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* Dairy Science Department, Virginia Tech, Blacksburg 24061-0315 and
and
Division of Animal and Veterinary Science, West Virginia University, Morgantown, 26506-6108
2 Correspondence:
phone: 540-231-4766; fax, 540-231-5014;
E-mail:herbeinj{at}vt.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Key Words: Dactylis glomerata • Digestion • Hydrogenation • Microbial Yield • Trans Fatty Acids • Trifolium pratense
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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-18:3n-3 in the rumen is stearic acid (18:0), biohydrogenation also yields a variety of monounsaturated, dienoic, or trienoic fatty acid intermediates with cis or trans double bonds. After isomerization of 18:2n-6 to cis9,trans11-18:2 (Kepler and Tove, 1967), sequential reductions of double bonds at carbons 9 and 11 yield trans11-18:1 and 18:0 (Polan et al., 1964). Biohydrogenation of -18:3n-3 also requires an initial isomerization to form a conjugated triene (cis9,trans11,cis15-18:3), followed by sequential reductions of double bonds at carbons 9, 15, and 11 to yield trans11,cis15-18:2, trans11-18:1, and 18:0 (Wilde and Dawson, 1966). Amounts of biohydrogenation intermediates produced in the rumen influence their concentrations in tissues or milk. Concentrations of trans11-18:1 and cis9,trans11-18:2, for example, are greater in milk (Loor et al., 2002b) or meat (Nuernberg et al., 2002) from grazing cattle. Trans10-18:1 and trans10,cis12-18:2 concentration in milk fat increases when high-grain diets are fed (Loor et al., 2002b; Piperova et al., 2002). Trans vaccenic acid and conjugated linoleic acids (CLA) in meat and milk are examples of hydrogenation intermediates that may have beneficial implications in human health (Jayan and Herbein, 2000; Lawson et al., 2001).
Continuous culture fermenters were used to characterize the effects of ionophores on biohydrogenation of unsaturated fatty acids to total trans-18:1, total CLA, and 18:0 (Fellner et al., 1997). Individual profiles of hydrogenation intermediates, however, likely vary due to nature and amount of unsaturated fatty acids. The primary objective of this study was to evaluate outputs of cis and trans isomers of 18:1 and 18:2 during incubations of fresh orchardgrass or red clover in continuous culture fermenters. In addition, ground corn replaced portions of the daily forage DM fed to fermenters to evaluate the effects of added starch on biohydrogenation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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The dual-flow continuous culture system was described previously (Stern and Hoover, 1990). Ruminal contents were obtained weekly for 6 wk (six fermentation periods) from two dry, grazing Holstein cows via a ruminal cannula. Grass hay and a concentrate containing (DM basis) dry shelled corn (75%), soybean meal (24%), and a vitamin/mineral mix (1%) were provided to supplement pasture intake. At each collection time, ruminal contents from both cows were strained through one layer of cheesecloth and pooled. One liter of pooled ruminal fluid plus 200 mL of warm buffer (2.2 g/L of Na2HPO4, 5 g/L of NaHCO3 , 0.6 g/L of KCl, 1.6 g/L of KHCO3, and 0.2 g/L of urea) were added to each of eight fermenters maintained at 39°C. The liquid turnover rate was maintained at 0.18/h using the buffer solution. The solids turnover rate was 0.07/h, which provided a mean solids retention time of 14.3 h. The ruminal liquid and solids turnover rates were similar to those previously determined for cows grazing pasture with or without corn grain supplementation (Berzaghi et al., 1996). Use of cannulated cows was approved by the West Virginia University Animal Care and Use Committee (protocol No. ACUC 9702-02).
Forages for Fermenter Diets
Red clover and orchardgrass were harvested in October (fall) 1996 and May (spring) 1997 from separate pastures at the Virginia Tech Dairy Center. After visual inspection and removal of unwanted plants to assure collection of only red clover or only orchardgrass for processing, forages were frozen (-40°C). Frozen forage was mixed with dry ice, ground through a 5-mm screen in a Wiley Mill (Thomas-Wiley Laboratory Mill, Arthur H. Thomas, Philadelphia, PA), and kept frozen during transport to West Virginia University. Upon arrival, samples of each type of frozen forage were obtained from containers (six per forage) used for shipping. The six samples of each type of forage were composited, and each composite remained frozen until its chemical composition (Table 1
) was determined.
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). After allowing 7 d for equilibration, daily effluent output was collected for 3 d in containers held at 4°C.
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Chemtrix pH meters (type 45 AR, Chemtrix, Hillsboro, OR) were used to monitor pH at 2-h intervals for 10 h after feeding at 0800. After daily effluent collection in an ice bath, 1-L samples from each of the three days were composited for analysis. After completion of the last daily effluent collection, contents of fermenters were allowed to settle, and two samples (250 mL) of the fluid layer were collected to isolate bacteria. After an initial centrifugation at 200 x g for 20 min to remove protozoa and feed particles, fluid then was centrifuged at 30,000 x g for 15 min. The resulting bacterial pellet was suspended, first with saline, and then with 50% methanol, and centrifuged twice at 30,000 x g for 15 min. The final pellet was dispersed in water and lyophilized. Microbial DM flow was calculated by measuring the diaminopimelic acid-N for each fermenter on the effluent flow and the bacterial pellet as described by Webster et al. (1990).
Dry matter content of ground corn and composited samples of forage and effluent was determined by drying at 102°C for 24 h. Effluent (40 g) was centrifuged at 30,000 x g for 45 min to obtain a pellet for drying. Concentrations of ADF and NDF in forage, ground corn, and effluent were determined as described by Van Soest et al. (1991). Nonstructural carbohydrate (NSC) content was estimated using the enzymatic procedure of Smith (1969), with modifications for use of ferricyanide as a colorimetric indicator. Total N content of forages, corn, effluents, and bacteria was determined by the Kjeldahl procedure (No. 988.05; AOAC, 1990). Digestibility (percentage) of dietary DM, NDF, ADF, CP, and NSC was calculated with the following equation:
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Lipids were extracted from 500 mg of freeze-dried effluent, ground corn, and forage using chloroform:methanol (2:1, vol/vol) followed by 6 N HCl as described by Fellner et al. (1995). Fatty acids in extracted lipids were methylated with 0.5 N NaOH in methanol and 14% boron trifluoride in methanol (Loor and Herbein, 2001). Undecenoate (Nu-Check Prep, Elysian, MN) was used as the internal standard. Samples were injected by auto-sampler into a Hewlett-Packard 5890A gas chromatograph equipped with a flame ionization detector (Hewlett-Packard, Sunnyvale, CA). Methyl esters of fatty acids in 0.5 µL of hexane (injected using a 70:1 split ratio) were separated on a 100 m x 0.25 mm i.d. fused silica capillary column (CP-Sil 88, Chrompack, Middelburg, The Netherlands). The injector temperature was maintained at 250°C, and the detector temperature at 255°C. The initial oven temperature was 70°C (held for 1 min) and was programmed to increase by 5°C/min to 100°C (held for 2 min), 10°C/min to 175°C (held for 40 min), and 5°C/min to a final temperature of 225°C (held for 15 min). Ultra-pure hydrogen was the carrier gas. Inlet pressure was held constant at 158.6 kPa.
To identify peaks and to determine response factors for individual fatty acids, known quantities of pure methyl esters were combined to obtain a calibration standard mixture with a total of 52 fatty acids. A custom preparation (Virginia Tech DaSc479, Nu-Check Prep) designed to resemble a typical milk fat and containing a total of 25 pure methyl esters (4:0 to 22:5n-3) was used as a base to which individual 18:1 and 18:2 isomers were added. Pure trans9-18:1 (cat. No. U-47-M), trans11-18:1 (cat. No. U-49-M), cis9-18:1 (cat. No. U-46-M), and cis11-18:1 (cat. No. U-48-M) methyl esters were purchased from Nu-Check Prep. Trans6-18:1 (cat. No. P5526), trans7-18:1 (cat. No. O2133), trans12-18:1 (cat. No. O2633), cis12-18:1 (cat. No. O9881), and cis13-18:1 (cat. No. O9256) were purchased from Sigma (St. Louis, MO). Trans13-18:1 (cat. No. 4-6909), trans15-18:1 (currently discontinued), and cis15-18:1 (cat. No. 4-6953) were purchased from Supelco Inc. (Bellefonte, PA). The nonconjugated 18:2 isomer mixture (cat. No. L8404) was purchased from Sigma, and contained trans9,trans12-18:2, cis9,trans12-18:2, trans9,cis12-18:2, and cis9,cis12-18:2. The conjugated linoleic acid mixture (cat. No. UC-59-M; Nu-Check Prep) contained cis9,trans11-18:2, trans8,cis10-18:2, cis11,trans13-18:2, trans10,cis12-18:2, cis9,cis11-18:2, cis10,cis12-18:2, cis11,cis13-18:2, trans11,trans13-18:2, and trans,trans-18:2. Trans10-18:1, trans16-18:1, and trans11,cis15-18:2 were not available commercially. Both trans-18:1 isomers were identified by order of elution according to Juaneda (2002). Trans11,cis15-18:2 was identified by order of elution with respect to cis9,cis12-18:2 as described by Ulberth and Henninger (1994). The response factor for 18:0 was used to quantify both trans-18:1 isomers and trans11,cis15-18:2.
Fatty acid output (mg/d) was calculated using fatty acid concentration (µg/g of DM) in effluent DM output. When compared with input from DM in forage or forage plus corn, total fatty acid recovery ([output/input] x 100) for all diet combinations averaged 101%.
Statistical Analyses
Only eight fermenters were available at the time the study was conducted. Given this constraint, it required six "periods" to achieve the desired number of fermentations (Table 2
). Treatments were randomly assigned to obtain four replicates for each combination of forage and corn grain. Data for digestibility, microbial yield, apparent biohydrogenation, and fatty acid output are reported as least squares means ± SEM. Replicate fermentations of each treatment were analyzed as a completely randomized design using the MIXED procedure (SAS Inst., Inc., Cary, NC). The statistical model included fermenter, season (fall or spring), forage (orchardgrass or red clover), corn level (0, 8, or 16 g/d), incubation period, two-way and three-way interactions between season, forage, and corn, and residual error. Fixed effects in the SAS model were season, forage, corn, forage x corn, forage x season, season x corn, and season x forage x corn interactions. The random effect was incubation period. Linear and quadratic contrasts for the interactions of season (fall or spring) x corn (0, 8, or 16 g/d) and forage (orchardgrass or red clover) x corn also were calculated. Compound symmetry (CS) was the covariate structure used for statistical analysis, as it best fit our experimental design. The statistical model for analysis of pH between feeding times included fermenter, season, forage, corn level, incubation period, hour, season x corn, season x forage x corn x hour, and residual error. The random effect was incubation period. Compound symmetry was the covariate structure used for statistical analysis. Overall differences due to season, forage, corn level, linear or quadratic effects due to corn level, and two-way interactions between forage or season and corn level were considered significant at P 
0.05. However, all P values are presented in Tables. Only those linear or quadratic interactions that were significant are indicated in the Tables.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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), but the CP content of both forages was similar regardless of season. Total fatty acid concentration in orchardgrass was approximately 35 to 50% higher than that of red clover in fall and spring. Oleic acid (cis9-18:1) and 18:2n-6 accounted for averages of 4 and 18% of total fatty acids in forages compared with 24% and 63% in corn grain. Linolenic acid (18:3n-3), however, accounted for 45% of total fatty acids in forages compared with only 1% in corn grain. Compared with orchardgrass, concentrations of cis9-18:1 and 18:2n-6 in red clover were numerically higher regardless of season, whereas concentration of 18:3n-3 was numerically lower. Overall, fatty acid profiles of forages were consistent with previous reports. Total fatty acid content of pasture species were highest during the primary growth stage (spring), decreased steadily through the stemmy regrowth period, and increased sharply during the next leafy regrowth stage (fall) (Bauchart et al., 1984; Dewhurst et al., 2001). Concentration of 18:3n-3 followed the same trend as total fatty acids during growth, whereas 18:2n-6 concentration was less variable. Grasses, however, contain more fatty acids than legumes at any stage of growth (Dewhurst et al., 2001; Loor et al., 2002c).
Due to replacement of forage DM with corn grain DM (8 or 16 g/d) in fermenter diets, daily NDF and CP inputs decreased as input of starch increased (Tables 3 and 4). In addition, 18:3n-3 input from forage decreased as cis9-18:1 and 18:2n-6 input from corn increased. Similar changes may occur in a typical grazing situation when cows reduce their voluntary intake of pasture to accommodate a fixed amount of supplemental corn grain.
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Overall, DM, NDF, and ADF in spring forages were digested to a greater extent than those in fall forages, but digestion of CP and NSC were not affected by season (Table 5). Digestibility of NSC also was not affected by type of forage in the diet. However, digestibility of NDF, ADF, and CP was higher when orchardgrass was the forage source (65, 61, and 73%, respectively) compared with red clover (52, 56, and 57%). Enhanced digestion of orchardgrass NDF and ADF was associated with greater microbial DM yield and efficiency of microbial N production, apparently in response to the greater extent of orchardgrass CP digestion. At similar maturity, the soluble CP fraction in orchardgrass compared with red clover was more degradable (45 vs. 35%) (Hoffman et al., 1993). Lignin content of orchardgrass is lower than that of red clover (Hoffman et al., 1993), and this may be an additional factor accounting for the higher rate of fiber digestion when orchardgrass was the forage source in the fermenters.
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), but reductions in pH due to increasing supplementation of corn grain plus fall orchardgrass, spring orchardgrass, fall clover, or spring clover were variable (-0.17, +0.01, -0.07, or -0.22, respectively). Overall, the pattern of pH observed between feeding times in the present study (Figure 1) resembled diurnal variations in pH reported in vivo for grazing cows supplemented with various forms or levels of grain (Reis and Combs, 2000a,b). Regardless of season, the combination of clover or orchardgrass plus 16 g of corn appeared to cause the most pronounced reductions in pH measured at 1000 (2 h postfeeding). Red clover fermentations, in particular, maintained the lowest pH up to 4 h after initial feeding then gradually increased (Figure 1).
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Estimates of apparent biohydrogenation (percentage) of individual dietary unsaturated fatty acids assumes that total 18-carbon fatty acid output from the rumen equals 18-carbon fatty acid input (Wu and Palmquist, 1991). When total fatty acid input and output are similar, as in the present study, proportions of 18:0, 18:1, 18:2, or 18:3 flowing to the small intestine reflect the extent of microbial hydrogenation of unsaturated 18-carbon fatty acids. The stage of growth of fresh forage appears to influence the overall process of biohydrogenation in the rumen, because rates of lypolysis and hydrogenation of 18:2n-6 were greater in ruminal fluid from sheep fed immature compared with mature ryegrass (Gerson et al., 1986).
Apparent biohydrogenation (Table 6) of total (cis9-18:1 + 18:2n-6 + 18:3n-3) unsaturated fatty acids was greater when fermenters were fed orchardgrass (95%) compared with red clover (84%). The overall seasonal effect on total unsaturated fatty acid apparent biohydrogenation resulted primarily from differences for spring red clover (86%) compared with fall red clover (82%) (significant forage x season interaction effect). Although overall effect of season was not significant for biohydrogenation of 18:2n-6, a significant forage x season interaction was found. Biohydrogenation of 18:2n-6 was greater with fall red clover (87%) compared with spring red clover (83%). With orchardgrass, however, biohydrogenation of 18:2n-6 was greater in spring (96%) compared with fall (95%). A marked seasonal effect was found for apparent biohydrogenation of 18:3n-3. Red clover harvested in spring (92%) resulted in greater biohydrogenation of 18:3n-3 compared with fall (85%). Apparent biohydrogenation of cis9-18:1 varied considerably, but it was consistently lower than that for 18:2n-6 or 18:3n-3. Hydrogenation of cis9-18:1 to 18:0 in other in vitro studies ranged from 40 to 90%, but some organisms could not hydrogenate it to any extent (Polan et al., 1964; Kemp et al., 1975; Kemp and Lander, 1984). Overall, apparent biohydrogenation of cis9-18:1 was greater when fermenters were fed red clover (65%) compared with orchardgrass (62%). Apparent biohydrogenation of 18:2n-6 and 18:3n-3, however, was greater for orchardgrass fermentations.
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) was greater when fermenters were fed orchardgrass as the sole source of DM, hydrogenation efficiency of microbes may have been near maximum when incremental portions of corn were fed. This is substantiated by the fact that there was no forage x corn interaction effect for 18:0 output (Table 7). More pronounced outputs of total trans-18:1 and total conjugated 18:2 isomers (significant forage x corn interaction effect; Table 7) in response to red clover, compared with orchardgrass, suggest that incomplete biohydrogenation was greater with this forage. These results differ from previous experiments conducted with batch cultures of mixed ruminal fluid in which higher inputs of unsaturated fatty acids resulted in greater accumulation of trans-18:1 and conjugated 18:2 intermediates (Polan et al., 1964). Continuous culture conditions certainly reflect more closely ruminal biohydrogenation and lipid metabolism.
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Apparent Biohydrogenation and pH
Across the range of pH measured in the present study, oleic acid biohydrogenation varied substantially with orchardgrass or red clover as the forage (Figure 2A). Its biohydrogenation, however, seemed to decrease to a similar extent with both forages as pH increased. Linoleic acid and linolenic acid biohydrogenation when orchargdrass was the forage appeared relatively constant at different pH (Figure 2B,C). When red clover was fed, however, biohydrogenation of 18:2n-6 seemed to gradually decrease as the pH increased from 6.73 to 6.98 (Figure 2B). Although linolenic acid biohydrogenation varied substantially with red clover across the range of pH measured, it appeared to increase between pH 6.73 and 6.98 (Figure 2C). We are not aware of previous in vivo or in vitro data comparing changes in biohydrogenation of unsaturated fatty acids from pasture alone or pasture plus grain across different pH. In batch culture, however, it was reported that biohydrogenation of soybean oil was reduced as pH decreased (Van Nevel and Demeyer, 1996).
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). Because a mixed population of microorganisms is necessary for biohydrogenation to proceed to completion (Jenkins, 1993), above a certain pH this balance may be upset. Shifts in microbial populations when cows are switched from high-forage to high-grain diets may be associated with reduced rates of biohydrogenation (Latham et al., 1972).
Early evidence indicated that species such as Ruminococcus, Butyrivibrio, Bacteroides, and gram-positive to gram-variable rods predominated in ruminal fluid from cows deriving 100% of their DMI from grazing (Wolstrup et al., 1974). Feeding increasing proportions of rolled oats in place of fresh herbage, however, resulted in a 90% decrease in Butyrivibrio, 32% decrease in Bacteroides, 69% decrease in gram-positive to gram-variable rods and a 52% increase in Ruminococcus colony counts. Counts of Megasphera elsdenii, lactobacillus, and gram-positive to gram-variable vibrios increased by 113, 100, and 105%, respectively, (Wolstrup et al., 1974). Subtle changes in pH with grain supplementation may be sufficient to alter the "balance" of microorganisms capable of carrying out the various steps of biohydrogenation. Under such circumstances, production of biohydrogenation intermediates may be altered. Outputs of trans10-18:1 in particular, but also trans10,cis12-18:2, appeared sensitive to pH in the present study (Figure 3).
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Outputs of Medium-Chain Fatty Acids and Total Biohydrogenation Intermediates
Overall, total fatty acid output in effluents from fermenters given orchardgrass was 52% higher compared with those from red clover (Table 7), which was consistent with the higher total fatty acid content of orchardgrass (Table 3). Recoveries (output/input x 100) of total 18-carbon fatty acids in effluent DM averaged 101% when forages were the inputs, regardless of season, and 95% when the daily DM contained 8 or 6 g of corn DM. Duodenal fatty acid flow in ruminants fed fresh pasture ranged between -33 to 120% of input (Bauchart et al., 1984; Doreau and Poncet, 2000). Negative flows were associated with a higher intake of forage fatty acids from immature pasture during the spring, but positive flows were found when mature pasture contained 67% fewer fatty acids (Bauchart et al., 1984).
Ruminal microbes can synthesize fatty acids containing 10 to 16 carbons from acetate or glucose, and 15:0 and 17:0 from propionate or valerate (Mackie et al., 1991; Jenkins, 1993; Kristensen, 2001). In addition, 14:1 and 16:1 isomers can be synthesized via an anaerobic pathway (Mackie et al., 1991). Relative enrichment of 13C from ruminally infused [2-13C]-acetate in 6:0, 12:0, 14:0, anteiso-15:0, iso-15:0, 15:0, iso-16:0, anteiso-17:0, and iso-17:0 was markedly greater than in 16:0, 17:0, or 18:0 (Kristensen, 2001). Overall, outputs of 10:0, 12:0, and 14:0 were greater for spring vs. fall forages, due primarily to high outputs from spring red clover (Table 7). Forages were the primary source of 14:0 in the total DM input during fermentation (Tables 3 and 4). Thus, type of forage and season affected 14:0 output. Compared with 14:0 input, however, overall output of 14:0 was 23% higher. All of the 15:0 in the effluent apparently was synthesized during fermentation. Output of 15:0 varied due to season and type of forage, but numerical differences were small.
Our results suggest that a portion of 16:0 in the DM input was lost due to desaturation and/or degradation (Mackie et al., 1991). Disappearance of dietary 16:0 from orchardgrass during fermentation in vitro was previously observed (Wu and Palmquist, 1991). End products of 16:0 oxidation by ruminal microbes could include isomers of 15-, 13-, or 11-carbon fatty acids in addition to short-chain volatile fatty acids (Mackie et al., 1991). Although outputs of cis9-14:1 or cis9-16:1 were not affected by forage, output of trans9-16:1 was greater for orchardgrass compared with red clover, primarily due to input of fall orchardgrass. Anaerobic bacteria can synthesize cis9-16:1 by dehydration of de novo synthesized ß-OH-10:0, via ß,
-dehydratase, to cis3-10:1 followed by elongation to cis9-16:1 (Mackie et al., 1991).
Overall, the profiles of 14:0, 15:0, 16:0, and 17:0 in effluents regardless of forage or season were similar to those found previously in ruminal fluid from cows fed fresh grass/clover pasture (Hawke and Robertson, 1964). Addition of corn to the DM input led to greater outputs of 14:0, 15:0, 16:0, trans9-16:1, and 17:0, suggesting de novo microbial fatty acid synthesis was not affected by the higher input of dietary fatty acids.
Across forage and season, cis9-18:1 plus 18:2n-6 plus 18:3n-3 accounted for 66% and 18:0 for 3% of total fatty acid input (Table 4). Due to biohydrogenation, cis9-18:1, 18:2n-6, and 18:3n-3 were converted primarily to 18:0 and trans-18:1 isomers (Table 7). Outputs of total cis-18:1 isomers accounted for approximately 4% (orchardgrass) or 8% (red clover) of total fatty acid output. Similarly, total nonconjugated 18:2 isomers accounted for 4% (orchardgrass) or 12% (red clover) of total fatty acid output. Conjugated 18:2 isomers, however, accounted for only 1% of total fatty acid output.
Outputs of trans-18:1 and 18:0 in effluents from fermenters fed orchardgrass as the input, were 80 and 134% greater compared with those fed red clover. In contrast, outputs of cis-18:1, nonconjugated 18:2, and 18:3n-3 were 12, 158, and 83%, respectively, greater when red clover was fed. Input of spring forages, but not fall forages, resulted in higher outputs of trans-18:1, but input of spring red clover also resulted in greater output of nonconjugated 18:2 isomers (higher trans11,cis15-18:2 output). These data confirm that the efficiency of apparent hydrogenation was greater when orchardgrass, due to higher 18:2n-6 and 18:3n-3 input, was the source of DM input. Stearic acid and trans-18:1 isomers were the major end products of hydrogenation of 18:2n-6 and 18:3n-3 in ryegrass. However, there was small accumulation of cis-18:1, total 18:2, and 18:3n-3 (Singh and Hawke, 1979).
Replacing portions of either forage with corn grain, regardless of season, caused an average increase of 64% in the output of 18:0 (Table 7). Because 18:3n-3 input decreased as corn replaced portions of forage (Table 4), the increase in 18:0 output most likely resulted from complete hydrogenation of cis9-18:1 and 18:2n-6 in the corn and forage. The lack of change in output of nonconjugated 18:2 isomers despite increasing 18:2n-6 input as corn replaced portions of forage, suggested most of the additional 18:2n-6 input was partially or completely hydrogenated. In contrast, total output of cis-18:1 isomers increased as the amount of corn grain (the primary source of cis9-18:1) in the daily input of DM increased.
Although input of 18:2n-6 increased due to corn, output of total trans-18:1 isomers and conjugated 18:2 isomers only increased by 9 and 34% with orchardgrass compared with increases of 65 and 117% when corn was added with red clover. This response was more pronounced when spring forages plus corn were fed. The higher efficiency of hydrogenation of 18:2n-6 from orchardgrass plus corn (96%) compared with red clover plus corn (85%), resulted in greater 18:0 output, but only moderate production of trans-18:1 and conjugated 18:2 isomers.
To further evaluate production of intermediates in biohydrogenation pathways, nonconjugated 18:2 isomers, conjugated 18:2 isomers, cis-18:1 isomers, and trans-18:1 isomers are listed individually in Tables 8 and 9.
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Although 18:2n-6 was the sole nonconjugated 18:2 isomer provided as input, it (cis9,cis12-18:2) accounted for only 25% of total nonconjugated 18:2 isomers in the effluent when orchardgrass or red clover was fed (Table 8). This was equivalent to 3% of total fatty acids in effluents. Overall, output of cis9,cis12-18:2 was greater (22 vs. 10 mg/d) from red clover compared with orchardgrass. Effluents also contained cis9,trans12-18:2 (0.5 to 0.6 mg/d), trans9,cis12-18:2 (0.9 to 2 mg/d), and trans9,trans12-18:2 (0.5 to 0.6 mg/d), but they accounted for only 2 to 9% of total nonconjugated 18:2 isomers. These cis/trans, trans/cis, and trans/trans isomers were most likely produced as a result of the isomerization of dietary cis9,cis12-18:2 during fermentation (Kemp et al., 1975).
Output of trans11,cis15-18:2 did not result from cis9,cis12-18:2 isomerization because it is an intermediate in the isomerization and hydrogenation of dietary 18:3n-3 (Wilde and Dawson, 1966). The percentage of total radioactivity found in trans11,cis15-18:2 when 14C--18:3n-3 was incubated with a wide range of ruminal bacteria ranged from 25 to 100% (Hazlewood et al., 1976). Also, incubating increasing amounts of 18:3n-3 with ruminal fluid more than doubled trans11,cis15-18:2 concentration (Body, 1976). In our study, output represented 65 to 73% of total nonconjugated 18:2 isomers or 3 to 12% of total fatty acid output. To our knowledge, this is the first study reporting outputs of trans11,cis15-18:2 during long-term digestion of fresh forage. Compared with orchardgrass, clover yielded greater output of trans11,cis15-18:2 (68 vs. 24 mg/d).
Input of increasing levels of corn grain with either forage increased the output of cis9,cis12-18:2 linearly, but decreased the output of trans11,cis15-18:2. At the highest level of corn input with orchardgrass or red clover, cis9,cis12-18:2 output had increased by 6 or 29 mg/d. In contrast, output of trans11,cis15-18:2 decreased by 9 to 29 mg/d when orchardgrass or red clover were fed with corn. Similar to our results, a greater proportion of the 18:2n-6 input was recovered during in vitro incubations of ruminal contents with increasing levels of 18:2n-6 (Polan et al., 1964). Overall, our results indicated that trans11,cis15-18:2 was the major 18:2 isomer produced during hydrogenation of 18:3n-3 in forages. Dietary 18:2n-6, whether derived from forage or ground corn, was isomerized to nonconjugated isomers, hydrogenated significantly to 18:0, but also led to substantial accumulation of conjugated 18:2 isomers.
Outputs of Conjugated Isomers of 18:2
Formation of a cis9,trans11 conjugated 18:2 intermediate is a prerequisite for the biohydrogenation of cis9,cis12-18:2 to 18:0 (Kepler and Tove, 1967), but trace amounts could also arise during hydrogenation of cis9,cis12,cis15-18:3 to 18:0 (Wilde and Dawson, 1966). Linoleate-cis12,trans11-isomerase (EC 5.2.1.5) is responsible for this isomerization step and has a specific requirement for the cis9,cis12 diene configuration (Kepler et al., 1971). Conjugated 18:2 isomers, however, appear to be transient intermediates during hydrogenation because their concentrations are very low (0.3 to 1.3%) compared with trans-18:1 or 18:0 (Fellner et al., 1997). During incubations with pure cultures of ruminal bacteria or ruminal fluid, hydrogenation of cis9,cis12-18:2 yielded 18:0 (40 to 70%) and trans11-18:1 (14 to 100%) as major end products (Kemp et al., 1975; Fellner et al., 1997). In contrast, hydrogenation of cis9,cis12,cis15-18:3 in mixed ruminal fluid resulted in equal proportions of trans-18:1 (40%) and 18:0 (38%) as major products (Wilde and Dawson, 1966; Singh and Hawke, 1979).
Outputs of conjugated 18:2 isomers in the present study (Table 8) accounted for 0.7 to 1.0% of total fatty acid output and were double those previously reported for effluents from fermenters fed a mixture of hay and concentrate (Fellner et al., 1997). Among the conjugated 18:2 isomers, cis9,cis11-18:2 and trans11,trans13-18:2 were predominant. Overall, their outputs accounted for 28 to 39% of total conjugated 18:2 isomers when forage was the only DM input. The output of a mixture of trans,trans-18:2 isomers ( t r a n s7 ,t r a n s9 - 1 8 : 2 + t r a n s8 ,t r a n s1 0 - 1 8 : 2 + t r a n s 9 ,t r a n s1 1 - 1 8 : 2 + t r a n s1 0 ,t r a n s1 2 -1 8 : 2 ) represented 9 to 15% of total conjugated 18:2 isomer output. In an earlier study, it was reported that approximately 41% of total conjugated isomers consisted of a mixture of trans,trans-18:2 isomers when fermenters were fed a mixed diet (Fellner et al., 1997). It could be possible that the trans,trans-18:2 isomer reported by Fellner et al. (1997) also contained the trans11,trans13-18:2, as well as other isomers with a conjugated trans,trans configuration. Cis9,trans11-18:2 output accounted for 9% of total conjugated 18:2 isomers derived from orchardgrass and 23% for red clover. Red clover fermentations resulted in greater output of cis9,trans11-18:2 (1.3 mg/d compared with 0.62 mg/d for orchardgrass). Grazing bulls, compared with bulls fed a concentrate diet, had greater concentrations of trans11,trans13-18:2 in intramuscular fat from longissimus muscle (Nuernberg et al., 2002). However, conjugated isomers with a cis,cis- configuration were not reported. Our results indicate cis9,cis11-18:2 and trans11,trans13-18:2 are the major CLA isomers produced in the rumen during grazing.
Trans10,cis12-18:2 was previously reported to account for 17% of total conjugated 18:2 isomers in fermenter effluents fed a mixed diet (Fellner et al., 1997). However, in our study, it accounted for 7% of total conjugated 18:2 isomers when either forage was fed. Concentration of trans10,cis12-18:2 averaged 0.83% of total CLA in intramuscular fat from grazing or concentrate-fed bulls and was not affected by diet (Nuernberg et al., 2002). Production of conjugated isomers other than cis9,trans11-18:2 suggests that the ruminal ecosystem contains cis,trans isomerases in addition to the cis12,trans11-isomerase. The trans10,cis12-18:2 could arise from isomerization of 18:2n-6 via a cis9,trans10-isomerase. Under "normal" conditions, however, only trace amounts of this isomer accumulate in the rumen even when cows are fed supplemental high-linoleic acid oil (Loor et al., 2002a). Low ruminal pH and availability of unsaturated fatty acids may be the major factors responsible for the synthesis of isomers with a trans10-double bond.
Outputs of trans10-18:1 and trans10,cis12-18:2 appeared to decrease as effluent pH increased from 6.73 to 6.98 (Figure 3A,B). This response was obvious for trans10-18:1 regardless of forage type. However, the relationship between increased pH and reduced output of trans10,cis12-18:2 (Figure 3A) was only apparent when red clover was the forage. This response may be explained by the fact that reductions in pH were more consistent with the combination of red clover and corn grain (Table 5, Figure 1).
Corn input with either forage increased the overall outputs of cis9,trans11-18:2, trans10,cis12-18:2, and trans,trans-18:2 by 201, 187, and 171%, respectively. In contrast, the overall output of cis9,cis11-18:2 decreased 33% when corn was added to orchardgrass, but not red clover. Concentrations of cis9,trans11-18:2 in effluents from forage plus corn were similar to those found in omasal contents of grazing cows deriving 40% of daily DMI from supplemental grain (Wu et al., 1998). In the present study, corn grain provided additional 18:2n-6, a rapidly fermentable starch, and decreased effluent pH, conditions which may have stimulated production of trans10,cis12-18:2 (Figures 3A and 4A). When high-concentrate/low forage (80:20) diets were fed, the ratios of cellulolytic (primarily B. fibrisolvens) to propionogenic, lactogenic, and amylolytic bacteria in the rumen were severely reduced (Latham et al., 1972). In vitro, strains of Megasphaera elsdenii (when enriched with DL-lactate and trypticase) isolated from a cow fed a 90% corn grain diet produced more trans10,cis12-18:2 from pure linoleic acid than controls (Kim et al., 2002). Colony counts of this species were drastically increased in the rumen of cows during the transition from grazing to concentrate feeding (Wolstrup et al., 1974). The overall extent to which pH was decreased in our study in response to corn grain (average of -0.11 pH unit) was modest, but may have favored the growth of starch fermenting bacteria as noted previously in grazing cows supplemented with increasing amounts of corn grain (Elias et al., 1996). Corn addition to either forage increased bacterial DM yield by an average of 53% in the present study (Table 5). Amylolytic and/or propionogenic bacteria may have accounted for a proportion of this increase. Taken together, our results indicate that under similar experimental conditions in vivo red clover pastures may result in greater production of biohydrogenation intermediates with a trans10 double bond.
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Oleic acid was the only 18:1 isomer detected in forages and corn grain (Table 1), and the amount of cis9-18:1 entering the fermenters (Table 4) varied with the forage:corn grain ratio. The amount of cis9-18:1 in effluents was greater during red clover fermentation, especially fall red clover, compared with orchardgrass (Table 9). The overall output of cis9-18:1, however, accounted for only 29 to 45% of total cis-18:1 isomers in effluents from orchardgrass or red clover. Although not affected by forage type or corn, overall outputs of cis11–, cis13-, and cis15-18:1 accounted for up to 53% of total cis-18:1 isomers. Output of cis13-18:1 and cis15-18:1, however, was greater when spring forages were fermented. In vitro studies with pure cultures indicated that only a small fraction of cis9-18:1 was isomerized to other cis-18:1 isomers (Kemp and Lander, 1984). In contrast, isomerization and hydrogenation of cis9, cis12-18:2 or cis9, cis12, cis15-18:3 can potentially result in the formation of several cis-18:1 isomers. With 18:2n-6 as the substrate, cis11-18:1 and cis12-18:1 accounted for 44 and 5% of total fatty acids recovered (Hazlewood et al., 1976). Although, cis-18:1 isomers were less than 3% of total fatty acids after incubation of 18:2n-6 or 18:3n-3 with pure cultures of ruminal bacteria, cis10-, cis9-, and cis11-18:1 accounted for 45, 35, and 20%, respectively, of total cis-18:1 isomers (Kemp et al., 1975). A major isomer produced during hydrogenation of 18:3n-3 in ruminal fluid was cis15-18:1, which increased from 0% to 32% of total fatty acid end products and was proportional to the amount of 18:3n-3 provided as substrate for incubations (Body, 1976).
Outputs of Trans Isomers of 18:1
Eight trans-18:1 isomers were identified in effluents, and they accounted for 25 to 30% of total fatty acid output from red clover or orchardgrass fermentations. Trans11-18:1, however, represented 61 to 66% of total trans-18:1 isomers. Each of the remaining isomers accounted for 2 to 12% of total trans-18:1 isomers. Consistent with the greater apparent hydrogenation of 18:2n-6 or 18:3n-3 (Table 6), outputs of all trans-18:1 isomers, except trans15-18:1, were greater from orchardgrass than from red clover fermentations. In addition, spring forages resulted in greater output of all trans-18:1 isomers, except trans16-18:1, compared with fall forages. The difference due to season, however, was more evident when red clover was fermented.
Corn grain addition to red clover, regardless of season, resulted in greater outputs of all trans-18:1 isomers. Increases in outputs of trans9-, trans10-, and trans12-18:1, however, also were evident when corn was added to orchardgrass. The greater outputs of all trans-18:1 isomers from red clover were a function of higher input and apparent hydrogenation of cis9-18:1 and 18:2n-6. It appears, however, that trans9-, trans10–, and trans12-18:1 are major trans isomers resulting from isomerization of cis9-18:1 in the rumen. Trans10-18:1 represented 25% of total fatty acids during incubation of cis9-18:1 in vitro with a pseudomonad strain, and its concentration increased more than 2.5-fold when the pH decreased gradually from 7 to 5 (Mortimer and Niehaus, 1972). Recently, it was conclusively shown that cis9-18:1 in the rumen could be isomerized to most trans-18:1 isomers (Mosley et al., 2002). Strikingly, the percentage of carbon in trans10-18:1 derived from 13C-oleic acid was 84% after only 12 h of incubation.
Our data demonstrated that output of trans10-18:1 was linearly related to cis9-18:1 and 18:2n-6 input from corn grain (Figure 5). Taking into account that production of trans10,cis12-18:2 also increased in response to 18:2n-6 input (Table 8), incomplete hydrogenation of trans10, cis12-18:2 may have contributed to the output of trans10-18:1. Ruminal bacteria capable of growing with starch as the sole substrate hydrogenated linoleic or linolenic acid mainly to trans-18:1 (95% of total end products) (Kemp et al., 1975). Under those conditions, trans10-18:1 accounted for up to 70% of the total trans-18:1. Although cis9,trans11-18:2 was found in small amounts during incubations with linoleic acid, trans10,cis12-18:2 was not detected. A common pathway whereby 18:2n-6 or 18:3n-3 may result in synthesis of trans10-18:1 would be from isomerization of a cis9-18:1 intermediate.
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) suggests that formation of trans10-18:1 from the conjugated isomer may have been affected by differences in pH due to the combination of corn grain and forage type. Despite an apparent greater accumulation of trans10,cis12-18:2 at lower pH with red clover fermentations (Figure 3A), biohydrogenation of trans10,cis12-18:2 to trans10-18:1 did not seem to occur to a significant extent (Figure 4B). With orchardgrass fermentations, output of trans10,cis12-18:2 remained constant (Figure 3A) and hydrogenation of linoleic acid (Figure 2B) remained high across pH. This could mean that production of trans10-18:1 from trans10,cis12-18:2 was very rapid and may explain the high outputs of trans10-18:1 at low outputs of trans10,cis12-18:2 (Figure 4B).
Output of trans10-18:1 also was closely associated with output of cis9-18:1 (Figure 4C). Isomerization of oleic acid, derived from the diet or during hydrogenation of 18:2n-6 or 18:3n-3, to trans10-18:1 could potentially explain the positive relationship between output of cis9-18:1 and trans10-18:1 (Figure 4C). Particularly because hydrogenation of oleic acid to 18:0 (Figure 2A), although variable, was lower than linoleic or linolenic acid along the range of pH values observed. Hence, more "substrate" was available for isomerization. Although not reported by Mosley et al. (2002), judging from their incubation conditions, an acidic pH does not seem to be necessary for trans10-18:1 formation from oleic acid in the rumen. Other factors must be taken into account to explain the observed differences in the trans10-18:1 and cis9-18:1 relationship due to feeding red clover or orchardgrass. It could be possible that isomerization of oleic acid, rather than hydrogenation of trans10,cis12-18:2, was a greater factor driving output of trans10-18:1 when orchardgrass and corn grain were fed (Figure 4B,C).
The greater input of 18:2n-6 with corn grain was a driving force leading to greater outputs of trans-18:1 isomers, particularly trans11-18:1. Upon hydrogenation of 18:2n-6 and 18:3n-3 in pure cultures of ruminal bacteria, concentrations of trans9-18:1 through trans15-18:1 were significantly increased (Kemp et al., 1975; Hazlewood et al., 1976). However, trans11-18:1 was by far (20 to 100% of total fatty acids) the predominant fatty acid produced during hydrogenation. In our study, outputs of trans-18:1 isomers were probably enhanced by the rapid rate of liquid and solid outflow from fermenters meant to resemble ruminal kinetics during grazing. Trans6/7/8-, trans9-, trans10-, trans11–, trans12-, trans13/14-, trans15-, and trans16-18:1 accounted for 2, 2, 4, 57, 6, 14, 7, and 9%, respectively, of total trans-18:1 duodenal flow (8.3 g/d) in goats fed soybean oil (Bickerstaffe et al., 1972). In duodenal digesta from cows fed a high-concentrate diet, trans10-18:1 and trans11-18:1 accounted for 26 and 31% of total trans-18:1 flow (Loor et al., 2002d; Piperova et al., 2002). In terms of proportions, outputs of individual trans-18:1 isomers in our study were comparable with these values.
Greater microbial DM yields were associated with higher digestibility of DM, NDF, CP, and NSC. The efficiency of apparent hydrogenation of cis9-18:1, 18:2n-6, or 18:3n-3 during fermentation varied with type of forage and season, partly due to differences in types and amounts of unsaturated fatty acids. Efficiency of hydrogenation was greater in response to orchardgrass, whereas incomplete hydrogenation was more pronounced with red clover. Biohydrogenation of 18:2n-6 and 18:3n-3 in response to orchardgrass fermentations was relatively constant across the range of pH observed. However, 18:2n-6 hydrogenation with red clover fermentations decreased as pH increased. Oleic acid hydrogenation, regardless of forage, appeared to decrease as pH increased. Linolenic acid was the primary substrate for hydrogenation when orchardgrass or red clover were fed and resulted in greater production of trans11,cis15-18:2 and trans11-18:1 during fermentation. Replacing corn (starch) for portions of forage provided more cis9-18:1 plus 18:2n-6 during fermentation, and these became the primary substrates for hydrogenation. As a result, the outflow of trans11,cis15-18:2 from fermenters decreased and outflows of trans10-18:1, trans11-18:1, cis9,trans11-18:2, or trans10,cis12-18:2 increased. Negative flows of trans10-18:1 were associated with increased pH regardless of forage. A similar response was observed for trans10,cis12-18:2, but only with red clover.
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Received for publication August 9, 2002. Accepted for publication December 16, 2002.
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