Role of the protozoan Isotricha prostoma, liquid-, and solid-associated bacteria in rumen biohydrogenation of linoleic acid

From the simultaneous accumulation of hydrogenation intermediates and the disappearance of Isotricha prostoma after algae supplementation, we suggested a role of this ciliate and/or its associated bacteria in rumen biohydrogenation of unsaturated fatty acids. The experiments described here evaluated the role of I. prostoma and/or its associated endogenous and exogenous bacteria in rumen biohydrogenation of C18:2n-6 and its main intermediates CLA c9t11 and C18:1t11. Fractions of I. prostoma and associated bacteria, obtained by sedimentation of rumen fluid sampled from a monofaunated sheep, were used untreated, treated with antibiotics or sonicated to discriminate between the activity of I. prostoma and its associated bacteria, the protozoan or the bacteria, respectively. Incubations were performed in triplicate during 6 h with unesterified C18:2n-6, CLA c9t11 or C18:1t11 (400 μg/ml) and 0.1 g glucose/cellobiose (1/1, w/w). I. prostoma did not hydrogenate C18:2n-6 or its intermediates whereas bacteria associated with I. prostoma converted a limited amount of C18:2n-6 and CLA c9t11 to trans monoenes. C18:1t11 was not hydrogenated by either I. prostoma or its associated bacteria but was isomerized to C18:1c9. A phylogenetic analysis of clones originating from Butyrivibrio-specific PCR product was performed. This indicated that 71% of the clones from the endogenous and exogenous community clustered in close relationship with Lachnospira pectinoschiza. Additionally, the biohydrogenation activity of solid-associated bacteria (SAB) and liquid-associated bacteria (LAB) was examined and compared with the activity of the non-fractioned I. prostoma monofaunated rumen fluid (LAB + SAB). Both SAB and LAB were involved in rumen biohydrogenation of C18:2n-6. SAB fractions performed the full hydrogenation reaction to C18:0 while C18:1 fatty acids, predominantly C18:1t10 and C18:1t11, accumulated in the LAB fractions. SAB and LAB sequence analyses were mainly related to the genera Butyrivibrio and Pseudobutyrivibrio with 12% of the SAB clones closely related to the C18:0 producing B. proteoclasticus branch. In conclusion, this work suggests that I. prostoma and its associated bacteria play no role in C18:2n-6 biohydrogenation, while LAB convert C18:2n-6 to a wide range of C18:1 fatty acids and SAB produce C18:0, the end product of rumen lipid metabolism.     

9 cis,11 trans conjugated linoleic acid (CLA) is synthesised and desaturated into conjugated 18:3 in bovine adipose tissues

Although endogenous synthesis of conjugated linoleic acid (CLA) in the mammary gland of lactating cows has been already well documented, no study has determined so far as to which tissue and/or organ is involved in CLA synthesis in the growing ruminant except one study showing that CLA synthesis does not occur in ruminant liver. In this context, adipose tissue appears to be a good candidate for endogenous synthesis of CLA in the growing ruminant. The aim of this study was to compare the respective metabolisms of 11trans 18:1 (vaccenic acid, VA) and 9cis,11trans 18:2 (rumenic acid) to that of stearic acid (the preferential substrate of Δ9 desaturase) in adipose tissues (subcutaneous, SC and intermuscular, IM) of six Charolais steers by using the in vitromethod of incubated tissue slices. Samples of SC and IM adipose tissues were incubated at 37°C for 16 h under an atmosphere of 95% O2/5% CO2 in a medium supplemented with 0.75 mM of fatty acid (FA) mixture (representative of circulating non-esterified FA) and 186 μM [1-14C]-18:0 or 58.6 μM [1-14C]-VA or 56 μM [1-14C]-9cis,11trans CLA. Viability of explants was verified by measuring metabolic functions (glucose uptake and glucose-6-phosphate dehydrogenase activity). After 16 h of incubation, FA uptake was similar for all FA (18:0, VA and 9cis,11trans 18:2) in both SC and IM adipose tissues (around 40%). Once in adipose tissue, all FA were preferentially esterified (>80% of cell FA) favouring neutral lipid synthesis (around 90% of esterified FA). Stearic acid was highly (27%) desaturated into oleic acid in SC adipose tissue whereas this desaturation was much lower (6.8%) in IM adipose tissue (P < 0.0001). VA was desaturated into 9cis,11trans CLA at a low extent of about 2.5% to 4.4% in both adipose tissues probably because of a limited affinity of Δ9 desaturase for VA. 9cis,11trans CLA was itself converted by desaturation into 6cis, 9cis,11trans 18:3 at the intensity of 10.8% and 14.5% of cell 9cis,11trans CLA in SC and IM adipose tissues, respectively. In conclusion, bovine adipose tissues of the growing ruminant were especially involved in the endogenous synthesis of CLA from VA and in its desaturation into conjugated derivative, mainly 6cis, 9cis,11trans 18:3, of which biological properties need to be elucidated.     

Effect of silage type and concentrate level on conjugated linoleic acids, trans-C18:1 isomers and fat content in milk from dairy cows

The objective of the study was to examine how the fatty acid composition of milk especially concentrations of conjugated linoleic acids (CLA) and trans-C18:1 isomers and milk fat percentage were affected by silage type and concentrate level. Forty dairy cows were blocked and randomly assigned to one of four diets in a 2 x 2 factorial arrangement of treatments and a six week experimental period. Treatments were total mixed rations with maize (M) or grass (G) silage differing in polyunsaturated fatty acid (PUFA) profile and starch content, combined with a high (H) or a low (L) level of concentrate (with or without grain). Treatments had no significant effect on milk, protein and lactose yield, but energy corrected milk yield, milk fat percentage and fat yield was lower and protein percentage higher for maize compared with grass silage diets. Overall, maize silage diets resulted in higher concentrations of CLA isomers compared with grass silage diets, but there was a significant interaction between silage type and concentrate level for concentrations of cis9,trans11-CLA; trans10,cis12-CLA; trans11-C18:1 and trans10-C18:1. A high level of concentrate increased trans10,cis12-CLA and trans10-C18:1 and reduced cis9,trans11-CLA and trans11-C18:1 when maize but not grass silage was provided. The results suggest that high levels of concentrate (grain) do not significantly alter the pattern of PUFA biohydrogenation in the rumen, the concentration of CLA and trans-C18:1 isomers in milk or cause milk fat depression unless combined with forage naturally high in starch and C18:2n-6 such as maize silage.     

Effects of olive and fish oil Ca soaps in ewe diets on milk fat and muscle and subcutaneous tissue fatty-acid profiles of suckling lambs

Enhancing healthy fatty acids (FAs) in ewe milk fat and suckling lamb tissues is an important objective in terms of improving the nutritional value of these foods for the consumer. The present study examined the effects of feeding-protected lipid supplements rich in unsaturated FAs on the lipid composition of ewe milk, and subsequently in the muscle and subcutaneous adipose tissues of lambs suckling such milk. Thirty-six pregnant Churra ewes with their new-born lambs were assigned to one of three experimental diets (forage/concentrate ratio 50 : 50), each supplemented with either 3% Ca soap FAs of palm (Control), olive (OLI) or fish (FO) oil. The lambs were nourished exclusively by suckling for the whole experimental period. When the lambs reached 11 kg BW, they were slaughtered and samples were taken from the Longissimus dorsi and subcutaneous fat depots. Although milk production was not affected by lipid supplementation, the FO diet decreased fat content (P<0.001), whereas the OLI milk FA profile resembled that of the Control diet. In contrast, although FO drastically diminished the contents of stearic and oleic acids (P<0.001), all the saturated even-numbered carbon FAs from 6:0 to 14:0 increased (P<0.05). FO also produced the highest levels of c9,t11-18:2 (2.21%) and n-3 FAs, 20:5 n-3 (0.58%), 22:5 n-3 (0.48%) and 22:6 n-3 (0.40%). The high levels of trans-11 18:1 (7.10%) obtained from the FO diet would suggest that Ca soaps only confer partial protection in the rumen. In contrast, the lack of significant differences in trans-10 18:1 levels (P>0.05) and other trans-FAs between Control and FO treatments would indicate that FO treatment does not alter rumen biohydrogenation pathways under the assayed conditions. Changes in dam milk FA composition induced differences in the FA profiles of meat and fat depots of lambs, preferentially incorporated polyunsaturated FAs into the muscle rather than storing them in the adipose tissue. In the intramuscular fat of the FO treatment, all the n-3 FAs reached their highest concentrations: 0.97 (18:3 n-3), 2.72 (20:5 n-3), 2.21 (22:5 n-3) and 1.53% (22:6 n-3). In addition, not only did FO intramuscular fat have the most cis-9, trans-11 18:2 (1.66%) and trans-11 18:1 (3.75%), but also the lowest n-6/n-3 ratio (1.80) and saturated FA content were not affected. Therefore, FO exhibited the best FA profile from a nutritional point of view.     

Online prediction of fatty acid profiles in crossbred Limousin and Aberdeen Angus beef cattle using near infrared reflectance spectroscopy

The objective of this study was to examine the online use of near infrared reflectance (NIR) spectroscopy to estimate the concentration of individual and groups of fatty acids (FA) as well as intramuscular fat (IMF) in crossbred Aberdeen Angus (AA×) and Limousin (LIM×) cattle. This was achieved by direct application of a fibre-optic probe to the muscle immediately after exposing the meat surface in the abattoir at 48 h post mortem. Samples of M. longissimus thoracis from 88 AA× and 106 LIM× were scanned over the NIR spectral range from 350 to 1800 nm and samples of the M. longissimus lumborum were analysed for IMF content and FA composition. Statistically significant differences (P < 0.001) were observed in most FA between the two breeds studied, with FA concentration being higher in AA× meat mainly. NIR calibrations, tested by cross-validation, showed moderate to high predictability in LIM× meat samples for C16:0, C16:1, C18:0, trans11 C18:1, C18:1, C18:2 n-6, C20:1, cis9, trans11 C18:2, SFA (saturated FA), MUFA (monounsaturated FA), PUFA (polyunsaturated FA) and IMF content with R(2) (SE(CV), mg/100 g muscle) of 0.69 (146), 0.69 (28), 0.71 (62), 0.70 (8.1), 0.76 (192), 0.65 (13), 0.71 (0.9), 0.71 (2.9), 0.68 (235), 0.75 (240), 0.64 (17) and 0.75 (477), respectively. FA such as C14:0, C18:3 n-3, C20:4 n-6, C20:5 n-3, C22:6 n-3, n-6 and n-3 were more difficult to predict by NIR in these LIM× samples (R(2) = 0.12 to 0.62; SECV = 0.5 to 26 mg/100 g muscle). In contrast, NIR showed low predictability for FA in AA× beef samples. In particular for LIM×, the correlations of NIR measurements and several FA in the range from 0.81 to 0.87 indicated that the NIR spectroscopy is a useful online technique for the early, fast and relatively inexpensive estimation of FA composition in the abattoir.     

Effect of replacing calcium salts of palm oil distillate with incremental amounts of conventional or high oleic acid milled rapeseed on milk fatty acid composition in cows fed maize silage-based diets

Based on potential benefits to human health, there is increasing interest in altering the composition of ruminant-derived foods. Including rapeseeds in the dairy cow diet is an effective strategy for replacing medium-chain saturated fatty acids (SFA) with cis-monounsaturated fatty acids (MUFA) in bovine milk, but there is limited information on the optimum level of supplementation. Decreases in SFA due to plant oils are also accompanied by increases in milk trans fatty acid (FA) content and it is possible that high oleic acid rapeseeds may result in a higher enrichment of cis-9 18:1 and lower increases in trans FAs in milk compared with conventional varieties. Seven multiparous lactating Holstein-Friesian cows were allocated to one of seven treatments in an incomplete Latin square design with five 28-day experimental periods, to evaluate the effect of replacing calcium salts of palm oil distillate (CPO; 41 g/kg diet dry matter, DM) with 128, 168 or 207 g/kg diet DM of conventional (COR) or a high oleic acid (HOR) rapeseed fed as a supplement milled with wheat. Rapeseed variety and inclusion level had no effect (P > 0.05) on DM intake, milk yield and composition. Both rapeseed varieties decreased linearly (P < 0.001) milk fat SFA content, which was partially compensated for by a linear increase (P < 0.001) in cis-9 18:1 concentration. Reductions in milk SFA were also associated with increases (P < 0.05) in trans 18:1 and total trans FA content, with no difference (P > 0.05) between rapeseed varieties. Replacing CPO in the diet with milled rapeseeds had no effect (P > 0.05) on total milk conjugated linoleic acid (CLA) concentration. Relative to a COR, inclusion of a high oleic acid variant in the diet increased (P = 0.01) the ratio of trans-MUFA : trans-polyunsaturated fatty acids in milk that may have implications with respect to cardiovascular disease risk in humans. In conclusion, data indicated that replacing CPO with milled rapeseeds at levels up to 1150 g oil/day could be used as a nutritional strategy to lower milk SFA content without inducing adverse effects on DM intake and milk production. HOR reduced milk fat SFA content to a greater extent than a conventional variety, but did not minimise associated increases in trans FA concentrations. However, the high oleic acid variant did alter the relative abundance of specific trans 18:1, CLA and trans 18:2 isomers compared with conventional rapeseeds.     

Using microbial fatty acids to improve understanding of the contribution of solid associated bacteria to microbial mass in the rumen

This study sought to distinguish liquid-(LAB) and detached (SAB₁) and undetached (SAB₂) solid-associated bacteria through their fatty acid (FA) and purine base (PB) profiles. Fatty acids and PB were also evaluated as internal microbial markers for estimating microbial biomass associated with rumen particles. Four merino rams fitted with rumen cannulae and fed dehydrated alfalfa pellets provided rumen contents. In 3 consecutive weeks, rumen contents were collected and samples of LAB and SAB₁, total rumen content (TRC), washed rumen particles (WRP) and rumen particles after SAB₁ extraction (ERP) were obtained and analysed for PB and FA. The SAB₂ biomass composition was estimated from the non-NDF organic matter (OM) remaining in ERP. The concentration of total SAB biomass in particles was estimated using both PB and odd and branched-chain fatty acids (OBCFA). Concentrations of PB and OBCFA were highly correlated among the different rumen fractions. Marked differences between LAB and SAB populations occurred with LAB having higher PB content, lower FA content and a higher proportion (g/100 g fatty acids) of OBCFA than did SAB. The chemical composition of SAB₁ and SAB₂ was similar, except for the 15% higher crude protein content of the latter. The concentration of OBCFA (mg/g microbial OM) did not differ between bacterial fractions. The PB/OBCFA ratio (mg/mg) was higher in LAB (2.08) than in SAB (0.94). The ratio between branched-chain and odd-linear-chain FA was higher in LAB (2.26) than in SAB (1.46). Extraction of PB and OBCFA from WRP with our SAB detachment procedure was 61% and 31%, respectively. Estimated SAB₁ and total SAB biomass (mg OM/g WRP) were 158 and 266, and 47 and 164, respectively, using PB and OBCFA as microbial markers. This study suggests that the OBCFA have potential as internal microbial markers in rumen ecosystem studies.     

Effects of supplementation of maize silage diets with extruded linseed, vitamin E and plant extracts rich in polyphenols, and morning v. evening milking on milk fatty acid profiles in Holstein and Montbéliarde cows

The aim of this study was to evaluate the effects on dairy performance and milk fatty acid (FA) composition of (i) supplementation with extruded linseed (EL), (ii) supplementation with synthetic or natural antioxidants, namely vitamin E and plant extracts rich in polyphenols (PERP), (iii) cow breed (Holstein v. Montbéliarde) and (iv) time of milking (morning v. evening). After a 3-week pre-experimental period 24 lactating cows (12 Holstein and 12 Montbéliarde) were divided up into four groups of six cows: the first group received a daily control diet (diet C) based on maize silage. The second group received the same diet supplemented with EL (diet EL, fat level approximately 5% of dietary dry matter (DM)). The third group received the EL diet plus 375 IU/kg diet DM of vitamin E (diet ELE). The fourth group received the ELE diet plus 10 g/kg diet DM of a PERP mixture (diet ELEP). Compared with the diet C, feeding EL-rich diets led to lower concentrations of total saturated FA (SFA) and higher concentrations of stearic and oleic acids, each trans and cis isomer of 18:1 (except c12-18:1), non-conjugated isomers of 18:2, some isomers (c9t11-, c9c11- and t11t13-) of conjugated linoleic acid (CLA), and 18:3n-3. The vitamin E supplementation had no effect on milk yield, milk fat or protein percentage and only moderate effects on milk concentrations of FA (increase in 16:0, decreases in 18:0 and t6/7/8-18:1). The addition of PERP to vitamin E did not modify milk yield or composition and slightly altered milk FA composition (decrease in total saturated FA (SFA) and increase in monounsaturated FA (MUFA)). The minor effects of vitamin E may be partly linked to the fact that no milk fat depression occurred with the EL diet. During both periods the Holstein cows had higher milk production, milk fat and protein yields, and milk percentages of 4:0 and 18:3n-3, and lower percentages of odd-branched chain FA (OBCFA) than the Montbéliarde cows. During the experimental period the Holstein cows had lower percentages of total cis 18:1, and c9,c11-CLA, and higher percentages of 6:0, 8:0, t12-, t16/c14- and t13/14-18:1, and 18:2n-6 than Montbéliarde cows because of several significant interactions between breed and diet. Also, the total SFA percentage was higher for morning than for evening milkings, whereas those of MUFA, total cis 18:1, OBCFA and 18:2n-6 were lower. Extruded linseed supplementation had higher effect on milk FA composition than antioxidants, breed or time of milking.     

Influence of an increase in diet structure on milk conjugated linoleic acid content of cows fed extruded linseed

This experiment studied the effect of a modest difference in diet structure value (SV) on milk conjugated linoleic acid (CLA) contents of cows fed diets supplemented with extruded linseed, in situations where the diets provided enough SV and therefore did not induce milk fat depression. Six lactating Holstein cows were used in a crossover design with two treatments ('SV 1.50' and 'SV 1.73') and two periods of 21 days. The 'SV 1.50' diet contained 59% maize silage, 13% soya bean meal, 13% sugar beet pulp and 14% Nutex Compact (containing 56% extruded linseed) (dry matter (DM) basis) and was offered as a restricted total mixed ration. For the 'SV 1.73' diet, 8% wheat straw (DM basis) was added to the 'SV 1.50' diet as an additional structure source. The two diets had a forage-to-concentrate ratio of 59 : 41 and 62 : 38. The inclusion of straw in the diet resulted in an additional intake of NDF (+1110 g/day), which accounted for 90% of the additional intake of OM, whereas additional intakes of the other nutrients were minor. Milk yield and composition did not differ among treatments. The inclusion of straw in the diet did not affect the milk levels of t10-18:1, 18:2n-6, c9-16:1, c9-18:1, c11-18:1, 6:0, 8:0, 20:4 and 20:5. It decreased the milk levels of c9,t11-CLA (2.13% v. 3.03% of fatty acids (FA) reported, P < 0.001), t11-18:1 (4.99% v. 7.10% of FA reported, P < 0.001), 18:3n-3, t9-16:1 and t9-18:1, while it increased the milk levels of 6:0-14:0 (20.90% v. 19.69% of FA reported, P < 0.01), 16:0 (26.55% v. 25.25% of FA reported, P < 0.01), 18:0 (13.54% v. 12.59% of FA reported, P < 0.001), 17:0, 20:0 and 22:5. Regarding the ratio between FA, the inclusion of straw increased the 18:0/total C18 FA ratio (37.74% v. 32.07%, P < 0.001), whereas it decreased the total trans-C18 FA/total C18 FA ratio (15.46% v. 20.34%, P < 0.001), the t11-18:1/total C18 FA ratio (13.70% v. 17.95%, P < 0.01) and the c9,t11-CLA/total C18 FA ratio (5.82% v. 7.64%, P < 0.001). We conclude from this experiment that even a modest increase in SV to a diet supplemented with extruded linseed, yet already providing enough SV, alters the rumen lipid metabolism and, hence, CLA levels in milk fat.     

Effect of botanical composition of silages on rumen fatty acid metabolism and fatty acid composition in longissimus muscle and subcutaneous fat of lambs

To study the effect of feeding silages with different botanical composition, on rumen and lamb fat, 30 male lambs were assigned to five different silage groups for 11 weeks: botanically diverse silage (BDS); white clover silage (WCS); red clover silage (RCS), intensive English ryegrass silage (IRS) and crushed linseed and maize silage (MSL). Besides the silages, animals received organic wheat and barley and the MSL group additionally received bicarbonate (15 g/day). Silages were sampled when the bales were opened and analysed for fatty acid (FA) content and chemical composition. At slaughter, ruminal contents were sampled and 24 h after slaughter, longissimus muscle and subcutaneous (SC) fat were sampled. All samples were analysed for FA composition. The MSL group ingested the highest amount of FA (35.8 g/day v. 13.5, 19.4, 17.2 and 30.4 g/day for MSL v. BDS, WCS, RCS and IRS, respectively) and the sum of the major polyunsaturated FA, C18:2 n-6 and C18:3 n-3, was similar for groups BDS, WCS, RCS and MSL (61.3 g/100 g, 62.3 g/100 g, 62.3 g/100 g, 63.7 g/100 g of FA methylesters (FAME), respectively), while group IRS ingested higher proportions of these FA (74.5 g/100 g of FAME). Rumen data showed that animals fed BDS presented higher proportions of biohydrogenation intermediates, particularly C18:1 t11 and CLA c9t11, suggesting partial inhibition of rumen biohydrogenation. In the MSL group, the content of C18:3 n-3 in the rumen was highest, most probably due to reduced lipolysis and hence biohydrogenation through the combined effect of esterified C18:3 n-3 and seed protection. Additionally, C18:3 n-3 proportions were higher in rumen contents of RCS animals compared with WCS animals, which could be due to the activity of the polyphenol oxidase enzyme in the RC silages. Proportions of C18:3 n-3 were similar between treatments both for SC and intramuscular (IM) fat, whereas CLA c9t11 content was higher in the SC fat of BDS animals and lower in the IM fat of IRS animals compared with the other forage groups. No differences were found for C20:4 n-6, C20:5 n-3, C22:5 n-3 and C22:6 n-3 in the IM fat of the animals. Nevertheless, indices for desaturation and elongation activity in muscle of BDS animals suggest some stimulation of the first three steps of desaturation and elongation (Δ6-desaturase, elongase and Δ5-desaturase) of long-chain FA.     

Effects of Nitrogen fertilisation and regrowth period on fatty acid concentrations in perennial ryegrass (Lolium perenne L.)

Information on lipids in forages is scarce, and effects of nitrogen (N) application level and regrowth period on the fatty acid (FA) concentration and composition of perennial ryegrass (Lolium perenne L.) were studied. N was applied at 0, 45 and 100 kg ha^?1, and swards were cut after various regrowth periods, resulting in six treatments designed as randomised blocks with three replicates. The stages of development ranged from vegetative to elongating swards, with herbage yield levels from 1.9 to 4.2 t dry matter (DM) ha^?1. Concentrations of individual FA were determined by gas chromatography, and canopy characteristics and herbage quality were assessed. The treatments resulted in canopies with contrasting DM yields and protein concentrations. Five FAs, representing 98% of total FAs, were studied in detail. On an average, the concentration of these major FAs in fresh grass was 15.1 g kg^?1 DM, and 69% of the major FAs consisted of C18:3. Regrowth period affected (P < 0.05) the total FA concentration, and significantly (P < 0.01) lower concentrations of C18:3 and C16:1 were found after a longer period of regrowth. N application resulted in higher (P < 0.001) concentrations of all FAs. The FA composition was not affected by N application, but a longer regrowth period significantly (P < 0.001) decreased the proportion of C18:3 and increased those of C18:2 and C16:0. A strong, positive overall linear relation was found between the concentrations of total FAs and C18:3 with the crude protein concentration in the herbage. These studies demonstrate opportunities to affect the FA concentration and composition of FA in herbage through management strategies, which could affect milk FA composition.     

Ruminal fatty acid outflow in dry cows fed different sources of linoleic acid: reticulum and omasum as alternative sampling sites to abomasum

This study was designed to determine the rumen outflow of fatty acids (FA) and biohydrogenation (BH) extent using alternative sampling sites (reticulum and omasum) to abomasum in dry cows fed different sources of FA. Four Holstein non-pregnant dry cows (≥3 parturitions, and 712 ± 125 kg BW), cannulated in the rumen and abomasum, were randomly assigned to a 4 × 4 Latin square design experiment, containing the following treatments: 1) control (CON); 2) soya bean oil (SO), dietary inclusion at 30 g/kg; 3) whole raw soya beans (WS), dietary inclusion at 160 g/kg; and 4) calcium salts of FA (CSFA), dietary inclusion at 32 g/kg. Rumen outflow of nutrients was estimated using the three markers reconstitution system (cobalt-EDTA, ytterbium chloride, and indigestible neutral detergent fibre [NDF]). Diets with FA sources decreased feed intake and increased FA intake. No differences in nutrient intake and digestibility were detected among cows fed diets supplemented with different FA sources. Diets with FA sources reduced the rumen outflow of DM and NDF, hence decreasing their passage rates. In addition, SO diet reduced the ruminal outflow of DM and NDF in comparison with WS and CSFA. Omasal sampling yielded the highest values of rumen outflow of NDF and potentially degradable NDF (pdNDF), whereas the reticular and abomasal samplings yielded intermediate and least values, respectively. The interaction effect between diet and sampling site was observed for rumen outflow of majority FA (except for C16:0, C18:0, and C18:2 trans-10, cis-12) and BH extension of C18:1 cis, C18:2, and C18:3. Calculations derived from abomasal sampling revealed that WS and CSFA diets had lower BH extent of C18:1 cis and C18:2 in comparison with SO, whereas cows fed CSFA had greater BH extent of C18:3 and lower BH extent of C18:1 cis compared to those fed WS. However, the latter results were not similar when calculations were performed based on the reticular and omasal samplings. Thus, there is evidence that neither reticular nor omasal samplings are suitable for estimating rumen outflow of FA in dry cows. In addition, WS and CSFA diets can increase the abomasal flow of polyunsaturated FA in dry cows.     

A novel unsaturated fatty acid hydratase toward C16 to C22 fatty acids from Lactobacillus acidophilus

Hydroxy FAs, one of the gut microbial metabolites of PUFAs, have attracted much attention because of their various bioactivities. The purpose of this study was to identify lactic acid bacteria with the ability to convert linoleic acid (LA) to hydroxy FAs. A screening process revealed that a gut bacterium, Lactobacillus acidophilus NTV001, converts LA mainly into 13-hydroxy-cis-9-octadecenoic acid and resulted in the identification of the hydratase responsible, fatty acid hydratase 1 (FA-HY1). Recombinant FA-HY1 was purified, and its enzymatic characteristics were investigated. FA-HY1 could convert not only C18 PUFAs but also C20 and C22 PUFAs. C18 PUFAs with a cis carbon-carbon double bond at the Δ12 position were converted into the corresponding 13-hydroxy FAs. Arachidonic acid and DHA were converted into the corresponding 15-hydroxy FA and 14-hydroxy FA, respectively. To the best of our knowledge, this is the first report of a bacterial FA hydratase that can convert C20 and C22 PUFAs into the corresponding hydroxy FAs. These novel hydroxy FAs produced by using FA-HY1 should contribute to elucidating the bioactivities of hydroxy FAs.     

The influence of addition of gallic acid, tannic acid, or quebracho tannins to alfalfa hay on in vitro rumen fermentation and microbial protein synthesis

Tannins may reduce rumen degradability of protein, increase the proportion of feed protein reaching the lower digestive tract for enzymatic digestion and thereby increase the efficiency of protein utilization. The objective was to assess the effects of different types and levels of tannins on rumen in vitro gas production and its kinetics, in vitro true degradability (IVTD) and rumen degradability of protein (IVRDP), and microbial protein synthesis by incubating alfalfa (Medicago sativa L.) hay in buffered rumen fluid. Alfalfa was incubated in buffered rumen fluid with and without the addition of different levels of gallic acid (GA), quebracho tannin (QT), or tannic acid (TA). Tannins at the lower inclusion levels had minimal effects on fermentation products compared to the higher levels. Addition of QT and TA reduced ammonium-N (NH₄⁺-N) concentration. Addition of QT at 20, 40, and 60 g/kg DM decreased NH₄⁺-N by 2, 7, and 12% compared with control whereas addition of TA reduced NH₄⁺-N by 5, 6, and 12% when added at 20, 40 and 60 g/kg DM, respectively. In experiment 2, addition of QT at 50, 100, and 150 g/kg DM, resulted in reduction of NH₄⁺-N by 12, 30, and 51%, respectively, compared with the control. Addition of TA at 50, 100, and 150 g/kd DM reduced NH₄⁺-N by 14, 26, and 47% compared with control. Inclusion of QT at 50, 100, 150 DM reduced IVRDP by 13, 30, and 36% compared with control whereas at these levels of inclusion, TA resulted in reduction of IVRDP by 14, 25, and 48%. Rate of gas production decreased (P<0.001), while asymptotic gas production increased (P<0.0001) with increasing level of GA and TA. Quebracho tannin decreased (P<0.0001) both the rate and asymptotic of gas production. Gallic acid had a positive effect on fermentation as indicated by increased gas production and total short chain fatty acids (SCFA) production. Quebracho tannin decreased 24 h gas production, IVTD, and total SCFA production. Acetate to propionate ratio increased with the addition of GA and but decreased when QT was added. Addition of tannins did not markedly increase total purines but numerical values tended to be higher in the presence of tannins compared with the control. Efficiency of microbial growth was lower in the presence of GA and unaltered by TA, but higher in the presence of QT compared with the control. The effect of tannins on rumen fermentation and protein degradation varied with type and level of tannins. In vivo studies will be conducted to validate the in vitro results.     

A 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus enhances acyl exchange between acyl-CoA and phosphatidylcholine

The gene encoding a 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus was over-expressed in developing seeds of Arabidopsis thaliana . Biochemical analysis of T₂ and T₃ A. thaliana seeds revealed a significant increase in polyunsaturated fatty acids (FAs) (18:2 ^{cis Δ9,12} and 18:3 ^{cis Δ9,12,15}) at the expense of very long monounsaturated FA (20:1 ^{cis Δ11}) and saturated FAs. In vitro assays demonstrated that recombinant B. napus ACBP (rBnACBP) strongly increases the formation of phosphatidylcholine (PC) in the absence of added lysophosphatidylcholine in microsomes from ΔYOR175c yeast expressing A. thaliana lysophosphatidylcholine acyltransferase ( AthLPCAT ) cDNA or in microsomes from microspore-derived cell suspension cultures of B. napus L. cv. Jet Neuf. rBnACBP or bovine serum albumin (BSA) were also shown to be crucial for AthLPCAT to catalyse the transfer of acyl group from PC into acyl-CoA in vitro . These data suggest that the cytosolic 10-kDa ACBP has an effect on the equilibrium between metabolically active acyl pools (acyl-CoA and phospholipid pools) involved in FA modifications and triacylglycerol bioassembly in plants. Over-expression of ACBP during seed development may represent a useful biotechnological approach for altering the FA composition of seed oil.     

Effects of flaxseed,raw soybeans and calcium salts of fatty acids on apparent total tract digestibility,energy balance and milk fatty acid profile of transition cows

Oilseeds offer some protection to the access of ruminal microorganisms and may be an alternative to calcium salts of fatty acids (FA), which are not fully inert in the ruminal environment. This study aimed to evaluate the effects of different sources of FA supplementation on apparent total tract nutrient digestibility, milk yield and composition, and energy balance (EB) of cows during the transition period and early lactation. We compared diets rich in C18:2 and C18:3 FA. Multiparous Holstein cows were randomly assigned to receive one of the four diets: control (n=11); whole flaxseed (WF, n=10), 60 and 80 g/kg (diet dry matter (DM) basis) of WF during the prepartum and postpartum periods, respectively; whole raw soybeans (WS, n=10), 120 and 160 g/kg (diet DM basis) of WS during the prepartum and postpartum periods, respectively; and calcium salts of unsaturated fatty acids (CSFA, n=11), 24 and 32 g/kg (diet DM basis) of CSFA during the prepartum and postpartum periods, respectively. Dry cows fed WF had higher DM and net energy of lactation (NE_L) intake than those fed WS or CSFA. The FA supplementation did not alter DM and NDF apparent total tract digestibility, dry cows fed WF exhibited greater NDF total tract digestion than cows fed WS or CSFA. Feeding WS instead of CSFA did not alter NE_L intake and total tract digestion of nutrients, but increased milk fat yield and concentration. Calculated efficiency of milk yield was not altered by diets. FA supplementation increased EB during the postpartum period. Experimental diets increased long-chain FA (saturated and unsaturated FA) in milk. In addition, cows fed WS and CSFA had higher C18:1 trans-11 FA and C18:2 cis, and lower C18:3 FA in milk than those fed WF. Furthermore, cows fed CSFA had higher C18:1 trans-11 and cis-9, trans-11 FA than cows fed WS. Although supplemental C18:2 and C18:3 FA did not influence the milk yield of cows, they positively affected EB and increased unsaturated long-chain FA in milk fat.     

Milk and meat fatty acids from sheep fed a plantain–chicory mixture or a grass-based permanent sward

Plantain and chicory are interesting forage species since they present good nutritional quality and are more resistant to drought than many temperate grasses. The fatty acid (FA) profile in milk and meat is related to a growing concern for the consumption of healthy foods, that is, with a lower content of saturated FA, higher polyunsaturated FA (PUFA) and a favourable n-6 : n-3 FAs ratio. Our objective was to evaluate the FA content in ewe’s milk and lamb’s meat fed a plantain–chicory mixture (PCH) or a grass-based permanent sward (GBS) dominated by perennial ryegrass. Eighteen Austral ewes in mid-lactation were allocated to PCH and GBS treatments. Milk samples were obtained during September (spring). Thirty weaned lambs were finished on both treatments from November to December (7 weeks), slaughtered and their meat sampled. Fat from milk and meat samples was extracted and stored until analysed by gas chromatography. Milk fat from GBS was higher than from PCH (P < 0.05) in C18:0 (11 385 v. 5874 mg/100 g FA), 9c-18:1 (15 750 v. 8565 mg/100 g FA), 11 t-18:1 (4576 v. 2703 mg/100 g FA) and 9c,11 t-18:2 (1405 v. 921 mg/100 g FA) and lower in 18:2n-6 (827 v. 1529 mg/100 g FA) and 18:3n-3 (943 v. 1318 mg/100 g FA) FA. Total mono-unsaturated FA was higher in GBS than PCH (P < 0.05). Meat fat from PCH swards presented a higher (P < 0.05) content than GBS for 18:2n-6 (46.8 v. 28.2 mg/100 g FA), linolenic (24.6 v. 14.2 mg/100 g FA), polyunsaturated FA (119.7 v. 73.4 mg/100 g FA), n-6 (65.9 v. 40.8 mg/100 g FA) and n-3 (53.8 v. 32.5 mg/100 g FA), respectively. No effect of treatment (P > 0.05) was detected for 9c-18:1 (283.9 v. 205.8 mg/100 g FA), 11 t-18:1 (26.2 v. 19.3 mg/100 g FA) and 9c,11 t-18:2 (10.1 v. 7.6 mg/100 g FA), for PCH and GBS. These results suggest that grazing a PCH mixture results in a higher concentration of PUFA in ewes’ milk and in lambs’ fat, as compared to a GBS sward.     

Rumen fermentation,nutrient flow to the duodenum and digestibility in bulls fed calcium soaps of rapeseed fatty acids and soya bean meal coated with calcium soaps

Three Polish Friesian bulls fitted with rumen and duodenal canulas were used in a 3 × 3 Latin square experiment to study the effect of Ca soaps of rapeseed fatty acids (CSRFA) on rumen fermentation, nutrient and fatty acid (FA) flow to the duodenum, and to investigate in vivo the possibility of using CSRFA as a means of protecting soya bean meal protein against degradation in the rumen. Treatments were (1) control, no fat (2) CSRFA, at 2% of dietary DM and (3) SBM protein coated with CSRFA (CSRFA:SBM, 1:1, wt/wt), at 4% of dietary DM. The animals were fed isonitrogenous diets, containing 45% meadow hay, 10% fodder beet and 45% concentrate mixture on a DM basis. Intake was limited to 80 g DM d⁻¹ kg^−0.75. There were no treatment effects on rumen fermentation, rumen liquid turnover rate or volume, suggesting that CSRFA were inert in the rumen. A slight decrease in ammonia concentration in the rumen fluid from the CSRFA:SBM diet and the degradability data do not confirm the protection of SBM protein by CSRFA. Treatments did not affect apparent OM digestibility in the rumen, total N and NAN (non-ammonia nitrogen) flow into the duodenum or microbial protein synthesis. However, rumen degradability of protein was increased by feeding CSRFA. Feeding CSRFA significantly increased the duodenal flows of C18:0, C18:1 and total FA, but when expressed as a percentage of intake, there were no significant treatment differences. The average duodenal flow of total FA was 81.7% of intake. Extents of biohydrogenation of unsaturated FA were decreased in diets containing CSRFA. Estimated net biohydrogenation of C18:2, C18:3 and total unsaturated C18 FA of CSRFA was 43.3, 60.4 and 59.4%, respectively. Postrumen and total tract digestibility of total and individual FA were not different between treatments     

Effect of soybean oil availabilities on rumen biohydrogenation and duodenal flow of fatty acids in beef cattle fed a diet with crude glycerine

Soybean oil with different ruminal availability (whole soybeans (WS), soybean oil (SO) and calcium salts (CS)) was used to evaluate the fatty acid (FA) intake, rumen biohydrogenation (BH) and duodenal flow of FA in Nellore steers fed diets with crude glycerine (CG). Eight castrated Nellore steers were fitted with a ruminal and duodenal silicone cannula, and distributed in a double, simultaneous, Latin square 4 × 4 design with four diets and four experimental periods. Concentrates contained ground maize, urea, mineral salts, CG (100 g/kg DM) and soybean products with different availability of soybean oil: (1) no additional fat (CO), (2) WS, (3) SO or (4) CS. Fat supplementation was fixed to obtain 50 g ether extract/kg DM. Experimental treatments had no effect on DM intake, DM duodenal flow or ruminal turnover rate of C:16 FA. However, fat addition increased C:18 and turnover rates of total FA rumen (p < 0.05). CS resulted in lower C:18 turnover rates and lower ruminal BH of monounsaturated and unsaturated FA (UFA) than WS (p < 0.05). SO resulted in a greater duodenal flow of C18:0 (stearic acid), C18:1t-11 (vaccenic acid) and saturated FA than the WS and CS diets (p < 0.05). CS resulted in a higher duodenal flow of C18:3n-3 (linolenic acid) than WS (p < 0.05). The association of CG and calcium salts in Nellore steers was the best nutritional strategy to increase duodenal flow of healthier UFA, which may increase the deposition of these FA in meat. However, SO associated with CG association increased the duodenal flow of vaccenic acid, which is main precursor of endogenous synthesis of conjugated linoleic acids in tissues.