Biohydrogenation Of Unsaturated Fatty Acids Research Articles

Nutritional strategies to improve the lipid composition of meat, with emphasis on Thailand and Asia

This article reviews opportunities for enriching the lipids of meat with n−3 fatty acids and conjugated linoleic acids (CLAs), both considered beneficial to human health. Special focus is put on feeds available and research carried out in Thailand. A differentiated consideration concerning the value of different n−3 fatty acids and isomers of CLAs is necessary. In ruminants, it is difficult to enrich the meat with n−3 fatty acids due to the extensive ruminal biohydrogenation of unsaturated fatty acids, but several possibilities to enhance the proportion of the most desired CLA isomer, rumenic acid, exist. By contrast, pork and poultry meat can be easily enriched with n−3 fatty acids. With purified CLA sources, CLAs also can be enhanced, but it is difficult to achieve this exclusively for rumenic acid. An interesting approach might consist in supplementing the CLA precursor vaccenic acid instead. Possible constraints for meat quality and in the fatty acid levels achieved are outlined.

Application of esterase inhibitors: A possible new approach to protect unsaturated fatty acids from ruminal biohydrogenation

General nutritional guidelines recommend reducing the consumption of fats originated from ruminant products. This is due to the ruminal biohydrogenation of unsaturated fatty acids (UFAs) which leads to the presence of unhealthy saturated or trans fats in ruminant products. Here, for the first time to our knowledge, we have focused on the main biochemical process which results in the saturation of UFAs. Rumen lipolytic activity (RLA) generates non‐esterified fatty acids (NEFAs) in the rumen, which are a prerequisite for the biohydrogenation process to occur. We have examined different concentrations of pyridostigmine bromide (PB), of reversible cholinesterase inhibitor, in batch cultures containing 80 mg soybean oil, as a source of triglyceride. PB is the main active compound of an FDA approved drug for the treatment of myasthenia gravis and also pretreatment against nerve gas in humans. Our hypothesis was to evaluate PB as an inhibitor for RLA. In normal conditions, soon after triglycerides enter the rumen, they are hydrolyzed as a result and free fatty acids undergo the biohydrogenation process. Our results indicated that no significant (P > 0.01) reduction in linoleic acid (C18:2, ω6) after 6 h incubation was observed for cultures containing PB with concentrations above 0.052 g/dL. We concluded that PB has the potential to inhibit RLA in cultures after 6 h of incubation. Such findings suggest the potential of PB to be utilized in vivo as a feed additive to inhibit biohydrogenation of unsaturated fatty acids in the rumen.Practical applications: The biohydrogenation of health benefitial unsaturated fatty acids in the rumen cause ruminant products, such as milk and meat, to contain highly saturated fats. Inhibition of rumen microbial lipolytic activity in vivo could increase the flow of unsaturated fatty acids for absorption and therefore, would have the potential to improve fatty acid composition of ruminant milk and meat. This would also have benefits for the animal.In normal ruminal fermentation, unsaturated fatty acids are hydrolyzed as a result of rumen lipolytic activity. Hydrolysis of lipids and production of non‐esterified fatty acids are prerequisite for biohydrogenation of unsaturated fatty acids. We have applied esterase inhibitors in vitro to investigate whether it is possible to block rumen lipolytic activity or not. Our objective was to introduce a new method to bypass unsaturated fatty acids to small intestine.

Evaluation of biohydrogenation rate of canola vs. soya bean seeds as unsaturated fatty acids sources for ruminants insitu.

An experiment was conducted to study disappearance of C14 to C18 fatty acids, lag times and biohydrogenation (BH) rates of C18 fatty acids of ground soya bean and canola seeds insitu. Three ruminally fistulated Dallagh sheep were used to determine ruminal BH of unsaturated fatty acids (UFAs). Differences in the disappearance of fatty acids through the bags and lag times were observed between the oilseeds. We saw that the longer the incubation time of the oilseeds in the rumen, the lower the content of C18:2 and C18:3. Significantly higher lag times for both C18:2 and C18:3 were observed in ground canola compared to ground soya bean. BH rates of C18:2 and C18:3 fatty acids in soya bean were three times higher than those of canola. These results suggest that the fatty acid profile of fat source can affect the BH of UFAs by rumen micro-organisms. So that UFAs of canola had higher ability to escape from ruminal BH. It seems that fatty acid profile of ruminant products is more affected by canola seed compared to soya bean seed.

RUMINANT NUTRITION SYMPOSIUM: How to use data on the rumen microbiome to improve our understanding of ruminant nutrition.

Metagenomics and high-throughput sequencing have greatly expanded our knowledge of the rumen microbiome. Surveys of all 4 cellular microbial groups (bacteria, archaea, protozoa, and fungi) reveal profound diversity. Even so, evidence exists for core members to perform key degradative or fermentative roles for the host. Some core members are functionally similar yet taxonomically diverse, and noncore members are particularly diverse and probably vary among diets, animals, and over time after feeding. Gains in functional knowledge are being made and offer much potential not only to improve fiber digestibility, decrease methane emissions, and improve efficiency of nitrogen usage but also to help explain the differences in nutrient digestibility or feed efficiency among animals fed the same diet. Integrated research using metagenomics, bioinformatics, traditional ruminant nutrition, and statistical inferences have provided opportunities for ruminant nutritionists and rumen microbiologists to work synergistically to improve nutrient utilization efficiency while minimizing output of wastes and emissions of methane and ammonia. Examples we highlight include residual feed intake, rumen biohydrogenation of unsaturated fatty acids, and dietary inclusion of ionophores. However, there are still some quantitative limitations in approaches being used. This review addresses knowledge gained and current limitations and challenges that remain.

Glycerol combined with oils did not limit biohydrogenation of unsaturated fatty acid but reduced methane production in vitro

Three runs of in vitro incubations were conducted to evaluate the effect of glycerol (0 or 150g/kg DM) combined with three different diets: Tifton 85 hay at 850g/kg DM without oil seeds (HWO), Tifton 85 hay+80g of soybean oil/kg DM (HSO) and Tifton 85 hay+80g of linseed oil/kg DM (HLO) incubated for 0, 6, 12 and 24h on fatty acid composition and ruminal fermentation parameters. Real-time PCR was used to quantify microbial population at 24h. Methanogens, fibrolitic and lipolityc bacteria were expressed as a proportion of total rumen bacterial 16S rDNA. Separately, kinetic of gas production was assessed at 2, 4, 6, 8, 10, 12, 14, 18, 22, 24, 36, 42 and 48h. In vitro true digestibility, g/kg (IVTD) and CH4 (%/g DMD) production were evaluated at 48h. The experimental design for fatty acid composition and ruminal parameters was a randomized block in a factorial arrangement 2×3×4, glycerol, diets and time, respectively. Microbial quantification, IVTD and methane were evaluated with the same design but without time as a factor. The pH value and ammonia concentration were lower in HWO compared with HSO and HLO diets, independent of glycerol addition. Ruminococcus albus, Ruminococcus flavefaciens, Butyrivrio vaccenic acid and stearic acid subgroup did not change with glycerol and oil addition (P>0.05). Among all cellulolytic bacteria, Fibrobacter succinogenes was the most sensitive to the addition of vegetable oil in the diet. Methane production decreased in HSO diet combined with glycerol and HLO diets with or without glycerol addition. Lag time (h) from the gas production kinetics decreased in HWO and HSO diets combined with glycerol. The C2:C3 ratio decreased in diets with glycerol addition. Glycerol inclusion did not reduce biohydrogenation of unsaturated fatty acids in vitro. Glycerol was ineffective to reduce the potentially negative effects of vegetable oils on ruminal fermentation and digestibility in vitro. Indeed, the proportion of Anaerovibrio lipolytica increased in all diets with glycerol added and unsaturated fatty acids profile remained unchanged.

Subcutaneous adipose fatty acid profiles and related rumen bacterial populations of steers fed red clover or grass hay diets containing flax or sunflower-seed.

Steers were fed 70∶30 forage∶concentrate diets for 205 days, with either grass hay (GH) or red clover silage (RC), and either sunflower-seed (SS) or flaxseed (FS), providing 5.4% oil in the diets. Compared to diets containing SS, FS diets had elevated (P<0.05) subcutaneous trans (t)-18:1 isomers, conjugated linoleic acids and n-6 polyunsaturated fatty acid (PUFA). Forage and oilseed type influenced total n-3 PUFA, especially α-linolenic acid (ALA) and total non-conjugated diene biohydrogenation (BH) in subcutaneous fat with proportions being greater (P<0.05) for FS or GH as compared to SS or RC. Of the 25 bacterial genera impacted by diet, 19 correlated with fatty acids (FA) profile. Clostridium were most abundant when levels of conjugated linolenic acids, and n-3 PUFA's were found to be the lowest in subcutaneous fat, suggestive of their role in BH. Anerophaga, Fibrobacter, Guggenheimella, Paludibacter and Pseudozobellia were more abundant in the rumen when the levels of VA in subcutaneous fat were low. This study clearly shows the impact of oilseeds and forage source on the deposition of subcutaneous FA in beef cattle. Significant correlations between rumen bacterial genera and the levels of specific FA in subcutaneous fat maybe indicative of their role in determining the FA profile of adipose tissue. However, despite numerous correlations, the dynamics of rumen bacteria in the BH of unsaturated fatty acid and synthesis of PUFA and FA tissue profiles require further experimentation to determine if these correlations are consistent over a range of diets of differing composition. Present results demonstrate that in order to achieve targeted FA profiles in beef, a multifactorial approach will be required that takes into consideration not only the PUFA profile of the diet, but also the non-oil fraction of the diet, type and level of feed processing, and the role of rumen microbes in the BH of unsaturated fatty acid.

Changes in fatty acid composition of various full fat crushed oilseeds and their free oils when incubated with rumen liquor in vitro

The fatty acid pattern of dietary lipids can be modified during rumen biohydrogenation (BH). The objective of the present study was to assess changes in the FA pattern of different oilseed products supplied either as crushed full fat oilseed or as free oil after in vitro incubation with buffered rumen liquor. The FA patterns were determined at the beginning and compared with those measured after 24 h of incubation. The contents of fatty acids (FA) < C18 increased (p < 0.05) in nearly all treatments, eventually due to microbial de novo synthesis and fermentation of carbohydrates and proteins during incubation. In contrast, the contents of the dominating C18 FA, (oleic acid – C18:1c9, linoleic acid – C18:2c9,12, linolenic acid – C18:3c9,12,15) were reduced due to BH, resulting in the accumulation of characteristic BH intermediates, such as conjugated linoleic acid (CLA) isomer C18:2c9t11 (rumenic acid). However, both for crushed full fat oilseeds and their free oils the process of BH was not completed at the end of incubation. The disappearance was highest for C18:3c9,12,15, followed by C18:2c9,12 and C18:1c9. The rate of BH of unsaturated FA was higher in the crushed form compared to the oil form. Higher amounts of BH intermediates accumulated in the crushed form. Obviously, the physical form affects the degree of BH in vitro. The current results suggest that feeding crushed full fat seeds instead of their free oils to dairy cows might stimulate the formation of beneficial BH intermediates such as CLA in the rumen.

Lipid oxidation products of heated soybeans as a possible cause of protection from ruminal biohydrogenation

AbstractHeating oilseeds has been shown to improve the milk fatty acid profile when given to dairy cows, compared to raw oilseeds. However, results from published studies are conflicting. The conditions of heating and storage of the oilseeds could be responsible for these differences, probably partly through their effects on lipid oxidation, the products of which could act on ruminal biohydrogenation (BH). Thus, 15 different treatments were applied to ground soybeans: three levels of heating (no heating, 30 min at 110 or 150°C) × 5 ambient storage durations (0, 1, 2, 4, or 6 months). Soybeans were incubated in vitro with ruminal fluid for 6 h. Triacylglycerol (TAG) polymers, hydroperoxides and hydroxyacids (HOA), aldehydes, and fatty acids were assayed in soybeans and ruminal culture. No TAG polymer was detected in any treatment. Soybeans stored for a long time had a high content of HOA, whereas those heated at 150°C, whatever the storage duration, had high aldehyde contents. The percentage disappearance of cis‐9,cis‐12 18:2 and cis‐9,cis‐12,cis‐15 18:3 in incubates decreased significantly in cultures with heated soybeans, especially at 150°C, suggesting that this partial protection of polyunsaturated fatty acids (PUFA) from BH was at least in part linked to the aldehyde content of the heated soybeans.Practical applications: Oilseeds given to ruminants are often heated, and heat treatment is known to generate oxidation products. Knowing what oxidation products influence ruminal biohydrogenation of unsaturated fatty acids could result in technological processes allowing a better transfer of unsaturated fatty acids from oilseeds to ruminant products.

Identification of C18 Intermediates Formed During Stearidonic Acid Biohydrogenation by Rumen Microorganisms In Vitro

In vitro batch incubations were used to study the rumen biohydrogenation of unsaturated fatty acids. An earlier study using increasing supplementation levels of stearidonic acid (18:4n-3), revealed that the rumen microbial population extensively biohydrogenates 18:4n-3 after 72h of in vitro incubation, though several intermediates formed were not completely characterized. Therefore, in the present study, samples were reanalyzed in order to identify the 18:2, 18:3 and 18:4 biohydrogenation intermediates of 18:4n-3. Gas-liquid chromatography coupled to mass spectrometry was used to characterize these intermediates. The acetonitrile chemical ionization mass spectrometry of the fatty acid methyl esters derivatives enabled the discrimination of fatty acids as non-conjugated or conjugated biohydrogenation intermediates. In addition, the acetonitrile covalent adduct chemical ionization tandem mass spectrometry yielded prominent ions indicative of the double bond position of the major 18:3 isomers, i.e. Δ5,11,15 18:3. Furthermore, the 4,4-dimethyloxazoline derivatives prepared from the fatty acid methyl esters enabled the structure of novel 18:2, 18:3 and 18:4 biohydrogenation intermediates to be elucidated. The intermediates accumulated in the fermentation media after 72h of incubation of 18:4n-3 suggest that similar to the biohydrogenation pathways of linoleic (18:2n-6) and α-linolenic (18:3n-3) acids, the pathway of the 18:4n-3 also proceeds with the formation of conjugated fatty acids followed by hydrogenation, although no conjugated dienes were found. The formation of the novel biohydrogenation intermediates of 18:4n-3 seems to follow an uncommon isomerization pattern with distinct double bond migrations.

Prefermented cereals containing fungal gamma-linolenic acid and their effect on rumen metabolism in vitro

The application of Thamnidium elegans fungal strain CCF 1456 (TE) for effective utilization of various agroindustrial materials creates new perspectives for animal cereal diets enriched with microbial &amp;gamma;-linolenic acid (GLA). Diets consisting of lucerne hay (LH) plus prefermented cereals (wheat bran/spent malt grains, WB+TE or WB+TE enriched with sunflower oil, WB+SO+TE in the first experiment and ground maize grains, GC+TE in the second experiment) were used in the artificial rumen. We examined their effect on the rumen fermentation pattern and lipid metabolism. The diet affected the results of degradability of dry matter, organic matter, neutral detergent fibre and acid detergent fibre of LH+WB diets (P &amp;lt; 0.05 and P &amp;lt; 0.01). The GLA daily output of prefermented diet substrates LH+WB+TE and LH+WB+SO+TE, or LH+GC+TE was higher compared to the non-prefermented LH+WB or LH+GC, respectively (P &amp;lt; 0.05 and P &amp;lt; 0.01). Daily outputs of trans11 oleic (TVA) of the LH+GC+TE diet were higher versus the non-prefermented LH+GC (P &amp;lt; 0.01). The biohydrogenation of fatty acids (C18:1 cis9 oleic, C18:2 linoleic, C18:3n-3 alpha-linolenic, C18:3n-6 GLA and total FA) of prefermented cereal diets was not influenced. Cereal diets containing microbial GLA might positively enhance GLA daily outputs in the RUSITEC effluent, but they are not effective enough to decrease the biohydrogenation of unsaturated fatty acids.

Effect of linseed oil and fish oil alone or as an equal mixture on ruminal fatty acid metabolism in growing steers fed maize silage-based diets1

Because of the potential benefits to human health, there is interest in increasing 18:3n-3, 20:5n-3, 22:6n-6, and cis-9,trans-11 CLA in ruminant foods. Four Aberdeen Angus steers (406 ± 8.2 kg of BW) fitted with ruminal and duodenal cannulas were used in a 4 × 4 Latin square experiment with 21-d periods to examine the potential of fish oil (FO) and linseed oil (LO) in the diet to increase ruminal outflow of trans-11 18:1 and total n-3 PUFA in growing cattle. Treatments consisted of a control diet (60:40; forage:concentrate ratio, on a DM basis, respectively) based on maize silage, or the same basal ration containing 30 g/kg of DM of FO, LO, or a mixture (1:1, wt/wt) of FO and LO (LFO). Diets were offered as total mixed rations and fed at a rate of 85 g of DM/(kg of BW(0.75)/d). Oils had no effect (P = 0.52) on DMI. Linseed oil had no effect (P > 0.05) on ruminal pH or VFA concentrations, whereas FO shifted rumen fermentation toward propionate at the expense of acetate. Compared with the control, LO increased (P < 0.05) 18:0, cis 18:1 (Δ9, 12-15), trans 18:1 (Δ4-9, 11-16), trans 18:2, geometric isomers of 9,11, 11,13, and 13,15 CLA, trans-8,cis-10 CLA, trans-10,trans-12 CLA, trans-12,trans-14 CLA, and 18:3n-3 flow at the duodenum. Inclusion of FO in the diet resulted in greater (P < 0.05) flows of cis-9 16:1, trans 16:1 (Δ6-13), cis 18:1 (Δ9, 11, and 13), trans 18:1 (Δ6-15), trans 18:2, 20:5n-3, 22:5n-3, and 22:6n-3, and decreased (P < 0.001) 18:0 at the duodenum relative to the control. For most fatty acids at the duodenum, responses to LFO were intermediate of FO and LO. However, LFO resulted in greater (P = 0.04) flows of total trans 18:1 than LO and increased (P < 0.01) trans-6 16:1 and trans-12 18:1 at the duodenum compared with FO or LO. Biohydrogenation of cis-9 18:1 and 18:2n-6 in the rumen was independent of treatment, but both FO and LO increased (P < 0.001) the extent of 18:3n-3 biohydrogenation compared with the control. Ruminal 18:3n-3 biohydrogenation was greater (P < 0.001) for LO and LFO than FO, whereas biohydrogenation of 20:5n-3 and 22:6n-3 in the rumen was marginally less (P = 0.05) for LFO than FO. In conclusion, LO and FO at 30 g/kg of DM altered the biohydrogenation of unsaturated fatty acids in the rumen, causing an increase in the flow of specific intermediates at the duodenum, but the potential of these oils fed alone or as a mixture to increase n-3 PUFA at the duodenum in cattle appears limited.

Enhancing fatty acid composition of milk and meat through animal feeding

In ruminants, extensive ruminal biohydrogenation of unsaturated fatty acids (FA) results in numerous cis and trans isomers of 18:1 and of conjugated and non-conjugated 18:2, the incorporation of which into ruminant products depends on the composition of the diet (forage vs concentrate) and of dietary lipid supplements. The low amount of 18:3n-3 (α-linolenic acid) absorbed explains its limited incorporation in meat and milk lipids. Its protection against hydrogenation has been an objective for several decades, but only encapsulation in a protein matrix is efficient. In non-ruminants, the FA composition of products is determined by dietary FA, despite minor differences in digestibility and in metabolic activity. Physicochemical differences in intestinal absorption processes between ruminants and non-ruminants can explain the lower FA digestibility in non-ruminants, especially for saturated FA. Unlike in non-ruminants, FA digestibility in ruminants does not depend on FA intake, except for 18:0. The decrease in cow butterfat, especially with concentrate diets, is generally attributed to t10–18:1 or t10,c12–18:2, but the regulation is probably more complex. Differences in terms of butterfat content and FA composition of milk between cow, ewe and goat responses to the amount and composition of ingested lipids are due to between-species variations in mammary metabolism. In animals bred for meat production, dietary 18:3n-3 results in increases in this FA and in n-3 long-chain polyunsaturated FA (20:5n-3, 22:5n-3) in muscles. The extent of this increase depends both on animal and nutritional factors. Grass is a source of 18:3n-3, which contributes to increased 18:3n-3 in muscle of ruminants as well as of pigs. Conjugated linoleic acids are mainly present in fat tissues and milk due to t11–18:1 desaturation. Their concentration depends on tissue type and on animal species. Non-ruminants fed synthetic conjugated linoleic acids incorporate them in significant amounts in muscle, depending on the isomer. All dietary manipulations favouring polyunsaturated FA incorporation in milk and meat lipids increase the risk of lipoperoxidation, which can be efficiently prevented by use of dietary combined hydro- and lipophilic antioxidants in the diet. Putative effects on organoleptic and technological quality of products deserve further studies.

Effect of different levels of monensin in diets containing whole cottonseed on milk production and composition of lactating dairy cows

Summary The objective of this study was to determine the effects of feeding different levels of monensin on feed intake, milk production and composition, and milk fatty acid profile in lactating Holstein cows. Four multiparous cows averaging 517 ± 47 (SD) kg in body weight and 101 ± 19.8 (SD) days in milk were housed individually in tie-stalls. The study was conducted as a 4 × 4 Latin square design for four periods (14-d for adaptation and 7-d for sampling). Cows were offered four dietary treatments (0, 10, 20, or 30 mg of monensin/kg of DM) as total mixed ration, twice daily. Dry matter (DM) intake was similar among treatments. Monensin supplementation significantly increased (P<0.05) milk yield and 4% fat corrected milk (FCM). Milk fat and protein percentages were not affected by monensin supplementation, but fat yield was increased. Monensin reduced the percentage of the short-chain and saturated fatty acids in milk fat, but had no effect on the percentages of medium- and long-chain fatty acids. Monensin supplementation increased (P<0.05) unsaturated fatty acids concentrations in milk fat. Based on the results of this study, feeding monensin was effective in inhibiting the biohydrogenation of unsaturated fatty acids in the rumen, and consequently increased the percentage of unsaturated fatty acids in milk fat, which improves the health characteristics of milk for human consumption.

Ruminal biohydrogenation of unsaturated fatty acids in vitro as affected by chitosan

Because of the health-promoting effects of conjugated linoleic acid (CLA) isomers in humans, there is growing interest in increasing the content of C18:1 t11 in the rumen in order to enhance the final CLA level in ruminant milk and meat products. Modifying ruminal microflora populations has been viewed as a means to increase their C18:1 t11 content. Therefore, the objective of this experiment was to identify whether chitosan, a natural antimicrobial agent, could be used to modify the biohydrogenation intermediates of two fat sources. The study used a Rusitec unit consisting of eight vessels, and the diets were grass hay and a concentrate mixture (10:90) formulated using either sunflower or rapeseed meal as a fatty acid (FA) source. The incubation experiment consisted of a 6-day adaptation followed by a 5-day chitosan dosing period (750 mg/d), sampling for FA profile and pH determination on the last 3 days. Chitosan interfered in FA biohydrogenation, decreasing total C18:0 (48.7 g/100 g FA versus 23.2 g/100 g FA; P < 0.01) and increasing total C18:1 (5.4 g/100 g FA versus 25.8 g/100 g FA; P < 0.05), C18:1 t11 (4.5 g/100 g FA versus 19.6 g/100 g FA; P < 0.05), total CLA (0.17 g/100 g FA versus 0.37 g/100 g FA; P < 0.05), t9t11 CLA (0.10 g/100 g FA versus 0.21 g/100 g FA; P < 0.05), and the C18:1 t11/C18:0 ratio (0.09 g/100 g FA versus 0.89 g/100 g FA; P < 0.05). Compared to sunflower meal, when rapeseed meal was incubated as the fat source in the concentrate mixture, only total C16:0 was decreased (22.9 g/100 g FA versus 20.6 g/100 g FA; P < 0.05). Chitosan supplementation increased proportions of C18:1 c9, C18:1 c11, and t10c12 CLA only when rapeseed meal was used as the fat source in the concentrate mixture. Chitosan was very effective at inhibiting biohydrogenation in vitro by increasing C18:1 t11 and total CLA proportions and lowering the proportion of saturated FA, regardless of the FA source. However, chitosan increased other biohydrogenation intermediates to a greater extent with rapeseed meal as the dietary fat source.

Investigating unsaturated fat, monensin, or bromoethanesulfonate in continuous cultures retaining ruminal protozoa. I. Fermentation, biohydrogenation, and microbial protein synthesis

Methane is an end product of ruminal fermentation that is energetically wasteful and contributes to global climate change. Bromoethanesulfonate, animal-vegetable fat, and monensin were compared with a control treatment to suppress different functional groups of ruminal prokaryotes in the presence or absence of protozoa to evaluate changes in fermentation, digestibility, and microbial N outflow. Four dual-flow continuous culture fermenter systems were used in 4 periods in a 4×4 Latin square design split into 2 subperiods. In subperiod 1, a multistage filter system (50-μm smallest pore size) retained most protozoa. At the start of subperiod 2, conventional filters (300-μm pore size) were substituted to efflux protozoa via filtrate pumps over 3 d; after a further 7 d of adaptation, the fermenters were sampled for 3 d. Treatments were retained during both subperiods. Flow of total N and digestibilities of NDF and OM were 18, 16, and 9% higher, respectively, for the defaunated subperiod but were not different among treatments. Ammonia concentration was 33% higher in the faunated fermenters but was not affected by treatment. Defaunation increased the flow of nonammonia N and bacterial N from the fermenters. Protozoal counts were not different among treatments, but bromoethanesulfonate increased the generation time from 43.2 to 55.6h. Methanogenesis was unaffected by defaunation but tended to be increased by unsaturated fat. Defaunation did not affect total volatile fatty acid production but decreased the acetate:propionate ratio; monensin increased production of isovalerate and valerate. Biohydrogenation of unsaturated fatty acids was impaired in the defaunated fermenters because effluent flows of oleic, linoleic, and linolenic acids were 60, 77, and 69% higher, and the ratio of vaccenic acid:unsaturated FA ratio was decreased by 34% in the effluent. This ratio was increased in both subperiods with the added fat diet, indicating an accumulation of intermediates of biohydrogenation. However, the flow of 18:2 conjugated linoleic acid was unaffected by defaunation or by treatments other than added fat. The flows of trans-10, trans-11, and total trans-18:1 fatty acids were not affected by monensin or faunation status.

Manipulating the fatty acid composition of lipids in the digital cushion of the bovine claw

The fatty acid (FA) composition of animal tissues is influenced by many factors including age, dietary forage to concentrate ratio, breed and energy status. Despite a high degree of bio-hydrogenation of unsaturated FA’s in the rumen, another crucial influence on tissue FA composition is the FA composition of the diet. Feeding linseed has been shown to increase omega-3 (n-3) polyunsaturated fatty acids (PUFA) in beef. Altering the fat content and composition of the bovine digital cushion has potential implications for lameness in cattle. This study aimed to test the hypothesis that cattle offered extruded linseed would show a marked difference in FA composition in the lipids of the digital cushion compared to cattle offered a diet with no linseed.

Role of the protozoan Isotricha prostoma, liquid-, and solid-associated bacteria in rumen biohydrogenation of linoleic acid

From the simultaneous accumulation of hydrogenation intermediates and the disappearance of Isotricha prostoma after algae supplementation, we suggested a role of this ciliate and/or its associated bacteria in rumen biohydrogenation of unsaturated fatty acids. The experiments described here evaluated the role of I. prostoma and/or its associated endogenous and exogenous bacteria in rumen biohydrogenation of C18:2n-6 and its main intermediates CLA c9t11 and C18:1t11. Fractions of I. prostoma and associated bacteria, obtained by sedimentation of rumen fluid sampled from a monofaunated sheep, were used untreated, treated with antibiotics or sonicated to discriminate between the activity of I. prostoma and its associated bacteria, the protozoan or the bacteria, respectively. Incubations were performed in triplicate during 6 h with unesterified C18:2n-6, CLA c9t11 or C18:1t11 (400 μg/ml) and 0.1 g glucose/cellobiose (1/1, w/w). I. prostoma did not hydrogenate C18:2n-6 or its intermediates whereas bacteria associated with I. prostoma converted a limited amount of C18:2n-6 and CLA c9t11 to trans monoenes. C18:1t11 was not hydrogenated by either I. prostoma or its associated bacteria but was isomerized to C18:1c9. A phylogenetic analysis of clones originating from Butyrivibrio-specific PCR product was performed. This indicated that 71% of the clones from the endogenous and exogenous community clustered in close relationship with Lachnospira pectinoschiza. Additionally, the biohydrogenation activity of solid-associated bacteria (SAB) and liquid-associated bacteria (LAB) was examined and compared with the activity of the non-fractioned I. prostoma monofaunated rumen fluid (LAB + SAB). Both SAB and LAB were involved in rumen biohydrogenation of C18:2n-6. SAB fractions performed the full hydrogenation reaction to C18:0 while C18:1 fatty acids, predominantly C18:1t10 and C18:1t11, accumulated in the LAB fractions. SAB and LAB sequence analyses were mainly related to the genera Butyrivibrio and Pseudobutyrivibrio with 12% of the SAB clones closely related to the C18:0 producing B. proteoclasticus branch. In conclusion, this work suggests that I. prostoma and its associated bacteria play no role in C18:2n-6 biohydrogenation, while LAB convert C18:2n-6 to a wide range of C18:1 fatty acids and SAB produce C18:0, the end product of rumen lipid metabolism.

Effect of In Vitro Docosahexaenoic Acid Supplementation to Marine Algae-Adapted and Unadapted Rumen Inoculum on the Biohydrogenation of Unsaturated Fatty Acids in Freeze-Dried Grass

The objective of this study was to examine the ruminal biohydrogenation of linoleic (18:2n-6) and linolenic (18:3n-3) acid during in vitro incubations with rumen inoculum from dairy cattle adapted or not to marine algae and with or without additional in vitro docosahexaenoic acid (DHA, 22:6n-3) supplementation. Treatments were incubated in 100-mL flasks containing 400mg of freeze-dried grass, 5mL of strained ruminal fluid, and 20mL of phosphate buffer. Ruminal fluid was collected just before the morning feeding from 3 cows receiving a control diet (49% ryegrass silage, 39% corn silage, 1% straw, and 11% concentrate, fresh-weight basis) supplemented with marine algae for 21 d (adapted rumen fluid, aRF) or from the same cows receiving the control diet only for 14 d after marine algae supplementation was stopped (unadapted rumen fluid, uRF). In half of the incubation flasks, pure DHA (5mg) was added as an oil-ethanol solution (100mL). Incubations were carried out during 0, 0.5, 1, 2, 4, 6, and 24h. After 24h, in vitro addition of DHA resulted in greater amounts (mg/incubation) of 18:3n-3 (0.23, 0.43, 0.26, and 0.34 for aRF, aRF+DHA, uRF, and uRF+DHA), 18:2n-6 (0.14, 0.22, 0.15, and 0.20 for aRF, aRF+DHA, uRF, and uRF+DHA) and trans-11, cis-15-18:2 (0.27, 2.40, 0.06, and 2.21 for aRF, aRF+DHA, uRF, and uRF+DHA), whereas no effect of inoculum source was observed. Trans-11-18:1 accumulated after 24h when aRF was incubated irrespective of in vitro DHA supplementation, whereas in incubations with uRF, accumulation of trans-11-18:1 only occurred when DHA was added (6.40, 4.35, 1.06, and 3.91 for aRF, aRF+DHA, uRF, and uRF+DHA). The increased amounts of trans-11-18:1 were due to the strong inhibition of the reduction to 18:0 because no 18:0 was formed when trans-11-18:1 accumulated after 24h. The results of the current experiment shows hydrogenation of trans-11, cis-15-18:2 occurred in the absence of in vitro DHA only, whereas substantial hydrogenation of trans-11-18:1 to 18:0 only took place in incubations without DHA and with unadapted rumen inoculum, confirming the higher sensitivity of the latter process to DHA.

BOARD-INVITED REVIEW: Recent advances in biohydrogenation of unsaturated fatty acids within the rumen microbial ecosystem1

Recent advances in chromatographic identification of CLA isomers, combined with interest in their possible properties in promoting human health (e.g., cancer prevention, decreased atherosclerosis, improved immune response) and animal performance (e.g., body composition, regulation of milk fat synthesis, milk production), has renewed interest in biohydrogenation and its regulation in the rumen. Conventional pathways of biohydrogenation traditionally ignored minor fatty acid intermediates, which led to the persistence of oversimplified pathways over the decades. Recent work is now being directed toward accounting for all possible trans-18:1 and CLA products formed, including the discovery of novel bioactive intermediates. Modern microbial genetics and molecular phylogenetic techniques for identifying and classifying microorganisms by their small-subunit rRNA gene sequences have advanced knowledge of the role and contribution of specific microbial species in the process of biohydrogenation. With new insights into the pathways of biohydrogenation now available, several attempts have been made at modeling the pathway to predict ruminal flows of unsaturated fatty acids and biohydrogenation intermediates across a range of ruminal conditions. After a brief historical account of major past accomplishments documenting biohydrogenation, this review summarizes recent advances in 4 major areas of biohydrogenation: the microorganisms involved, identification of intermediates, the biochemistry of key enzymes, and the development and testing of mathematical models to predict biohydrogenation outcomes.

Effects of fatty acid sources on conjugated linoleic acid (CLA) and other fatty acids in dairy milk

Conjugated linoleic acid (CLA) is aniticarcinogenic, antiatherogenic and antidiabetogenic actives. Research has therefore focused on methods of increasing CLA content in milk fat. Amount of CLA in milk fat was highly related to biohydrogenation of unsaturated fatty acid of rumen microbes. (Bauman et al., 1999). Linoleic acid (C18:2) were the precursors of CLA synthesis. The CLA was also synthesized in the mammary gland of lactating ruminants, using oleic acid (C18:1) as a precursor and activity of delta 9-desaturase (Griinari and Bauman, 1999). Linoleic acid is high in soybean oil (SO) (54.4%) and tuna oil (TO) (20.3%) while oleic acid is high in pork oil (PO) (43.5%) and groundnut oil (GO) (40.7%). Therefore, the objective of this experiment was to compare the increasing of CLA and fatty acid composition in milk fat form cows fed dietary oils obtained from either animal or plant sources.