Progress 10/01/99 to 09/30/04
Outputs
In a previous in vitro study, mixed ruminal microorganisms converted oleic acid to a variety of trans monenes when grown in batch cultures under constant environmental conditions. To determine if a similar conversion occurs under environmental conditions more typical of the rumen, conversion of 13C-labeled oleic acid to biohydrogenation intermediates was determined in ruminal microorganisms grown in continuous culture at two pH (5.5 and 6.5) and liquid dilution rates (0.05 and 0.10 h-1) arranged factorially. At 6.5 pH and 0.10 h-1 dilution rate, 13C enrichment was detected in all trans monoenes by GC-MS. However, when pH or dilution rate in fermentors was lowered, no 13C enrichment was detected in any trans monoene having a double bond position between carbons 11 to 16 of the acyl chain. The 13C enrichment of trans-10 was reduced under low pH and dilution rate conditions. The 13C enrichment also was found in two additional peaks identified as 10-hydroxy stearic acid
and 10-keto stearic acid. The results of this study show that ruminal microorganisms are capable of converting oleic acid to trans monoenes, and 10-hydroxy and 10-keto stearic acids and that conversion was reduced under low pH and dilution rate conditions. Additional in vitro studies were done in batch cultures to examine the biohydrogenation of docosahexaenoic acid (C22:6, DHA) and eicosapentaenoic acid (C20:5, EPA). After 24 h of incubation, 4.1, 4.1, 4.0, and 3.3 mg of DHA disappeared from the 5, 10, 15, and 20 mg of DHA cultures, respectively. In the second experiment, 5, 8.3, and 7.1 mg of EPA disappeared after 24 h of incubation for the 5-, 10-, and 15-mg EPA cultures, respectively. Addition of DHA to cultures increased trans-C18:1 fatty acid accumulation by 105, 91, 82, and 74% for the 5, 10-, 15-, and 20-mg cultures, respectively, compared with control. The addition of EPA increased trans-C18:1 fatty acid accumulation by 56, 64, and 55% for the 5-, 10-, and 15-mg EPA cultures,
respectively, compared with control. Addition of DHA and EPA to cultures caused a reduction in C18:1 n-9 and C18:2 n-6 biohydrogenation compared with control. Results from these experiments clearly demonstrate the ability of ruminal microorganism to transform DHA and EPA to other fatty acids causing their disappearance from cultures. An additional in vitro study was done with the goal of determining if DHA is the active component that promotes trans-C18:1 FA accumulation in ruminal contents when dairy cows are fed a blend of soybean and fish oils. Treatments consisted of control, control plus 5 mg of DHA (DH), control plus 30 mg of soybean oil (SBO), and control plus 5 mg of DHA and 30 mg of SBO (DHSBO). After 24 h of incubation, the level of trans-C18:1 FA (14.1 and 11.7 mg/culture) and VA (13.0 and 10.2 mg/culture) increased more with added DHA than with added SBO, respectively. Combining DHA and SBO yielded higher quantities of trans-C18:1 FA (21.3 mg/culture) and VA (19.8
mg/culture) in the cultures than either fat source alone. These data suggest that DHA is the component in fish oil that promotes VA accumulation when incubated with linoleic acid.
Impacts
This project has provided several key steps in the biohydrogenation pathways for important dietary omega fatty acids in the feed of dairy cows. Identification of intermediates produced during biohydrogenation is important to determine fatty acid derivatives from the gastrointestinal tract that are deposited in meat and milk and then enter the human food chain where they possible have health effects. The project also has shwon that conversion of unsaturated fatty acids to amides can reduce their susceptibility to biohydrogenation.
Publications
- AbuGhazaleh, A. A., and T. C. Jenkins. 2004. Disappearance of docosahexaenoic acid and eicosapentaenoic acids from cultures of mixed ruminal microorganisms. J. Dairy Sci. 87:645-651.
- AbuGhazaleh, A. A., and T. C. Jenkins. 2004. Short Communication: Docosahexaenoic acid promotes vaccenic acid accumulation in mixed ruminal cultures when incubated with linoleic acid. J. Dairy Sci. 87:1047-1050.
- Lundy III, F. P., E. Block, W. C. Bridges Jr., J. A. Bertrand, and T. C. Jenkins. 2004. Ruminal biohydrogenation in Holstein cows fed soybean fatty acids as amides or calcium salts. J. Dairy Sci. 87:1038-1046.
- AbuGhazaleh, A. A., M. B. Riley, and T. C. Jenkins. 2004. The effect of dilution rate and pH on the conversion of stable isotopically labelled oleic acid to trans monoenes in continuous culture. J. Dairy Sci. 87 (Suppl. 1):337.
|
Progress 01/01/03 to 12/31/03
Outputs
Fatty amides of high-oleate fats and calcium salts of palm oil were reported to resist biohydrogenation by ruminal microorganisms. This study was conducted to determine if converting polyunsaturated fat sources to amides and calcium salts had equal ability to resist biohydrogenation. A total mixed ration consisting of forage and concentrate contained either (dry basis); 1) 2.45% soybean oil (SBO), 2) 2.75% calcium salt of SBO, 3) 2.75% amide of SBO, or 4) 2.75% of a mixture of the calcium salt and amide (80:20, wt/wt.) of SBO. The four diets were fed ad-libitum to four multiparious lactating Holstein cows fitted with ruminal cannulae in a 4 x 4 Latin square with 21-d periods. Omasal samples were taken to measure postruminal fatty acid content and determine the extent of ruminal biohydrogenation. Adding SBO to the diets as either calcium salts or amides had little effect on reducing biohydrogenation of dietary C18:2 (n-6) by ruminal microorganisms. Biohydrogenation of
dietary C18:1 decreased slightly when SBO was fed to cows as calcium salts, but decreased extensively when SBO was fed as amides. Adding the soybean amide to the diet more than doubled the delivery of C18:1 (n-9) to the omasum of lactating cows, but it also increased trans fatty acid production in the rumen accompanied by milk fat depression. In this study, ruminal biohydrogenation of linoleic acid in lactating cows decreased slightly by adding SBO to the diet as either amide or calcium salts. Biohydrogenation of oleic acid in the diets decreased dramatically by feeding SBO as the amide. The biohydrogenation of fish oil fatty acids were examined in a second in vitro study. Previous studies showed conflicting results regarding the ability of ruminal microorganisms to hydrogenate docosahexaenoic acid (C22:6, DHA) and eicosapentaenoic acid (C20:5, EPA). To determine the disappearance of DHA and EPA from mixed ruminal cultures, two ruminal in vitro experiments were conducted using graded
levels of DHA and EPA. The first experiment examined DHA added at 0, 5, 10, 15, and 20 mg / culture flask. In the second experiment, EPA was added at 0, 5, 10, and 15 mg / culture flask. After 24 h of incubation, 4.1, 4.1, 4.0, and 3.3 mg of DHA disappeared from the 5, 10, 15, and 20 mg DHA cultures, respectively. In the second experiment, 5, 8.3, and 7.1 mg of EPA disappeared after 24 h of incubation for the 5, 10, and 15 mg EPA cultures, respectively. Addition of DHA to cultures increased trans- C18:1 fatty acid accumulation by 105, 91, 82, and 74% for the 5, 10, 15, and 20 mg cultures, respectively, compared with control. The addition of EPA increased trans- C18:1 fatty acid accumulation by 56, 64, and 55 % for the 5, 10, and 15 mg EPA cultures, respectively compared with control. Results from these experiments clearly demonstrate the ability of ruminal microorganism to transform DHA and EPA to other fatty acids causing their disappearance from cultures.
Impacts
Unsaturated fatty acids having important nutritional and marketing benefits for milk products are destroyed by bacteria in the rumen of cows before reaching the mammary gland. These studies demonstrate that conversion of an unsaturated fatty acid to either a calcium salt or an amide reduces their destruction by ruminal bacteria. The protection from bacterial destruction is greater for oleic acid than for linoleic acid. Also, the destruction of fish oil fatty acids by ruminal bacteria is constant regardless of amount fed, showing that percentage loss by biohydrogenation is reduced as levels increase in the diet.
Publications
- Palmquist, D. L., and T. C. Jenkins. 2003. Challenges with fats and fatty acid methods. J. Anim. Sci. 81:3250-3254.
- Jenkins, T. C., V. Fellner, and R. K. McGuffey. 2003. Monensin by fat interactions on trans fatty acids in cultures of mixed ruminal microbes grown in continuous fermenters fed corn or barley. J. Dairy Sci. 86:324-330.
- Jenkins, T. C., and A. AbuGhazaleh. 2003. The challenges of supplying omega fatty acids to body tissues of cattle to meet critical metabolic and physiologic functions. J. Dairy Sci. 86 (Suppl. 1): 146.
- Lundy, III, F. P., and T. C. Jenkins. 2003. The ability of amide versus calcium salts of soybean oil to increase unsaturated fatty acid concentration in omasal or continuous culture samples. J. Dairy Sci. 86 (Suppl. 1): 34.
|
Progress 01/01/02 to 12/31/02
Outputs
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. Unlabelled and labelled (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 6 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. Other studies showed that oleamide was protected from ruminal biohydrogenation and increased C18:1(n-9) concentration in milk when fed to lactating dairy cows. To appraise if this protection extended to linoleamide, a rumen in vitro experiment was conducted to determine biohydrogenation of linoleamide followed by two sheep experiments to evaluate if linoleamide could increase C18:2 (n-6) concentration in duodenal contents. Treatments for the in vitro and sheep studies consisted of three diets containing no added lipid (control), linoleic acid, or linoleamide. The concentrations of C18:2 (n-6) (corrected for C18:2 (n-6) in the control cultures) at 0, 24, and 48 h were 2.51, 0.38, and 0.11 mg/5 mL for the linoleic acid cultures compared to 2.10, 1.35, and 1.08 mg/5
mL for the linoleamide cultures. Three sheep with duodenal cannulae were fed the three diets in two separate 3 x 3 Latin squares each with 2-wk periods. The two squares only differed in the amount of added lipid (1.5 vs 5% of the ration DM). When the lipids were added at 1.5% of the ration DM, they had little effect on duodenal C18:2 (n-6) concentration (2.8, 3.6 and 4.3 mg/g DM for the control, linoleic acid, and linoleamide treatments, respectively). At 5% of the ration DM, both lipid supplements increased duodenal C18:2 (n-6) concentration over the control diet with a greater response observed for linoleamide (2.5, 12.2, and 16.8 mg/g DM for the control, linoleic acid, and linoleamide treatments, respectively). This study demonstrates reduced biohydrogenation of linoleamide based on its ability to maintain a higher concentration of C18:2 (n-6) in ruminal cultures and in duodenal contents of sheep compared to free linoleic acid.
Impacts
Specific information about the factors controlling the bioavailability of unsaturated fatty acids in cattle will be used to control the fatty acid composition of milk, which can improve both its nutritional and manufacturing properties.
Publications
- Martin, S. A. and Jenkins, T. C. 2002. Factors affecting conjugated linoleic acid and trans-vaccenic acid production by mixed ruminal bacteria. J. Anim. Sci. (In Print).
- Proell, J. M., Mosley, E. E., Powell, G. L. and Jenkins, T. C. 2002. Isomerization of stable isotopically labeled elaidic acid to cis and trans monoenes by ruminal microbes. J. Lipid Res. 43:2072-2076.
- Loor, J. J., Herbein, J. H., and Jenkins, T. C. 2002. Nutrient digestion, biohydrogenation, and fatty acid profiles in blood plasma and milk fat from lactating Holstein cows fed canola oil or canolamide. Anim. Feed Sci. and Tech. 97:65-82.
- Vaughan, J. M., Bertrand, J. A., Jenkins, T. C., and Pinkerton, B. W. 2002. Effects of feeding time on nitrogen capture by lactating dairy cows grazing rye pasture. J. Dairy Sci. 85:1267-1272.
- Jenkins, T. C., and Adams, C. S. 2002. The biohydrogenation of linoleamide in vitro and its effects on linoleic acid concentration in duodenal contents of sheep. J. Anim. Sci. 80:533-540.
- Mosley, E. E., Powell, G. L., Riley, M. B., and Jenkins, T. C. 2002. Microbial biohydrogenation of oleic acid to trans isomers in vitro. J. Lipid Res.43:290-296.
- Jenkins, T. C. 2002. Lipid transformations by the rumen microbial ecosystem and their impact on fermentative capacity. pp 103-117 in Gastrointestinal Microbiology in Animals, S. A. Martin (Ed.), Research Signpost, Kerala, India.
|
Progress 01/01/01 to 12/31/01
Outputs
This study was conducted to trace the metabolism of trans-9 C18:1 isomers upon its incubation with mixed ruminal microbes. Unlabelled and labelled (13C-18- trans-9 C18:1) elaidic acid were each added to three in vitro batch cultures of mixed ruminal microbes. Samples were taken at 0, 12, 24, and 48 h and analyzed for 13C enrichment in component fatty acids by gas chromatography-mass spectroscopy. Enrichments were corrected for natural 13C abundance in unlabelled cultures and then triplicate enrichments were analyzed by t-test to determine if they differed from zero. At 0 h of incubation, enrichment was 37% (P < 0.01) for trans-9 C18:1, but not significant for stearic acid or any trans C18:1 isomer. By 48 h of incubation, 13C enrichment was 18% (P < 0.01) for stearic acid and ranged from 7 to 30% (P < 0.01) for all trans C18:1 isomers having double bonds between carbons 6 through 16. Two additional cultures were run to determine if movement of the double bond from
elaidic acid to other trans C18:1 isomers required the presence of the ruminal microbes. One culture contained unlabelled elaidic acid and the other contained labelled (13C-18- trans-9 C18:1) elaidic acid, but neither were inoculated with ruminal microbes. After 48 h, 13C enrichment was only detected in the original elaidic acid. In previous studies, monensin (M) and unsaturated plant oils independently increased trans fatty acid concentrations in cultures of mixed ruminal microbes. This study was conducted to determine if combining M with plant oil yielded interactions on trans fatty acid concentrations in cultures of mixed ruminal microbes or their effects were additive. Four continuous fermenters were fed 14 g of dry feed per day (divided equally between two feedings) consisting of alfalfa hay pellets (30% of DM) and either a high corn (HC) or a high barley (HB) concentrate (70% of DM) in each of two fermenters. Within each grain type, one fermenter was supplemented with M (25 ppm)
and the other fermenter was supplemented with 5% soybean oil (SBO) during d 5 to 8. Monensin and fat were added together in all fermenters during d 9 to 12. Samples were taken at 2 h after the morning feeding on the last day of each period and analyzed for fatty acids by gas chromatography. A second run of the fermenters followed the same treatment sequence to give additional replication. Average pH across all treatments was 6.15, which was reduced (P < 0.01) by M but not affected by SBO. Monensin reduced (P < 0.05) the ratio of acetate to propionate, which averaged 2.03 across all treatments; fat decreased the acetate to propionate ratio in cultures not receiving M but increased it in the presence of M. Monensin and SBO altered the concentration of several trans fatty acids, but the only interaction was a grain x M x SBO interaction for trans-10 C18:1. The increase in trans-10 C18:1 by the M and SBO combination exceeded the sum of increases in trans-10 C18:1 for each individual feed
additive, but only for HB. For the HC diet, M increased (P < 0.05) trans-10 C18:1 more than fat alone and more than the M and SBO combination.
Impacts
This study shows that ruminal microbes transform elaidic acid to stearic acid consistent with the process of biohydrogenation. Unexpectedly, the 13C label from elaidic acid was also found in a multitude of trans C18:1 isomers suggesting that ruminal microbes have the capacity to move the trans double bond in elaidic acid from carbon 9 to other carbon positions. Because double bond movement only occurred in the presence of ruminal microbes, the movement is more likely an enzymatic process involving one or more microbial isomerases than it is a nonenzymatic process such as double bond migration. The results of this project also showed that monensin and soybean effects are additive for all trans fatty acids except for trans-10 C18:1. In the case of trans-10 C18:1, monensin and soybean oil interacted to give higher trans-10 C18:1 concentrations in ruminal contents than would be expected simply by adding their individual effects, but only for high-barley diets. Because
some trans fatty acid isomers have been associated with milk fat depression in dairy cows, these results suggest more severe depressions in milk fat content when cows are fed monensin along with unsaturated plant oils.
Publications
- 2001. White, S. L., J. A. Bertrand, M. R. Wade, S. P. Washburn, J. T. Green, Jr., and T. C. Jenkins. Comparison of fatty acid content of milk from Jersey and Holstein cows consuming pasture or a total mixed ration. J. Dairy Sci. 84:2295-2301.
- 2001. T. C. Jenkins, E. J. Thies, and E. E. Mosley. Direct methylation procedure for converting fatty amides to fatty acid methyl esters in feed and digesta samples. J. Agric. Food Chem. 49:2142-2145.
- 2001. Mosley, E. E., T. C. Jenkins, and G. L. Powell. The biohydrogenation of oleic acid to trans monoenes by ruminal microbes in vitro. J. Dairy Sci. 84 (Suppl. 1):8.
- 2001. Martin, S. A., and T. C. Jenkins. Factors affecting conjugated linoleic acid production by mixed ruminal microbes. J. Dairy Sci. 84 (Suppl. 1):159.
- 2001. Jenkins, T. C., S. A. Mosley, and J. A. Bertrand. Trans fatty acids in milk of Holostein cows fed soybean oil or two forms of conjugated linoleic acid. J. Dairy Sci. 84 (Suppl. 1):353.
|
Progress 01/01/00 to 12/31/00
Outputs
An in vitro study was initiated in 2000 to test the hypothesis that biohydrogenation of oleic acid by ruminal microbes results in the formation of trans monoenes, rather than being converted directly to stearic acid as commonly reported. Microbial cultures were done in 125 mL Erlenmeyer flasks containing 10 mL inoculum collected from a ruminally-fistulated Holstein cow, 40 mL medium as described by Goering and Van Soest (1970), and 500 mg of substrate. The ground hay substrate contained either 10% oleic acid or 10% 1-13C-oleic acid. Each substrate was run in quadruplicate. Samples (5 mL) of culture contents were taken at 0, 24, and 48 hours, then freeze-dried and methylated. Trans monoenes in the fatty acid methyl esters were isolated on silver-loaded cation exchange columns. Monoenes were converted to dimethyl disulfide adducts with location of the double bond and 13C enrichment determined by gas chromatography-mass spectroscopy. No 13C enrichment was detected in
palmitic, myristic, or linoleic acids (Table 1). Enrichment in oleic acid was 85% at all sampling times. Stearic acid enrichment averaged 10.9, 44.1, and 42.1% at 0, 24, and 48 hours. Enrichment exceeded 70% in trans-10 and trans-11 monomers at 24 and 48 hours. Enrichment for the trans-11 isomer averaged 0.2, 39.9, and 47.4% at 0, 24, and 48 hours, respectively. The results show that biohydrogenation of oleic acid leads to the formation of several trans monoene intermediates similar to the pattern of biohydrogenation of polyunsaturated fatty acids. Two in vitro studies demonstrated reduced biohydrogenation of amides of linoleic acid and cis-9, trans-11 C18:2 by mixed ruminal microbes. As a follow-up, 4 lactating Holstein cows were used in a 4 x 4 Latin square design with 2 week periods to examine amides of CLA versus other fat sources. The four diets evaluated in the study were a control with no added fat, and three diets containing 3% added fat from soybean oil (SBO), CLA (Conlinco,
Inc.), or amide of CLA. Dry matter intakes and milk yields were not affected by diet, although both were numerically lower for the CLAamide diet. Both the CLA and CLAamide diets reduced (P < 0.05) milk fat and increased milk protein compared to the SBO diet. All fat supplements increased (P < 0.05) milk C18:0 and C18:1 at the expense of C4 to C16 fatty acids. The CLAamide diet increased (P < 0.05) milk C18:1 compared to the CLA diet. The SBO diet increased (P < 0.05) cis-9, trans-11 C18:2 concentration in milk from 0.38 to 0.63% of total fatty acids. Both the CLA and CLAamide supplements further increased (P < 0.05) milk cis-9, trans-11 C18:2 concentration to an average of 1.26%. The results show that both the unprotected and protected forms of CLA markedly increased milk CLA concentration when fed at 3% of the ration dry matter.
Impacts
Trans fatty acids were recently shown to have significant physiological effects, including reducing fat accumulation in body tissues and milk. However, the pathways for synthesis of the trans fatty acids have not been elucidated. These results show that trans monoenes arise in ruminal contents from the biohydrogenation of oleic acid, which is counter to many published reports that oleic acid is directly hydrogenated to stearic acid. Also, these results indicate that trans fatty acids can be markedly increased in milk of dairy cows by feeding CLA supplements, which subsequently causes severe milk fat depression.
Publications
- Jenkins, T. C., C. E. Thompson, and W. C. Bridges, Jr. 2000. Site of administration and duration of feeding oleamide to cattle on feed intake and ruminal fatty acid concentrations. J. Anim. Sci. 78:2745-2753.
- Beam, T. M., T. C. Jenkins, P. J. Moate, R. A. Kohn, and D. L. Palmquist. 2000. Effects of level and source of fat on the rates of lipolysis and biohydrogenation of fatty acids in ruminal contents. J. Dairy Sci. 83:2564-2573.
- Jenkins, T. C. 2000. Feeding oleamide to lactating Jersey cows. 1. Effects on lactation performance and milk fatty acid composition. J. Dairy Sci. 83:332-337.
- DeLuca, D. D., and T. C. Jenkins. 2000. Feeding oleamide to lactating Jersey cows. 2. Effects on nutrient digestibility, plasma fatty acids, and hormones. J. Dairy Sci. 83:569-576.
|
|