Effect of CLA and other C18 unsaturated fatty acids on DGAT in bovine milk fat biosynthetic systems.

Production of dairy products with increased amounts of nutraceutic FA such as conjugated linoleic acid (CLA) represents a recent approach for dairy producers and processors to increase the value of their products. The effect of CLA and other FA on the expression of diacylglycerol acyltransferase-1 (DGAT-1) and DGAT-2, and DGAT activity were investigated in bovine mammary gland epithelial (MAC-T) cells. DGAT gene expression analyses were also conducted using bovine mammary gland tissue from dairy cows. In the studies with MAC-T cells, there were no significant effects of CLA isomers or other FA on DGAT1 expression, whereas all FA tested showed enhanced DGAT2 expression (P < 0.05 to P < 0.001), with alpha-linolenic acid (alpha-18:3) having the greatest effect. Additionally, DGAT2 expression was co-ordinated with expression of lysophosphatidic acid acyltransferase (LPAAT), an observation that was also apparent in mammary gland from lactating dairy cows. In contrast, treatment of MAC-T cells with trans-10, cis-12 18:2 or alpha-18:3 resulted in a significant (P < 0.05) decrease in overall DGAT enzyme activity, although the mechanisms resulting in these effects are unclear. Competition assays using microsomes from bovine mammary gland tissue and 1-[(14)C]oleoyl-CoA suggested that DGAT activity was more selective for oleoyl (cis-9 18:1)-CoA than cis-9, trans-11 18:2-, trans-10, cis-12 18:2- or cis-9, cis-12 18:2-CoA. Collectively, the results suggest the relationship between trans-10, cis-12 18:2 and reduced TAG production in bovine milk is not linked to the production of DGAT1 or DGAT2 transcripts, but probably involves effects of this CLA isomer at events beyond transcription, such as post-translational and/or enzyme activity effects.

Onset of lactation in the bovine mammary gland: gene expression profiling indicates a strong inhibition of gene expression in cell proliferation.

The mammary gland undergoes dramatic functional and metabolic changes during the transition from late pregnancy to lactation. To better understand the molecular events underlying these changes, we analyzed expression profiles of approximately 23,000 gene transcripts in bovine mammary tissue about day 5 before parturition and day 10 after parturition. At the cutoff criteria of the signed fold change >or=2 or

Effect of conjugated linoleic acid on bovine mammary cell growth, apoptosis and stearoyl Co-A desaturase gene expression.

The effects of the primary biologically active conjugated linoleic acid (CLA) isomers (cis-9, trans-11 and trans-10, cis-12; 15-150microM) on growth and survival of the bovine mammary cell-line, Mac-T, were evaluated using cell enumeration and TUNEL assay. Previous studies have shown that high concentrations of CLA induced severe milk fat depression and have had negative effects on milk yield and composition whereas the impact of lower doses has been a modest depression in milk fat percent. In this study, we show that increasing concentrations of both CLA isomers had negative impacts on cell growth, including reduced cell number at concentrations of 35microM and above (P<0.05) and a two-fold increase in induction of apoptosis in the mammary cells. Changes in cell morphology occurred with large vacuole-like structures in the cytoplasm, nuclear shrinkage and changes of nuclear shape to kidney shape. Insulin did not significantly affect apoptosis in CLA-treated cells. In addition, the effect of increased doses of CLA and the interaction of CLA and insulin on the bovine stearoyl Co-A desaturase (Scd) gene promoter was also analyzed. While a significant difference in the Scd promoter transcriptional activity was not observed in cells treated with different concentrations of CLA, insulin significantly enhanced Scd promoter activity in CLA-treated cells. Our in vitro data support the hypothesis that high levels of CLA may induce in vivo apoptosis in the mammary gland.

Characterization and regulation of the bovine stearoyl-CoA desaturase gene promoter.

The bovine stearoyl-CoA desaturase (Scd) gene plays an important role in the bovine mammary gland where substrates such as stearic and vaccenic acids are converted to oleic acid and conjugated linoleic acid (CLA), respectively. Up to 90% of the CLA in bovine milk is formed due to the action of this enzyme in the mammary gland. The areas of the bovine promoter of importance in regulating this key enzyme were examined and an area of 36bp in length was identified as having a critical role in transcriptional activation and is designated the Scd transcriptional enhancer element (STE). Electrophoretic mobility shift assay detected three binding complexes on this area in Mac-T cell nuclear extracts. Treatment of cells with CLA caused a significant reduction in transcriptional activity, with this effect being mediated through the STE region. The bovine Scd gene promoter was up-regulated by insulin and down-regulated by oleic acid.

Effects of feeding or abomasal infusion of canola oil in Holstein cows. 1. Nutrient digestion and milk composition.

We determined the effects of feeding canola oil or infusing it into the abomasum on rumen fermentation, nutrient digestibility, duodenal flows of fatty acids, and milk composition in Holstein cows. Five ruminally and duodenally cannulated Holstein cows in late lactation were used in a 3 x 5 incomplete Latin square design. Treatments were 1) CONTROL: basal diet (CON), 2) CONTROL+supplementation of canola oil at 1 kg/d in the feed (FED), and 3) CONTROL+abomasal infusion of canola oil at 1 kg/d (INF). Compared with CON, feed intake, ruminal fermentation characteristics, ruminal and total tract digestibilities of nutrients were not significantly affected by FED treatment but duodenal flows and milk concentrations of fatty acids (FA) such as trans-11 18:1 and cis-9 trans-11 18:2 (conjugated linoleic acid, CLA) were increased. In contrast to the effects of FED, INF reduced feed intake, total VFA production, intestinal flows of nutrients, FA digestibility and yields of milk and milk fat. Both FED and INF significantly reduced the proportions of saturated and medium-chain FA, and increased cis 18:1 in milk. Concentrations of 18:2n-6 and 18:3n-3 in milk were increased nearly 2-fold with INF relative to CON. Dietary or postruminal supplementation of canola oil to late-lactation cows reduced saturated FA and increased unsaturated C18 in milk but nutrient digestion was adversely affected with abomasal infusion of canola oil.

Effects of feeding or abomasal infusion of canola oil in Holstein cows. 2. Gene expression and plasma concentrations of cholecystokinin and leptin.

We determined the relative importance of cholecystokinin (CCK), leptin, and fatty acid concentrations in plasma in mediating the satiety effects of supplemental fat in lactating cows. Five ruminally and duodenally cannulated Holstein cows in late lactation were used in a 3 x 5 incomplete Latin square design with three treatments: 1) CONTROL: basal diet (CON), 2) CONTROL+supplementation of canola oil at 1 kg/d in the feed (FED) and 3) CONTROL+abomasal infusion of canola oil at 1 kg/d (INF). Relative to CON, feed intake was reduced by INF but not by FED. We provide evidence that both FED and INF treatments stimulated CCK gene expression in the duodenum and elevated plasma CCK concentrations. However, our results did not support a role for CCK in mediating satiety through an endocrine mechanism of action. We speculate that CCK might be acting either through paracrine and/or neurocrine routes to influence feed intake in cattle. Both FED and INF had no effect on the mRNA abundance of leptin, lipoprotein lipase, or acetyl-CoA carboxylase in adipose tissue. Plasma concentrations of leptin, insulin and IGF-I were not altered by FED or INF, indicating that these signals may not be involved in mediating short-term hypophagic effects of dietary fat. Plasma concentrations of 18:1n-9 and 18:2n-6 were significantly greater for INF than for FED or CON. We conclude that the hypophagic effects of supplemental fat in cattle depend on the amount of unsaturated fatty acids reaching the intestine and that this satiety effect is mediated through CCK, oleic acid and (or) linoleic acid, but leptin is not involved.

Response of plasma leptin concentration to jugular infusion of glucose or lipid is dependent on the stage of lactation of Holstein cows.

In this study we investigated the hormonal and metabolite responses to isoenergetic jugular infusions of glucose or lipid in early- and late-lactation Holstein cows. Six Holstein cows were used in a replicated Latin square design with jugular infusions of either 1) control (CON; saline), 2) glucose (GLU; 50% dextrose) or 3) lipid (LIP; 20% Intralipid). Treatments did not affect dry matter intake, with the exception of a hypophagic effect of LIP in late lactation. The GLU-induced hyperglycemia and hyperinsulinemia were greater in late-lactation than in early-lactation cows. The GLU treatment did not affect plasma leptin and insulin-like growth factor-1 (IGF-1) concentrations in early-lactation cows, but it increased them in late-lactation cows. The LIP treatment did not affect plasma leptin, insulin and IGF-1 concentrations in early-lactation cows, despite a marked LIP-induced increase in plasma nonesterified fatty acid and beta-hydroxybutyrate concentrations and a reduction in growth hormone (GH) concentration. Compared with the delayed leptin response to GLU, the stimulatory effect of LIP on leptin secretion in late-lactation cows was relatively rapid and occurred in the absence of any significant changes in plasma insulin, IGF-1 or GH. We propose that insulin-mediated glucose metabolism may be involved in the stimulatory effects of glucose on leptin secretion in late-lactation animals but that the stimulatory effects of lipid are independent of insulin or IGF-1. In early-lactation animals a strong inhibitory effect of GH on leptin expression and release, in addition to low adipose reserves and/or energy balance, might override any short-term stimulatory effect of glucose or lipid on leptin secretion.

Addition of protected and unprotected fish oil to diets for dairy cows. I. Effects on the yield, composition and taste of milk.

Thirty Holstein cows in mid-lactation (158+/-20 DIM) were given a total mixed ration based on grass silage, maize silage and rolled barley. After a preliminary period of 1 week, this diet was supplemented with nothing (control), unprotected fish oil (3.7% of dry matter, DM), or two levels of glutaraldehyde-protected microcapsules of fish oil (1.5% and 3.0% of DM, respectively). Unprotected and protected supplements contained, respectively, 74% and 58% of DM as lipids. Cows given the unprotected supplement reduced their feed intake by > 25%. Consequently, these cows lost body weight and produced less milk. DM intake, body weight, and milk yield were unaffected by protected fish oil. Fish oil reduced both milk fat and protein percentages, and decreased the proportion of short-chain fatty acids, stearic, and oleic acids in milk fat. Milk trans C18:1 fatty acids increased in cows given both unprotected and protected fish oil. Milk fat content of very-long-chain n3 polyunsaturated fatty acids, including C20:5 and C22:6, increased with fish oil in the diet. Accordingly, the peroxide index increased and a taste panel was able to detect unusual taste in milk from cows consuming the higher level of protected fish oil and disliked the milk from cows given unprotected fish oil. In conclusion, when lactating cows consumed fish oil, milk concentration of long-chain n3 fatty acids increased and mammary de novo synthesis of fatty acids decreased, but milk yield and milk protein content were reduced, and the milk was more susceptible to oxidation and its taste was adversely affected.

Addition of fish oil to diets for dairy cows. II. Effects on milk fat and gene expression of mammary lipogenic enzymes.

Sixteen Holstein cows in mid-lactation were used to determine whether alterations of mammary fatty acid metabolism are responsible for the milk fat depression associated with consumption of fish oil. Cows were given a total mixed ration with no added fish oil (control), unprotected fish oil (3.7 % of dry matter), or glutaraldehyde-protected microcapsules of fish oil (1.5% or 3.0% of dry matter) for 4 weeks. Milk samples were taken once a week and a mammary biopsy was taken from a rear quarter at the end of the treatment period. Milk fat content was lower in cows given unprotected fish oil (26.0 g/kg), 1.5% protected fish oil (24.6 g/kg) and 3% protected fish oil (20.4 g/kg) than in cows fed the control diet (36.0 g/kg). This was mainly due to a decrease in the synthesis of short-chain fatty acids. Consumption of protected fish oil decreased the abundance of lipogenic enzymes mRNA in the mammary gland. Acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase mRNAs for cows given 3% protected fish oil averaged only 30%, 25% and 25% of control values, respectively. Dietary addition of unprotected fish oil slightly decreased mRNA abundance of these enzymes but markedly reduced the amount of lipoprotein lipase mRNA. Milk fat content was significantly correlated with gene expression of acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase but not lipoprotein lipase. These results suggest that fish oil reduces milk fat percentage by inhibiting gene expression of mammary lipogenic enzymes.