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* Department of Animal Science, Food and Nutrition, Southern Illinois University-Carbondale, Carbondale 62901
Department of Dairy Science, South Dakota State University, Brookings 57006
1 Corresponding author: aabugha{at}siu.edu
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ABSTRACT |
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Key Words: docosatrienoic acid • eicosatrienoic acid • vaccenic acid
Conjugated linoleic acid (CLA) refers to a collection of positional and geometric isomers of octadecadienoic acid with conjugated double bonds. The cis-9,trans-11 CLA can be synthesized in the rumen as an intermediate in the biohydrogenation of linoleic acid and in animal tissues by 
9-desaturase from vaccenic acid (trans-11 C18:1, VA), another intermediate in ruminal biohydrogenation (Griinari and Bauman, 1999). In animal models, cis-9,trans-11 CLA reduced the incidence and growth of tumors, enhanced immune function, and prevented diabetes (Belury, 1995; Parodi, 1997). Recent studies (Corl et al., 2001; Piperova et al., 2002) have indicated that the majority of milk cis-9,trans-11 CLA (78 to 93%) is derived from 9-desaturation of VA. Therefore, increasing VA production in the rumen would be the most feasible approach to enhance milk fat cis-9,trans-11 CLA.
Production of VA in the rumen can be enhanced by changes in the diet, especially through utilization of diets with high concentrations of unsaturated fat. When fed at similar concentrations, fish oil was more efficient than polyunsaturated plant oils in increasing milk VA concentration, even though fish oil contains only small amounts of the primary precursors (linoleic acid, linolenic acid) of VA (Offer et al., 1999; Whitlock et al., 2002). These findings led AbuGhazaleh et al. (2001) to hypothesize that a component in fish oil may have stimulated ruminal VA production from other sources of unsaturated fatty acids. To test this hypothesis, AbuGhazaleh et al. (2002) fed fish oil from fish meal with or without extruded soybeans (linoleic acid source) to dairy cows. They observed that feeding the fish meal and extruded soybeans diet increased the concentration of milk cis-9,trans-11 CLA and VA more than feeding the fish meal or extruded soybeans separately. Recently, AbuGhazaleh and Jenkins (2004a) identified docosahexaenoic acid (C22:6n-3; DHA) as an active component in fish oil that promotes VA accumulation in ruminal cultures. Because fish oil contains additional n-3 fatty acids suspected of playing a role in ruminal trans-C18:1 production, in particular VA, we hypothesize that eicosatrienoic (C20:3n-3) and do-cosatrienoic acid (C22:3n-3), n-3 fatty acids found in fish oil (approximately 2% of total fatty acids), also promote VA accumulation when incubated with linoleic acid.
Eicosatrienoic acid, C22:3n-3, and linoleic acid (>99% purity) were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO), dissolved in ethanol, and stored at –20°C until used. Ruminal contents were collected 3 h after morning feeding (0600 h) from a ruminally fistulated Holstein cow fed a TMR composed of 50% concentrate mix, 25% corn silage, 12.5% alfalfa haylage, and 12.5% alfalfa hay (DM basis). The rumen contents were brought to the laboratory in a plastic bag under anaerobic conditions, strained through 2 layers of cheesecloth, and used within 15 min. Treatments consisted of control (no added fatty acids; CONT), control plus 5 mg of C20:3n-3 (ETA), control plus 5 mg of C22:3n-3 (DTA), control plus 15 mg of linoleic acid (LA), control plus 5 mg of C20:3n-3 and 15 mg of LA (ETALA), and control plus 5 mg of C22:3n-3 and 15 mg of LA (DTALA). Eicosatrienoic, C22:3n-3, and LA in 100 µL of ethanol were added directly into the treatment cultures.
Cultures were maintained in 125-mL Erlenmeyer flasks containing 500 mg of the cow TMR diet dried and ground through a 2-mm screen, 10 mL of strained ruminal fluid, 40 mL of media, and 2 mL of reducing solution according to Goering and VanSoest (1970). All buffer solutions were prewarmed at 39°C and flushed with CO2. Cultures were run in triplicate at 39°C under anaerobic conditions. A 5-mL sample was taken from each culture flask at 0 and 24 h while being stirred with a magnetic bar under a stream of CO2, placed immediately in an ice bath, and then stored at –20°C.
Samples were freeze-dried and then methylated according to Kramer et al. (1997) and analyzed for fatty acids by GLC. Methylated fatty acids were separated on a Shimadzu GC-2010 gas chromatograph equipped with a flame-ionization detector and a Supelco 100-m SP-2560 fused-silica capillary column (0.25 mm i.d. x 0.2-µm film thickness, Supelco, Bellefonte, PA). The helium carrier gas was maintained at a linear velocity of 23 cm/s. The oven temperature was programmed for 170°C for 50 min, then increased at 5°C/min to 249°C and held for 10 min. The injector and detector temperatures were set at 255°C. Heptadecanoic acid (C17:0) was added to all samples as an internal standard. Peaks were identified by comparing the retention times with those of the corresponding standards (Nu-Chek Prep, Elysian, MN; Supelco; and Larodan Fine Chemicals, Malmo, Sweden).
Data were analyzed using the mixed model procedure of SAS (SAS Inst., Inc., Cary, NC) using treatment as the fixed effect, and replicate as the random effect. All results were expressed as least squares means. Preplanned contrasts were 1) CONT vs. ETA, 2) CONT vs. DTA, 3) LA vs. ETALA, and 4) LA vs. DTALA. Significance was declared at P < 0.05.
If C20:3n-3 or C22:3n-3 were the components in fish oil that promote trans-C18:1 accumulation when incubated with other unsaturated fat sources, combining these 2 fatty acids with linoleic acid would maximize trans-C18:1 concentration, VA in particular. After 24 h of incubation, the concentration of trans-C18:1 was similar (P > 0.05), with CONT, ETA, and DTA cultures averaging 0.87, 0.88, and 0.99 mg/culture, respectively (Table 1
). Additions of C20:3n-3 or C22:3n-3 to ruminal cultures did not increase VA (0.56 and 0.62 mg/culture, respectively) concentration compared with CONT cultures (0.52 mg/culture). As expected, addition of linoleic acid to ruminal cultures increased trans-C18:1 and VA concentrations approximately 5- and 8-fold respectively, compared with CONT. However, combining C20:3n-3 or C22:3n-3 with linoleic acid did not further increase (P > 0.05) trans-C18:1 and VA concentrations compared with LA cultures (Table 1). The concentrations of trans-C18:1 (5.51, 5.41, and 4.91 mg/culture) and VA (4.78, 4.62, and 4.19 mg/culture) were similar (P > 0.05) for LA and ETALA, and DTALA, respectively. Previously, AbuGhazaleh and Jenkins (2004b) showed that combining DHA with a linoleic acid source resulted in greater trans-C18:1 and VA concentrations and VA:C18:0 ratio than when they were added separately in cultures. AbuGhazaleh and Jenkins (2004a) concluded that DHA or its derivatives promoted trans-C18:1 and VA accumulations, possibly by inhibiting the reductase enzyme in ruminal microorganisms that is responsible for the terminal hydrogenation of trans-C18:1 to C18:0. In the current study, addition of C20:3n-3 or C22:3n-3 to ruminal cultures had no effect on the VA:C18:0 ratio (Table 1).
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) when compared with CONT, indicating that EPA and DTA were not converted into any of these fatty acids. However, some unidentified peaks were detected around the ETA and DTA peaks in cultures. Similar unidentified peaks around DHA and eicosapentaenoic acid (C20:5) peaks were seen previously (AbuGhazaleh and Jenkins, 2004b). The lack of effect of C20:3n-3 and C22:3n-3 on accumulation of trans-C18:1 and VA and on VA:C18:0 ratio in ETA, DTA, ETALA, and DTALA cultures compared with CONT and LA cultures, respectively, indicates that C20:3n-3 and C22:3n-3 are not the active components in fish oil that promote VA accumulation when incubated with linoleic acid.
Received for publication March 13, 2006. Accepted for publication May 22, 2006.
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TOPABSTRACTREFERENCES |
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9-desaturase in the production of cis-9,trans-11 CLA. J. Nutr. Biochem. 12:622–630.[Medline]
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