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Department of Dairy Science Virginia Tech Blacksburg, VA 24061-0315
Corresponding author:
J. H. Herbein; e-mail:
herbeinj{at}vt.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Key Words: rumenic acid • biohydrogenation • milk fat • unsaturated oil
Abbreviation key: CLA = conjugated linoleic acid, OLE = high-oleic sunflower oil, LIN = high-linoleic safflower oil, 9/11CLA = cis9,trans11-18:2 infusion, 10/12CLA = trans10,cis12-18:2 infusion, TG+FFA = fatty acids in blood plasma triglycerides plus FFA fractions.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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9 desaturase (EC 1.14.99.5), in the mammary gland of the cow (see review by Bauman et al., 2001) or human tissues (see review by Pariza et al., 2001). Concentrations of trans11-18:1 and cis9,trans11-18:2 in milk fat can be enhanced by feeding diets containing unsaturated oil with a high linoleic acid content (Chilliard et al., 2000; Bauman et al., 2001). Feeding greater amounts of grain or grain plus unsaturated oil in place of forage causes production of milk fat with lower concentrations of trans11-18:1 and greater concentrations of total trans-18:1, due to a substantial increase in trans10-18:1 primarily (Piperova et al., 2000; Piperova et al., 2002; Loor et al., 2002b). In some cases trans10,cis12-18:2 also increased by feeding high-grain diets with (Piperova et al., 2000) or without high-linoleic oil (Piperova et al., 2002). A review of initial studies using dietary CLA mixtures (primarily cis9,trans11-18:2 plus trans10,cis12-18:2) indicated CLA isomers had potential anticarcinogenic, antidiabetic, and antilipogenic properties in laboratory animals (Pariza et al., 2001). Subsequent work indicated the cis9,trans11-18:2 in bovine milk fat was more effective for inhibition of growth of human mammary cancer cells than synthetic trans10,cis12-18:2 (O’Shea et al., 2000), whereas trans10,cis12-18:2 was identified as the isomer responsible for reduced lipogenesis (Pariza et al., 2001).
Administration of mixtures of CLA isomers to lactating cows via abomasal infusion reduced de novo fatty acid synthesis and fat yield (Loor and Herbein, 1998; Chouinard et al., 1999a). Reduced concentrations of products of desaturation reactions, however, indicated the CLA mixtures also tended to reduce desaturation of long-chain fatty acids. Subsequently, relatively pure (>90%) sources of CLA isomers allowed Baumgard et al. (2000) to determine that trans10, cis12-18:2 was responsible for reduced milk fat percentage and reduced concentrations of saturated medium-chain fatty acids in milk fat. Baumgard et al. (2000, 2001) also found an increase in the ratios (substrate to product) of fatty acid pairs associated with 9 desaturase activity in response to abomasal infusion of trans10,cis12-18:2. Infused cis9,trans11-18:2 increased the ratio of 18:0 to cis9-18:1 compared with basal (Baumgard et al., 2000). In rat liver homogenates trans10,cis12-18:2 decreased 9 desaturase activity, whereas cis9,trans11-18:2 decreased 6 desaturase (EC 1.14.99.25) activity (Pariza et al., 2001). In the mammary gland, portions of diet- or rumen-derived 18:0, trans11-18:1, 18:2n-6, and 18:3n-3 may be used as substrates for endogenous synthesis of cis9-18:1, cis9,trans11-18:2, 20:4n-6, and 20:5n-3, respectively, via 9, 5, and 6 desaturase activity (Hermansen et al., 1995; Enjalbert et al., 1998; Bauman et al., 2001).
As indicated above, diets containing an unsaturated oil as a source of linoleic acid can be used as a practical means for enhancing the cis9,trans11-18:2 content of bovine milk fat. However, the addition of a rumen-protected CLA mixture (Giesy et al., 2002) or purified trans10,cis12-18:2 to the diet appears to be the only practical method for substantial enhancement of the trans10,cis12-18:2 content of milk fat. The concentration of trans10,cis12-18:2 in milk fat was proportional to the daily quantity of trans10,cis12-18:2 entering the rumen of cows fed a typical TMR (Loor and Herbein, 2001). As expected, however, milk fat yield and apparent desaturation of fatty acids were inversely proportional to trans10,cis12-18:2 input. It is not known whether a supplemental supply of unsaturated fatty acids in the diet would alleviate the inhibitory effect of trans10,cis12-18:2 on desaturation of fatty acids, such as 18:0, trans11-18:1, 18:2n-6, or 18:3n-3, in the mammary gland. Thus, the objective of this study was to evaluate desaturation of these fatty acids in lactating cows fed high-oleic oil or high-linoleic oil and infused with cis9,trans11-18:2 or trans10,cis12-18:2 via the abomasum.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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) in equal amounts at 1400 and 0200 h. The amount of TMR offered was enough to allow 5 to 10% feed refusal, which was weighed at 1400 h. Cows initially were fed a basal diet (similar to OLE and LIN, but without oil), which was replaced incrementally (0, 25, 50, 75, then 100%) with a mixture of equal parts OLE and LIN to allow cows to adapt to a diet containing oil. During 4 d before the start of the first period and 4 d between each of 15-d periods, the incremental replacement procedure was used to provide a transition from the previous diet (equal parts OLE and LIN for all cows before the first period) to the assigned diet for the next period (OLE or LIN in the following periods). Intake of DM was measured every 12 h during d 11 through d 15. Continuous infusion of 9/11CLA or 10/12CLA via the abomasum began at 1400 h on d 11 and continued for 48 h. Milk samples were obtained at each milking from d 11 through 15 (-12 to 96 h relative to the start of the 48-h infusion). Milk was collected in a stainless steel bucket, weighed, and thoroughly mixed before sampling. The experimental protocol was reviewed and approved by the Virginia Polytechnic Institute and State University Animal Care Committee.
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), were emulsified in skim milk. Due to differences in purity of CLA mixtures, it required 16.5 g of 9/11CLA and 15.6 g of 10/12CLA to obtain a dose of 15 g of each isomer. Emulsions were prepared the day before an infusion by combining 15 g of CLA with 3.5 g of glycerol (Eastman Kodak Co., Rochester, NY) and 1.8 g of soy lecithin powder (Refined, Alfa, Ward Hill, MA) in 975 ml of skim milk at room temperature. The mixture was homogenized at 12,000 rpm for 2 min with a Polytron homogenizer (PT 10/35, Brinkmann Instruments, Westbury, NY). Emulsions were dispensed into 1 L Viaflex plastic bags (Baxter Corporation, Deerfield, IL) and stored at 4°C. During infusion, bags were attached to a platform on a wrist-action shaker (Burrell Corporation, Pittsburgh, PA) set at low speed. Emulsions flowed through Tygon tubing (1.6 mm i.d., 0.8-mm wall; Fisher Scientific Co., Pittsburgh, PA) to a Harvard Peristaltic pump (55-1762; Harvard Apparatus, South Natick, MA). Flow from the pump was via Tygon tubing (3.2 mm i.d., 1.6-mm wall) passed through a rumen cannula, rumen, and omasum before terminating in the abomasum. A perforated Nalgene plastic bottle (60 ml) was attached to the end of the tubing to secure it in the abomasum. The tubing was primed with 15 ml of emulsion at the start of infusion, and flow rate was set at 41.7 ml/h. After 24 h, the empty bag was replaced with another bag containing 1 L of emulsion with 15 g of CLA.
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). In preparation for analyses, dried forages and concentrates were ground first through a 2-mm screen (Thomas-Wiley Laboratory Mill, Arthur H. Thomas, Philadelphia, PA), then through a 1-mm screen in a Cyclotec mill (Tecator 1093, Hoganas, Sweden). Ground samples were analyzed for ADF and NDF (Van Soest et al., 1991), total N (AOAC, 1990), and fatty acid content. Two 50-ml aliquots of milk were collected at -12, 0, 12, 24, 36, 48, 60, 72, 84, and 96 h relative to the start of infusion. The first aliquot containing Bronopol (D & F Control Systems, San Ramon, CA) was stored at 4°C until analyzed for fat, protein, SNF, and lactose (AOAC, 1990) by infrared analysis with a four-channel spectrophotometer (Multispec, Foss Food Technology Corp., Eden Prairie, MN) at the Virginia Dairy Herd Improvement Association laboratory. The second aliquot was stored at -20°C until the end of the study, thawed, and centrifuged at 10,000 x g for 1 h to harvest milk fat for fatty acid analysis.
Plasma total fatty acid profiles were determined using blood samples (10 ml) obtained from the coccygeal artery/vein after collection of milk samples. Profiles of fatty acids in arterial blood plasma lipid fractions and estimated fatty acid extraction ratios were determined using coccygeal artery and subcutaneous mammary vein samples (10 ml) obtained at 2-h intervals from -12 to 0 h and 36 to 48 h relative to the start of a CLA infusion. Blood was transferred to tubes containing 286 IU of heparin in 100 µl of sterile saline and centrifuged at 3,000 x g for 15 min for harvesting plasma. An equal volume of plasma from each of the six arterial or six venous samples obtained before (-12 to 0 h) and during (36 to 48 h) CLA infusion was pooled for isolation of plasma lipid fractions. Plasma was stored at -20°C until lipid extraction.
Total lipids were extracted from all plasma samples with chloroform/methanol (2:1, vol/vol). Lipid fractions (FFA, phospholipids, cholesterol esters, and triglycerides) in arterial and venous samples obtained before and during CLA infusion were isolated (Ågren et al., 1992) using 500 mg of Bond Elut aminopropyl columns (Varian, Walnut Creek, CA). Fatty acids in forages, concentrates, milk fat, blood plasma (total fatty acid profiles), and plasma lipid fractions were methylated by in situ transesterification with 0.5 N methanolic NaOH in methanol followed by 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 an autosampler into a Hewlett-Packard 5890A gas chromatograph equipped with a flame ionization detector (Hewlett-Packard, Sunnyvale, CA). Methyl esters were separated on a 100 m x 0.25 mm i.d. fused silica capillary column (CP-Sil 88, Chrompack, Middleburg, The Netherlands).
To identify peaks and 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, Elysian, MN), designed to resemble a typical milk fat, 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, trans11-18:1, cis9-18:1, and cis11-18:1 methyl esters were purchased from Nu-Check Prep (Elysian, MN). Trans6-18:1, trans7-18:1, trans12-18:1, cis12-18:1, and cis13-18:1 were purchased from Sigma Chemical Co. (St. Louis, MO). Trans13-18:1 and cis15-18:1 were purchased from Supelco Inc. (Bellefonte, PA). The nonconjugated 18:2 isomer mixture was purchased from Sigma Chemical Co. (St. Louis, MO), and contained trans9,trans12-18:2, cis9,trans12-18:2, trans9,cis12-18:2, and cis9,cis12-18:2. The conjugated linoleic acid mixture (Nu-Check Prep, Elysian, MN) 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. They were identified by order of elution as described in Griinari et al. (1998) and Ulberth and Henninger (1994). The response factor for 18:0 was used to quantify these fatty acids.
An 80 to 1 split ratio was used for injection of 0.5 µl hexane containing methyl esters of fatty acids from forage, concentrate, or milk fat samples. The carrier gas was ultrapure hydrogen, and inlet pressure was maintained at 23.1 psi. Injector temperature was maintained at 250°C, and detector temperature was maintained at 255°C. The initial oven temperature was 70°C (held for 1 min), increased 5°C/min to 100°C (held for 2 min), increased 10°C/min to 175°C (held for 40 min), and increased 5°C/min to 225°C (held for 15 min).
Injections of 0.5 µl methyl esters in hexane (splitless) were used for arterial plasma (total fatty acid profiles). The purge valve was closed for 0.8 min after injection. Injections of 2.5 µl were used for arterial and venous plasma lipid fractions, and the purge valve was closed for 1.5 min after injection. For both analyses, the injector and detector temperatures were 250 and 275°C. The initial column temperature was 40°C (held for 1.5 min), increased 40°C/min to 100°C (held for 10 min), increased 20°C/min to 175°C (held for 45 min), and increased 10°C/min to 220°C (held for 25 min).
Mammary gland extraction ratios for individual fatty acids were estimated using the sum of the amount of a fatty acid in the triglyceride fraction plus the amount in the FFA fraction (TG+FFA) of arterial and venous samples (Enjalbert et al., 1998). Extraction (%) of fatty acids from arterial plasma was calculated as [(arterial - venous concentration)/arterial concentration] x 100.
Statistical Analysis
Data for DMI, milk production, milk composition, fatty acid intake, milk fatty acids, and ratios of milk fatty acids were analyzed as a Latin square with factorial arrangement of treatments and repeated measures using the MIXED procedure of SAS (2000). Compound symmetry was the covariate structure used for all repeated measures analysis. The statistical model included cow, period, oil supplement, CLA isomer, time, CLA isomer x oil interaction, oil x time interaction, CLA isomer x time interaction, oil x CLA isomer x time interaction, and residual error. Fixed effects in the model included period, oil supplement, CLA isomer, time, oil x isomer interaction, and oil x isomer x time interaction. Cow was the random effect. Data for fatty acid profiles in blood plasma and mammary fatty acid extraction were analyzed as a Latin square with factorial arrangement of treatments without repeated measures using the MIXED procedure of SAS (2000). The statistical model included cow, period, oil supplement, CLA isomer, oil x isomer interaction, and residual error. Fixed effects in the model included: period, oil supplement, CLA isomer, and oil x isomer interaction. Cow was the random effect. One cow in the high-oleic oil group receiving 9/11CLA was omitted from all statistical analyses from that period, because the infusion line inadvertently was dislodged from the abomasum into the rumen. Overall differences between treatment least squares means were considered significant at P 
0.05, but all P values are presented in tables.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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). Percentages (data not shown) and yields (Table 3) of protein, lactose, and SNF in milk also did not differ. Milk fat percentage from 24 to 96 h, however, was substantially reduced by infusion of 10/12CLA, regardless of diet (Figure 1). The lower overall fat concentration in response to 10/12CLA reduced overall milk fat yield (Table 3) by 25% compared with 9/11CLA.
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). Intakes of 14:0, 16:0, cis9-16:1, and 18:0 were slightly, but significantly, higher when LIN was fed. As expected, the primary fatty acid in the DM was cis9-18:1 when OLE was fed and 18:2n-6 when LIN was fed. Concentration of total fatty acids in blood plasma at the end of the 48-h infusion period (1,957 µg/ml) was similar for all treatments (Table 5). When cows were fed OLE, concentrations of cis9-18:1, trans6/7/8-18:1, trans9-18:1, and 18:3n-3 were greater compared with feeding LIN. When cows were fed LIN, concentrations of 18:2n-6 and the primary biohydrogenation intermediates, cis9,trans11-18:2 and trans11-18:1 were elevated in plasma. In addition, concentrations of cis12-18:1, trans12-18:1, and trans16-18:1 were elevated when LIN was fed. Concentrations of cis9,trans11-18:2 in plasma increased in response to 9/11CLA infusion, whereas trans10-18:1 and trans10,cis12-18:2 were elevated in response to 10/12CLA infusion.
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). Concentrations (milligrams per gram total fatty acids in each lipid fraction) of cis9-18:1 and 18:2n-6 in all lipid fractions reflected the amount of each fatty acid contained in OLE or LIN. Oleic acid concentration in phospholipids, cholesteryl esters, triglycerides, and FFA when OLE was fed averaged 100, 32, 97, and 96 mg/g, respectively, compared with 53, 19, 50, and 53 mg/g when LIN was fed. In contrast, 18:2n-6 concentration in phospholipids, cholesteryl esters, triglycerides, and FFA due to feeding LIN averaged 450, 880, 100, and 100 mg/g, respectively, compared with 370, 850, 50, and 50 mg/g when OLE was fed.
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). Overall, the concentrations of individual CLA isomers in triglycerides and FFA increased in proportion to the amount of isomer infused. Trans10,cis12-18:2 was detectable only when 10/12CLA was infused, and averaged 2, 11, and 6 mg/g, respectively, in phospholipids, triglycerides, and FFA. The elevated concentrations of CLA isomers in blood plasma lipids at 36 to 48 h corresponded with the peak in their concentrations in milk fat (data not shown).
Fatty Acids in Arterial Plasma Triglycerides plus Free Fatty Acids
To estimate the primary pool of fatty acids available to the mammary gland for uptake and incorporation into milk fat, the sum of individual fatty acids in plasma TG+FFA was determined. Concentration of total TG+FFA in coccygeal blood plasma at 48 h averaged 99 µg/ml and did not differ across treatments (Table 6). When cows were fed OLE concentrations of cis9-18:1 and 18:3n-3 in TG+FFA were 76 and 38% greater compared with feeding LIN. In contrast, feeding LIN resulted in elevated concentrations of 18:2n-6 and cis12-, cis15-, trans10-, trans11-, trans12-, trans13/14-, and trans16-18:1. Infusing 10/12CLA, regardless of diet, elevated concentrations of cis11-, cis12-, cis13-, trans6/7/8-, trans9-, trans10-, and trans12-18:1. Concentrations of 14:0 and 18:3n-3 in TG+FFA also were elevated by 10/12CLA infusion. Greater concentrations of cis9,trans11-18:2 or trans10,cis12-18:2 in TG+FFA was expected in response to infusion of either CLA isomer, but alterations in concentrations of the cis- and trans-isomers of 18:1, 14:0, or 18:3n-3 were not. The cause(s) of the response may be due to reduced need of exogenous fatty acids for milk fat synthesis or the effects of 10/12CLA on tissues other than the mammary gland.
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). Despite greater concentrations of several fatty acids in TG+FFA in response to OLE or LIN, extraction ratios for most fatty acids did not differ due to treatment. The exception was extraction of 18:2n-6, which was greater when cows were fed LIN compared with OLE. Infusion of 10/12CLA resulted in lower extraction of 18:0 and higher extraction of cis9,trans11-18:2, regardless of diet. Extraction of trans10,cis12-18:2 during 10/12CLA infusion averaged 81%, which was numerically higher than the extraction ratios for all other fatty acids flowing to the mammary gland in blood plasma.
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), regardless of diet, decreased 19% from 0 to 60 h in response to 10/12CLA then remained low until 96 h. Overall yields of fatty acids with 4 to 16 carbons were reduced when 10/12CLA was infused (Table 8). Reductions in the yields of the short- and medium-chain fatty acids were the primary cause of the 19% reduction in the yield of total fatty acids in response to 10/12CLA.
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). Similarly, the basal concentration of trans11-18:1 was greater when LIN was fed compared with OLE (Figure 3). During 10/12CLA infusion, concentration of cis9,trans11-18:2 in milk fat was reduced by 40% when cows were fed LIN. As a result, yield of cis9,trans11-18:2 decreased with 10/12CLA infusion.
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). As a result of the lower yield of milk fat in response to 10/12CLA infusion, however, the yield of 18:0 from 0 to 60 h did not differ due to type of infusion (Table 8). The concentration of cis9-18:1 in milk fat did not change during infusion of 9/11CLA when cows were fed OLE or LIN (data not shown). However, when LIN was fed and 10/12CLA was infused, concentration of cis9-18:1 was 12% lower from 24 to 60 h (significant diet x isomer x time interaction). The greater availability of dietary cis9-18:1 when OLE was fed, compared with LIN, probably masked the potential effects of 10/12CLA infusion on desaturation of 18:0. Indicators of inhibition of desaturase activity are discussed in the "normalized ratios" section below.
Cows fed LIN had higher basal concentrations of 18:2n-6 (Figure 3) and 20:4n-6 (16 vs. 13 mg/g) (data not shown). Similar to the response in 18:0 concentration, infusion of 10/12CLA increased the concentration of 18:2n-6 and decreased 20:4n-6 concentration (data not shown). As noted above, however, the depressed yield of nearly all fatty acids (Table 8) masked the divergent changes in concentrations of substrate/product fatty acid pairs for desaturase reactions.
Normalized Ratios of Milk Fatty Acids
Normalized ratios (mg/g product ([mg/g substrate + mg/g product]) were estimated to assess the extent of desaturation of specific fatty acids during milk fat synthesis (Sol-Morales et al., 2000). The basal ratios of cis9-14:1/14:0, cis9-16:1/16:0, cis9-18:1/18:0, cis9,trans11-18:2/trans11-18:1, and 20:4n-6/18:2n-6 were higher when cows were fed OLE compared with LIN (Table 9). Higher ratios indicated cows desaturated more of the substrate fatty acids when they were fed OLE compared with LIN. Compared with 9/11CLA, however, infusion of 10/12CLA decreased the above ratios, regardless of diet. Lower ratios were evident after only 24 h of 10/12CLA infusion, regardless of diet, and were maintained until 72 h (data not shown). The decline in the ratios suggested exogenous trans10,cis12-18:2 reduced the amount and(or) activity of 6, 5, and 9 desaturases in the mammary gland.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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). Differences in intakes of cis9-18:1 and 18:2n-6 due to type of oil fed led to major changes in the profiles of most fatty acids in blood plasma lipids. Oleic acid accounted for 10% of total fatty acids in triglycerides, FFA, and phospholipids when high-oleic oil was fed (Figure 2). Linoleic acid also accounted for 10% of triglycerides and FFA but was 42% of total fatty acids in phospholipids when high-linoleic oil was fed. These three lipid fractions contained 53% of total fatty acids in blood plasma. Plasma cholesteryl esters also contained more cis9-18:1 or 18:2n-6 in response to intake of high-oleic or high-linoleic oil, but 18:2n-6 averaged 85% of total fatty acids regardless of oil type. Concentrations of isomers derived from isomerization and hydrogenation of dietary cis9-18:1 or 18:2n-6 also were proportional to intake of both fatty acids from the diet. For example, feeding high-oleic oil increased concentrations of trans6/7/8-18:1 and trans9-18:1 by 86 and 57% in blood plasma compared with feeding high-linoleic oil. Concentrations of cis12-18:1 and trans10-, trans11-, trans12-, or trans16-18:1, however, were 69, 31, 82, 42, and 60%, respectively, greater in response to feeding high-linoleic oil compared with high-oleic oil. Trans11-18:1 was the primary trans-18:1 isomer in blood plasma, and it accounted for 42 or 55% of total trans-18:1 when cows were fed high-oleic or high-linoleic oil, respectively.
Among lipid fractions, triglycerides and FFA contained 2 to 4% trans11-18:1, whereas phospholipids contained 0.8% trans11-18:1 (Figure 2). Overall, trans isomers of 18:1 were primarily found in triglycerides (7% of total fatty acids), FFA (5%), and phospholipids (2%). Greater flow of trans-18:1 isomers to the duodenum during hydrogenation of supplemental 18:2n-6 in the rumen increased absorption and incorporation of 18:1 and 18:2 isomers into blood plasma triglycerides and phospholipids (Bickerstaffe et al., 1972; Loor et al., 2002c). Thus, upon hydrolysis of triglycerides and phospholipids, the plasma FFA pool also contains more trans-18:1 isomers. Our results confirmed previous in vivo and in vitro observations, indicating ruminal hydrogenation of cis9-18:1 and 18:2n-6 gives rise to geometrical isomers of 18:1 with double bonds at positions 6 through 16 of the carbon chain (Bickerstaffe et al., 1972; Kemp et al., 1975; Loor et al., 2002a). Ruminal isomerization of oleic acid was associated with production of several trans18:1 but primarily trans6/7/8-18:1, trans9-18:1, and trans10-18:1 (Loor et al., 2002a; Mosley et al., 2002). Under normal circumstances, however, trans11-18:1 is by far the predominant trans-18:1 isomer resulting from hydrogenation of 18:2n-6 in the rumen.
Whereas cis9,trans11-18:2 in plasma was proportional to dietary 18:2n-6 intake, trans10,cis12-18:2 was detectable only after 10/12CLA was infused (Table 5). Infused 9/11CLA was primarily incorporated into plasma FFA and triglycerides, where it averaged 7 to 13 mg/g of total fatty acids. During infusion of 10/12CLA, regardless of diet, concentration of trans10,cis12-18:2 increased from nondetectable levels to 15 mg/g in total blood plasma (Table 5). Its concentration in triglycerides, FFA, and phospholipids averaged 11, 6, and 2 mg/g after infusion of 10/12CLA (Figure 2). The triglyceride and FFA fractions contained approximately 3% of total fatty acids in plasma, whereas the phospholipid fraction contained approximately 48% of total plasma fatty acids. However, absolute amounts of cis9,trans11-18:2 or trans10,cis12-18:2 in the three fractions during infusion of 9/11CLA or 10/12CLA was similar. To our knowledge, these are the first data available on incorporation of trans10,cis12-18:2 in bovine plasma lipids. The increase in the proportions of cis9,trans11-18:2 or trans10,cis12-18:2 in triglycerides and FFA indicated they were readily available to the mammary gland. The low transfer efficiency (8% at 48 h) of infused 10/12CLA into milk fat found in the present and previous (Baumgard et al., 2000, 2001) studies may be explained by its greater incorporation (total micrograms per milliliter plasma) into plasma phospholipids, which are not readily taken up by the mammary gland.
Availability of cis9-18:1, trans9-18:1, and 18:3n-3 for extraction from TG+FFA was greater when high-oleic oil was fed compared with high-linoleic oil. In contrast, feeding high-linoleic oil increased the availability of cis12-, cis15-, trans10- through trans16-18:1, and 18:2n-6. There were minor but significant increases in the concentrations of 14:0 and some cis- or trans-isomers of 18:1 and 18:3n-3 in response to 10/12CLA infusion compared with 9/11CLA (Table 6). This response to exogenous 10/12CLA was associated with an overall 30% increase in total fatty acids in the FFA fraction (data not shown). Higher concentrations of total plasma FFA were previously found during abomasal infusions of CLA mixtures (Loor and Herbein, 1998) or various doses (2 to 10 g/d) of trans10,cis12-18:2 (Baumgard et al., 2000).
Despite the increase in concentrations of the various cis- and trans-18:1 isomers in plasma TG+FFA due to oils or 10/12CLA, extraction ratios for these fatty acids did not change. Assuming that mammary blood flow remained constant, the extent of the increases in concentration apparently was not large enough to influence extraction. Only the extraction of 18:2n-6 was greater when high-linoleic oil was fed, regardless of isomer, because its concentration in TG+FFA was 147% higher compared with feeding high-oleic oil, and it accounted for 13% of total TG+FFA. Although concentrations of 18:0 in TG+FFA were constant across treatments, infusion of 10/12CLA decreased the extraction ratio for 18:0 by 6 percentage units regardless of diet. In contrast, the extraction ratio for cis9,trans11-18:2 increased due to 10/12CLA infusion. Our values are the first estimates of extraction of the major CLA isomers from blood plasma TG+FFA by the mammary gland of lactating cows.
Compared with 9/11CLA, infusion of 10/12CLA decreased milk fat concentration from 3.5% before infusion to 2.1% at 72 h (Figure 1). This represented a 40% reduction in concentration and led to an overall 25% reduction in yield (Table 3). Basal concentrations of saturated fatty acids with 6 to 16 carbons averaged 370 mg/g of total fatty acids, and were 34% lower than typically seen in milk fat from cows fed diets without supplemental oil (Palmquist et al., 1993). A large portion of the reduction in de novo synthesis due to feeding unsaturated oils occurs as a result of greater uptake and secretion of dietary and ruminally derived fatty acids (Palmquist et al., 1993). Exogenous fatty acids compete for esterification with newly synthesized short-chain fatty acids in mammary cells and could lead to feedback inhibition of lipogenic enzymes (Palmquist et al., 1993). Results from a recent study indicated that supplemental cis9-18:1 was preferentially incorporated into the sn-2 position of the milk fat triglyceride at the expense of 16:0 (DePeters et al., 2001). The net effect was lower concentration of 16:0 but higher cis9-18:1 concentration in milk fat.
Because of the small amount of trans10,cis12-18:2 required to reduce milk fat percentage (Figure 1A) and fatty acid yield (Table 8; Figure 4A), this isomer appears to depress de novo fatty acid synthesis in a manner distinct from that caused by dietary unsaturated fatty acids. Despite lower concentrations of 16:0 in milk fat when 18:2n-6 was infused into the abomasum, only the infusion of a CLA mixture reduced 16:0 and milk fat concentrations (Loor and Herbein, 1998). It seems that a trans-10 double bond in the CLA is required for inhibition of lipogenesis. For example, infusions of trans10,cis12-18:2 or cis8,trans10-18:2 (in combination with cis9,trans11-18:2) reduced milk fat synthesis to a similar extent (Loor and Herbein, 1998; Chouinard et al., 1999b). A common response to CLA mixtures or 10/12CLA not observed when unsaturated oils are fed is a marked accumulation of 18:0 in milk fat.
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) and yields of saturated 6:0 to 16:0 (Table 8) corresponded with the gradual increase in concentration of trans10,cis12-18:2 in milk fat from 0 to 48 h during 10/12CLA infusion (data not shown), and accounted for the overall reduction in milk fat yield. The temporal nature of the decrease in lipogenesis we observed with very small concentrations (6 mg/g) of trans10,cis12-18:2 were consistent with a sequence of events, which may have begun with reductions in synthesis of mRNA for acetyl-CoA carboxylase and fatty acid synthase. Hence, between 0 and 48 h of 10/12CLA infusion, reductions in yields of saturated 4:0-16:0 accounted for more than 50% of the decrease in total fatty acid yield (Figure 5). The present study confirmed initial evidence (Baumgard et al., 2000, 2001; Peterson et al., 2002) indicating trans10,cis12-18:2 at very small concentrations is extremely effective in reducing milk fat percentage and de novo fatty acid synthesis in dairy cows.
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). After uptake by the mammary gland, 18:0 becomes the primary substrate for 9 desaturase (Enjalbert et al., 1998). Trans11-18:1 derived from the rumen also is a substrate for 9 desaturase and leads to endogenous synthesis of cis9,trans11-18:2 in the mammary gland (Bauman et al., 2001). As indicated by normalized ratios (Table 9), the extent of desaturation of 16:0, 18:0, and trans11-18:1 before infusion of any CLA was higher when high-oleic oil was fed compared with high-linoleic oil. However, the marked increase in milk fat 18:0 and trans11-18:1 concentrations from 24 through 48 h when 10/12CLA was infused (Figure 3A,B), regardless of diet, suggested desaturation of fatty acids derived from plasma was severely decreased by exogenous trans10,cis12-18:2. Desaturation of trans11-18:1 to cis9,trans11-18:2 apparently was very sensitive to exogenous 10/12CLA (Figure 4B), because yield of cis9,trans11-18:2 in milk fat was reduced by 50% regardless of diet (Figure 5). The depressed ratios of cis9-14:1/14:0 and cis9-16:1/16:0, despite their lower concentrations in milk fat, provide evidence that desaturation of endogenously synthesized fatty acids also is sensitive to 10/12CLA.
Reduced desaturation, resulting in accumulation of 18:0 in the mammary gland during infusion of 10/12CLA, might have lowered 18:0 extraction from plasma TG+FFA. In contrast, the reduction in desaturation of trans11-18:1 to cis9,trans11-18:2 may have decreased endogenously synthesized cis9,trans11-18:2 concentration in the mammary gland. The lower amount of cis9,trans11-18:2 in the gland could have enhanced its extraction from TG+FFA when 10/12CLA was infused (Table 7). The reduction in yield of cis9,trans11-18:2, despite greater extraction from blood, in combination with the lower normalized ratio for cis9,trans11-18:2/trans11-18:1 during 10/12CLA infusion provides additional evidence (Bauman et al., 2001) that endogenous synthesis (via desaturation) may be the primary source of cis9,trans11-18:2 in milk fat.
Expression of 9 desaturase activity is markedly reduced by trans10,cis12-18:2, but not cis9,trans11-18:2, in rodent adipose, liver, and mammary gland tissue (Pariza et al., 2001; Lin, 2000). The negative effect of trans10,cis12-18:2 on desaturation activity in the bovine mammary gland might be mediated by reductions in the transcription of the 9 desaturase gene (Lin, 2000). We postulate that a large portion of the reduction in milk fat concentration and yield due to trans10,cis12-18:2, and not observed when unsaturated oils are fed, is a consequence of its acute effects on 9 desaturase. Even when availability of trans11-18:1 and 18:0 were elevated due to supplemental oils, both cis9,trans11-18:2 and cis9-18:1 concentrations in milk fat were negatively correlated with trans10,cis12-18:2 (Figure 4B and C). Nearly 40% of the reduction in total fatty acid yield between 0 and 48 h of infusion was due solely to oleic acid (Figure 5). With mice with a null mutation in the 9 desaturase gene, it was conclusively shown that triglyceride synthesis in the liver relies heavily on endogenous synthesis of oleic acid (Miyazaki et al., 2001). Results from the present study provide evidence of a similar mechanism present in bovine mammary tissue.
Plasma-derived 18:2n-6, through elongation and desaturation via 5 and 6 desaturases (Hermansen et al., 1995), is the major source of 20:3n-6 and 20:4n-6 in milk fat. Before infusion, feeding high-linoleic oil compared with high-oleic oil resulted in greater extraction of 18:2n-6 from TG+FFA (Table 7) and led to greater concentration (data not shown) and yield of 20:4n-6 in milk fat (Table 8). However, from 60 through 84 h after infusion of 10/12CLA, the concentration of 18:2n-6 in milk fat (Figure 3) increased but 20:4n-6 decreased (data not shown) regardless of diet. Similar responses were not found when 18:2n-6 was infused into the abomasum of lactating cows (Loor and Herbein, 1998), suggesting that the presence of a trans10-double bond in the CLA is associated with the reduction in 20:4n-6 concentration and yield.
As mentioned earlier, the presence of the trans10-double bond (either in CLA or as trans10-18:1) may be required to induce lower milk fat synthesis in the mammary gland of dairy cows. It has been speculated that trans10,cis12-18:2 is an intermediate in the hydrogenation of 18:2n-6, which accumulates when high-grain low-forage diets are fed (Bauman et al., 2001). In response to a high-grain diet plus 5% soybean oil, trans10-18:1 accounted for 60% of all trans-18:1 isomers (16% of total milk fatty acids) but trans10,cis12-18:2 represented only 10% of total CLA isomers (1% of total milk fatty acids) (Piperova et al., 2000). Others did not detect (Griinari et al., 1998) or found no correlation (Loor et al., 2002b) between trans10,cis12-18:2 and reduced milk fat percentage in response to high-concentrate diets with or without unsaturated oil. Thus, the involvement of trans10,cis12-18:2 in diet-induced milk fat depression is still unclear. Greater production of trans10-18:1 (and to a lesser extent trans10,cis12-18:2) in the rumen depressed milk fat percentage and yield by inhibiting the activity and mRNA abundance for acetyl-CoA carboxylase and fatty acid synthase (Piperova et al., 2000). Desaturation of 18:0 did not seem to be affected, as the concentrations of 18:0 and cis9-18:1 in milk fat were similar compared with controls. At least in mouse liver, trans10-18:1 did not inhibit 9 desaturase activity compared with trans10,cis12-18:2 (Park et al., 2000). Hence, trans10,cis12-18:2 and trans10-18:1 might affect overall lipogenesis in the bovine mammary gland by different mechanisms. In this regard, profiles of stearic plus oleic acid (along with trans10-18:1 and trans10,cis12-18:2) in milk fat from cows with depressed milk fat content could be used to assess the relative involvement of 18:1 and CLA isomers with a trans 10-double bond in diet-induced milk fat depression.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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9 desaturase. Transfer of dietary trans10,cis12-18:2 into milk fat may be low due to its preferential incorporation into plasma phospholipids. However, if diet were capable of enhancing rumen-outflow of trans10,cis12-18:2, its uptake by the mammary gland could decrease de novo fatty acid synthesis and desaturation of long-chain fatty acids. Reduced milk fat production due to trans10,cis12-18:2 is closely associated with a lack of endogenously synthesized oleic acid for triglyceride formation.
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FOOTNOTES |
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2 Current address: Department of Animal Sciences, University of Illinois, 206 ERML, Urbana, IL, 61801; E-mail: jloor{at}uiuc.edu.
Received for publication August 14, 2002. Accepted for publication October 8, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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