Based on the potential benefits of cis-9, trans-11 conjugated linoleic acid (CLA) for human health, there is a need to develop effective strategies for enhancing milk fat CLA concentrations. Levels ...of cis-9, trans-11 CLA in milk can be increased by supplements of fish oil (FO) and sunflower oil (SO), but there is considerable variation in the response. Part of this variance may reflect time-dependent ruminal adaptations to high levels of lipid in the diet, which lead to alterations in the formation of specific biohydrogenation intermediates. To test this hypothesis, 16 late lactation Holstein-British Friesian cows were used in a repeated measures randomized block design to examine milk fatty acid composition responses to FO and SO in the diet over a 28-d period. Cows were allocated at random to corn silage-based rations (8 per treatment) containing 0 (control) or 45g of oil supplement/kg of dry matter consisting (1:2; wt/wt) of FO and SO (FSO), and milk composition was determined on alternate days from d 1. Compared with the control, the FSO diet decreased mean dry matter intake (21.1 vs. 17.9kg/d), milk fat (47.7 vs. 32.6g/kg), and protein content (36.1 vs. 33.3g/kg), but had no effect on milk yield (27.1 vs. 26.4kg/d). Reductions in milk fat content relative to the FSO diet were associated with increases in milk trans-10 18:1, trans-10, cis-12 CLA, and trans-9, cis-11 CLA concentrations (r2=0.74, 0.57, and 0.80, respectively). Compared with the control, the FSO diet reduced milk 4:0 to 18:0 and cis 18:1 content and increased trans 18:1, trans 18:2, cis-9, trans-11 CLA, 20:5 n-3, and 22:6 n-3 concentrations. The FSO diet caused a rapid elevation in milk cis-9, trans-11 CLA content, reaching a maximum of 5.37g/100g of fatty acids on d 5, but these increases were transient, declining to 2.35g/100g of fatty acids by d 15. They remained relatively constant thereafter. Even though concentrations of trans-11 18:1 followed the same pattern of temporal changes as cis-9, trans-11 CLA, the total trans 18:1 content of FSO milk was unchanged because of the concomitant increases in the concentration of other isomers (Δ4–10 and Δ12–15), predominantely trans-10 18:1. In conclusion, supplementing diets with FSO enhances milk fat cis-9, trans-11 CLA content, but the high level of enrichment declines because of changes in ruminal biohydrogenation that result in trans-10 replacing trans-11 as the major 18:1 biohydrogenation intermediate formed in the rumen.
Conjugated linoleic acid (CLA) refers to a mixture of conjugated octadecadienoic acids of predominantly ruminant origin. The main isomer in bovine milk fat is the cis-9, trans-11 CLA. Interest in CLA ...increased after the discovery of its health-promoting properties, including potent anticarcinogenic activity. Two experiments were conducted to evaluate dietary strategies aimed at increasing the concentration of CLA in bovine milk fat. Both experiments were organized as a randomized complete block design with a repeated measures treatment structure. In Experiment 1, 28 Holstein cows received either a control diet or one of 3 treatments for a period of 2 wk. The control diet consisted of 60% forage (barley silage, alfalfa silage, and alfalfa hay) and 40% concentrate on a dry matter (DM) basis, fed as a total mixed ration (TMR). The concentrate was partially replaced in the treatment groups with 24ppm of monensin (MON), 6% of DM safflower oil (SAFF), or 6% of DM safflower oil plus 24ppm of monensin (SAFF/M). Average cis-9, trans-11 CLA levels in milk fat after 2 wk of feeding were 0.45, 0.52, 3.36, and 5.15% of total fatty acids for control, MON, SAFF, and SAFF/M, respectively. In Experiment 2, 62 Holstein cows received either a control diet or one of 5 treatment diets for a period of 9 wk. The control diet consisted of 60% forage (barley silage, alfalfa silage, and alfalfa hay) and 40% concentrate on a DM basis, fed as a TMR. The concentrate was partially replaced in the treatment groups with 6% of DM safflower oil (SAFF), 6% of DM safflower oil plus 150 IU of vitamin E/kg of DM (SAFF/E), 6% of DM safflower oil plus 24ppm of monensin (SAFF/M), 6% of DM safflower oil plus 24ppm of monensin plus 150 IU of vitamin E/kg of DM (SAFF/ME), or 6% of DM flaxseed oil plus 150 IU of vitamin E/kg of DM (FLAX/E). Average cis-9, trans-11 CLA levels during the treatment period were 0.68, 4.12, 3.48, 4.55, 4.75, and 2.80% of total fatty acids for control, SAFF, SAFF/E, SAFF/M, SAFF/ME, and FLAX/E, respectively. The combination of safflower oil with monensin was particularly effective at increasing milk fat CLA. The addition of vitamin E to the diet partially prevented the depression in milk fat associated with oilseed feeding, but had no significant effect on the concentration of CLA in milk.
The potential of dietary fish oil (FO) supplements to increase milk 20:5n-3 and 22:6n-3 concentrations and the associated effects on milk fatty acid (FA) composition, intake, and milk production were ...examined. Four multiparous lactating cows offered a grass silage-based diet (forage:concentrate ratio 58:42, on a dry matter basis) supplemented with 0, 75, 150, or 300g of FO/d (FO0, FO75, FO150, and FO300, respectively) were used in a 4×4 Latin square with 28-d experimental periods. Milk FA composition was analyzed by complementary silver-ion thin-layer chromatography, gas chromatography-mass spectrometry, and silver-ion HPLC. Supplements of FO decreased linearly dry matter intake, yields of energy-corrected milk, milk fat and protein, and milk fat content. Compared with FO0, milk fat content and yield were decreased by 30.1 and 40.6%, respectively, on the FO300 treatment. Supplements of FO linearly increased milk 20:5n-3 and 22:6n-3 concentrations from 0.07 to 0.18 and 0.03 to 0.10g/100g of FA, respectively. Enrichment of 20:5n-3 and 22:6n-3 was accompanied by decreases in 4- to 18-carbon saturated FA and increases in total conjugated linoleic acid (CLA), trans FA, and polyunsaturated FA concentrations. Fish oil elevated milk fat cis-9,trans-11 CLA content in a quadratic manner, reaching a maximum on FO150 (from 0.61 to 2.15g/100g of FA), whereas further amounts of FO increased trans-10 18:1 with no change in trans-11 18:1 concentration. Supplements of FO also resulted in a dose-dependent appearance of 37 unique 20- and 22-carbon intermediates in milk fat. Concentrations of 16-, 18-, 20-, and 22-carbon trans FA were all increased by FO, with enrichment of trans 18:1 and trans 18:2 being quantitatively the most important. Decreases in milk fat yield to FO were not related to changes in milk trans-10,cis-12 CLA concentration or estimated milk fat melting point. Partial least square regression analysis indicated that FO-induced milk fat depression was associated with changes in the concentrations of multiple FA in milk. Even though a direct cause and effect could not be established, a decrease in 18:0 supply in combination with increased mammary uptake of cis-11 18:1, trans-10 18:1, and trans 20- and 22-carbon FA may contribute. In conclusion, dietary FO supplements enrich 20:5n-3 and 22:6n-3 in milk, but also elevate mono- and polyenoic trans FA concentrations, and in high amounts alter the distribution of individual trans FA isomers.
Diets causing milk fat depression (MFD) are known to alter ruminal lipid metabolism leading to the formation of specific biohydrogenation intermediates that exert antilipogenic effects. Several ...isomers of conjugated linoleic acid (CLA), namely trans-10, cis-12 CLA, cis-10, trans-12 CLA, and trans-9, cis-11 CLA, inhibit mammary lipogenesis in the lactating cow, but ruminal outflow of these biohydrogenation intermediates does not account entirely for the reductions in milk fat synthesis during diet-induced MFD. Milk fat trans-10 18:1 concentrations are consistently increased on diets that cause MFD, suggesting a possible role in the regulation of milk fat secretion. Three rumen-fistulated cows in mid lactation were used in a 3 x 3 Latin square to evaluate the effects of a mixture of 18:1 fatty acid methyl esters (FAME) on milk fat synthesis. Experimental treatments consisted of abomasal infusions of ethanol (control), 6 g/d of trans-10, cis-12 CLA (positive control; CLA), or 247 g/d of a mixture of 18:1 FAME containing (% fatty acids) cis-9 (9.45), cis-12 (3.35), trans-10 (37.3), trans-11 (37.4), and trans-12 (2.66) as major isomers (T181 treatment). Administration of the T181 treatment supplied 92.1 g/d of trans-10 18:1. Infusions were conducted over a 5-d period with a 9-d interval between treatments. Treatments had no effect on dry matter intake, milk yield, or milk protein. Relative to the control, abomasal infusion of T181 and trans-10, cis-12 CLA treatments reduced milk fat secretion by 19.5 and 41.5%, respectively. Even though a direct cause and effect on mammary lipogenesis could not be established, comparisons with published data and considerations of the relative abundance of constituent FAME in treatment T181 implicated trans-10 18:1 as the isomer responsible. In conclusion, current data suggest that trans-10 18:1 potentially exerts antilipogenic effects and may contribute to the reduction in milk fat synthesis during diet-induced MFD in the lactating cow.
Certain diets cause a marked reduction in milk fat production in ruminants. Commonly referred to as milk fat depression (MFD), the mechanism involves an interrelationship between rumen microbial ...processes and tissue metabolism. Numerous theories to explain this interrelationship have been proposed and investigations offer little support for theories that are based on a limitation in the supply of lipogenic precursors. Rather, the basis involves alterations in rumen biohydrogenation of dietary polyunsaturated fatty acids and a specific inhibition of mammary synthesis of milk fat. The biohydrogenation theory proposes that under certain dietary conditions, typical pathways of rumen biohydrogenation are altered to produce unique fatty acid intermediates that inhibit milk fat synthesis. Trans-10, cis-12 conjugated linoleic acid (CLA) has been identified as one example that is correlated with the reduction in milk fat. Investigations with pure isomers have shown that trans-10, cis-12 CLA is a potent inhibitor of milk fat synthesis, and similar to diet-induced MFD, the mechanism involves a coordinated reduction in mRNA abundance for key enzymes involved in the biochemical pathways of fat synthesis. A more complete identification of these naturally produced inhibitors of fat synthesis and delineation of cellular mechanisms may offer broader opportunities for application and understanding of the regulation of lipid metabolism.
Under certain dietary situations, rumen biohydrogenation results in the production of unique fatty acids that inhibit milk fat synthesis. The first of these to be identified was trans-10, cis-12 ...conjugated linoleic acid (CLA), but others are postulated to contribute to diet-induced milk fat depression (MFD). Our objective was to examine the potential role of trans-9, cis-11 CLA in the regulation of milk fat. In a preliminary study, we used gas-liquid and high-performance liquid chromatography techniques to examine milk fat samples from a diet-induced MFD study and found that an increase in trans-9, cis-11 CLA corresponded to the decrease in milk fat yield. We investigated this further using a CLA enrichment of 9, 11 isomers to examine the biological effect of trans-9, cis-11 CLA on milk fat synthesis. Four rumen-fistulated Holstein cows were randomly assigned in a 4 x 4 Latin square experiment involving 5-d treatment periods and abomasal infusion of 1) ethanol (control), 2) a 9, 11 CLA mix (containing 32% trans-9, cis-11, 29% cis-9, trans-11, and 17% trans-9, trans-11), 3) a trans-9, trans-11 CLA supplement, and 4) a trans-10, cis-12 CLA supplement (positive control). The trans-9, trans-11 CLA and trans-10, cis-12 CLA supplements were of high purity (>90%), and all supplements were infused at a rate to provide 5 g/d of the CLA isomer of interest. Milk yield and dry matter intake did not differ among treatments. Compared with the control treatment, milk fat yield was reduced by 15% for the 9, 11 CLA mixture and by 27% for the trans-10, cis-12 CLA treatment. We also found that trans-9, trans-11 CLA had no effect on milk fat yield, and previous research has shown that milk fat yield is unaltered when cows are infused with cis-9, trans-11 CLA. When all treatments were considered, results suggested that trans-9, cis-11 was the CLA isomer in the 9, 11 CLA mix responsible for the reduction in milk fat synthesis, although the magnitude was less than that observed for trans-10, cis-12 CLA. Interestingly, trans-9, trans-11 CLA altered the milk fat desaturase index, further demonstrating that alterations in desaturase can occur independently of effects on milk fat synthesis. Overall, our investigations identified that an increase in milk fat content of trans-9, cis-11 CLA was associated with diet-induced MFD and provided evidence of a role for this isomer in MFD based on the 15% reduction in milk fat yield with abomasal infusion of a CLA enrichment that supplied 5 g/d of trans-9, cis-11 CLA.
Based on the potential benefits of cis-9, trans-11-conjugated linoleic acid (CLA) for human health there is interest in developing sustainable nutritional strategies for enhancing the concentration ...of this fatty acid in ruminant-derived foods. Most evidence to date suggests that endogenous synthesis is the major source of cis-9, trans-11 in milk fat and ruminal outflow is limited and largely independent of dietary 18 : 2n-6 supply. Four lactating cows fitted with a rumen cannula were used in a 4 x 4 Latin square with 14 d experimental periods to examine the effects of sunflower-seed oil (SFO) as a source of 18 : 2n-6 on ruminal lipid metabolism. Cows were offered grass silage-based diets supplemented with 0, 250, 500 or 750 g SFO/d. Supplements of SFO had no effect on DM intake, milk fat or protein secretion, but increased linearly (P < 0·01) milk yield and milk lactose output and shifted (P < 0·001) rumen fermentation towards propionate at the expense of acetate. SFO supplements increased linearly (P < 0·05) the flow of 18 : 0, 18 : 1, 18 : 2n-6 and total CLA at the omasum and enhanced ruminal cis-9-18 : 1, 18 : 2n-6 and 18 : 3n-3 metabolism. Flows of all-trans- (delta 4-16) and cis- (delta 9-16) 18 : 1 isomers were elevated, while increases in ruminal CLA outflow were confined to trans-8, trans-10 and geometric 9,11 and 10,12 isomers. It is concluded that supplementing grass silage-based diets with plant oils rich in 18 : 2n-6 enhances ruminal outflow of trans-11-18 : 1 and cis-9, trans-11-CLA in lactating cows.
We examined the role of trans-octadecenoic acids in milk fat depression when low fiber diets were fed. The study consisted of four experimental periods with a 2 x 2 factorial arrangement of ...treatments to test the effects of dietary fat (saturated vs. unsaturated) and rumen fermentation (high fiber diets vs. low fiber diets) on milk fat depression. Dietary fiber concentration and type of fat had significant effects on milk fat. Effects were most pronounced when unsaturated fat was added to the low fiber diet. When the low fiber diet plus unsaturated fat was fed, milk fat percentage and yield were decreased by 30 and 35%, respectively, compared with the percentage and yield when the high fiber diet plus saturated fat was fed. Alterations in rumen fermentation caused by differences in dietary fiber concentrations had little effect on the amount of trans-octadecenoic acids in milk fat, and the total amount did not correlate with changes in milk fat percentage. Further examination of the isomeric profile of trans-octadecenoic acid revealed substantial differences among the dietary treatments. Although the addition of unsaturated fat resulted in marked increases in the milk fat content of trans-11-octadecenoic acid, regardless of dietary fiber concentration, the low fiber diet plus unsaturated fat increased the content of trans-10-octadecenoic acid. This combination was also associated with a significant decrease in milk fat content and yield. When the low fiber diets were fed, circulating insulin concentrations were elevated, regardless of the type of fat supplement. However, marked milk fat depression occurred only when the low fiber diet was supplemented with unsaturated fat
Dietary unsaturated fatty acids are extensively hydrogenated in the rumen, resulting in the formation of numerous intermediates that may exert physiological effects and alter the fat composition of ...ruminant-derived foods. A batch culture method was used to characterize the hydrogenation of linoleic acid (LeA) by strained rumen fluid in vitro. Incubations (n = 5) were performed in 100-mL flasks maintained at 39 °C containing 400mg of grass hay, 50 mL of buffered rumen fluid, and incremental amounts of LeA (0, 1.0, 2.5, 5.0, or 10.0mg) for 0, 1.5, 3.0, 4.5, 6.0, and 9.0 h. The fatty acid composition of flask contents was determined using complimentary silver-ion thin-layer chromatography, gas chromatography mass-spectrometry, and silver-ion high-performance liquid chromatography. Linoleic acid was extensively (98.1, 97.6, 98.0, and 89.8% for additions of 1.0, 2.5, 5.0, and 10.0mg of LeA, respectively) hydrogenated over time. Complete reduction of LeA to 18:0 was inhibited in direct relation to the amount of added substrate, the extent of which was greatest for the highest amount of LeA addition. Recoveries of 1.0, 2.5, 5.0, and 10.0mg of added LeA as 18:0 averaged 73.6, 65.0, 57.3, and 10.7%, respectively. Incubation of incremental amounts of LeA resulted in a time-dependent accumulation of geometric isomers of 9,11 and 10,12 conjugated linoleic acid, several nonconjugated 18:2 isomers, and a wide range of cis 18:1 and trans 18:1 intermediates. Several unusual intermediates including cis-6,cis-12 18:2; cis-7,cis-12 18:2; and cis-8,cis-12 18:2, were found to accumulate in direct relation to the amount of added LeA, providing the first indications that hydrogenation of LeA by ruminal bacteria may also involve mechanisms other than hydrogen abstraction or isomerization of the cis-12 double bond. Fitting of single-pool, first-order kinetic models to experimental data indicated that the rate of LeA disappearance decreased with increases in substrate availability. Reduction of 18:1 and 18:2 intermediates occurred at much lower rates compared with conjugated linoleic acid and nonconjugated 18:2 isomer formation. In conclusion, the extent of LeA biohydrogenation in vitro was shown to be time- and dose-dependent with evidence that LeA is hydrogenated by ruminal bacteria via several distinct metabolic pathways. The accumulation of several unusual 18:2 isomers indicates that biohydrogenation of LeA also proceeds via mechanisms other than isomerization of the cis-12 double bond.
Conjugated linoleic acid (CLA) is a naturally occurring anticarcinogen found in milk fat and body fat of ruminants. Although CLA is an intermediate in ruminal biohydrogenation of linoleic acid, we ...hypothesized that its primary source was from endogenous synthesis. This would involve Delta(9)-desaturase and synthesis from trans-11 18:1, another intermediate in ruminal biohydrogenation. Our first experiment supplied lactating cows (n = 3) with trans-11 18:1 by abomasal infusion and examined the potential for endogenous synthesis by measuring changes in milk fat CLA. By d 3, infusion of trans-11 18:1 resulted in a 31% increase in concentration of cis-9, trans-11 CLA in milk fat, demonstrating that an active pathway for endogenous synthesis of CLA exists. Our second experiment examined the quantitative importance of endogenous synthesis of CLA in lactating cows (n = 3) by abomasally infusing a putative stimulator (retinol palmitate) or an inhibitor (sterculic oil) of Delta(9)-desaturase. Infusion of retinol palmitate had no influence on milk fatty acid desaturation, and yield of CLA in milk fat was not altered. However, sterculic oil infusion decreased the concentration of CLA in milk fat by 45%. Consistent with Delta(9)-desaturase inhibition, the sterculic oil treatment also altered the milk fat concentration of other Delta(9)-desaturase products as indicated by the two- to threefold increase in the ratios of 14:0 to 14:1(,) 16:0 to 16:1 and 18:0 to cis-18:1. Using changes in the ratio of 14:0 to 14:1 as an indication of the extent of Delta(9)-desaturase inhibition with the sterculic oil treatment, an estimated 64% of the CLA in milk fat was of endogenous origin. Overall, results demonstrate that endogenous synthesis of CLA from trans-11 18:1 represented the primary source of CLA in milk fat of lactating cows.
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