The Journal of Nutrition Vol. 128 No. 12 December 1998, pp. 2411-2419

Exogenous Conjugated Linoleic Acid Isomers Reduce Bovine Milk Fat Concentration and Yield by Inhibiting De Novo Fatty Acid Synthesis^1,2

Juan J. Loor and Joseph H. Herbein³

Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060-0315

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Conjugated linoleic acid (CLA) is a potent anticarcinogen secreted in milk of ruminants, but it inhibits de novo fatty acid synthesis and desaturation in mammary cell cultures. The potential for increasing CLA content of milk fat and the effects of elevated CLA availability on milk fat secretion were investigated. Four Holstein cows were used in a single crossover design with repeated measures to determine milk fatty acid concentration in response to a 24-h infusion of 200 g linoleic acid (LA) or a mixture of 100 g LA plus 100 g CLA (LCLA). Milk and blood samples were obtained 12 h before infusion and at 12-h intervals from 0 to 72 h. Compared with LA infusion, total CLA concentration in blood plasma at 24 h in response to LCLA was elevated fivefold, whereas CLA content of plasma triglycerides was increased 10-fold. Milk fat yield from 24 to 72 h was ~34% lower in response to LCLA compared with LA, due primarily to reduced yield of fatty acids with six to 16 carbons. Amount of CLA in milk increased from 0.5 g/100 g total fatty acids at 0 h to 3.3 g/100 g at 36 h in response to LCLA. Concentration of stearic acid in milk fat at 36 h in response to LCLA was nearly double the stearic acid concentration in response to LA. Oleic and arachidonic acid concentrations in milk declined as stearic acid increased in response to LCLA. Results indicated CLA content of milk fat reflects the amount available for absorption from the small intestine, and CLA appeared to be a potent inhibitor of de novo fatty acid synthesis and desaturation in the mammary gland.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Conjugated linoleic acid (CLA)⁴ refers to a mixture of positional and geometrical isomers of linoleic acid with conjugated double bonds (Ip et al. 1994). The ⁹ cis, ¹¹ trans isomer of CLA has received much attention in recent years, because research indicates it is effective as an anticarcinogenic agent (Belury 1995). In the bovine, CLA is formed during the enzymatic hydrogenation of linoleic acid by rumen microorganisms. The initial step involves the isomerization of linoleic acid to the ⁹ cis, ¹¹ trans conjugated dienoic form. However, much of the CLA produced in the rumen is used for preferential reduction of the ⁹ cis double bond to form trans-vaccenic acid (Fujimoto et al. 1993).

Parodi (1977) reported that the primary CLA (90%) in bovine milk fat is the ⁹ cis, ¹¹ trans isomer, but the concentration in milk fat can vary substantially. Griinari et al. (1995) indicated a range of 2.4-18.0 mg CLA/g of fat in milk samples from New York herds. Griinari et al. (1996) and Kelly et al. (1998) also observed that a dietary supply of unsaturated fatty acids was needed to increase concentrations of CLA in milk. McGuire et al. (1996) reported that CLA concentration increased from 2 to 6.8 mg/g fat when corn oil supplementation increased from 3 to 7.2% of the diet dry matter.

Trans isomers of unsaturated fatty acids, whether derived from the diet or from incomplete biohydrogenation of unsaturated fatty acids, depress milk fat percentage (Wonsil et al. 1994). Abomasal infusion of trans-vaccenic acid also decreased milk fat percentage from 4.1 to 3.1% and yield from 1.4 to 1.1 kg/d (Romo et al. 1996). Dawson and Herbein (1996) reported that bovine mammary cell cultures incorporated CLA in an amount proportional to the amount available in the medium. Furthermore, de novo synthesis of palmitic acid and desaturation of stearic acid decreased as CLA uptake by the mammary cells increased. The objectives of this study were to evaluate the effects of enhanced CLA availability in the small intestine on CLA transport in blood plasma lipid fractions, milk fatty acid composition and rate of milk fat secretion by Holstein cows.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and experimental design.  Four multiparous Holstein cows from the Virginia Tech dairy herd (between 168 and 230 d postpartum), each with a cannula in their rumen and proximal duodenum, were fed a basal diet (Table 1) throughout each of two periods in a single crossover design with repeated measures. The basal diet was formulated using Dair4 (Stallings et al. 1985) to meet nutrient requirements of cows producing 30 kg milk and consuming 22 kg of diet dry matter daily. Treatments were 24-h abomasal infusions of 200 g of linoleic acid (LA) (United States Biochemical, Cleveland, OH) or a mixture of 100 g linoleic acid plus 100 g CLA (LCLA) (donated by Dr. Clement Ip, Roswell Park Cancer Institute) (Table 2). Two cows were randomly assigned to LA followed by LCLA, and two cows to LCLA followed by LA. Abomasal infusion of LA and LCLA began at 1400 h. There were 7 d between treatments. Cows were housed in a tie-stall barn during the experiment, and the basal diet was prepared and offered in equal amounts at 0200 and 1400 h daily. Feed refusals were weighed at 1400 h. Cows were milked at 0100 and 1300 h. Milk was collected in a stainless steel bucket, weighed, and thoroughly mixed prior to obtaining samples at each milking from 12 to 72 h relative to the start of infusion. The experimental protocol was reviewed and approved by the Virginia Polytechnic Institute and State University Animal Care Committee.

View larger version (14K): [in this window] [in a new window]   Fig 1. Concentration of linoleic acid (A) and conjugated linoleic acid (CLA) isomers (9 cis, 11 trans isomer plus 10 trans, 12 cis isomer) (B) in blood plasma of Holstein cows at 12-h intervals after the initiation of abomasal infusion of linoleic acid (LA) or an equal mixture of linoleic and conjugated linoleic acid (LCLA) for 24 h. Values are means ± pooled SEM for four cows at each 12-h interval. Asterisks denote significant differences (P < 0.05) due to treatments.

View larger version (25K): [in this window] [in a new window]   Fig 2. Distribution of conjugated linole.ic acid (CLA) isomers (9 cis, 11 trans isomer plus 10 trans, 12 cis isomer) in blood plasma free fatty acids (FFA), phospholipids (PL), cholesterol esters (CE) and triglycerides (TG) of Holstein cows at 24 h after the initiation of abomasal infusion of linoleic acid (LA) or an equal mixture of linoleic and conjugated linoleic acid (LCLA) for 24 h. Values are means ± pooled SEM for four cows. Asterisks denote significant differences (P < 0.05) due to treatments.

View larger version (13K): [in this window] [in a new window]   Fig 3. Concentration of conjugated linoleic acid (CLA) isomers (9 cis, 11 trans isomers plus 10 trans, 12 cis isomer) (A) and palmitic acid (B) in milk fat from Holstein cows at 12-h intervals after the initiation of abomasal infusion of linoleic acid (LA) or an equal mixture of linoleic and conjugated linoleic acid (LCLA) for 24 h. Values are means ± pooled SEM for four cows at each 12-h interval. Asterisks denote significant differences (P < 0.05) due to treatments.

Infusion procedures.  Fatty acids were infused via Tygon tubing (1.6 mm i.d., 0.8 mm wall; Fisher Scientific, Pittsburgh, PA) that passed through 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) that passed through the rumen cannula, rumen and omasum, and into 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 infusate at the start of infusion, and flow rate was set at 8.2 mL/h.

Digesta marker.  Cr-mordanted fecal fibers were used as a digesta marker to determine digestibility of dry matter, organic matter and crude protein. Cr was attached (Uden et al. 1980) to washed fecal fibers (4.3 g Cr/100 g fecal fibers) that were collected from cows fed only orchardgrass hay. A 15-g dose of Cr-mordanted fecal fibers was placed in the rumen at 0600 and 1800 h daily, starting 10 d before the first infusion.

Measurements and sampling.  Samples of the total mixed diet were collected during each infusion, dried to constant weight in a forced-air oven at 60°C, and stored at room temperature until analyzed. Duodenal digesta (200 mL) and fecal-grab samples were collected from each cow at 12 and 0 h before infusion, 12 and 24 h during infusion, and 36 and 48 h after infusion. Fecal samples were dried to constant weight at 60°C, and duodenal digesta was stored at 20°C. At the end of the experiment, digesta samples were thawed, ground with a homogenizer (Polytron PT 10/35, Brinkmann Instruments, Westbury, NY) and then lyophilized (Dura-Top freeze dryer, FTS sytems, Stone Ridge, NY).

Two 50-mL aliquots of milk were collected at 12 and 0 h before infusion, 12 and 24 h during infusion, and 36, 48, 60 and 72 h after infusion. One aliquot containing Bronopol (D & F Control Systems, San Ramon, CA) was stored at 4°C until analyzed for fat, protein, solids-not-fat and lactose by infrared analysis with a 4-channel spectrophotometer (Virginia Dairy Herd Improvement Association). The second aliquot was stored at 20°C until the end of the experiment, then thawed and centrifuged at 10,000 × g for 1 h to harvest milk fat for fatty acid analysis.

Blood samples (10 mL) were obtained from the coccygeal artery by venipuncture after each milking from 12 to 72 h. Blood was transferred to tubes containing 286 IU heparin in 100 µL of sterile saline then centrifuged at 3000 × g for 15 min for harvesting plasma. Plasma was stored at 20°C until lipid extraction and fatty acid analysis.

Sample analysis.  Lyophilized and oven-dried samples were ground through a 1 mm screen in a Cyclotec mill (Tecator 1093, Hoganas, Sweden) prior to total nitrogen and organic matter analysis (AOAC 1984). Aliquots of duodenal and fecal samples also were wet-ashed with HNO₃ and HClO₄, as described by Sandell (1950), to determine chromium content by atomic absorption spectrophotometry (Perkin Elmer 3300, Norwalk, CT). Chromium concentration was determined at 357.9 nm using a hollow cathode lamp (Starna Cells, Atascadero, CA) and acetylene/N₂O₂ as flame gases.

Lipids were extracted from plasma (2 mL) with chloroform/methanol (2:1, v/v) (Folch et al. 1957). Subsequently, blood plasma lipid fractions (free fatty acids, phospholipids, cholesterol esters and triglycerides) were isolated (Kaluzny et al. 1985) using Bond Elut aminopropyl disposable columns (500 mg) in a Vac Elut system (Analytichem International, Harbor City, CA). Fatty acids in feed, blood plasma, blood plasma lipid fractions and milk fat were transmethylated by in situ transterification (Park and Goins 1994). 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 were separated on a 30 m × 0.25 mm i.d. fused silica capillary column (SP-2380, Supelco, Bellafonte, PA). The injector temperature was maintained at 225°C and the detector temperature at 275°C. The initial column temperature was 205°C (held for 12 min) and was programmed to increase 2°C per min to a final temperature of 220°C (held for 2 min). Peak identification was based on relative retention times of a commercial standard (Nu-Check Prep, Elysian, MN).

Statistical analysis.  Data are reported as least squares means ± pooled SEM. Data for total plasma fatty acids, milk yield and composition, and individual fatty acids in milk fat were analyzed by repeated measures analysis of covariance in a single crossover design using the GLM procedure of SAS (SAS/STAT Version 6, 1988). Observations obtained at 12 and 0 h before infusion were averaged and served as preinfusion reference values (covariate) for comparison with observations obtained at 12, 24, 36, 48, 60 and 72 h. Duodenal digesta and feces were collected in the same sequence described above, but up to 48 h only. Data derived from analyses of feed, duodenal digesta, and feces were averaged over the 48 h following the start of fatty acid infusion into the abomasum, then subjected to analysis of covariance without repeated measures. Fatty acid distributions in blood plasma lipid fractions were determined only at 0 and 24 h after infusion, and resulting data also were analyzed without repeated measures. Treatment means, time by treatment interactions and covariates were designated as significantly different at P < 0.05. A regression of CLA in milk fat (g/100 g total fatty acids) at 36 h on CLA in plasma triglycerides (g/100 g total fatty acids) at 24 h was used to estimate the apparent transfer of CLA from plasma triglycerides into milk fat. In addition, milk fat percentages from 0 to 36 h were regressed on the corresponding CLA concentrations (g/100 g total fatty acids) in milk fat.

	RESULTS

Abstract Introduction Methods Results Discussion References

Intake and digestibility of basal diet.  Intakes of dry matter (22 ± 4 kg/d), organic matter (21 ± 4 kg/d), crude protein (3 ± 1 kg/d) and total fatty acids (559 ± 18 g/d) during the 48 h following start of infusion were similar for both treatments and did not differ from preinfusion values. Apparent digestibility of dry matter, organic matter and crude protein in the rumen (46 ± 1, 51 ± 6 and 45 ± 2%) and total tract (70 ± 3, 70 ± 3 and 68 ± 4%) did not differ from preinfusion values or due to treatment.

Plasma fatty acid concentration.  Concentration of total and most individual fatty acids in blood plasma during the 72 h following start of the 24-h infusion was similar for both treatments (Table 3). Infusion of LA, however, resulted in greater concentration (g/100 g total fatty acids) of linoleic acid at 12 and 36 h compared with LCLA infusion (Fig. 1A). Plasma CLA concentration increased from 0 to 0.57 g/100 g within 24 h, then decreased to 0.13 g/100 g by 72 h when LCLA was infused (Fig. 1B). Time by treatment interactions for plasma stearic and eicosatrienoic acids (Table 3) indicated concentrations of these fatty acids decreased in response to LA compared with LCLA. Infusion of LA also reduced arachidonic acid concentration compared with LCLA, resulting in an overall treatment effect and a time by treatment response which approached significance (P = 0.06).

Fatty acid distribution in plasma lipid fractions.  To determine the blood plasma lipid fractions in which infused CLA was distributed for transport to the mammary gland, free fatty acid, phospholipid, cholesterol ester and triglyceride fractions were isolated from samples obtained at 0 and 24 h (Table 4). Although cows differed (significant covariate effect) in concentration of total fatty acids in their free fatty acid, phospholipid and cholesterol ester fractions at 0 h, only total fatty acid content of the free fatty acid fraction increased at 24 h in response to LCLA compared with LA. The increase was associated with a greater stearic acid percentage. There were no differences in percentages of fatty acids in the phospholipid or cholesterol ester fractions due to treatment, but percentage of oleic acid in the triglyceride fraction at 24 h was lower in response to LCLA compared with LA.

The percentage of CLA in free fatty acid and cholesterol ester fractions at 24 h was similar for both treatments (Fig. 2). Infusion of LCLA, however, increased the proportion of CLA in phospholipids and triglycerides. The elevated percentages of CLA in phospholipids and triglycerides at 24 h corresponded with its peak concentration in total plasma lipids (Fig. 1B). Because the phospholipid fraction accounted for ~10 times more fatty acids than the triglyceride fraction, the majority of the supplemental CLA apparently was contained in phospholipids.

Milk production and composition.  Milk yield was not affected by treatments (Table 5). Concentration of fat was substantially reduced by infusion of LCLA from 12 to 48 h, and it remained lower by 72 h compared with LA. Lower fat concentration in response to LCLA reduced milk fat yield compared with LA. A moderate, but significant, reduction of protein and solids-not-fat concentrations in milk was observed when LCLA was infused compared with the LA infusion, whereas lactose concentration was similar for both infusions throughout the 72-h period. Yields of protein, lactose and solids-not-fat were not affected by treatment.

Fatty acid composition of milk fat.  Total fatty acid yield (Table 6) and yield of fatty acids with six to 16 carbons (data not shown) decreased from 12 to 48 h, then remained lower until 72 h when LCLA was infused compared with LA. Concentration of CLA (g/100 g total fatty acids) in milk fat (Fig. 3A) peaked at 36 h then remained elevated in milk from cows infused with LCLA. Percentages of palmitic acid, the primary fatty acid in milk fat, initially declined until 36 h in response to both infusates but remained lower at 48 to 72 h following LCLA compared with LA (Fig. 3B). When LA was infused, the greater concentration and yield (data not shown) of oleic, linoleic and linolenic acid at 12 and 36 h when LA was infused partially compensated for the decrease in palmitic acid concentration and yield, such that total fatty acid yield remained constant. In contrast, the observed reduction in milk fatty acid yield during 12 to 72 h in response to LCLA infusion was accounted for by the lower overall percentages and yields of palmitic and caproic acids, coupled with lower percentages (significant time by treatment interaction effect) and yields of lauric and myristic acids. The above responses indicated de novo synthesis of saturated fatty acids with 16 or fewer carbons was reduced by CLA in the mammary gland following the 24-h LCLA infusion, thus lowering fatty acid yield.

Stearic acid percentage (Fig. 4A) and yield (data not shown) increased for 36 h in response to LCLA but not LA. In contrast, an increase in the percentage of oleic acid (Fig. 4B) and yield during the first 36 h occurred in response to LA but not LCLA. The changes suggested CLA inhibited desaturation of stearic acid in the mammary gland. In addition, elongation and desaturation of linoleic acid may have been inhibited by CLA, as indicated by changes in eicosatrienoic acid (Table 6) and arachidonic acid (Fig. 4C) content of milk fat.

View larger version (14K): [in this window] [in a new window]   Fig 4. Concentration of stearic acid (A), oleic acid (B) and arachidonic acid (C) in milk fat from Holstein cows at 12-h intervals after the initiation of abomasal infusion of linoleic acid (LA) or an equal mixture of linoleic and conjugated linoleic acid (LCLA) for 24 h. Values are means ± pooled SEM for four cows at each 12-h interval. Asterisks denote significant differences (P < 0.05) due to treatments.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Mixtures of long-chain unsaturated fatty acids (438 g/d) or trans-vaccenic acid (from 624 to 750 g/d) depressed dry matter intake (DMI) when infused into the abomasum of lactating cows (Drackley et al. 1992, Gaynor et al. 1994, Romo et al. 1996). In the present study, infusion of only 200 g of primarily linoleic acid or CLA isomers for 24 h did not reduce DMI. In agreement with previous studies, digestibilities of dry matter, crude protein and organic matter in the rumen and total tract were not affected by abomasal infusion of unsaturated fatty acids (Christensen et al. 1994, Drackley et al. 1992).

Total fatty acid concentration in blood plasma did not differ due to treatment (Table 3), but linoleic acid content of plasma was greater following LA compared with LCLA infusion (Fig. 1A). Linoleic acid is preferentially incorporated into plasma phospholipids and cholesterol esters (Christie 1980), but extensive incorporation into triglycerides also can occur (Harrison et al. 1974). Our data indicate linoleic acid accounted for ~78% of cholesterol esters and 37% of phospholipids, and each of these fractions contained 47% of the total fatty acids in plasma.

The CLA in plasma total fatty acids were detectable only after LCLA was infused (Fig. 1B). However, we were able to detect CLA in all major plasma lipid fractions (Fig. 2) before and 24 h after the start of infusion of either treatment, suggesting that isolation of plasma lipid fractions is a more suitable method to determine CLA content of blood plasma. The free fatty acid fraction contained ~3% of total fatty acids in plasma, whereas the phospholipid fraction contained ~47% of total plasma fatty acids. However, absolute amount of CLA in the two fractions (0.01 and 0.02 g/L, respectively) was similar. The proportion of CLA in plasma triglycerides and phospholipids was increased ~10-fold and threefold, respectively, after 24 h of LCLA infusion, indicating CLA was readily available to the mammary gland via lipoprotein lipase activity. During passage of blood through the udder, it was estimated that 70% of blood plasma triglycerides were hydrolyzed and their fatty acids were available for milk fat synthesis (Glascock and Welch 1974). At high arterial concentrations, phospholipids also provided fatty acids for milk fat synthesis (Nielsen and Jakobsen 1994).

Trans-vaccenic acid is preferentially concentrated in the sn-1 position of plasma triglycerides (Christie and Moore 1971) where it can be rapidly hydrolyzed and extracted by tissues. Thompson and Christie (1990) determined that mammary extraction of trans-vaccenic acid from plasma triglycerides was ~74%. During LCLA infusion in the present study, there was a positive relationship between the percentage of plasma triglyceride CLA (TGCLA) at 24 h and peak CLA concentration in milk fat (MCLA) at 36 h (MCLA = 0.57 TGCLA + 0.83; r² = 0.70; P < 0.01). Thus, similar to trans-vaccenic acid, CLA bound to plasma triglycerides apparently was rapidly absorbed by mammary cells and secreted in milk.

Infusion of LA, but not LCLA, decreased the concentration of eicosatrienoic acid in total plasma fatty acids causing a time by treatment interaction effect. Arachidonic acid in total plasma fatty acids was also reduced by LA infusion, resulting in an overall treatment effect as well as a time by treatment interaction effect. Linoleic acid was previously reported to inhibit the activity of ⁶ desaturase in adipose tissue (Chang et al. 1992, Kinsella et al. 1990). The major site of linoleic acid desaturation and elongation in nonlactating ruminants is adipose tissue (St. John et al. 1991), and reduced activity of the desaturase in response to infusion of LA may have been responsible for the lower eicosatrienoic and arachidonic acid in blood plasma (Table 3). This effect, however, was not evident in the mammary gland as discussed below.

Plasma stearic acid concentration was lower in LA-infused cows compared with LCLA (Table 3), as indicated by the significant time by treatment interaction effect. Similarly, oleic acid in the triglyceride fraction (Table 4) was elevated in response to infusion of LA compared with LCLA. This response was consistent with a higher amount of oleic acid infused as part of the LA (54 g/24 h) treatment compared with LCLA (26 g/24 h) and its preferred esterification into blood plasma triglycerides (La Count et al. 1994). However, it is also possible that desaturation of stearic acid via ⁹ desaturase in the intestine (Chang et al. 1992) or adipose tissue may have contributed to the decrease in plasma stearic acid and the subsequent increase in oleic acid when LA was infused.

Compared with LA, infusion of LCLA decreased milk fat concentration from 35.6 g/L at 0 h to 23.4 g/L at 48 h, which was a 34% reduction in concentration and a 27% reduction in yield. Lower concentrations and yields of fatty acids with six to 16 carbons accounted for the reduction in fat yield when LCLA was infused, indicating depressed de novo synthesis in the mammary gland. A negative relationship between milk fat percentage (MFP) and CLA content of milk fat (MFP = 0.33 MCLA + 3.6; r² = 0.70; P < 0.01) substantiated our findings. Trans fatty acids derived from rumen biohydrogenation of unsaturated fatty acids have been reported to inhibit mammary secretion of milk fat. The work of Wonsil et al. (1994) demonstrated a negative linear relationship between trans-vaccenic acid flow to the duodenum and milk fat percentage. Their data, along with that of Gaynor et al. (1994) and Romo et al. (1996), also confirmed that milk fat depression was proportional to the availability of trans-vaccenic acid for absorption in the small intestine. The mechanism by which trans fatty acids depress milk fat secretion has not been elucidated. Recent work in our laboratory indicated that bovine mammary cell cultures incorporated trans-vaccenic acid and CLA from the medium in proportion to the amount present (Dawson and Herbein 1996). Fatty acid synthase activity and ⁹ stearoyl-CoA desaturase activity were inversely related with CLA uptake (Jayan et al. 1998).

The initial reaction in de novo synthesis of fatty acids in animal tissues is catalyzed by acetyl-CoA carboxylase (Wakil et al. 1983), and it appears to be a point at which control can be exerted. Emken et al. (1987) reported that linoleic acid and its trans isomer (⁹ trans, ¹² trans) reduced the activity of acetyl CoA carboxylase and other lipogenic enzymes in mouse liver. In contrast, incubating mouse adipocytes for 3 d with CLA did not reduce fatty acid synthase activity (Park et al. 1997), for which short-term regulation is not well documented (Wakil et al. 1983). Our data suggested that LA and LCLA may have decreased acetyl-CoA carboxylase activity in the mammary gland for 36 h. However, the extent of inhibition of de novo synthesis of fatty acids with six to 16 carbons was greater and continued until 72 h when LCLA was infused.

We observed an increase in milk stearic acid concentration and yield from 0 through 36 h when LCLA was infused. Associated with the increase was reduced oleic acid content. Similar responses were not reported when linoleic acid (Christensen et al. 1994, Drackley et al. 1992) or trans-vaccenic acid (Romo et al. 1996) where infused into the abomasum of lactating cows. Kinsella (1970) demonstrated that mammary cells isolated from the lactating bovine actively desaturated stearic acid to oleic acid via ⁹ stearoyl-CoA desaturase, esterified both fatty acids into triglycerides, and secreted the triglycerides into culture media. Based on the linear increase in stearic acid and the slight decrease in the proportion of oleic acid from 0 to 36 h, it appears that CLA may have reduced the activity of ⁹ stearoyl-CoA desaturase activity in the mammary gland. Infusion of LA apparently increased oleic acid in milk between 0 and 36 h. However, this increase could be ascribed to the higher concentration of oleic acid in the LA mixture (Table 2).

Belury and Kempa-Steczko (1997) demonstrated that mice fed increasing amounts of CLA in the diet (0 to 1.5% by weight) contained lower amounts of arachidonic acid, and greater CLA in hepatic neutral lipids. The authors suggested that CLA depressed ⁶ desaturase activity, thus leading to reduced arachidonic acid synthesis. Although -linolenic acid is a substrate for arachidonic acid synthesis, the main route of eicosatrienoic and arachidonic acid synthesis in animal tissues is desaturation of linoleic acid to -linolenic acid via ⁶ desaturase, elongation to eicosatrienoic acid, and a further desaturation to arachidonic acid via ⁵ desaturase (Gurr 1984). Our data indicated that LCLA infusion reduced the concentration of eicosatrienoic (Table 6) and arachidonic acid (Fig. 4C) in milk fat despite the enhanced availability of linoleic acid in the infusate. Infusion of LA was associated with an increase in the concentration of both fatty acids from 0 to 36 h, thus indicating that supplemental linoleic acid was actively desaturated when the supply of CLA remained at the basal level. Hermansen et al. (1995) showed that infusion (0-600 g/d) of primrose oil (72% linoleic acid) into the abomasum raised the proportions of eicosatrienoic and arachidonic acid in milk from Holstein cows. In contrast, feeding a high grain diet to lactating cows increased trans-vaccenic acid in milk fat globule membrane but did not affect arachidonic acid concentration (Palmquist and Schanbacher 1991). Similar results were observed in milk from sows fed partially hydrogenated fish oil (28% trans-vaccenic acid) (Pettersen and Opstvedt 1991). Thus, it appears that CLA, but not trans-vaccenic acid, may play an inhibitory role in the desaturation of stearic acid and polyunsaturated fatty acids by mammary gland ⁹, ⁶ and ⁵ desaturase enzymes.

Overall, the data reported herein suggested that increasing delivery of CLA to the small intestine of lactating cows for absorption is not expected to affect DMI or milk yield. Increased flow of CLA to the duodenum raised the level of this fatty acid in blood plasma triglycerides, which then could be rapidly taken up by the mammary gland for milk triglyceride synthesis. Absorption of CLA by the mammary gland, however, may be detrimental for the synthesis of fatty acids with 16 or fewer carbons, stearic acid desaturation, and eicosatrienoic and arachidonic acid synthesis, possibly due to direct inhibition of the enzymes involved.

	FOOTNOTES

Manuscript received 15 April 1998. Initial reviews completed 8 June 1998. Revision accepted 25 August 1998.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• Association of Official Analytical Chemists (1980) Official Methods of Analysis, 13th ed. AOAC, Washington, DC.
• Belury M. A. Conjugated dienoic linoleate. A polyunsaturated fatty acid with unique chemoprotective properties. Nutr. Rev. 1995; 53:83-89[Medline]
• Belury M. A., Kempa-Steczko A. Conjugated linoleic acid modulates hepatic lipid composition in mice. Lipids 1997; 32:199-204[Medline]
• Chang J.H.P., Lunt D. K., Smith S. B. Fatty acid composition and fatty acid elongase and stearoyl-CoA desaturase activities in tissues of steers fed high oleate sunflower seed. J. Nutr. 1992; 122:2074-2080
• Christensen R. A., Drackley J. K., La Count, D. W., Clark J. H. Infusion of four long-chain fatty acid mixtures into the abomasum of lactating dairy cows. J. Dairy Sci. 1994; 77:1052-1069[Abstract]
• Christie, W. W. (1980) The effect of diet and other factors on the lipid composition of ruminant tissues and milk. In: Lipid Metabolism in Ruminant Animals (Christie, W. W., ed.), pp. 193-226. Pergamon Press, England.
• Christie W. W., Moore J. H. Structures of triglycerides isolated from various sheep tissues. J. Sci. Food Agric. 1971; 22:120-124
• Dawson, S. E. & Herbein, J. H. (1996) Influence of exogenous unsaturated fatty acids on de novo synthesis of saturated fatty acids in mouse and bovine mammary cell cultures. Va. J. Sci. 47: 138 (abs.).
• Drackley J. K., Klusmeyer T. H., Trusk A. M., Clark J. H. Infusion of long-chain fatty acids varying in saturation and chain length into the abomasum of lactating dairy cows. J. Dairy Sci. 1992; 75:1517-1526[Abstract]
• Emken E. A., Abraham S., Lin C. Y. Metabolism of cis-12-octadecenoic acid and trans-9, trans-12-octadecadienoic acid and their influence on lipogenic enzyme activities in mouse liver. Biochim. Biophys. Acta 1987; 919:111-121[Medline]
• Folch J., Lees M., Stanley G.H.S. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957; 226:477-480
• Fujimoto K., Kimoto H., Shishikura M., Endo Y., Ogimoto K. Biohydrogenation of linoleic acid by anaerobic bacteria isolated from the rumen. Biosci. Biotechnol. Biochem. 1993; 57:1026-1031
• Gaynor P. J., Erdman R. A., Teter B. B., Sampugna J., Capuco A. V., Waldo D. R., Hamosh M. Milk fat yield and composition during abomasal infusion of cis or trans octadecenoates in Holstein cows. J. Dairy Sci. 1994; 77:157-165[Abstract]
• Glascock R. F., Welch V. A. Contribution of the fatty acids of three low density serum lipoproteins to bovine milk fat. J. Dairy Sci. 1974; 57:1364-1370
• Griinari, J. M., Bauman, D. E. & Jones, L. R. (1995) Low milk fat in New York Holstein herds. In: Proceedings of the Cornell Nutrition Conference, pp. 96-105.
• Griinari, J. M., Dwyer, D. A., McGuire, M. A. & Bauman, D. E. (1996) Partially hydrogenated fatty acids and milk fat depression. J. Dairy Sci. 79(suppl. 1): 177 (abs.).
• Gurr, M. I. (1984) The chemistry and biochemistry of plant fats and their nutritional importance. In: Fats in Animal Nutrition (Wiseman, J. ed.), pp. 3-22. Butterworths, London.
• Harrison F. A., Leat W. M. F., Forster A. Absorption of maize oil infused into the duodenum of the sheep. Proc. Nutr. Soc. 1974; 33:101A-102A[Medline]
• Hermansen J. E., Jonsbo F., Andersen J. O., Michaelsen K. F., Weisbjerg M. R. On the transfer of -linolenic acid into milk fat and its possible elongation to arachidonic acid by cows. Milchwissenschaft 1995; 50:3-5
• Ip C., Scimeca J. A., Thompson H. J. Conjugated linoleic acid : a powerful anticarcinogen from animal fat sources. Cancer 1994; 74:1051-1054
• Jayan, G. C., Herbein, J. H., Wong, E. A. & Keenan, T. W. (1998) Influence of unsaturated fatty acids on de novo fatty acid synthesis in mouse mammary epithelial cell cultures. FASEB J. 12: A553 (abs.).
• Kaluzny M. A., Duncan L. A., Merritt M. V., Epps D. E. Rapid separation of lipid classes in high yield and purity using bonded phase columns. J. Lipid Res. 1985; 26:135-140[Abstract]
• Kelly M. L., Berry J. R., Dwyer D. A., Griinari J. M., Chouinard P. Y., VanAmburg M. E., Bauman D. E. Dietary fatty acid sources affect conjugated linoleic acid concentrations in milk from lactating dairy cows. J. Nutr. 1998; 128:881-885[Abstract/Free Full Text]
• Kinsella J. E. Stearic acid metabolism by mammary cells. J. Dairy Sci. 1970; 53:1757-1765
• Kinsella J. E., Broughton K. S., Whelan J. W. Dietary unsaturated fatty acids: interactions and possible needs in relation to eicosanoid synthesis. J. Nutr. Biochem. 1990; 1:123-141[Medline]
• La Count, D. W., Drackley J. K., Laesch S. O., Clark J. H. Secretion of oleic acid in milk fat in response to abomasal infusions of canola or high-oleic sunflower fatty acids. J. Dairy Sci. 1994; 77:1372-1385[Abstract]
• McGuire, M. A., McGuire, M. K., Guy, M. A., Sanchez, W. K., Shultz, T. D., Harrison, L. Y., Bauman, D. E. & Griinari, J. M. (1996) Effect of dietary lipid concentration on content of conjugated linoleic acid (CLA) in milk from dairy cattle. J. Anim. Sci. 74(suppl. 1): 266 (abs.).
• Nielsen, M. O. & Jacobsen, K. (1994) Changes in mammary uptake of free fatty acids, triglyceride, cholesterol, and phospholipid in relation to milk synthesis during lactation in goats. Comp. Biochem. Physiol. 109A: 857-867.
• Palmquist D. L., Schanbacher F. L. Dietary fat composition influences fatty acid composition of milk fat globule membrane in lactating cows. Lipids 1991; 26:718-722[Medline]
• Park P. W., Goins R. E. In situ preparation of fatty acid methyl esters for analysis of fatty acid composition in foods. J. Food Sci. 1994; 59:1262-1266
• Park Y., Albright K. J., Liu W., Storkson J. M., Cook M. E., Pariza M. W. Effect of conjugated linoleic acid on body composition in mice. Lipids 1997; 32:853-858[Medline]
• Parodi P. W. Conjugated octadecadienoic acids of milk fat. J. Dairy Sci. 1977; 60:1550-1553[Abstract/Free Full Text]
• Pettersen J., Opstvedt J. Trans fatty acids. 4. Effects on fatty acid composition of colostrum and milk. Lipids 1991; 26:711-717[Medline]
• Romo G. A., Casper D. P., Erdman R. A., Teter B. B. Abomasal infusion of cis or trans fatty acid isomers and energy metabolism of lactating dairy cows. J. Dairy Sci. 1996; 79:2005-2015[Abstract]
• Sandell, E. B. (1950) Colorimetric Determinations of Trace Metals. Interscience, New York, NY.
• SAS Institute (1988) SAS User's Guide: Statistics, Version 6, 4th ed. Cary, NC.
• Stallings C. C., Kroll G., Kelley J. C., McGuilliard M. L. A computer ration evaluation program for heifers, dry cows, and lactating cows. J. Dairy Sci. 1985; 68:1015-1019[Abstract/Free Full Text]
• St. John L. C., Lunt D. K., Smith S. B. Fatty acid elongation and desaturation enzyme activities of bovine liver and subcutaneous adipose tissue microsomes. J. Anim. Sci. 1991; 69:1064-1073[Abstract]
• Thompson G. E., Christie W. W. Extraction of plasma triacylglycerols by the mammary gland of the lactating cow. J. Dairy Res. 1990; 58:251-255
• Uden P., Colucci P. E., Van Soest P. J. Investigation of chromium, cerium, and cobalt as markers in digesta. Rate of passage studies. J. Sci. Food Agric. 1980; 31:625-632[Medline]
• Wakil J., Stoops J. K., Joshi V. C. Fatty acid synthesis and its regulation. Ann. Rev. Biochem. 1983; 52:537-579[Medline]
• Wonsil B. J., Herbein J. H., Watkins B. A. Dietary and ruminally derived trans-18:1 fatty acids alter bovine milk lipids. J. Nutr. 1994; 124:556-565

0022-3166/98 $3.00 ©1998 American Society for Nutritional Sciences