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.     

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.     

Differential effects of n-3 and n-6 fatty acids on prostaglandin F2alpha production by bovine endometrial cells

Recent studies have implicated n-3 polyunsaturated fatty acids in the reduction of eicosanoid production in the bovine uterus. The objective of this study was to determine whether the effect of eicosapentaenoic acid (EPA; C_20:5, n-3) on PGF_2α production by bovine endometrial (BEND) cells is influenced by the quantity of linoleic acid (C_18:2, n-6) in the incubation medium. Confluent BEND cells were incubated in the absence (control) or presence of 100 μMof EPA for 24 h. After incubation, cells were rinsed and then stimulated with phorbol 12,13-dibutyrate (PDBu; 100 ng/mL) for 6 h. Additional sets of culture dishes were treated with a combination of EPA and increasing n-6/n-3 fatty acid ratios for 24 h and then challenged with PDBu for 6 h. The PDBu stimulated PGF_2α secretion and upregulated steady-state concentrations of prostaglandin endoperoxide synthase-2 and peroxisome proliferator-activated receptor delta mRNA within 6 h. Preincubation of BEND cells with EPA for 24 h decreased PGF_2α response to phorbol ester, but had no detectable effects on prostaglandin endoperoxide synthase-2 or peroxisome proliferator-activated receptor delta mRNA abundance in PDBu-stimulated BEND cells. The inhibitory effect of EPA on PGF_2α production was reverted in BEND cells treated with an increasing n-6-to-n-3 fatty acid ratio. Findings indicate that the net inhibition of endometrial PGF_2α bioynthesis by n-3 fatty acids may vary depending on the ratio of n-6 to n-3 fatty acids in the uterus.     

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 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.     

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.     

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.     

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 pH and level of concentrate in the diet on the production of biohydrogenation intermediates in a dual-flow continuous culture

Milk fat depression in cows fed high-grain diets has been related to an increase in the concentration of trans-10 C_18:1 and trans-10,cis-12 conjugated linoleic acid (CLA) in milk. These fatty acids (FA) are produced as a result of the alteration in rumen biohydrogenation of dietary unsaturated FA. Because a reduction in ruminal pH is usually observed when high-concentrate diets are fed, the main cause that determines the alteration in the biohydrogenation pathways is not clear. The effect of pH (6.4 vs. 5.6) and dietary forage to concentrate ratios (F:C; 70:30 F:C vs. 30:70 F:C) on rumen microbial fermentation, effluent FA profile, and DNA concentration of bacteria involved in lipolysis and biohydrogenation processes were investigated in a continuous culture trial. The dual-flow continuous culture consisted of 2 periods of 8 d (5 d for adaptation and 3 d for sampling), with a 2 × 2 factorial arrangement of treatments. Samples from solid and liquid mixed effluents were taken for determination of total N, ammonia-N, and volatile fatty acid concentrations, and the remainder of the sample was lyophilized. Dry samples were analyzed for dry matter, ash, neutral and acid detergent fiber, FA, and purine contents. The pH 5.6 reduced organic matter and fiber digestibility, ammonia-N concentration and flow, and crude protein degradation, and increased nonammonia and dietary N flows. The pH 5.6 decreased the flow of C_18:0, trans-11 C_18:1 and cis-9, trans-11 CLA, and increased the flow of trans-10 C_18:1, C_18:2n-6, C_18:3n-3, trans-11,cis-15 C_18:2 and trans-10,cis-12 CLA in the 1 h after feeding effluent. The pH 5.6 reduced Anaerovibrio lipolytica (32.7 vs. 72.1 pg/10 ng of total DNA) and Butyrivibrio fibrisolvens vaccenic acid subgroup (588 vs. 1,394 pg/10 ng of total DNA) DNA concentrations. The high-concentrate diet increased organic matter and fiber digestibility, nonammonia and bacterial N flows, and reduced ammonia-N concentration and flow. The high-concentrate diet reduced trans-11 C_18:1 and trans-10 C_18:1, and increased C_18:2n-6, C_18:3n-3 and trans-10,cis-12 CLA proportions in the 1 h after feeding effluent. The increase observed in trans-10,cis-12 CLA proportion in the 1 h after feeding effluent due to the high-concentrate diet was smaller that that observed at pH 5.6. Results indicate that the pH is the main cause of the accumulation of trans-10 C_18:1 and trans-10, cis-12 CLA in the effluent, but the trans-10,cis-12 CLA proportion can be also affected by high levels of concentrate in the diet.     

Effect of in vitro docosahexaenoic acid supplementation to marine algae-adapted and unadapted rumen inoculum on the biohydrogenation of unsaturated fatty acids in freeze-dried grass

The objective of this study was to examine the ruminal biohydrogenation of linoleic (18:2n-6) and linolenic (18:3n-3) acid during in vitro incubations with rumen inoculum from dairy cattle adapted or not to marine algae and with or without additional in vitro docosahexaenoic acid (DHA, 22:6n-3) supplementation. Treatments were incubated in 100-mL flasks containing 400 mg of freeze-dried grass, 5 mL of strained ruminal fluid, and 20 mL of phosphate buffer. Ruminal fluid was collected just before the morning feeding from 3 cows receiving a control diet (49% ryegrass silage, 39% corn silage, 1% straw, and 11% concentrate, fresh-weight basis) supplemented with marine algae for 21 d (adapted rumen fluid, aRF) or from the same cows receiving the control diet only for 14 d after marine algae supplementation was stopped (unadapted rumen fluid, uRF). In half of the incubation flasks, pure DHA (5 mg) was added as an oil-ethanol solution (100 mL). Incubations were carried out during 0, 0.5, 1, 2, 4, 6, and 24 h. After 24 h, in vitro addition of DHA resulted in greater amounts (mg/incubation) of 18:3n-3 (0.23, 0.43, 0.26, and 0.34 for aRF, aRF+DHA, uRF, and uRF+DHA), 18:2n-6 (0.14, 0.22, 0.15, and 0.20 for aRF, aRF+DHA, uRF, and uRF+DHA) and trans-11, cis-15-18:2 (0.27, 2.40, 0.06, and 2.21 for aRF, aRF+DHA, uRF, and uRF+DHA), whereas no effect of inoculum source was observed. Trans-11-18:1 accumulated after 24 h when aRF was incubated irrespective of in vitro DHA supplementation, whereas in incubations with uRF, accumulation of trans-11-18:1 only occurred when DHA was added (6.40, 4.35, 1.06, and 3.91 for aRF, aRF+DHA, uRF, and uRF+DHA). The increased amounts of trans-11-18:1 were due to the strong inhibition of the reduction to 18:0 because no 18:0 was formed when trans-11-18:1 accumulated after 24 h. The results of the current experiment shows hydrogenation of trans-11, cis-15-18:2 occurred in the absence of in vitro DHA only, whereas substantial hydrogenation of trans-11-18:1 to 18:0 only took place in incubations without DHA and with unadapted rumen inoculum, confirming the higher sensitivity of the latter process to DHA.     

Efficacy of a novel whey protein gel complex to increase the unsaturated fatty acid composition of bovine milk fat

A novel whey protein emulsion gel (WPEG) complex was developed to protect dietary unsaturated fatty acids from rumen biohydrogenation with the goal of modifying the fatty acid composition of milk fat. Three experiments were conducted with WPEG complexes made from either whey protein concentrate containing 80% crude protein, whey protein isolate, or whey protein concentrate high-gel capacity. Each experiment lasted 3 wk. All cows received a basal total mixed ration (TMR). During wk 1 and 3, all cows received only the TMR. During wk 2, 3 control cows received 330 g/d of soybean oil added to the TMR, and the other 3 cows received 330 g/d of soybean oil in one of the WPEG complexes. During wk 2, C18:2 increased from 3.29 to 5.88 g/100 g of fat in Experiment 1, 2.91 to 7.42 g/100 g of fat in Experiment 2, and 3.57 to 6.56 g/100 g of fat in Experiment 3 for WPEG cows. Fatty acid C18:3 increased from 0.51 to 0.84, 0.52 to 1.15, and 0.51 to 0.97 g/100 g of fat for Experiments 1, 2, and 3, respectively, for WPEG cows. Higher proportions of C18:1 trans-9 in milk fat of control cows compared with WPEG cows were seen in all experiments. The proportion of C18:1 trans-11 was also higher in control cows in Experiments 1 and 2, but not in Experiment 3. The WPEG complexes successfully protected unsaturated fatty acids from rumen biohydrogenation and resulted in an increase in the unsaturated fatty acid composition of milk fat produced by Holstein cows without increasing the trans 18-carbon monoenes.     

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.     

Effects of trans fatty acids on markers of inflammation in bovine mammary epithelial cells

Trans fatty acids (tFA) contribute to inflammation. The objective was to investigate the effects of tFA on mRNA expression of proinflammatory markers in cultured bovine mammary epithelial cells (MAC-T cell line). Bovine mammary epithelial cells were grown in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum. Cells were then subcultured in a medium lacking fetal bovine serum, to which incremental concentrations (up to 90 μM) of elaidic acid (trans-9 C18:1) or linoleidic acid (trans-9, trans-12 C18:2) were added. Bovine serum albumin (fatty acid-free) solutions were added and cells were collected at specific time points over 48 h. Then, RNA was extracted and converted to complementary DNA for quantitative real-time PCR analysis of proinflammatory gene expression. Presence of elaidic acid caused increases in mRNA expression of interleukin (IL)-1β (3.4-fold; dose-independently over a 6-h period) and intercellular adhesion molecule (ICAM)-1 (up to 1.4-fold) relative to that for cells treated with no tFA, whereas expression of IL-6 and IL-8 was reduced 0.75- and 0.85-fold, respectively. Presence of linoleidic acid reduced mRNA expression of IL-6 and IL-8 relative to that for control (0.95- and 0.87-fold, respectively). Trans mono- and dienoic fatty acids upregulated mRNA expression of IL-1β and ICAM-1, whereas expression of IL-6 and IL-8 was downregulated in MAC-T cells. Because these genes are ultimately involved in inflammation, elaidic or linoleidic acid, either directly fed or formed in the rumen during biohydrogenation, may alter the risk for mastitis in vivo.     

Dilution rate and pH effects on the conversion of oleic acid to trans C18:1 positional isomers in continuous culture

In a previous in vitro study, mixed ruminal microorganisms converted oleic acid to a variety of trans monenes when grown in batch cultures under constant environmental conditions. To determine whether a similar conversion occurs under environmental conditions more typical of the rumen, conversion of ¹³C-labeled oleic acid to biohydrogenation intermediates was determined in ruminal microorganisms grown in continuous culture at two pH (5.5 and 6.5) and liquid dilution rates (0.05 and 0.10/h) arranged factorially. After each morning feeding of the dual-flow continuous cultures, 250 mg of oleic acid in 5 mL of ethanol were injected into each culture. On d 10, 250 mg of oleic-1-¹³C replaced the unlabelled oleic acid in ethanol. Trans fatty acids were isolated from culture samples by solid phase extraction, and ¹³C enrichment and identity of double bond position was determined by gas chromatography-mass spectroscopy. At pH 6.5 and 0.10/h dilution rate, ¹³C enrichment was detected in all trans-C_18:1 isomers having double bond positions from C₆ through C₁₆ in the acyl chain. However, when pH or dilution rate in fermentors was lowered, no ¹³C enrichment was detected in any trans isomer with a double bond position beyond C₁₀. Enrichment in stearic acid increased by reducing culture pH from 6.5 to 5.5, but decreased when dilution rate dropped from 0.10 to 0.05/h. The stearic acid carbons that originated from oleic acid biohydrogenation increased from 30 to 72% when pH dropped from 6.5 to 5.5. The ¹³C enrichment of trans-10 was reduced under low pH and dilution rate conditions. The results of this study confirm that ruminal microorganisms are capable of converting oleic acid to a wide variety of trans-C_18:1 positional isomers when ruminal conditions are favorable (such as the pH 6.5 and 0.10/h dilution rate treatment). However, at low pH and dilution rate, the conversion of oleic acid to trans-C_18:1 still occurs, but positional isomers produced are restricted to double bond positions from C₆ to C₁₀. Low pH conditions also increased the conversion of oleic acid to stearic acid.     

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.     

Effect of different rumen-inert fatty acids supplemented with a dietary antioxidant on performance and antioxidative status of early-lactation cows

The objective of the study was to evaluate the effect of diets supplemented with fatty acids of different degrees of saturation, in the absence or presence of an antioxidant (AOX; Agrado Plus, Novus International Inc., St. Charles, MO), on dairy cow lactation performance. Calcium salts of long-chain fatty acids were supplemented as a source of lower saturation fatty acid, and a palm acid product was supplemented as the higher saturation fatty acid source. Sixty early-lactation Chinese Holstein cows (100 ± 23 d in milk) were randomly allocated to 4 dietary treatments in a 2 × 2 factorial design: (1) lower saturation fatty acid (LS), (2) LS and AOX, (3) higher saturation fatty acid (HS), and (4) HS and AOX. The Ca salts of long-chain fatty acids and palm acid product were supplied at 1.8 and 1.5% on a dry matter basis, respectively, to form isoenergetic diets. The AOX was added at 0.025% in the ration. The experiment lasted 9 wk, including 1 wk for adaptation. Lactation performance was recorded and milk was sampled and analyzed weekly. Blood samples were taken from the coccygeal vein to determine metabolism parameters on d 16, 36, and 56 during the experiment. Neither fatty acid type nor AOX supplementation showed a significant effect on dry matter intake during the study. Milk yield was lower in the LS-fed cows compared with the cows fed HS. Milk fat and milk protein concentrations were not affected by fatty acid type or AOX supplementation. Adding AOX increased the yield of milk in the LS-fed cows, but did not affect those fed HS. Activity of plasma superoxide dismutase was significantly lower, plasma glucose tended to be lower, and plasma malondialdehyde was higher in the LS-fed animals compared with those fed HS. Addition of AOX decreased both plasma nonesterified fatty acids and hydrogen peroxide contents and increased total antioxidant capacity across the fatty acid types. Plasma β-hydroxybutyrate was not affected by fatty acid type or AOX treatment. Cows fed LS had higher cis-9 C_18:1 and trans-10, cis-12 C_18:2 in milk at the expense of C_18:0, whereas AOX addition increased milk cis-9 C_18:1 at the expense of milk C_12:0, C_16:0, and trans-10, cis-12 C_18:2. It is inferred that feeding LS resulted in inferior lactation performance, whereas addition of antioxidant partially alleviated these negative effects.     

Isolation and characterization of acid-sensitive mutants of Pediococcus acidilactici

Acid-sensitive mutants of Pediococcus acidilactici BCC 9545, a starter culture of the Thai fermented pork sausage nham, were isolated as spontaneous neomycin resistant mutants. The mutants generally produced less acid and acidified the culture media less than the parent strain in a 72 h culturing period. Interestingly, the ATPase activities of the mutants did not differ considerably from that of the parent strain in acidic conditions. It was also found that the internal pH values of the mutant strains were somewhat lower in neutral environment, while at pH 5.0 their internal pHs were significantly lower compared to the parent's. Inhibiting the H⁺-ATPase activities in energized cells by N,N′-dicyclohexyl carbodiimide also revealed that protons were leaking from the mutants at neutral pH, which increased under acidic conditions. In contrast, the parent strain exhibited a smaller proton leak and only under acidic conditions. The membrane fatty acid analysis of the mutants indicated that under acidic conditions the mutants had a significantly smaller major unsaturated/saturated fatty acids ratio ((C_18:1 + C_18:3n6)/(C_16:0 + C_18:0)) compared to the parent strain's membrane. Taken together, these observations suggest there is a reasonable possibility that the membrane fatty acid profile differences in the mutants resulted in their acid-sensitivity.     

A simple method for the analysis of trans fatty acid with GC–MS and AT-Silar-90 capillary column

The study was aimed to evaluate a simplified gas chromatography method based on the AOAC method 996.06 to analyze the trans fat content in food samples. The gas chromatograph was equipped with mass spectrometer and Alltech AT^™-Silar-90 capillary column. Ten kinds of the trans fatty acid standard were separated completely from the cis standard and the chemical composition of the peaks was verified by using the mass spectrum. Under the optimized conditions, the recovery rate for triheptadecanoin was 99.0%, the correlation coefficients of trans fatty acid calibration curve was 0.9998 or higher. It demonstrated that the methylation and hexane extraction procedures used in this method was effective and the result was consistent. The major fatty acids found in the shortening sample were 16:0, 18:0, trans-18:1, cis-18:1, cis-18:2, and cis-18:3. The total trans fat content in the sample was 283.6 ± 18.2 mg/g. The current method was more convenient. It is adequate for the routine analysis of trans fat content in food products with low free fatty acid content.     

Effect of abomasal infusions of a mixture of octadecenoic acids on milk fat synthesis in lactating cows

Diets causing milk fat depression (MFD) are known to alter ruminal lipid metabolism leading to the formation of specific biohydrogenation intermediates that exert antilipogenic effects. Several isomers of conjugated linoleic acid (CLA), namely trans-10, cis-12 CLA, cis-10, trans-12 CLA, and trans-9, cis-11 CLA, inhibit mammary lipogenesis in the lactating cow, but ruminal outflow of these biohydrogenation intermediates does not account entirely for the reductions in milk fat synthesis during diet-induced MFD. Milk fat trans-10 18:1 concentrations are consistently increased on diets that cause MFD, suggesting a possible role in the regulation of milk fat secretion. Three rumen-fistulated cows in mid lactation were used in a 3 × 3 Latin square to evaluate the effects of a mixture of 18:1 fatty acid methyl esters (FAME) on milk fat synthesis. Experimental treatments consisted of abomasal infusions of ethanol (control), 6 g/d of trans-10, cis-12 CLA (positive control; CLA), or 247 g/d of a mixture of 18:1 FAME containing (% fatty acids) cis-9 (9.45), cis-12 (3.35), trans-10 (37.3), trans-11 (37.4), and trans-12 (2.66) as major isomers (T181 treatment). Administration of the T181 treatment supplied 92.1 g/d of trans-10 18:1. Infusions were conducted over a 5-d period with a 9-d interval between treatments. Treatments had no effect on dry matter intake, milk yield, or milk protein. Relative to the control, abomasal infusion of T181 and trans-10, cis-12 CLA treatments reduced milk fat secretion by 19.5 and 41.5%, respectively. Even though a direct cause and effect on mammary lipogenesis could not be established, comparisons with published data and considerations of the relative abundance of constituent FAME in treatment T181 implicated trans-10 18:1 as the isomer responsible. In conclusion, current data suggest that trans-10 18:1 potentially exerts antilipogenic effects and may contribute to the reduction in milk fat synthesis during diet-induced MFD in the lactating cow.