Experiments were conducted to investigate: 1) production of trans-18:1 and cis/trans-18:2 isomers due to input of forage and corn grain in continuous culture fermenters, 2) concentrations of trans-18:1 and cis/trans-18:2 isomers in blood and milk fat of grazing cows fed a grain supplement containing solvent- or mechanically-extracted soybean meal, 3) plasma and milk fatty acid profiles of lactating cows in response to a conjugated linoleic acid (CLA) mixture infused into the rumen, and 4) effects of cis9,trans11-18:2 (9/11CLA) or trans10,cis12-18:2 (10/12CLA) on de novo synthesis and desaturation of milk fatty acids in lactating cows fed unsaturated oils. In the first study, rumen fermenters were fed (50 g DM/d) orchardgrass or red clover with 0, 8, or 16 g/d corn replacing equal portions of forage DM. Outflow of trans11,cis15-18:2 (11/15LA) in effluents was greater when DM input was clover or grass only. With each increment of corn, output of 9/11CLA increased until it was 205% greater compared with forage alone. Trans11-18:1 (TVA), an intermediate in 18:2n6 (LA) and 18:3n3 (LN) hydrogenation, output increased by 28% with corn addition. Outputs of trans10-18:1 and 10/12CLA nearly doubled as corn increment increased. In the second experiment, Holstein cows grazing mixed clover-grass pastures were fed a grain supplement (6.7 kg/d) containing 1.7 kg solvent-extracted soybean meal (SES, 15 mg LA/g of DM), 1.9 kg mechanically-extracted soybean meal (MES, 24 mg LA/g of DM), or 1.9 kg MES plus 30 g of liquid methionine hydroxy analog (MESM). Cows fed MES or MESM had greater concentrations of LA, TVA, 9/11CLA, and 11/15LA in blood compared with cows fed SES. Daily yields of 18:0 (SA), LA, LN, TVA, and 9/11CLA in milk fat also were greater for cows fed MES or MESM compared with SES. In experiment 3, four Holstein cows were used in a 4 x 4 Latin square to determine plasma and milk fatty acid profiles during infusion of a CLA mixture at 0, 45, 90, or 180 g/d for 48 h into the rumen. Relative to the control, infusion of 180 g CLA/d decreased milk fat percentage and yield. Lower milk fat yield resulted from depressed concentrations of saturated 6:0 to 16:0 medium-chain fatty acids (MCFA). Concentrations of TVA, trans10-18:1, and 10/12CLA in blood plasma, and yields in milk fat increased in response to each dose of CLA. Stearic acid yield also increased as dose of CLA increased. Yield of cis9-18:1 (OA) in milk fat, however, was lower at 180 g CLA/d. In experiment 4, four cows were fed high-oleic (HO) or high-linoleic (HL) (2.5% of DM) oil for 11 d prior to abomasal infusion (15 g/d) of 9/11CLA or 10/12CLA for 48 h (2 x 2 factorial). Milk fat percentage and yield decreased 25% due to infusion of 10/12CLA compared with 9/11CLA, regardless of diet. Lower fat yields resulted from lower MCFA concentrations and yields. Regardless of diet, concentration (but not yield) of SA increased 40% when 10/12CLA was infused compared with 9/11CLA. Concentrations and yields of OA, 9/11CLA, and 20:4n6 also were reduced by infusing 10/12CLA compared with 9/11CLA regardless of diet. Thus, in addition to inhibiting de novo fatty acid synthesis, 10/12CLA appeared to inhibit desaturation via delta-6 and delta-9 desaturases. Significant implications from the above studies include: 1) 11/15LA and TVA are the primary intermediates flowing out of the rumen during hydrogenation of pasture lipids, 2) replacing forage DM with starch, OA, and LA increases synthesis of trans10-18:1 and 10/12CLA in the rumen, 3) desaturation of TVA produced in the rumen provides an alternate source for 9/11CLA in milk fat, and 4) 10/12CLA decreases de novo synthesis and desaturation of milk fatty acids. / Ph. D.

Studies were conducted to investigate: 1) desaturation of dietary trans-vaccenic acid (TVA, trans11-18:1) to the cis9,trans11-18:2 isomer of conjugated linoleic acid (9/11CLA), 2) effects of two conjugated linoleic acid isomers [9/11CLA or trans10,cis12-18:2 (10/12CLA)] and TVA on enzyme activities and mRNA abundance for lipogenic enzymes, and 3) regulation of stearoyl-CoA desaturase (SCD) gene transcription. In the first study, lactating mice were fed 3% linoleic acid (LA), or 2% LA plus 1% stearic acid (SA), 1% TVA, or 1% CLA mixture. Dietary TVA enriched the 9/11CLA content of carcass, liver, and mammary tissue of lactating mice. A similar enhancement of 9/11CLA also was observed in liver, but not carcass, of suckling pups nursing TVA-fed dams. The CLA mixture decreased mammary acetyl-CoA carboxylase (ACC) activity compared with other treatments. However, total fatty acid content of mammary tissue was reduced only when compared with TVA. In the second experiment, lactating mice were fed 3% canola oil (OA), or 2% OA plus 1% SA, 1% TVA, 1% 9/11CLA, or 1% 10/12CLA. Dietary TVA, 9/11CLA, and 10/12CLA decreased mRNA abundance for ACC and fatty acid synthase (FAS) in mammary tissue, suggesting each had the potential to reduce de novo fatty acid synthesis. However, only the CLA isomers decreased ACC activity in mammary tissue and concentration of medium-chain fatty acids (MCFA = 12:0+14:0+16:0) in milk fat. The 10/12CLA isomer caused greater reductions in MCFA and milk fat percentage than the 9/11CLA, indicating that 10/12CLA is the primary CLA isomer affecting lipid metabolism in the mammary gland. Dietary TVA, 9/11CLA, or 10/12CLA decreased SCD enzyme activity and mRNA abundance in mammary tissue. In study 3, mouse (COMMA-D/MME) and bovine (Mac-T) mammary epithelial cells were transfected with the putative promoter (600 bp) of SCD gene. The 9/11CLA reduced SCD gene transcription in mouse cells, but not bovine cells. Transcription, however, was reduced in both cell lines by 10/12CLA, linoleic acid, and linolenic acid. Thus, reduced SCD transcription in response to the CLA isomers in mouse mammary cells in vitro may provide an explanation for reduced SCD enzyme activity and mRNA abundance in mammary tissue when lactating mice were fed either of the CLA isomers. In contrast, stearic acid, oleic acid, and TVA did not affect SCD transcription. Although TVA did not reduce SCD transcription in mouse mammary cells in vitro, it did reduce SCD enzyme activity and mRNA abundance in mammary tissue when fed to lactating mice. The results suggested TVA may influence SCD mRNA processing or stability in the nucleus after transcription. Despite the reduction in SCD mRNA and enzyme activity, however, substantial quantities of TVA were desaturated to the 9/11CLA isomer when TVA was fed to lactating mice in the first two studies. Thus, dietary TVA provides an alternate supply of the anticarcinogenic 9/11CLA isomer in tissues. / Ph. D.

The objective of the study was to examine the effects of trans-9-octadecenoic acid (elaidic) and trans-II-octadecenoic acid (trans-vaccenic) on uptake and alteration of exogenous fatty acids by mouse mammary epithelial cells. Cells from a subclone of the COMMA-D cell line were plated on uncoated plastic petri dishes and grown to confluence. Supplemental fatty acids bound to bovine serum albumin were added to the medium applied to the confluent cell cultures. Treatments included 200 JlM octadecanoic acid (CI8:0) as a control and 100 JlM CI8:0 with one of the following JlM ratios of cis-octadecenoic acid (cis-CI8:I) to elaidic or trans-vaccenic: 100:0, 75:25, 50:50, 0:100. In addition, all treatments were conducted with or without lactogenic hormone supplementation. The cellular protein to DNA ratio and total amount of fatty acids per mg protein were decreased (P < .05) by addition of lactogenic hormones. In treatments without hormone supplementation, however, the total amount of cellular fatty acids per mg protein was decreased (P < .05) by addition of either trans-CI8:I isomer. Results indicated a significant (P < .05) relationship between the concentration oftrans-C18:1 in the media and uptake of trans-C 18: 1 isomers, and retroconversion of trans-C 18: 1 to trans-C 16: 1. The slopes of the lines for cellular C16:0, cis-C16:1, and cis-CI8:1 were less (P < .05) than zero as concentration oftrans-CI8:1 in the media increased. However, trans-CI8:1 isomers did not influence the proportion of polar and nonpolar lipids synthesized by the cells. It appears that trans fatty acids may depress milk fat output by decreasing de novo fatty acid synthesis and cis-C 18: I content. / Master of Science

The effect of exogenous unsaturated fatty acids on cellular fatty acid biosynthesis in mammary cells was examined. Under normal situations, even though the diet of a dairy cow contains considerable amounts of unsaturated fatty acids, viz. oleic acid (18:1) and linoleic acid (18:2), the major 18-carbon fatty acid that enters the circulation post-ruminally for delivery to the mammary gland is saturated fatty acid, viz. stearic acid (18:0). This is due to extensive ruminal biohydrogenation of unsaturated fatty acids. Studies have indicated that saturated fatty acids such as 18:0 are enhancers and that certain unsaturated fatty acids are inhibitors of de novo fatty acid synthesis in tissues such as the liver and adipose tissue. The present study investigated the effect of cis and trans isomers of 18:1 and 18:2 on de novo fatty acid synthesis and desaturation in mouse and bovine mammary epithelial cell cultures, and compared it with the effect caused by 18:0. In the first experiment 12.5, 25, 50 or 100 micromoles stearic acid (SA), oleic acid (OA), elaidic acid (EA), trans-vaccenic acid (TVA), linoleic acid (LA) or conjugated linoleic acid (CLA) were supplemented in the media of mouse mammary epithelial (MME) cells that were grown to confluence in Dulbecco's modified Eagle's medium (DMEM). As indicated by cellular palmitic acid (16:0) content and fatty acid synthetase (FAS) activity, when compared with SA all unsaturated fatty acid treatments inhibited de novo fatty acid synthesis in MME cells. In addition, OA at all concentrations and LA and CLA at 50 and 100 micromoles inhibited cellular stearoyl-CoA desaturase (SCD) activity and mRNA abundance. However, EA and TVA, when compared with SA, enhanced SCD activity and mRNA abundance at 12.5 and 25 micromoles. In the second experiment 25, 50 or 100 micromoles SA, OA, TVA, LA or CLA were supplemented in the media of bovine mammary epithelial cells that were grown to confluence in DMEM. As indicated by cellular 16:0 content, acetyl-CoA carboxylase (ACC) activity and FAS activity, treatment with the unsaturated fatty acids inhibited de novo fatty acid synthesis at all concentrations, when compared with SA. Unsaturated fatty acid treatments also reduced the abundance of ACC and FAS mRNA in the cells. When compared with SA at all treatment-concentrations, OA and LA inhibited whereas TVA and CLA enhanced cellular SCD activity and mRNA abundance in the bovine cells. In both cell types, CLA and TVA appeared to be the most potent inhibitors of saturated fatty acid biosynthesis. / Ph. D.