness, efficacy and possible effects on fatty acid metabolism and on ruminal microorganisms and other areas of their metabolism are described, and areas of opportunity identified.     

Rates and efficiencies of reactions of ruminal biohydrogenation of linoleic acid according to pH and polyunsaturated fatty acids concentrations

Data from a previous study about the effects of pH and of linolenic acid (C18:3n-3) and linoleic acid (C18:2n-6) concentrations on C18:2n-6 biohydrogenation in ruminal cultures were used to calculate the rates and efficiencies of the three reactions of C18:2n-6 biohydrogenation (isomerisation of C18:2n-6 to CLA; reduction of CLA to trans-octadecenoic acids; reduction of trans-octadecenoic acids to stearic acid). First, low pH was confirmed to inhibit isomerisation and was shown to inhibit the second reduction, leading to an accumulation of vaccenic acid. This later effect had only been observed in some in vivo studies using high concentrate diets, because in in vitro experiments, the very low pH frequently used depresses isomerisation which consequently generates very low amount of substrates for reductions whose variations become difficult to ascertain. Second, C18:2n-6 at high concentration was confirmed to saturate its own isomerisation and the increase of CLA production due to high initial C18:2n-6 was shown to inhibit the two subsequent reductions. Third, C18:3n-3 at high concentrations was confirmed to inhibit C18:2n-6 isomerisation. Moreover, the second reduction was shown to be saturated, probably by all trans-octadecenoic acids intermediates of C18:2n-6 and C18:3n-3 biohydrogenation, leading to an accumulation of trans-octadecenoic acids, especially vaccenic acid. This fatty acid is partly desaturated into CLA in the mammary gland, which explains the synergy between C18:2n-6 and C18:3n-3 for milk CLA noticed by others in vivo. This approach helped explain the actions of pH and of C18:2n-6 and C18:3n-3 concentrations on C18:2n-6 biohydrogenation and allows some explanations about differences noticed between studies.     

Hydrogenation alternatives: effects of trans fatty acids and stearic acid versus linoleic acid on serum lipids and lipoproteins in humans.

The objective of this study was to compare the effects of linoleic acid (cis,cis-C18:2(n-6)) and its hydrogenation products elaidic (trans-C18:1(n-9)) and stearic acid (C18:0) on serum lipoprotein levels in humans. Twenty-six men and 30 women, all normolipemic and apparently healthy, completed the trial. Three experimental diets were supplied to every subject for 3 weeks each, in random order (multiple cross-over). The Linoleate-diet provided 12.0% of total energy intake as linoleic acid, 2.8% as stearic acid, and 0.1% as trans fatty acids. The Stearate-diet supplied 3.9 energy % as linoleic acid, 11.8% stearic acid, and 0.3% trans fatty acids. The Trans-diet provided 3.8 energy % as linoleic acid, 3.0% stearic acid, and 7.7% as monounsaturated trans fatty acids, largely elaidic acid (trans-C18:1(n-9)). Other nutrients were constant. Fasting blood was sampled at the end of each dietary period. Mean (+/- SD) serum LDL cholesterol was 109 +/- 24 mg/dl (2.83 +/- 0.63 mmol/l) on the Linoleate-diet. It rose to 116 +/- 27 mg/dl (3.00 +/- 0.71 mmol/l) on the Stearate-diet (change, 7 mg/dl or 0.17 mmol/l, P = 0.0008) and to 119 +/- 25 mg/dl (3.07 +/- 0.65 mmol/l) on the Trans-diet (change, 9 mg/dl or 0.24 mmol/l, P less than 0.0001). High density lipoprotein (HDL) cholesterol decreased by 2 mg/dl (0.06 mmol/l, P less than 0.0001) on the Stearate-diet and by 4 mg/dl (0.10 mmol/l, P less than 0.0001) on the Trans-diet, both relative to linoleic acid. Our findings show that 7.7% of energy (mean, 24 g/day) of trans fatty acids in the diet significantly lowered HDL cholesterol and raised LDL cholesterol relative to linoleic acid. Combination with earlier results (Mensink, R. P., and M. B. Katan. 1990. N. Engl. J. Med. 323: 439-445) suggests a linear dose-response relation. Replacement of linoleic acid by stearic acid also caused somewhat lower HDL cholesterol and higher LDL cholesterol levels. Hydrogenation of linoleic acid to either stearic or trans fatty acids produces fatty acids that may increase LDL and decrease HDL cholesterol relative to linoleic acid itself.     

Effect of soybean oil availabilities on rumen biohydrogenation and duodenal flow of fatty acids in beef cattle fed a diet with crude glycerine

Soybean oil with different ruminal availability (whole soybeans (WS), soybean oil (SO) and calcium salts (CS)) was used to evaluate the fatty acid (FA) intake, rumen biohydrogenation (BH) and duodenal flow of FA in Nellore steers fed diets with crude glycerine (CG). Eight castrated Nellore steers were fitted with a ruminal and duodenal silicone cannula, and distributed in a double, simultaneous, Latin square 4 × 4 design with four diets and four experimental periods. Concentrates contained ground maize, urea, mineral salts, CG (100 g/kg DM) and soybean products with different availability of soybean oil: (1) no additional fat (CO), (2) WS, (3) SO or (4) CS. Fat supplementation was fixed to obtain 50 g ether extract/kg DM. Experimental treatments had no effect on DM intake, DM duodenal flow or ruminal turnover rate of C:16 FA. However, fat addition increased C:18 and turnover rates of total FA rumen (p < 0.05). CS resulted in lower C:18 turnover rates and lower ruminal BH of monounsaturated and unsaturated FA (UFA) than WS (p < 0.05). SO resulted in a greater duodenal flow of C18:0 (stearic acid), C18:1t-11 (vaccenic acid) and saturated FA than the WS and CS diets (p < 0.05). CS resulted in a higher duodenal flow of C18:3n-3 (linolenic acid) than WS (p < 0.05). The association of CG and calcium salts in Nellore steers was the best nutritional strategy to increase duodenal flow of healthier UFA, which may increase the deposition of these FA in meat. However, SO associated with CG association increased the duodenal flow of vaccenic acid, which is main precursor of endogenous synthesis of conjugated linoleic acids in tissues.     

Fatty Acid Transport and Utilization for the Developing Brain

Abstract: To determine the transport and utilization of dietary saturated, monounsaturated, and n-6 and n-3 polyunsaturated fatty acids for the developing brain and other organs, artificially reared rat pups were fed a rat milk substitute containing the perdeuterated (each 97 atom% deuterium) fatty acids, i.e., palmitic, stearic, oleic, linoleic, and linolenic, from day 7 after birth to day 14 as previously described. Fatty acids in lipid extracts of the liver, lung, kidney, and brain were analyzed by gas chromatography-mass spectrometry to determine their content of each of the deuterated fatty acids. The uptake and metabolism of perdeuterated fatty acid lead to the appearance of three distinct groups of isotopomers: the intact perdeuterated, the newly synthesized (with recycled deuterium), and the natural unlabeled fatty acid. The quantification of these isotopomers permits the estimation of uptake and de novo synthesis of these fatty acids. Intact perdeuterated palmitic, stearic, and oleic acids from the diet were found in liver, lung, and kidney, but not in brain. By contrast, perdeuterated linoleic acid was found in all these organs. Isotopomers of fatty acid from de novo synthesis were observed in palmitic, oleic, and stearic acids in all tissues. The highest enrichment of isotopomers with recycled deuterium was found in the brain. The data indicate that, during the brain growth spurt and the prelude to myelination, the major saturated and monounsaturated fatty acids in brain lipids are exclusively produced locally by de novo biosynthesis. Consequently, the n-6 and n-3 polyunsaturated fatty acids must be transported and delivered to the brain by highly specific mechanisms.     

Multivariate approach to evaluating the fatty acid composition of seed oil in a doubled haploid population of winter oilseed rape (Brassica napus L.)

The fatty acid composition of oil of the zero erucic acid commercial Brassica napus L. is typical for this species. It is rich in oleic acid and contains moderate levels of linoleic and linolenic acid. For human nutrition, it is advantageous primarily to obtain the highest possible content of oleic acid and to maintain the 2:1 ratio of linoleic to linolenic acid, while preserving the average total content of saturated acids. Uni- and multivariate analyses of variance were used for evaluation of doubled haploid lines of winter oilseed rape in respect of five fatty acids: palmitic (C16:0), stearic (C18:0) oleic (C18:1), linoleic (C18:2) and linolenic (C18:3). Some proposals of studying doubled haploid (DH) lines with the use of canonical transformation were also given. In MANOVA, the five original variables (individual fatty acids) were replaced by three 'new' variables (combinations of these acids) and used to evaluate DH lines with respect to the requirements concerning the nutritional role of fatty acids. The first variable was the total content of the saturated acids (C16:0 + C18:0), the second (unchanging) was the content of the monounsaturated acid C18:1, and the third was the difference between polyunsaturated acids, i.e. between linoleic acid, and the doubled content of linolenic acid (C18:2 - 2 x C18:3).     

Detailed Dimethylacetal and Fatty Acid Composition of Rumen Content from Lambs Fed Lucerne or Concentrate Supplemented with Soybean Oil

Lipid metabolism in the rumen is responsible for the complex fatty acid profile of rumen outflow compared with the dietary fatty acid composition, contributing to the lipid profile of ruminant products. A method for the detailed dimethylacetal and fatty acid analysis of rumen contents was developed and applied to rumen content collected from lambs fed lucerne or concentrate based diets supplemented with soybean oil. The methodological approach developed consisted on a basic/acid direct transesterification followed by thin-layer chromatography to isolate fatty acid methyl esters from dimethylacetal, oxo- fatty acid and fatty acid dimethylesters. The dimethylacetal composition was quite similar to the fatty acid composition, presenting even-, odd- and branched-chain structures. Total and individual odd- and branched-chain dimethylacetals were mostly affected by basal diet. The presence of 18∶1 dimethylacetals indicates that biohydrogenation intermediates might be incorporated in structural microbial lipids. Moreover, medium-chain fatty acid dimethylesters were identified for the first time in the rumen content despite their concentration being relatively low. The fatty acids containing 18 carbon-chain lengths comprise the majority of the fatty acids present in the rumen content, most of them being biohydrogenation intermediates of 18∶2n−6 and 18∶3n−3. Additionally, three oxo- fatty acids were identified in rumen samples, and 16-O-18∶0 might be produced during biohydrogenation of the 18∶3n−3.     

Unsaturated fatty acid isomers: effects on the circadian rhythm of a fatty-acid-deficient Neurospora crassa mutant

The fatty acids oleic, linoleic, and linolenic, each of which has a cis double bond at the delta 9 position, are known to lengthen the circadian period of conidiation (spore formation) of strains of Neurospora crassa carrying the cel mutation. cel confers a partial fatty acid requirement on the organism and has been used to promote incorporation of exogenous fatty acids. To test whether a physical effect imparted by the cis double bonds, such as increased membrane fluidity, is critical for the perturbation of the rhythm, various isomers of these fatty acids were supplemented to the bd csp cel strain. Positional isomers of oleic acid, such as petroselinic (delta 6) and vaccenic (delta 11) acids, and longer-chain isomers, such as eicosenoic (delta 11) and erucic (delta 13) acids, did not lengthen the rhythm. The shorter-chain palmitoleic (delta 9) acid did not give a consistent lengthening of the rhythm; it may be elongated to vaccenic acid. In contrast, gamma-linolenic acid (delta 6,9,12) dramatically lengthened the period. Linoelaidic acid (the trans,trans isomer of linoleic acid) lengthened the period at 22 degrees C, but elaidic acid (the trans isomer of oleic acid) did not. Elaidic acid was shown to exert a lengthening effect, but only at lower temperatures. The data do not support a direct physical action as the source of the fatty acids' "chronobiotic" ability.     

Biohydrogenation of erucic acid (22:1 n-9 cis) in artificial rumen. I). Effect of octadecapolyenoic fatty acids and the incubation period

Normally dietary octadecapolyenoic fatty acids are anaerobically hydrogenated in ruminants, both "in vivo" in the rumen and "in vitro" with ruminal content. Here it has been investigated in artificial rumen the process concerning the erucic acid (22:1 n-9 cis) compared with and in presence of C 18 polyunsaturated acids at various incubation times. The results have shown that C 18 polyunsaturated acids hydrogenation with conversion into hydrogenation intermediates and stearic acid does always occur in contrast with erucic acid where it is never revealable, unrelated to the incubations conditions applied.     

In vitro biohydrogenation of four dietary fats

This study was designed to determine in vitro rates of biohydrogenation of dietary unsaturated fatty acids by a mixed population of rumen microbes. The four dietary fats [Alifet High-Energy^® (AHE), Alifet-Repro^® (AR), Megalac^® (MG), and Energy Booster^® (EB)] differ in method of preparation, fatty acid composition, or both of these factors. Dietary fats (20 mg) were incubated with 4 mL strained rumen fluid diluted with 16 mL of medium, 0.8 mL of reducing solution buffer, and 200 mg of a synthetic diet (370 g cellulose, 370 g starch, and 160 g casein per kg DM) at 37 °C. Total contents were collected after 0, 6, 12, 24, or 36 h and change in fatty acid content determined. Disappearance of oleic acid was minimal (0.05–0.20) in AR and MG but moderate (about 0.60) in AHE and EB after 36 h of incubation. Rate of biohydrogenation of linoleic and linolenic acids from AR were similar (0.025 ± 0.009 h⁻¹) and 0.65 of these fatty acids remained intact after 36 h. Rate of biohydrogenation of linoleic acid was four times greater than for oleic acid (0.040 ± 0.013 h⁻¹ versus 0.009 ± 0.002 h⁻¹) in MG. Thus, 0.65 of the linoleic acid but only 0.20 of the oleic acid had disappeared from MG after 36 h. Trans-11 and trans-12 were the predominant trans-isomers in AHE and AR cultures whereas trans-9 and trans-10 were the predominant trans-isomers in EB and MG cultures. None of the dietary fats contained conjugated linoleic acid (CLA) but CLA was present in the incubation inoculum. The amount of CLA decreased with time but this was not affected by source of dietary fat. Most (0.90–0.95) of the long-chain fatty acids eicosapentaenoic (EPA) and docosahexaenoic (DHA) in AR remained after 36 h of incubation. Results demonstrate that biohydrogenation varied among fatty acids and among source of dietary fat and indicate that AR can be used to increase post-ruminal supply of linolenic, EPA and DHA.