Docosahexaenoic acid elevates trans-18:1 isomers but is not directly converted into trans-18:1 isomers in ruminal batch cultures

Pathways of docosahexaenoic (DHA) biohydrogenation are not known; however, DHA is metabolized by ruminal microorganisms. The addition of DHA to the rumen alters the fatty acid profile of the rumen and milk and leads to increased trans-18:1 isomers, particularly trans-11 18:1. This study included 2 in vitro experiments to identify if the increase in trans-11 C18:1 was due to DHA being converted into trans-11 18:1 or if DHA stimulated trans-11 products from biohydrogenation of other fatty acids. In each experiment, ruminal microorganisms collected from a lactating Holstein cow were incubated in 10-mL batch cultures for 0, 6, 24, and 48 h and a uniformly ¹³C-labeled DHA was added to the cultures at 0 h as a metabolic tracer. Experiment 1 tested 0.5% DHA supplementation and experiment 2 examined 1, 2, and 3% DHA supplementation to determine if the level of DHA effected its conversion into trans-11 18:1. In both experiments, any fatty acid that was enriched with the ¹³C label was determined to arise from DHA. Palmitic (C16:0), stearic (C18:0), all trans-18:1, eicosanoic (C20:0), and docosanoic (C22:0) acids were examined for enrichment. In experiment 1, the amount of trans-18:1 isomers increased 0.415 mg from 0 to 48 h; however, no label was found in trans-18:1 at any time. Docosanoic acid was highly enriched at 24 h and 48 h to 20.2 and 16.3%. Low levels of enrichment were found in palmitic and stearic acids. In experiment 2, trans-18:1 isomers increased 185, 256, and 272% from 0 to 48 h when DHA was supplemented at 1, 2, and 3%, respectively; however, as in experiment 1, no enrichment occurred of any trans-18:1 isomer. In experiment 2, low levels of label were found in palmitic and stearic acids. Enrichment of docosanoic acid decreased linearly with increased DHA supplementation. These studies showed that trans-18:1 fatty acids are not produced from DHA, supporting that DHA elevates trans-18:1 by modifying biohydrogenation pathways of other polyunsaturated fatty acids.     

Effects of chemically or technologically treated linseed products and docosahexaenoic acid addition to linseed oil on biohydrogenation of C18:3n-3 in vitro

Rumen biohydrogenation kinetics of C18:3n-3 from several chemically or technologically treated linseed products and docosahexaenoic acid (DHA; C22:6n-3) addition to linseed oil were evaluated in vitro. Linseed products evaluated were linseed oil, crushed linseed, formaldehyde treated crushed linseed, sodium hydroxide/formaldehyde treated crushed linseed, extruded whole linseed (2 processing variants), extruded crushed linseed (2 processing variants), micronized crushed linseed, commercially available extruded linseed, lipid encapsulated linseed oil, and DHA addition to linseed oil. Each product was incubated with rumen liquid using equal amounts of supplemented C18:3n-3 and fermentable substrate (freeze-dried total mixed ration) for 0, 0.5, 1, 2, 4, 6, 12, and 24 h using a batch culture technique. Disappearance of C18:3n-3 was measured to estimate the fractional biohydrogenation rate and lag time according to an exponential model and to calculate effective biohydrogenation of C18:3n-3, assuming a fractional passage rate of 0.060/h. Treatments showed no differences in rumen fermentation parameters, including gas production rate and volatile fatty acid concentration. Technological pretreatment (crushing) followed by chemical treatment applied as formaldehyde of linseed resulted in effective protection of C18:3n-3 against biohydrogenation. Additional chemical pretreatment (sodium hydroxide) before applying formaldehyde treatment did not further improve the effectiveness of protection. Extrusion of whole linseed compared with extrusion of crushed linseed was effective in reducing C18:3n-3 biohydrogenation, whereas the processing variants were not different in C18:3n-3 biohydrogenation. Crushed linseed, micronized crushed linseed, lipid encapsulated linseed oil, and DHA addition to linseed oil did not reduce C18:3n-3 biohydrogenation. Compared with the other treatments, docosahexaenoic acid addition to linseed oil resulted in a comparable trans11,cis15-C18:2 biohydrogenation but a lesser trans10+11-C18:1 biohydrogenation. This suggests that addition of DHA in combination with linseed oil was effective only in inhibiting the last step of biohydrogenation from trans10+11-C18:1 to C18:0.     

Effects of fatty acid oxidation products (green odor) on rumen bacterial populations and lipid metabolism in vitro

This study investigated the effects of green odor fatty acid oxidation products (FAOP) from cut grass on lipid metabolism and microbial ecology using in vitro incubations of rumen microorganisms. These compounds have antimicrobial roles in plant defense, and we hypothesized that they may influence rumen lipid metabolism. Further, they may partially explain the higher levels of conjugated linoleic acid cis-9, trans-11 in milk from cows grazing pasture. The first of 2 batch culture experiments screened 6 FAOP (1 hydroperoxide, 3 aldehydes, 1 ketone, and 1 alcohol) for effects on lipid profile, and in particular C₁₈ polyunsaturated fatty acid biohydrogenation. Experiment 2 used the most potent FAOP to determine effects of varying concentrations and identify relationships with effects on microbial ecology. Batch cultures contained anaerobic buffer, rumen liquor, and FAOP to a final concentration of 100 μM for experiment 1. Triplicates for each compound and controls (water addition) were incubated at 39°C for 6 h. The hydroperoxide (1,2-dimethylethyl hydroperoxide, 1,2-DMEH) and the long chain aldehyde (trans-2 decenal) had the largest effects on lipid metabolism with significant increases in C_18:0 and C_18:1trans and reductions in C_12:0, C_14:0, C_16:0, C_18:1cis, C_18:2n-6, C_18:3n-3, C_20:0 and total branch and odd chain fatty acids compared with the control. This was associated with significantly higher biohydrogenation of C₁₈ polyunsaturated fatty acid. In experiment 2, 1,2-DMEH was incubated at 50, 100, and 200 μM for 2, 6, and 24 h. Increasing 1,2-DMEH concentration resulted in a significant linear increase in C_18:1trans-10, trans-11, conjugated linoleic acid, and C_18:0 and a linear decrease in C_18:2n-6 and C_18:3n-3, although the scale of this response declined with time. Microbial profiling techniques showed that 1,2-DMEH at concentrations of 100 and 200 μM changed the microbial community from as early as 2 h after addition, though microbial biomass remained similar. These preliminary studies have shown that FAOP can alter fatty acid biohydrogenation in the rumen. This change was associated with changes in the microbial population that were detected through DNA and branched- and odd-chain fatty acid profiling approaches.     

Effect of Dietary Starch or Micro Algae Supplementation on Rumen Fermentation and Milk Fatty Acid Composition of Dairy Cows

Two experiments with rumen-fistulated dairy cows were conducted to evaluate the effects of feeding docosahexaenoic acid (DHA; C22:6 n-3)-enriched diets or diets provoking a decreased rumen pH on milk fatty acid composition. In the first experiment, dietary treatments were tested during 21-d experimental periods in a 4 × 4 Latin square design. Diets included a control diet, a starch-rich diet, a bicarbonate-buffered starch-rich diet, and a diet supplemented with DHA-enriched micro algae [Schizochytrium sp., 43.0 g/kg of dry matter intake (DMI)]. Algae were supplemented directly through the rumen fistula. The total mixed ration consisted of grass silage, corn silage, soybean meal, and a standard or glucogenic concentrate. The glucogenic and buffered glucogenic diet had no effect on rumen fermentation and milk fatty acid composition because, unexpectedly, no reduced rumen pH was detected. The algae diet had no effect on rumen pH but provoked decreased butyrate and increased isovalerate molar proportions in the rumen. In addition, algae supplementation affected rumen biohydrogenation of linoleic and linolenic acid as reflected in the modified milk fatty acid composition toward increased conjugated linoleic acid (CLA) cis-9 trans-11, CLA trans-9 cis-11, C18:1 trans-10, C18:1 trans-11, and C22:6 n-3 concentrations. Concomitantly, on average, a 45% decrease in DMI and milk yield was observed. Based on these drastic and impractical results, a second animal experiment was performed for 20 d in which 9.35 g/kg of total DMI of algae were incorporated in the concentrate and supplemented to 3 rumen-fistulated cows. Algae concentrate feeding increased rumen pH, which was associated with decreased rumen short-chain fatty acid concentrations. Moreover, a different shift in rumen short-chain fatty acid proportions was observed compared with the first experiment because molar proportions of butyrate, isobutyrate, and isovalerate increased, whereas acetate molar proportion decreased. The milk fatty acid profile changed as in experiment 1. However, the decrease in DMI and milk yield was less pronounced (on average 10%) at this algae supplementation level, whereas milk fat percentage decreased from 47.9 to 22.0 g/kg of milk after algae treatment. In conclusion, an algae supplementation level of about 10 g/kg of DMI proved effective to reduce the milk fat content and to modify the milk fatty acid composition toward increased CLA cis-9 trans-11, C18:1 trans, and DHA concentrations.     

Effect of fish oil and sunflower oil on rumen fermentation characteristics and fatty acid composition of digesta in ewes fed a high concentrate diet

Studies in ruminants have shown that supplementing the diet with a mixture of fish oil (FO) and sunflower oil (SO) enhances the concentration of cis-9, trans-11 conjugated linoleic acid (CLA), 20:5 n-3, and 22:6 n-3 in milk because of alterations in ruminal biohydrogenation, but the intermediates formed under these conditions are not well characterized. Five ewes fitted with rumen cannula and fed a high concentrate diet were used to examine the effect of a mixture (30 g/kg of DM) of FO and SO (1:2, wt/wt) on temporal changes in rumen fermentation characteristics and the relative abundance of biohydrogenation intermediates in ruminal digesta collected after 0, 3, and 10 d on diet. Appearance and identification of biohydrogenation intermediates was determined based on complementary gas-liquid chromatography and Ag+-HPLC analysis of fatty acid methyl esters and gas chromatography-mass spectrometry analysis of corresponding 4,4-dimethyloxazoline derivatives. Inclusion of FO and SO in the diet had no effect on rumen pH, volatile fatty acid concentrations, or nutrient digestion, but altered the fatty acid composition of ruminal digesta, changes that were characterized by time-dependent decreases in 18:0 and 18:2 n-6 and the accumulation of trans 16:1, trans 18:1, 10-O-18:0, and trans 18:2. Lipid supplements enhanced the proportion of 20:5 n-3 and 22:6 n-3 in digesta and resulted in numerical increases in cis-9, trans-11 conjugated linoleic acid concentrations, but decreased the relative abundance of trans-10, cis-12 conjugated linoleic acid. Furthermore, detailed analysis revealed the appearance of several unique 20:1, 20:2, 22:1, 22:3, and 22:4 products in ruminal digesta that accumulated over time, providing the first indications of 20 and 22 carbon fatty acid intermediates formed during the biohydrogenation of long-chain unsaturated fatty acids in sheep. In conclusion, FO and SO in a high concentrate diet caused a time-dependent inhibition of the complete biohydrogenation of 16 and 18 carbon unsaturated fatty acids, and resulted in the accumulation of trans 16:1, trans 18:1, and trans 18:2, 20, and 22 carbon metabolites in ruminal digesta of sheep, with no evidence of a shift in ruminal biohydrogenation pathways toward trans-10 18:1 formation.     

Examination of the persistency of milk fatty acid composition responses to fish oil and sunflower oil in the diet of dairy cows

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 45 g 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. 17.9 kg/d), milk fat (47.7 vs. 32.6 g/kg), and protein content (36.1 vs. 33.3 g/kg), but had no effect on milk yield (27.1 vs. 26.4 kg/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 5.37 g/100 g of fatty acids on d 5, but these increases were transient, declining to 2.35 g/100 g 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 (Δ^4-10 and Δ^12-15), 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.     

Effect of chemical form,heating, and oxidation products of linoleic acid on rumen bacterial population and activities of biohydrogenating enzymes

Heating polyunsaturated fatty acids (PUFA) produces oxidation products, such as hydroperoxides, aldehydes, and oxypolymers, which could be responsible at least in part for modification of PUFA rumen biohydrogenation (BH). Three in vitro experiments were conducted to investigate the effects of linoleic acid (cis-9,cis-12-C18:2) oxidation products on BH. In the first experiment, we studied the effects of free linoleic acid (FLA), heated FLA (HFLA, at 150°C for 6 h), triacylglycerols of linoleic acid (TGLA), heated TGLA (HTGLA, at 150°C for 6 h), 13-hydroperoxide (13HPOD), trans-2-decenal (T2D), and hexanal (HEX) on BH in vitro after 6 and 24 h of incubation. In the second experiment, aldehydes differing in chain length and degree of unsaturation [pentanal, HEX, heptanal, nonanal, T2D, trans-2,trans-4-decadienal (T2T4D)] were incubated in vitro for 5 h in rumen fluid. In the third experiment, 9-hydroperoxide (9HPOD), 13HPOD, HEX, or T2T4D were incubated for 1 h in rumen fluid inactivated with chloramphenicol to investigate their effects on enzyme activity. In experiment 1, heat treatment of TGLA generated TGLA oxypolymers, did not affect cis-9,cis-12-C18:2 disappearance, but did decrease BH intermediates, especially trans-11 isomers. Heating FLA decreased cis-9,cis-12-C18:2 disappearance and cis-9,trans-11-CLA and trans-11-C18:1 production. Treatment with HEX and T2D did not affect cis-9,cis-12-C18:2 disappearance and barely affected production of BH intermediates. The bacterial community was affected by 13HPOD compared with FLA and HFLA, in parallel with an increase in trans-10 isomer production after a 6-h incubation. After 24 h of incubation, 13HPOD decreased trans-11 isomer production, but to a lesser extent than HFLA. In experiment 2, some weak but significant effects were observed on BH, unrelated to chain length or degree of unsaturation of aldehydes; the bacterial community was not affected. In experiment 3, 9HPOD inhibited Δ⁹-isomerization, and both 9HPOD and 13HPOD inhibited Δ¹²-isomerization. We concluded that oxypolymers did not affect cis-9,cis-12-C18:2 disappearance. Heating both esterified and free cis-9,cis-12-C18:2 greatly altered Δ¹²-isomerization. Aldehydes had few effects. Hydroperoxides are responsible, at least in part, for the effects of fat heating: 13HPOD increased trans-10 isomer production (probably by affecting the bacterial community) and decreased trans-11 isomer production by inhibiting Δ¹²-isomerase activity, whereas 9HPOD inhibited both isomerases.     

High-concentrate diets and polyunsaturated oils alter trans and conjugated isomers in bovine rumen, blood, and milk

Three Holstein cows were fed a high-concentrate diet (65:35 concentrate to forage) supplemented with either 5% sunflower oil (SO), 5% linseed oil (LO), or 2.5% fish oil (FO) to examine effects on biohydrogenation and fatty acid profiles in rumen, blood plasma, and milk. Diets were fed in a 3 × 3 Latin square with 4-wk periods with grass hay as the forage. Milk yield, dry matter intake, and percentages of milk fat (2.64) and protein (3.22) did not differ. All diets resulted in incomplete hydrogenation of unsaturated fatty acids as indicated by the profiles of 18:1 isomers, conjugated 18:2 isomers, nonconjugated 18:2 isomers, and 18:0 in ruminal fluid. Percentages of 8:0-14:0 and 16:0 in milk fat were greater with FO. Percentage and yield of trans10,cis12-18:2 were small and greater in cows fed SO (0.14%, 0.57 g/d) than FO (0.03%, 0.15 g/d) or LO (0.04%, 0.12 g/d). Percentage and yield of trans10-18:1, however, increased with FO (6.16%) and SO (6.47%) compared with LO (1.65%). Dietary FO doubled percentage of cis11-18:1 in rumen, plasma, and milk fat. Despite a lack of difference in ruminal percentage of trans11-18:1 (10.5%), cows fed FO had greater plasma trans11-18:1 (116 vs. 61.5 μg/mL) but this response did not result in greater trans11-18:1 percentage in milk fat, which averaged 5.41% across diets. Percentage (2.2%) and yield (14.3 g/d) of cis9,trans11-18:2 in milk fat did not differ due to oils. Unique responses to feeding LO included greater than 2-fold increases in percentages of trans13+14-18:1, trans15-18:1, trans16-18:1, cis15-18:1, cis9,trans12-18:2 and trans11,cis15 -18:2 in umen, plasma, and milk, and cis9,trans13-18:2 in plasma and milk. Ruminal 18:0 percentage had the highest positive correlation with milk fat content (r = 0.82) across all diets. When compared with previous data with cows fed high-concentrate diets without oil supplementation, results suggest that greater production of trans10-18:1, cis11-18:1, and trans11,cis15-18:2 coupled with low production of 18:0 in the rumen may be associated with low milk fat content when feeding high-concentrate diets and fish oil. In contrast, SO or LO could lead to low milk fat content by increasing ruminal trans10-18:1 (SO) or trans11,cis15-18:2 and trans9,trans12-18:2 (LO) along with a reduction in mammary synthesis of 8:0-16:0. Simultaneous increases in ruminal trans11-18:1 with fish oil, at a fraction of sunflower oil supplementation, may represent an effective strategy to maintain cis9,trans11-18:2 synthesis in mammary while reducing milk fat output and sparing energy.     

Milk fatty acids in dairy cows fed whole crude linseed, extruded linseed, or linseed oil, and their relationship with methane output

This experiment studied the effect of 3 different physical forms of linseed fatty acids (FA) on cow dairy performance, milk FA secretion and composition, and their relationship with methane output. Eight multiparous, lactating Holstein cows were assigned to 1 of 4 dietary treatments in a replicated 4 × 4 Latin square design: a control diet (C) based on corn silage (59%) and concentrate (35%), and the same diet supplemented with whole crude linseed (CLS), extruded linseed (ELS), or linseed oil (LSO) at the same FA level (5% of dietary dry matter). Each experimental period lasted 4 wk. Dry matter intake was not modified with CLS but was lowered with both ELS and LSO (−3.1 and −5.1 kg/d, respectively) compared with C. Milk yield and milk fat content were similar for LSO and ELS but lower than for C and CLS (19.9 vs. 22.3 kg/d and 33.8 vs. 43.2 g/kg, on average, respectively). Compared with diet C, CLS changed the concentrations of a small number of FA; the main effects were decreases in 8:0 to 16:0 and increases in 18:0 and cis-9 18:1. Compared with diet C (and CLS in most cases), LSO appreciably changed the concentrations of almost all the FA measured; the main effects were decreases in FA from 4:0 to 16:0 and increases in 18:0, trans-11 16:1, all cis and trans 18:1 (except trans-11 18:1), and nonconjugated trans 18:2 isomers. The effect of ELS was either intermediate between those of CLS and LSO or similar to LSO with a few significant exceptions: increases in 17:0 iso; 18:3n-3; trans-11 18:1; cis-9, trans-11 conjugated linoleic acid; and trans-11, trans-13 conjugated linoleic acid and a smaller increase in cis-9 18:1. The most positive correlations (r = 0.87 to 0.91) between milk FA concentrations and methane output were observed for saturated FA from 6:0 to 16:0 and for 10:1, and the most negative correlations (r = −0.86 to −0.90) were observed for trans-16+cis-14 18:1; cis-9, trans-13 18:2; trans-11 16:1; and trans-12 18:1. Thus, milk FA profile can be considered a potential indicator of in vivo methane output in ruminants.     

Effect of supplementation with fish oil or microalgae on fatty acid composition of milk from cows managed in confinement or pasture systems

The objective of this study was to examine the interaction between lipid supplement (LS) and management system (MS) on fatty acid (FA) composition of milk that could affect its healthfulness as a human food. Forty-eight prepartal Holstein cows were blocked by parity and predicted calving date and deployed across pasture (PAS; n = 23) or confinement (CONF; n = 25) systems. Cows within each system were assigned randomly to a control (no marine oil supplement) or to 1 of 2 isolipidic (200 g/d) marine oil supplements: fish oil (FO) or microalgae (MA) for 125 ± 5 d starting 30 d precalving. The experiment was conducted as a split-plot design, with MS being the whole-plot treatment and LS as the subplot treatment. Cows were housed in a tie-stall barn from −30 until 28 ± 10 d in milk (DIM) and were fed total mixed rations with similar formulations. The PAS group was then adapted to pasture and rotationally grazed on a perennial sward until the end of the experiment (95 ± 5 DIM). Milk samples were collected at 60 and 90 DIM for major components and FA analyses. Milk yield (kg/d) was lower in PAS (34.0) compared with CONF (40.1) cows. Milk fat percentage was reduced with MA compared with FO (3.00 vs. 3.40) and the control (3.56) cows. However, milk fat yield (kg/d) was not affected by lipid supplements. Compared with CONF, PAS cows produced milk fat with a lower content of 12:0 (−38%), 14:0 (−28%), and 16:0 (−17%), and more cis-9 18:1 (+32%), 18:3 n-3 (+30%), conjugated linoleic acid (CLA; +70%) and trans 18:1 (+34%). Both supplements, regardless of MS, reduced similarly the milk fat content of 16:0 (−12%) and increased CLA (+28%) and n-3 long-chain polyunsaturated FA (n-3 LC-PUFA; +150%). Milk fat content of trans 18:1 (trans-6 to trans-16) was increased with FO or MA, although the effect was greater with MA (+81%) than with FO (+42%). The interaction between MS and LS was significant only for trans-11 18:1 (vaccenic acid, VA) and cis-9,trans-11 CLA (rumenic acid). In contrast to CONF, feeding FO or MA to PAS cows did not increase milk fat content of VA and rumenic acid. We concluded that compared with CONF, milk from PAS cows had a more healthful FA composition. Feeding either FO or MA improved n-3 long-chain polyunsaturated FA and reduced levels of 16:0 in milk fat, regardless of MS, but concurrently increased the trans 18:1 isomers other than VA, at the expense of VA, particularly in grazing cows.     

Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage

Five multiparous Finnish Ayrshire cows fed red clover silage-based diets were used in a 5 × 5 Latin square with 21-d experimental periods to evaluate the effects of various plant oils or camelina expeller on animal performance and milk fatty acid composition. Treatments consisted of 5 concentrate supplements containing no additional lipid (control), or 29 g/kg of lipid from rapeseed oil (RO), sunflower-seed oil (SFO), camelina-seed oil (CO), or camelina expeller (CE). Cows were offered red clover silage ad libitum and 12 kg/d of experimental concentrates. Treatments had no effect on silage or total dry matter intake, whole-tract digestibility coefficients, milk yield, or milk composition. Plant oils in the diet decreased short- and medium-chain saturated fatty acid (6:0-16:0) concentrations, including odd- and branched-chain fatty acids and enhanced milk fat 18:0 and 18-carbon unsaturated fatty acid content. Increases in the relative proportions of cis 18:1, trans 18:1, nonconjugated 18:2, conjugated linoleic acid (CLA), and polyunsaturated fatty acids in milk fat were dependent on the fatty acid composition of oils in the diet. Rapeseed oil in the diet was associated with the enrichment of trans 18:1 (Δ4, 6, 7, 8, and 9), cis-9 18:1, and trans-7,cis-9 CLA, SFO resulted in the highest concentrations of trans-5, trans-10, and trans-11 18:1, Δ9,11 CLA, Δ10,12 CLA, and 18:2n-6, whereas CO enhanced trans-13-16 18:1, Δ11,15 18:2, Δ12,15 18:2, cis-9,trans-13 18:2, Δ11,13 CLA, Δ12,14 CLA, Δ13,15 CLA, Δ9,11,15 18:3, and 18:3n-3. Relative to CO, CE resulted in lower 18:0 and cis-9 18:1 concentrations and higher proportions of trans-10 18:1, trans-11 18:1, cis-9,trans-11 CLA, cis-9,trans-13 18:2, and trans-11,cis-15 18:2. Comparison of milk fat composition responses to CO and CE suggest that the biohydrogenation of unsaturated 18-carbon fatty acids to 18:0 in the rumen was less complete for camelina lipid supplied as an expeller than as free oil. In conclusion, moderate amounts of plant oils in diets based on red clover silage had no adverse effects on silage dry matter intake, nutrient digestion, or milk production, but altered milk fat composition, with changes characterized as a decrease in saturated fatty acids, an increase in trans fatty acids, and enrichment of specific unsaturated fatty acids depending on the fatty acid composition of lipid supplements.     

Uptake and utilization of trans octadecenoic acids in lactating dairy cows

Trans fatty acids (FA) arise in ruminant-derived foods as a consequence of rumen biohydrogenation and are of interest because of their biological effects and potential role in chronic human diseases. Our objective was to compare 2 trans FA, elaidic acid (EA; trans-9 18:1) and vaccenic acid (VA; trans-11 18:1), with oleic acid (OA; cis-9 18:1) relative to plasma lipid transport and mammary utilization for milk fat synthesis. Three ruminally cannulated, Holstein dairy cows, 259 ± 6 DIM (mean ± SEM), were randomly assigned in a 3 × 3 Latin square design. Treatments were a 4-d abomasal infusion of 1) OA (45.5 g/d), 2) EA (41.7 g/d), and 3) VA (41.4 g/d). Milk samples were collected at each milking and blood samples were collected at the start and end of each treatment period. The proportions of total plasma FA associated with each plasma lipid fraction at baseline (pretreatment) were 62.6 ± 0.6% phospholipids, 26.1 ± 0.6% cholesterol esters, 9.8 ± 0.4% triglycerides, and 1.5 ± 0.1% nonesterified fatty acids; these values were unaffected by treatment. There were striking differences in the FA composition of the individual plasma lipid fractions and in the distribution of specific 18-carbon FA among the lipid fractions. Infusion of treatment isomers caused their specific increase in the various plasma lipid fractions but had no effect on milk production variables, including milk fat yield and content. Transfer efficiency of infused OA, EA, and VA to milk fat averaged 65.5 ± 3.0%, 59.7 ± 1.5%, and 54.3 ± 0.6%, respectively. For the VA infusion, 24.6 ± 1.1% of the transfer was accounted for by the increased yield of cis-9, trans-11 conjugated linoleic acid in milk fat, consistent with its endogenous synthesis from VA via the mammary enzyme Δ⁹-desaturase. Notably, linoleic acid (18:2n-6) and linolenic acid (18:3n-3) accounted for 47.7% of total plasma FA, but only 2.6% of FA in milk. Overall, results demonstrate clear differences in plasma transport and mammary uptake and utilization of 18-carbon FA, and these relate to the location, orientation, and number of double bonds.     

Rumen biohydrogenation-derived fatty acids in milk fat from grazing dairy cows supplemented with rapeseed, sunflower, or linseed oils

The effects of supplementation with rapeseed, sunflower, and linseed oils (0.5 kg/d; good sources of oleic, linoleic, and linolenic acids, respectively) on milk responses and milk fat fatty acid (FA) profile, with special emphasis on rumen-derived biohydrogenation intermediates (BI), were evaluated in a replicated 4 × 4 Latin square study using 16 grazing dairy cows. The dietary treatments were 1) control diet: 20-h access to grazing pasture supplemented with 5 kg/d of corn-based concentrate mixture (96% corn; CC); 2) RO diet: 20-h access to grazing supplemented with 4.5 kg/d of CC and 0.5 kg of rapeseed oil; 3) SO diet: 20-h access to grazing supplemented with 4.5 kg/d of CC and 0.5 kg of sunflower oil; and 4) LO diet: 20-h access to grazing supplemented with 4.5 kg/d of CC and 0.5 kg of linseed oil. Milk fatty acids were converted to methyl esters and analyzed by gas-liquid chromatography and silver-ion HPLC. Dietary treatments had no effect on milk production or on milk protein content and milk protein production. Supplementation with rapeseed and sunflower oils lowered milk fat content and milk fat production, but linseed oil had no effect. Inclusion of dietary vegetable oils promoted lower concentrations of short-chain (including 4:0) and medium-chain FA (including odd- and branched-chain FA) and 18:3n-3, and higher concentrations of C₁₈ FA (including stearic and oleic acids). The BI concentration was higher with the dietary inclusion of vegetable oils, although the magnitude of the concentration and its pattern differed between oils. The RO treatment resulted in moderate increases in BI, including trans 18:1 isomers and 18:2 trans-7,cis-9, but failed to increase 18:1 trans-11 and 18:2 cis-9,trans-11. Sunflower oil supplementation resulted in the highest concentrations of the 18:1 trans-10, 18:1 cis-12, and 18:2 trans-10,trans-12 isomers. Concentrations of 18:1 trans-11 and 18:2 cis-9,trans-11 were higher than with the control and RO treatments but were similar to the LO treatment. Concentration of BI in milk fat was maximal with LO, having the highest concentrations of some 18:1 isomers (i.e., trans-13/14, trans-15, cis-15, cis-16), most of the nonconjugated 18:2 isomers (i.e., trans-11,trans-15, trans-11,cis-15, cis-9,cis-15, and cis-12,cis-15), and conjugated 18:2 isomers (i.e., trans-11,cis-13, cis-12,trans-14, trans-11,trans-13, trans-12,trans-14, and trans-9,trans-11), and all conjugated 18:3 isomers. The LO treatment induced the highest amount and diversity of BI without decreasing milk fat concentration, as the RO and SO treatments had, suggesting that the BI associated with 18:3n-3 intake may not be the major contributors to inhibition of mammary milk fat synthesis.     

In vitro response to EPA,DPA, and DHA: Comparison of effects on ruminal fermentation and biohydrogenation of 18-carbon fatty acids in cows and ewes

The modulation of milk fat nutritional quality through fish oil supplementation seems to be largely explained by the action of n-3 very long chain polyunsaturated fatty acids (PUFA) on ruminal biohydrogenation (BH) of C18 fatty acids (FA). However, relationships among this action, disappearance of those PUFA in the rumen, and potential detrimental consequences on ruminal fermentation remain uncertain. This study compared the effect of 20:5n-3 (eicosapentaenoic acid; EPA), 22:5n-3 (docosapentaenoic acid; DPA), and 22:6n-3 (docosahexaenoic acid; DHA) on rumen fermentation and BH of C18 FA and was conducted simultaneously in cows and sheep to provide novel insights into interspecies differences. The trial was performed in vitro using batch cultures of rumen microorganisms with inocula collected from cannulated cows and ewes. The PUFA were added at a dose of 2% incubated dry matter, and treatment effects on ruminal C18 FA concentrations, PUFA disappearances, and fermentation parameters (gas production, ammonia and volatile FA concentrations, and dry matter and neutral detergent fiber disappearances) were examined after 24 h of incubation. A principal component analysis suggested that responses to PUFA treatments explained most of the variability; those of ruminant species were of lower relevance. Overall, EPA and DHA were equally effective for inhibiting the saturation of trans-11 18:1 to 18:0 and had a similar influence on ruminal fermentation in cows and sheep (e.g., reductions in gas production and acetate:propionate ratio). Nevertheless, DHA further promoted alternative BH pathways that lead to trans-10 18:1 accumulation, and EPA seemed to have specific effects on 18:3n-3 metabolism. Only minor variations attributable to DPA were observed in the studied parameters, suggesting a low contribution of this FA to the action of marine lipids. Although most changes due to the added PUFA were comparable in bovine and ovine, there were also relevant specificities, such as a stronger inhibition of 18:0 formation in cows and a greater increase in 18:3n-3 metabolites in sheep. No direct relationship between in vitro disappearance of the incubated PUFA and effect on BH (in particular, inhibition of the last step) was found in either cows or ewes, calling into question a putative link between extent of disappearance and toxicity for microbiota. Conversely, an association between the influence of these PUFA on ruminal lipid metabolism and fermentation may exist in both species. In vivo verification of these findings would be advisable.     

Milk production, conjugated linoleic acid content, and in vitro ruminal fermentation in response to high levels of soybean oil in dairy ewe diet

Feeding vegetable oils rich in linoleic acid has been demonstrated to be an effective strategy to enrich milk with conjugated linoleic acid (CLA). However, high amounts of vegetable oil in the diet in free form could adversely affect animal performance, mainly in sheep. The aim of this work was to improve the ewe milk fatty acid profile by increasing potentially healthy acids such as CLA without any detrimental effects on milk production and ruminal fermentation with soybean oil (SBO) diet supplementation. Twenty-four ewes were assigned to 2 treatments and fed 2 diets (control or supplemented with 6% of SBO; 2 lots of 6 animals per treatment) and fed ad libitum for 4 wk. The forage:concentrate ratio was 20:80. Batch cultures of rumen microorganisms were used to study in vitro rumen fermentation. Changes in fatty acid profile were characterized as a reduction in C6:0 to C16:0 at the expense of an increase in C18:0, C18:1 isomers, and CLA concentrations. Proportions of milk CLA and trans-11 C18:1 (vaccenic acid) went from 1.04 to 3.44 and 2.08 to 6.20 g/100 g of total fatty acids, respectively. However, the SBO diet also increased trans-10 C18:1 and other trans C18:1 content. No significant decreases were found in the treatments for dry matter intake and milk production. The notable increases in trans-10, cis-12 and trans-9, cis-11 were not accompanied by fat level decreases in ewe milk. Concerning in vitro ruminal fermentation, no significant differences were found in the extent and rate of gas production, effective degradability, in vitro true digestibility, and volatile fatty acid production. The results demonstrate that dairy sheep milk CLA content can be substantially increased (more than 3-fold) by adding high levels of SBO in the diet as free oil, without any negative effects on animal performance.     

Effect of weaning status on lipids of Galician Blond veal: Total fatty acids and 18:1 cis and trans isomers

The effect of weaning at different ages (NW = not weaned, W5 = 5.5 months and W2 = 2 months) on fatty acids (FA) of the Longissimus thoracis (LT) muscle was studied in 36 Galician Blond (GB) calves. Total FA (TFA) were determined by gas chromatography (GC) and 18:1 isomers by a combination of reversed-phase high performance liquid chromatography (HPLC) and GC. NW group showed higher (P < 0.001) values of n-3 polyunsaturated FA (PUFA), conjugated linoleic acid (CLA) and 18:1trans-11 compared to LT from W5 and W2 calves. W2 calves showed the highest levels of n-6 PUFA (P < 0.01), 18:1trans-10 and 18:1trans-6/7/8 (P < 0.001). Generally, W5 calves had intermediate values for TFA and 18:1 isomers. As the suckling period was longer, GB milk and veal FA profiles became more similar, it seems that muscle FA were partially transmitted by the milk FA intake due to the persistence of the reticular grove closing reflex.     

Effect of induction of subacute ruminal acidosis on milk fat profile and rumen parameters

High-concentrate diets can lead to subacute ruminal acidosis and are known to result in changes of the ruminal fermentation pattern and mammary secretion of fatty acids. The objective of this paper is to describe modifications in milk fatty acid proportions, particularly odd- and branched-chain fatty acids and rumen biohydrogenation intermediates, associated with rumen parameters during a 6-wk subacute ruminal acidosis induction protocol with 12 ruminally fistulated multiparous cows. The protocol involved a weekly gradual replacement of a standard dairy concentrate with a wheat-based concentrate (610 g of wheat/kg of concentrate) during the first 5 wk and an increase in the total amount of concentrate in wk 6. Before the end of induction wk 6, cows were switched to a control diet because 7 cows showed signs of sickness. The pH was measured continuously by an indwelling pH probe. Milk and rumen samples were taken on d 2 and 7 of each week. Data were analyzed using a linear mixed model and by principal component analysis. A pH decrease occurred after the first concentrate switch but rumen parameters returned to the original values and remained stable until wk 5. In wk 5 and 6, rumen pH values were indicative of increasing acidotic conditions. After switching to the control diet in wk 6, rumen pH values rapidly achieved normal values. Odd- and branched-chain fatty acids and C18:1 trans-10 increased with increasing amount of concentrate in the diet, whereas C18:1 trans-11 decreased. Four fatty acids [C18:1 trans-10, C15:0 and C17:0+C17:1 cis-9 (negative loadings), and iso C14:0 (positive loading)] largely correlated with the first principal component (PC1), with cows spread along the PC1 axis. The first 4 wk of the induction experiment showed variation across the second principal component (PC2) only, with high loadings of anteiso C13:0 (negative loading) and C18:2 cis-9,trans-11 and C18:1 trans-11 (positive loadings). Weeks 5 and 6 deviated from PC2 and tended toward the negative PC1 axis. A discriminant analysis using a stepwise approach indicated the main fatty acids discriminating between the control and acidotic samples as iso C13:0, iso C16:0, and C18:2 cis-9,trans-11 rather than milk fat content or C18:1 trans-10, which have been used before as indicators of acidosis. This shows that specific milk fatty acids have potential in discriminating acidotic cases.     

Meta-analysis of relationships between enteric methane yield and milk fatty acid profile in dairy cattle

Various studies have indicated a relationship between enteric methane (CH₄) production and milk fatty acid (FA) profiles of dairy cattle. However, the number of studies investigating such a relationship is limited and the direct relationships reported are mainly obtained by variation in CH₄ production and milk FA concentration induced by dietary lipid supplements. The aim of this study was to perform a meta-analysis to quantify relationships between CH₄ yield (per unit of feed and unit of milk) and milk FA profile in dairy cattle and to develop equations to predict CH₄ yield based on milk FA profile of cows fed a wide variety of diets. Data from 8 experiments encompassing 30 different dietary treatments and 146 observations were included. Yield of CH₄ measured in these experiments was 21.5 ± 2.46 g/kg of dry matter intake (DMI) and 13.9 ± 2.30 g/kg of fat- and protein-corrected milk (FPCM). Correlation coefficients were chosen as effect size of the relationship between CH₄ yield and individual milk FA concentration (g/100 g of FA). Average true correlation coefficients were estimated by a random-effects model. Milk FA concentrations of C6:0, C8:0, C10:0, C16:0, and C16:0-iso were significantly or tended to be positively related to CH₄ yield per unit of feed. Concentrations of trans-6+7+8+9 C18:1, trans-10+11 C18:1, cis-11 C18:1, cis-12 C18:1, cis-13 C18:1, trans-16+cis-14 C18:1, and cis-9,12 C18:2 in milk fat were significantly or tended to be negatively related to CH₄ yield per unit of feed. Milk FA concentrations of C10:0, C12:0, C14:0-iso, C14:0, cis-9 C14:1, C15:0, and C16:0 were significantly or tended to be positively related to CH₄ yield per unit of milk. Concentrations of C4:0, C18:0, trans-10+11 C18:1, cis-9 C18:1, cis-11 C18:1, and cis-9,12 C18:2 in milk fat were significantly or tended to be negatively related to CH₄ yield per unit of milk. Mixed model multiple regression and a stepwise selection procedure of milk FA based on the Bayesian information criterion to predict CH₄ yield with milk FA as input (g/100 g of FA) resulted in the following prediction equations: CH₄ (g/kg of DMI) = 23.39 + 9.74 × C16:0-iso – 1.06 × trans-10+11 C18:1 – 1.75 × cis-9,12 C18:2 (R² = 0.54), and CH₄ (g/kg of FPCM) = 21.13 – 1.38 × C4:0 + 8.53 × C16:0-iso – 0.22 × cis-9 C18:1 – 0.59 × trans-10+11 C18:1 (R² = 0.47). This indicated that milk FA profile has a moderate potential for predicting CH₄ yield per unit of feed and a slightly lower potential for predicting CH₄ yield per unit of milk.     

In vitro biohydrogenation of 13C-labeled α-linolenic acid in response to ruminal alterations associated with diet-induced milk fat depression in ewes

The basis for marine lipid-induced milk fat depression (MFD) has not been established yet, but recent reports suggest the putative contribution of shifts in the ruminal metabolism of α-linolenic acid (ALA). To test this hypothesis, an isotopic tracer approach was used in batch cultures of rumen microorganisms with inoculum collected from cannulated ewes fed either a total mixed ration without lipid supplementation (control inoculum) or the same diet supplemented with 2% of fish oil, which is known to cause MFD in lactating sheep (FO-MFD inoculum). The [1-¹³C]ALA was added at a dose of 1% of incubated dry matter and the proportions of ¹³C-labeled fatty acids (FA) were examined after 24 h of incubation, using complementary gas chromatography and gas chromatography-combustion isotope ratio mass spectrometry (GC-C-IRMS) analyses. Expected differences in FA profiles were confirmed between control and FO-MFD inocula (e.g., large decreases in 18:0 and increases in most 18:1 and 18:2 intermediates, particularly trans isomers, to fish oil supply). The biohydrogenation of ¹³ALA was extensive and yielded multiple metabolites, with a total of 48 chromatographic peaks showing ¹³C enrichment, regardless of the inoculum type. However, although ALA was biohydrogenated through common pathways under standard or MFD conditions, large changes in the accumulation of ¹³C-labeled FA suggest important differences in the relative contribution of each specific route. First, increased accumulation of trans-11-containing FA in FO-MFD incubations was accompanied by a general repression of the trans-13/14 pathway (supported by lower trans-13+14 18:1 or trans-11,trans-13 18:2 proportions), together with a lower production of cis FA (e.g., cis-9, -12, and -15 18:1 and some cis,cis 18:2). Results also downplayed the relevance of the trans-11 to trans-10 shift as an effective marker of diet-induced MFD in sheep, and challenged the involvement of some trans-10 intermediates (e.g., trans-10 18:1 and trans-10,cis-15 18:2) in the low-fat milk syndrome in this species. Conversely, increased abundance of most 18:3 intermediates (including some unidentified isomers) might be representative of ruminal alterations related to fish oil supplementation in ewes. Further research is necessary to examine the potential association between these findings and MFD in lactating animals.     

Milk fatty acid profile and dairy sheep performance in response to diet supplementation with sunflower oil plus incremental levels of marine algae

In an attempt to develop strategies for enhancing the nutritional value of sheep milk fat, dairy ewe diet was supplemented with 3 incremental levels of marine algae (MA), in combination with sunflower oil, to evaluate the effects of these marine lipids on milk fatty acid (FA) profile and animal performance. Fifty Assaf ewes in mid lactation were distributed in 10 lots of 5 animals each and allocated to 5 treatments (2 lots per treatment): no lipid supplementation (control) or supplementation with 25 g of sunflower oil/kg of DM plus 0 (SO), 8 (SOMA₁), 16 (SOMA₂), or 24 (SOMA₃) g of MA (56.7% ether extract)/kg of DM. Milk production and composition, including FA profile, were analyzed on d 0, 3, 7, 14, 21, and 28 of treatment. Neither intake nor milk yield were significantly affected by lipid addition, but all MA supplements decreased milk fat content from d 14 onward, reaching a 30% reduction after 28 d on SOMA₃. This milk fat depression might be related not only to the joint action of some putative fat synthesis inhibitors, such as trans-9,cis-11 C18:2 and probably trans-10 C18:1, but also to the limited ability of the mammary gland to maintain a desirable milk fat fluidity, that would have been caused by the noticeable increase in trans-C18:1 together with the lowered availability of stearic acid for oleic acid synthesis through Δ⁹-desaturase. Furthermore, all lipid supplements, and mainly MA, reduced the secretion of de novo FA (C6:0-C14:0) without increasing the yield of preformed FA (>C16). Supplementation with sunflower oil plus MA resulted in larger increases in cis-9,trans-11 C18:2 than those observed with sunflower oil alone, achieving a mean content as high as 3.22% of total FA and representing a more than 7-fold increase compared with the control. Vaccenic acid (trans-11 C18:1) was also significantly enhanced (on average +794% in SOMA treatments), as was C22:6 n-3 (DHA) content, although the transfer efficiency of the latter, from the diets to the milk, was very low (5%). However, the highest levels of MA inclusion (SOMA₂ and SOMA₃) reduced the milk n-6:n-3 ratio, but MA supplements caused an important increase in trans-10 C18:1, which would rule out the possibility that this milk has a healthier fat profile before determining the specific role of each individual FA and ensuring that this trans-FA is at least innocuous in relation to cardiovascular disease risk.