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.     

Susceptibility to milk fat depression in dairy sheep and goats: Individual variation in ruminal fermentation and biohydrogenation

Small ruminants are susceptible to milk fat depression (MFD) induced by marine lipid supplementation. However, as observed in dairy cows, there is wide individual variation in the response to MFD-inducing diets, which may be due to individual differences in ruminal processes. Therefore, we compared the ruminal responses of goats and sheep with varying degrees of MFD extent to improve our understanding of this complex syndrome. Our specific aims were to attempt to elucidate whether pre-existing variations in ruminal fermentation and biohydrogenation determine a higher tolerance or susceptibility to MFD, and whether the severity of MFD depends exclusively on the response to the diet. The trial was conducted with 25 does and 23 ewes fed a basal diet without lipid supplementation for 3 wk (control period). Then, 2% fish oil (FO) was added to the same diet for 5 additional weeks (MFD period). Based on the extent of the elicited MFD (i.e., the percentage variation between milk fat concentrations recorded at the end of the control and MFD periods), the 5 most responsive (RESPON+) and the 5 least responsive (RESPON?) animals were selected within each species. On the last day of each period, ruminal fluid samples were collected to examine fermentation parameters and fatty acid profiles. In general, the individual degree of MFD in sheep and goats did not seem to be predetermined by traits related to ruminal fermentation and biohydrogenation, including fatty acids that may serve as biomarkers of microorganisms. Regarding differences in the response to FO, the results suggest no link between MFD susceptibility and concentration of biohydrogenation intermediates such as trans-10-containing C18, C20, and C22 metabolites. The explanation for individual responses based on a shortage of ruminal acetate and 18:0 for mammary uptake also seems to be dismissed, based on the lack of variation in these compounds between RESPON+ and RESPON?. However, the concentration of unsaturated fatty acids provided by FO (e.g., cis-9 16:1, cis-11 18:1, and 20:5n-3) was higher in the rumen of RESPON+ than RESPON? ewes and does. Thus, although further research is needed, the extent of biohydrogenation of these fatty acids might be associated with tolerance or susceptibility to MFD.     

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.     

Changes in milk fatty acid profile and animal performance in response to fish oil supplementation, alone or in combination with sunflower oil, in dairy ewes

Ruminant diet supplementation with sunflower oil (SO) and fish oil (FO) has been reported as a good strategy for enhancing some milk fat compounds such as conjugated linoleic acid (CLA) and n-3 polyunsaturated fatty acids in dairy cows, but no information is available regarding dairy sheep. In this work, ewe diet was supplemented with FO, alone or in combination with SO, with the aim of improving milk nutritional value and evaluating its effect on animal performance. Sixty-four Assaf ewes in mid lactation, fed a high-concentrate diet, were distributed in 8 lots of 8 animals each and assigned to 4 treatments (2 lots/treatment): no lipid supplementation (control) or supplementation with 20 g of SO/kg (SO), 10 g of FO/kg (FO), or 20 g of SO plus 10 g of FO/kg (SOFO). Milk production and composition, including a complete fatty acid profile, were analyzed on d 0, 3, 7, 14, 21, and 28 of treatments. Supplementation with FO tended to reduce dry matter intake compared with the control treatment (−15%), and its use in combination with SO (SOFO) resulted in a significant decrease in milk yield as well (−13%). All lipid supplements reduced milk protein content, and FO also reduced milk fat content by up to 21% alone (FO) and 27% in combination with SO (SOFO). Although the mechanisms involved in FO-induced milk fat depression are not yet well established, the observed increase in some milk trans-FA that are putative inhibitors of milk fat synthesis, such as trans-9,cis-11 CLA, and the 63% decrease in C18:0 (consistent with the theory of reduced milk fat fluidity) may be involved. When compared with the control, lipid supplementation remarkably improved the milk content of rumenic acid (cis-9,trans-11 CLA; up to 4-fold increases with SO and SOFO diets), whereas FO-containing diets also increased milk n-3 polyunsaturated fatty acids, mainly docosahexaenoic acid (with mean contents of 0.29 and 0.38% of total fatty acids for SOFO and FO, respectively), and reduced the n-6:n-3 FA ratio to approximately half the control value. All lipid supplements resulted in high levels of some trans-FA, mainly trans-11 C18:1 (vaccenic acid) but also trans-10 C18:1.     

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.     

Temporal changes in milk fatty acid composition during diet-induced milk fat depression in lactating cows

Diet-induced milk fat depression (MFD) in lactating cows has been attributed to alterations in ruminal lipid metabolism leading to the formation of specific fatty acid (FA) biohydrogenation intermediates that directly inhibit milk fat synthesis. However, the mechanisms responsible for decreased lipid synthesis in the mammary gland over time are not well defined. The aim of this study was to evaluate the effect of diet on milk FA composition and milk fat production over time, especially during MFD, and explore the associations between MFD and FA biohydrogenation intermediates in omasal digesta and milk. Four lactating Finnish Ayrshire cows used in a 4 × 4 Latin square with a 2 × 2 factorial arrangement of treatments and 35-d experimental periods were fed diets formulated to cause differences in ruminal and mammary lipid metabolism. Treatments consisted of an iso-nitrogenous total mixed ration based on grass silage with a forage to concentrate ratio of 65:35 or 35:65 without added oil, or with sunflower oil at 50 g/kg of diet dry matter. The high-concentrate diet with sunflower oil (HSO) induced a 2-stage drop in milk fat synthesis that was accompanied by specific temporal changes in the milk FA composition. The MFD on HSO was associated especially with trans-10 18:1 and also with trans-9,cis-11 conjugated linoleic acid (CLA) in milk and omasal digesta across all diets and was accompanied by the appearance of trans-10,cis-15 18:2. Trans-10,cis-12 CLA was increased in HSO, but milk fat secretion was not associated with omasal or milk trans-10,cis-12 CLA. The temporal changes in milk fat content and yield and milk FA composition reflect the shift from the predominant ruminal biohydrogenation pathway to an alternative pathway. The ambiguous role of trans-10,cis-12 CLA suggests that trans-10 18:1, trans-9,cis-11 CLA and trans-10,cis-15 18:2 or additional mechanisms contributed to the diet-induced MFD in lactating cows.     

Fatty acid composition and bacterial community changes in the rumen fluid of lactating sheep fed sunflower oil plus incremental levels of marine algae

Supplementation of ruminant diets with plant oils and marine lipids is an effective strategy for lowering saturated fatty acid (FA) content and increasing the concentration of cis-9,trans-11 conjugated linoleic acid and long-chain n-3 FA in ruminant milk. However, changes in populations of ruminal microorganisms associated with altered biohydrogenation of dietary unsaturated FA are not well characterized. Twenty-five lactating Assaf ewes were allocated at random to 1 of 5 treatments composed of dehydrated alfalfa hay and concentrates containing no additional lipid (control), or supplemented with 25 g of sunflower oil and 0 (SO), 8 (SOMA(1)), 16 (SOMA(2)), or 24 (SOMA(3)) g of marine algae/kg of diet dry matter. On d 28 on diet, samples of rumen fluid were collected for lipid analysis and microbial DNA extraction. Appearance and identification of biohydrogenation intermediates was determined based on complementary gas chromatography and Ag+-HPLC analysis of FA methyl esters. Total bacteria and the Butyrivibrio group were studied in microbial DNA by terminal RFLP analysis, and real-time PCR was used to quantify the known Butyrivibrio bacteria that produce trans-11 18:1 or 18:0. Dietary supplements of sunflower oil alone or in combination with marine algae altered the FA profile of rumen fluid, which was associated with changes in populations of specific bacteria. Inclusion of marine algae in diets containing sunflower oil resulted in the accumulation of trans 18:1 and 10-O-18:0 and a marked decrease in 18:0 concentrations in rumen fluid. At the highest levels of supplementation (SOMA(2) and SOMA(3)), marine algae also promoted a shift in ruminal biohydrogenation pathways toward the formation of trans-10 18:1 at the expense of trans-11 18:1. Changes in the concentration of biohydrogenation intermediates were not accompanied by significant variations in the abundance of known cultivated ruminal bacteria capable of hydrogenating unsaturated FA. However, certain bacterial groups detected by terminal RFLP (such as potentially uncultured Lachnospiraceae strains or Quinella-related bacteria) exhibited variations in their relative frequency consistent with a potential role in one or more metabolic pathways of biohydrogenation in the rumen.     

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.     

Effects of different forage:concentrate ratios in dairy ewe diets supplemented with sunflower oil on animal performance and milk fatty acid profile

The aim of this study was to evaluate the effect of different forage:concentrate (FC) ratios in dairy ewe diets supplemented with sunflower oil (SO) on animal performance and milk fatty acid (FA) profile, particularly focusing on trans C18:1 FA and conjugated linoleic acid (CLA). Sixty lactating Assaf ewes were randomly assigned to 6 treatments in a 3 × 2 factorial arrangement: 3 FC ratios (30:70, 50:50, and 70:30) and 2 levels of SO addition (0 and 20 g/kg of dry matter). Both the diet FC ratio and SO supplementation affected milk yield, but differences between treatments were small. Although the proportion of concentrate induced limited changes in milk FA profile, dietary SO significantly decreased saturated FA and enhanced total CLA. Furthermore, the incorporation of SO in ewe diets decreased the atherogenicity index value by about 25% and doubled the contents of potentially healthy FA such as trans-11 C18:1 and cis-9,trans-11 CLA. However, the inclusion of SO in a high-concentrate diet (30:70) could switch linoleic acid biohydrogenation pathways, resulting in a significant increase in trans-10 C18:1, trans-9,cis-11 C18:2, and trans-10,cis-12 C18:2 milk fat percentages.     

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 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.     

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.     

Changes in the rumen bacterial community in response to sunflower oil and fish oil supplements in the diet of dairy sheep

Rumen microbial biohydrogenation of dietary unsaturated fatty acids has a major effect on the process of developing healthier dairy products. This study aimed to investigate in vivo the effect of diet supplementation with sunflower (SO) and fish (FO) oils on the rumen bacterial community in dairy sheep. First, 32 lactating ewes, divided in 8 lots of 4 animals each (2 lots per treatment), were fed a high-concentrate total mixed ration supplemented with 0, 2% SO, 1% FO, or 2% SO plus 1% FO. After 21 d, rumen fluid samples were taken from each lot for DNA extraction and fluorescence in situ hybridization (FISH) analysis. In a second experiment, 5 cannulated ewes were first fed the same TMR, with the exception of a higher forage level, and then changed to the same diet supplemented with 2% SO plus 1% FO. After 0, 3, and 10 d, rumen content samples were taken for DNA extraction and FISH analysis (fluid). Total bacteria and the Butyrivibrio group were studied in microbial DNA by terminal RFLP analysis (T-RFLP), and real-time PCR was used to quantify Butyrivibrio bacteria that produce vaccenic acid or stearic acid. In rumen fluid samples, total bacteria and clostridial clusters IX and XIV were analyzed by FISH. Dietary supplementation with SO plus FO seemed to induce important changes in the total bacteria and Butyrivibrio populations, and a high interindividual variation was observed, and the speed of the effect of the lipid supplementation depended on the individual microbial composition. Analysis by T-RFLP and FISH showed increases in cluster IX bacteria with SO plus FO supplementation, presumably Quinella-like microorganisms. The abundances of vaccenic acid- and stearic acid-producing Butyrivibrio relative to total bacteria, estimated by real time PCR, were low (0.28 and 0.18%, respectively, in rumen fluid, and 0.86 and 0.81% in rumen contents) and only that of SA-producing bacteria seemed to be reduced by diets containing FO, although differences were only significant in lactating ewes. The T-RFLP analysis showed a variable effect of lipid supplementation on different bacteria of the family Lachnospiraceae, which includes the cultured bacteria known to be actively involved in rumen biohydrogenation. These results suggest that the latter bacteria do not play a dominant role in this process, and therefore other as-yet-uncultivated microorganisms might be more relevant.     

Starch and oil in the donor cow diet and starch in substrate differently affect the in vitro ruminal biohydrogenation of linoleic and linolenic acids

Trans isomers of fatty acids exhibit different health properties. Among them, trans-10,cis-12 conjugated linoleic acid has negative effects on milk fat production and can affect human health. A shift from the trans-11 to the trans-10 pathway of biohydrogenation (BH) can occur in the rumen of dairy cows receiving high-concentrate diets, especially when the diet is supplemented with highly unsaturated fat sources. The differences of BH patterns between linoleic acid (LeA) and linolenic acid (LnA) in such ruminal conditions remain unknown; thus, the aim of this work was to investigate in vitro the effects of starch and sunflower oil in the diet of the donor cows and starch level in the incubates on the BH patterns and efficiencies of LeA and LnA. The design was a 4 × 4 Latin square design with 4 cows, 4 periods, and 4 diets with combinations of 21 or 34% starch and 0 or 5% sunflower oil. The rumen content of each cow during each period was incubated with 4 substrates, combining 2 starch levels and either LeA or LnA addition. Capillary electrophoresis single-strand conformation polymorphism of incubates showed that dietary starch decreased the diversity of the bacterial community and the high-starch plus oil diet modified its structure. High-starch diets poorly affected isomerization and first reduction of LeA and LnA, but decreased the efficiencies of trans-11,cis-15-C18:2 and trans C18:1 reduction. Dietary sunflower oil increased the efficiency of LeA isomerization but decreased the efficiency of trans C18:1 reduction. An interaction between dietary starch and dietary oil resulted in the highest trans-10 isomers production in incubates when the donor cow received the high-starch plus oil diet. The partition between trans-10 and trans-11 isomers was also affected by an interaction between starch level and the fatty acid added to the incubates, showing that the trans-10 shift only occurred with LeA, whereas LnA was mainly hydrogenated via the more usual trans-11 pathway, whatever the starch level in the substrate, although the bacterial communities were not different between LeA and LnA incubates. In LeA incubates, trans-10 isomer production was significantly related to the structure of the bacterial community.     

Changes in fermentation and animal performance during recovery from classical diet-induced milk fat depression using corn with differing rates of starch degradability

Diet-induced milk fat depression (MFD) is a multifactorial disorder that can be triggered by a variety of conditions. Feeding high amounts of starch and unsaturated fatty acids has been shown to reduce milk fat yield and composition, as well as alter ruminal biohydrogenation patterns. However, little is known about how starch degradability in the rumen influences recovery from diet-induced MFD and if production of milk fat–inhibiting isomers will persist following an episode of MFD. The objective of this study was to evaluate production performance and ruminal fermentation in cows recovering from MFD when corn with a low or high starch degradability is fed. Six ruminally fistulated Holstein cows were used in a crossover design with 2 periods. During each period, MFD was induced for 10 d by feeding a diet with low fiber, high starch, and high unsaturated fatty acid. The polyunsaturated fatty acid concentration of the diet during the induction phase was modified primarily through inclusion of soybean oil. Following induction, cows were switched to either a high degradable starch recovery diet (HDS) or a low degradable starch recovery diet (LDS) for 18 d. The 7-h starch degradability was 66.5% for LDS and 87.8% for HDS. Milk was collected every 3 d for component and fatty acid analysis. On d 0, 4, 7, 10, 16, 22, and 28 of each period, ruminal pH and rumen fluid were collected every 2 h. Milk fat yield and composition was reduced during MFD induction and progressively increased by day in both HDS and LDS during recovery. Dry matter intake was similar among treatments and increased steadily over time during recovery. Preformed fatty acids were greater for HDS-fed animals, and de novo fatty acid in milk fat was greater for LDS-fed animals. Milk trans-10 C18:1 tended to be greater for HDS, and trans-10,cis-12 conjugated linoleic acid was significantly greater for HDS. cis-9,trans-11 conjugated linoleic acid was not affected by starch degradability during recovery. Total volatile fatty acids, butyrate, and valerate tended to differ or differed with recovery treatment, but ruminal pH and ammonia concentration were unaffected. The HDS diet responded similarly to the LDS diet during recovery with regard to milk fat percentage, but milk and fat yield tended to consistently be lower in HDS. When considering approaches to ameliorate diet-induced MFD, the degradability of the starch within rations should be evaluated. Although animal performance was similar, some trans fatty acid isomers were persistent in the milk through the recovery phase with HDS-fed animals, suggesting that milk fat synthesis might be potentially inhibited and biohydrogenation pathways modified in the rumen following an episode of MFD.     

Effect of replacing grass silage with red clover silage on ruminal lipid metabolism in lactating cows fed diets containing a 60:40 forage-to-concentrate ratio

Diets based on red clover silage (RCS) typically increase the concentration of polyunsaturated fatty acids (PUFA) in ruminant milk and meat compared with grass silages (GS), an effect that has been attributed to higher activity of polyphenol oxidase in red clover, promoting ruminal escape of dietary lipid. Four multiparous Finnish Ayrshire cows in mid lactation fitted with rumen cannulas were used in a 4 × 4 Latin Square design with 21-d experimental periods to evaluate the effects of incremental replacement of GS with RCS on ruminal lipid metabolism, using the omasal sampling technique in combination with Cr-EDTA, Yb acetate, and indigestible neutral detergent fiber as markers. Treatments comprised total mixed rations offered ad libitum containing 600 g of forage/kg of diet dry matter, with RCS replacing GS in a ratio of 0:100, 33:67, 67:33, and 100:0 on a dry matter basis. Silages contained a high proportion of lipid as nonesterified fatty acids (NEFA), with no difference between forage species (75 and 73% for GS and RCS, respectively). Substitution of GS with RCS had no influence on the intakes of NEFA, polar lipid, triacylglycerol, diacylglycerol, monoacylglycerol, or total fatty acids (FA), but altered the ingestion of specific FA. Replacing GS with RCS decreased linearly 18:3n-3 and increased linearly 18:2n-6 intakes. Changes in the proportion of RCS in the diet had no effect on the amounts or on the relative proportions of different lipid fractions at the omasum. On average, NEFA, polar lipid, triacylglycerol, diacylglycerol, and monoacylglycerol accounted for 80, 12, 4.4, 2.4, and 0.8% of total FA in omasal digesta, respectively. Replacement of GS with RCS increased linearly the amount of esterified and nonesterified 18:3n-3 at the omasum. Flows of cis-9 18:1 and 18:2n-6 were also increased linearly in response to RCS in the diet, whereas 3,7,11,15-tetramethyl-16:0 at the omasum was decreased. Replacing GS with RCS in the diet decreased linearly the lipolysis of dietary esterified lipids in the rumen from 85 to 70%. Effects on lipolysis due to forage species were also associated with linear decreases in apparent ruminal 18:3n-3 biohydrogenation from 93 to 85% and a trend toward lowered biohydrogenation of cis-9 18:1 and 18:2n-6 in the rumen. However, forage species had no effect on the flow of bound phenols formed as a consequence of polyphenol oxidase activity at the omasum. In conclusion, despite minimal differences in the extent of lipolysis in silo, lipid and constituent FA in RCS were less susceptible to ruminal lipolysis and biohydrogenation compared with GS.     

The effect of rumen digesta inoculation on the time course of recovery from classical diet-induced milk fat depression in dairy cows

Ten ruminally cannulated cows were used in a crossover design that investigated the effect of rumen digesta inoculation from non-milk fat-depressed cows on recovery from classical diet-induced milk fat depression (MFD) characterized by reduced fat yield, reduced de novo milk fat synthesis, and increased alternate trans isomers. Two additional cows fed a high-fiber and low-polyunsaturated fatty acid (FA) diet (31.8% neutral detergent fiber, 4.2% FA, and 1.2% C18:2) were used as rumen digesta donors. Milk fat depression was induced during the first 10 d of each period by feeding a low-fiber and high-polyunsaturated FA diet (induction; 26.1% neutral detergent fiber, 5.8% FA, and 1.9% C18:2), resulting in a 30% decrease in milk fat yield. A recovery phase followed where all cows were switched to the high-forage, low-polyunsaturated FA diet and were allocated to (1) control (no inoculation) or (2) ruminal inoculation with donor cow digesta (8 kg/d for 6 d). Milk yield and composition were measured every 3 d. Milk yield progressively decreased during recovery. Milk fat concentration increased progressively during the recovery phase and no effect of treatment existed at any time point. Also, no treatment effect of milk fat yield was detected. The concentration of milk de novo FA increased progressively during recovery for both treatments and was higher for inoculated compared with control cows on d 6. In agreement, milk fat concentration of trans-10,cis-12 conjugated linoleic acid decreased progressively in both treatments and was lower in inoculated cows on d 3 and 6. Ruminal inoculation from non-milk fat-depressed cows did not change milk fat yield, but slightly accelerated the rate of recovery of de novo FA synthesis and normal ruminal FA biohydrogenation, demonstrating a possible opportunity for other interventions that improve the ruminal environment to accelerate recovery from this condition.     

A canonical discriminant analysis to study the association between milk fatty acids of ruminal origin and milk fat depression in dairy cows

Although milk fat depression (MFD) has been observed and described since the beginning of the last century, all the molecular and biochemical mechanisms involved are still not completely understood. Some fatty acids (FA) originating during rumen biohydrogenation have been proposed as causative elements of MFD. However, contradictory results were obtained when studying the effect of single FA on MFD. An alternative could be the simultaneous evaluation of the effect of many FA using a multivariate approach. The aim of this study was to evaluate the relationship between individual milk FA of ruminal origin and MFD using canonical discriminant analysis, a multivariate technique able to distinguish 2 or more groups on the basis of a pool of variables. In a commercial dairy herd, a diet containing 26% starch on a DM basis induced an unintentional MFD syndrome in 14 cows out of 40. Milk yielded by these 14 animals showed a fat content lower than 50% of the ordinary value, whereas milk production and protein content were normal. The remaining 26 cows secreted typical milk fat content and therefore were considered the control group, even though they ate the same diet. The stepwise discriminant analysis selected 14 milk FA of ruminal origin most able to distinguish the 2 groups. This restricted pool of FA was used, as variables, in a run of the canonical discriminant analysis that was able to significantly discriminate between the 2 groups. Out of the 14 FA, 5 conjugated linoleic acid isomers (C18:2 trans-10,trans-12, C18:2 trans-8,trans-10, C18:2 trans-11,cis-13, C18:2 cis-9,cis-11, C18:2 cis-10,cis-12) and C15:0 iso were more related to the control group, whereas C18:2 trans-10,cis-12, C16:1 trans-6–7, C16:1 trans-9, C18:1 trans-6–8, C18:1 trans-9, C18:1 trans-10, C18:1 cis-11, and C18:3n-3 were positively associated with the MFD group, allowing a complete discrimination. On the basis of these results, we can conclude that (1) the shift of ruminal biohydrogenation from C18:1 trans-11 to C18:1 trans-10 seemed to be strongly associated with MFD; (2) at the same time, other C18:1 trans isomers showed a similar association; (3) on the contrary, conjugated linoleic acid isomers other than C18:2 trans-10,cis-12 seemed to be associated with a normal fat secretion. Results confirmed that MFD is the consequence of a combined effect of the outflow of many ruminal FA, which collectively affect mammary fat synthesis. Because the animals of the 2 groups were fed the same diet, these results suggested that factors other than diet are involved in the MFD syndrome. Feeding behavior (i.e., ability to select dietary ingredients in a total mixed ration), rumen environment and the composition of ruminal bacteria are additional factors able to modify the products of rumen biohydrogenation. Results of the present work confirmed that the multivariate approach can be a useful tool to evaluate a metabolic pathway that involves several parameters, providing interesting suggestions about the role of some FA involved in MFD. However, results about the MFD syndrome obtained in the present research require a deep molecular investigation to be confirmed.     

In vitro ruminal biohydrogenation of eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic acid (DHA) in cows and ewes: Intermediate metabolites and pathways

A great deal of uncertainty still exists about intermediate metabolites and pathways explaining the biohydrogenation (BH) of 20- and 22-carbon polyunsaturated fatty acids (PUFA). Therefore, this study was conducted to provide further insight into the ruminal metabolism of 20:5 n-3 (EPA), 22:5 n-3 (DPA), and 22:6 n-3 (DHA), the main n-3 PUFA present in the marine lipids used in dairy ruminant feeding, and to examine potential differences between bovine and ovine. To meet this aim, we investigated the 20- and 22-carbon metabolites accumulated during in vitro incubation of EPA, DPA, and DHA with rumen inocula from cows and ewes. The PUFA were added at a dose of 2% incubated dry matter and digesta samples were analyzed after 24 h of incubation using complementary gas-liquid chromatography of fatty acid methyl esters and gas chromatography-mass spectrometry of 4,4-dimethyloxazoline derivatives. Results suggested that the main BH pathway of EPA and DPA would proceed via the reduction of the double bond closest to the carboxyl group (cis-5 in EPA and cis-7 in DPA); curiously, this mechanism seemed of much lower importance for DHA. Thus, DPA would not be a major intermediate product of DHA and their BH might actually follow separate pathways, with the accumulation of numerous unique metabolites in each case. A principal component analysis supported this hypothesis, with a clear separation between PUFA treatments in the score and loading plots. Within EPA and DPA groups, cow and ewe samples loaded separately from each other but not distant. No conjugated 20:5, 22:5, or 22:6 isomer compatible with the initial product of EPA, DPA, or DHA metabolism, respectively, was identified in the ruminal digesta, although this would not unequivocally exclude their transient formation. In this regard, results from DPA incubations provided the first indication that the metabolism of this very long chain PUFA may involve the formation of conjugated double bond structures. The BH of EPA, DPA, and DHA resulted in the appearance of several tentative trans-10–containing metabolites, showing a general trend to be more abundant in the digesta of ewes than in that of cows. This finding was speculated to have some relationship with the susceptibility of dairy sheep to marine lipid-induced milk fat depression. Differences in the relative proportion of intermediate products would also suggest an influence of ruminant species on BH kinetics, with a process that would likely be slower and less complete in cows than in ewes.