Effect of fish oil on ruminal biohydrogenation of C18 unsaturated fatty acids in steers fed grass or red clover silages

Red clover and fish oil (FO) are known to alter ruminal lipid biohydrogenation leading to an increase in the polyunsaturated fatty acid (PUFA) and conjugated linoleic acid (CLA) content of ruminant-derived foods, respectively. The potential to exploit these beneficial effects were examined using eight Hereford × Friesian steers fitted with rumen and duodenal cannulae. Treatments consisted of grass silage or red clover silage fed at 90% of ad libitum intake and FO supplementation at 0, 10, 20 or 30 g/kg diet dry matter (DM). The experiment was conducted with two animals per FO level and treatments formed extra-period Latin squares. Flows of fatty acids at the duodenum were assessed using ytterbium acetate and chromium ethylene diamine tetra-acetic acid as indigestible markers. Intakes of DM were higher (P < 0.001) for red clover silage than grass silage (5.98 v. 5.09 kg/day). There was a linear interaction effect (P = 0.004) to FO with a reduction in DM intake in steers fed red clover silage supplemented with 30 g FO/kg diet DM. Apparent ruminal biohydrogenation of C18:2n-6 and C18:3n-3 were lower (P < 0.001) for red clover silage than grass silage (0.83 and 0.79 v. 0.87 and 0.87, respectively), whilst FO increased the extent of biohydrogenation on both diets. Ruminal biohydrogenation of C20:5n-3 and C22:6n-3 was extensive on both silage diets, averaging 0.94 and 0.97, respectively. Inclusion of FO in the diet enhanced the flow of total CLA leaving the rumen with an average across silages of 0.22, 0.31, 0.41 and 0.44 g/day for 0, 10, 20 or 30 g FO/kg, respectively, with a linear interaction effect between the two silages (P = 0.03). FO also showed a dose-dependent increase in the flow of trans-C18:1 intermediates at the duodenum from 4.6 to 15.0 g/day on grass silage and from 9.4 to 22.5 g/day for red clover silage. Concentrations of trans-C18:1 with double bonds from Δ4-16 in duodenal digesta were all elevated in response to FO in both diets, with trans-11 being the predominant isomer. FO inhibited the complete biohydrogenation of dietary PUFA on both diets, whilst red clover increased the flow of C18:2n-6 and C18:3n-3 compared with grass silage. In conclusion, supplementing red clover silage-based diets with FO represents a novel nutritional strategy for enhancing the concentrations of beneficial fatty acids in ruminant milk and meat.     

Effect of grazing pastures with different botanical composition by lambs on rumen fatty acid metabolism and fatty acid pattern of longissimus muscle and subcutaneous fat

In order to study the effect of grazing pastures with a different botanical composition on rumen and intramuscular fatty acid metabolism, 21 male lambs were assigned to three botanically different pastures: botanically diverse (BD) (consisting for 65% of a variety of grass species); Leguminosa rich (L) (consisting for 61% of Leguminosae) and intensive English ryegrass (IR) (with 69% Lolium perenne). Pastures were sampled weekly for 12 weeks for analysis of their fatty acid content and composition and on nine occasions to determine the botanical composition. Ruminal and abomasal contents were sampled at slaughter and muscle and subcutaneous fat 24 h after slaughter. All samples were prepared and analysed for fatty acid composition. The L pasture showed a higher fatty acid content (29.8 mg/g dry matter (DM) v. 18.5 and 25.5 mg/g DM, for BD and IR pastures, respectively), but the sum of the proportions of the major polyunsaturated fatty acids, C18:2 n-6 and C18:3 n-3, were similar for the three pastures (69.9, 69.4 and 71.1% of fatty acids methyl esters (FAME) for BD, L and IR pastures, respectively). The BD pasture was richer in C18:2 n-6 (18.2% of FAME), while IR pasture had a higher C18:3 n-3 content (57.2% of FAME). Rumen data showed that animals grazing the BD pasture presented higher proportions of biohydrogenation intermediates, mainly C18:1 t11, C18:2 t11c15 and CLA c9t11, suggesting an inhibition of biohydrogenation. These changes were associated with shifts in the rumen microbial population as indicated by differences in the rumen pattern of volatile fatty acids, microbial odd- and branched-chain fatty acids. In L pasture animals, the content of C18:2 n-6 and C18:3 n-3 in the abomasum and subcutaneous fat was higher. Finally, higher proportions of C20:4 n-6, C20:5 n-3 and C22:5 n-3 and higher indices for elongation and desaturation activity in the intramuscular fat of BD grazing animals suggest some stimulation of elongation and desaturation of long-chain fatty acids, although this also might have been provoked partially by reduced fat deposition (due to a lower growth rate of the animals).     

Evaluation of the effects of different diets on microbiome diversity and fatty acid composition of rumen liquor in dairy goat

Fat supplementation plays an important role in defining milk fatty acids (FA) composition of ruminant products. The use of sources rich in linoleic and α-linolenic acid favors the accumulation of conjugated linoleic acids isomers, increasing the healthy properties of milk. Ruminal microbiota plays a pivotal role in defining milk FA composition, and its profile is affected by diet composition. The aim of this study was to investigate the responses of rumen FA production and microbial structure to hemp or linseed supplementation in diets of dairy goats. Ruminal microbiota composition was determined by 16S amplicon sequencing, whereas FA composition was obtained by gas-chromatography technique. In all, 18 pluriparous Alpine goats fed the same pre-treatment diet for 40±7 days were, then, arranged to three dietary treatments consisting of control, linseed and hemp seeds supplemented diets. Independently from sampling time and diets, bacterial community of ruminal fluid was dominated by Bacteroidetes (about 61.2%) and Firmicutes (24.2%) with a high abundance of Prevotellaceae (41.0%) and Veillonellaceae (9.4%) and a low presence of Ruminococcaceae (5.0%) and Lachnospiraceae (4.3%). Linseed supplementation affected ruminal bacteria population, with a significant reduction of biodiversity; in particular, relative abundance of Prevotella was reduced (−12.0%), whereas that of Succinivibrio and Fibrobacter was increased (+50.0% and +75.0%, respectively). No statistically significant differences were found among the average relative abundance of archaeal genera between each dietary group. Moreover, the addition of linseed and hemp seed induced significant changes in FA concentration in the rumen, as a consequence of shift from C18 : 2n-6 to C18 : 3n-3 biohydrogenation pathway. Furthermore, dimethylacetal composition was affected by fat supplementation, as consequence of ruminal bacteria population modification. Finally, the association study between the rumen FA profile and the bacterial microbiome revealed that Fibrobacteriaceae is the bacterial family showing the highest and significant correlation with FA involved in the biohydrogenation pathway of C18 : 3n-3.     

Inclusion of sunflower seed and wheat dried distillers’ grains with solubles in a red clover silage-based diet enhances steers performance,meat quality and fatty acid profiles

The current study compared beef production, quality and fatty acid (FA) profiles of yearling steers fed a control diet containing 70 : 30 red clover silage (RCS) : barley-based concentrate, a diet containing 11% sunflower seed (SS) substituted for barley, and diets containing SS with15% or 30% wheat dried distillers’ grain with solubles (DDGS). Additions of DDGS were balanced by reductions in RCS and SS to maintain crude fat levels in diets. A total of two pens of eight animals were fed per diet for an average period of 208 days. Relative to the control diet, feeding the SS diet increased (P<0.05) average daily gain, final live weight and proportions of total n-6 FA, non-conjugated 18:2 biohydrogenation products (i.e. atypical dienes) with the first double bond at carbon 8 or 9 from the carboxyl end, conjugated linoleic acid isomers with the first double bond from carbon 7 to 10 from the carboxyl end, t-18:1 isomers, and reduced (P<0.05) the proportions of total n-3 FA, conjugated linolenic acids, branched-chain FA, odd-chain FA and 16:0. Feeding DDGS-15 and DDGS-30 diets v. the SS diet further increased (P<0.05) average daily gains, final live weight, carcass weight, hot dressing percentage, fat thickness, rib-eye muscle area, and improved instrumental and sensory panel meat tenderness. However, in general feeding DGGS-15 or DDGS-30 diets did not change FA proportions relative to feeding the SS diet. Overall, adding SS to a RCS-based diet enhanced muscle proportions of 18:2n-6 biohydrogenation products, and further substitutions of DDGS in the diet improved beef production, and quality while maintaining proportions of potentially functional bioactive FA including vaccenic and rumenic acids.     

Lipid metabolism in mixtures of red clover (Trifolium repens) and perennial ryegrass (Lolium perenne) in lab scale silages and in vitro rumen incubations

Most often, farmers consider red clover an unattractive forage because of its low ensilability. Nevertheless, several in vivo and in vitro experiments also showed advantages of red clover silages such as decreased rumen biohydrogenation of polyunsaturated fatty acids. This has been attributed to a possible protective role of protein-bound phenols, with polyphenol oxidase playing a key role in their formation. This enzyme is active in red clover, but not in other green forages, such as, for example, perennial ryegrass. Therefore, the aim was to study the lipid metabolism within red clover/ryegrass mixtures in lab scale silages and during in vitro rumen batch incubations. Ensilability of red clover increased with higher proportions of ryegrass in the silage mixture. However, the lipid-protecting mechanism of red clover does not seem to occur in the co-ensiled ryegrass as lipolysis of polar lipids linearly increased with increasing proportions of ryegrass (86.0%, 91.6%, 89.9%, 93.1% and 95.6% in 60-day-old silages with 100/0, 75/25, 50/50, 25/75 and 0/100 red clover/ryegrass, respectively). Rumen lipolysis and biohydrogenation of C18:3n-3 and C18:2n-6 were negatively related to red clover proportions in the silage mixtures. The lipid-protective mechanism in red clover silages is confirmed, but it seems not to be transferred to lipids in co-ensiled forages.     

Effect of selected plant species within biodiverse pasture on in vitro fatty acid biohydrogenation and tissue fatty acid composition of lamb

The effect of botanical diversity on supply of polyunsaturated fatty acids (PUFA) to ruminants in vitro, and the fatty acid (FA) composition of muscle in lambs was investigated. Six plant species, commonly grown as part of UK herbal ley mixtures (Trifolium pratense, Lotus corniculatus, Achillea millefolium, Centaurea nigra, Plantago lanceolata and Prunella vulgaris), were assessed for FA profile, and in vitro biohydrogenation of constituent PUFA, to estimate intestinal supply of PUFA available for absorption by ruminants. Modelling the in vitro data suggested that L. corniculatus and P. vulgaris had the greatest potential to increase 18:3n-3 supply to ruminants, having the highest amounts escaping in vitro biohydrogenation. Biodiverse pastures were established using the six selected species, under-sown in a perennial ryegrass-based sward. Lambs were grazed (~50 days) on biodiverse or control pastures and the effects on the FA composition of musculus longissimus thoracis (lean and subcutaneous fat) and musculus semimembranosus (lean) were determined. Biodiverse pasture increased 18:2n-6 and 18:3n-3 contents of m. semimembranosus (+14.8 and +7.2 mg/100 g tissue, respectively) and the subcutaneous fat of m. longissimus thoracis (+158 and +166 mg/100 g tissue, respectively) relative to feeding a perennial ryegrass pasture. However, there was no effect on total concentrations of saturated FA in the tissues studied. It was concluded that enhancing biodiversity had a positive impact on muscle FA profile reflected by increased levels of total PUFA.     

The effect of N-fertilisation rate or inclusion of red clover to timothy leys on fatty acid composition in milk of dairy cows fed a commercial silage : concentrate ratio

The aim of this experiment was to, under typical Swedish production conditions, evaluate the effects of grass silages subjected to different N-fertilisation regimes fed to dairy cows on the fatty acid (FA) composition of their milk, and to compare the grass silages in this respect to red clover-dominated silage. Grass silages made from first year Phleum pratense L. leys subjected to three N-fertilisation regimes (30, 90 and 120 kg N/ha, designated G-30, G-90 and G-120, respectively) and a mixed red clover–grass silage (Trifolium pratense L. and P. pratense L.; 60/40 on dry matter (DM) basis, designated RC–G) were produced. The experiment was conducted as a change-over design, including 24 primiparous and multiparous dairy cows of the Swedish Red breed, each of which was allocated to three of the four diets. The cows were offered 11 kg DM of silage and 7 kg concentrates. The silages had similar DM and energy concentrations. The CP concentration increased with increase in N-fertilisation level. There was a linear increase in DM intake of the different silages with increased N fertilisation. There were also differences in concentrations of both individual and total FAs amongst silages. The daily milk production (kg/day) did not significantly differ between treatments, but G-30 silage resulted in higher concentrations of 18:2n-6 in the milk compared with the other two grass silages. The highest concentrations of 18:3n-3 and cis-9, trans-11 18:2 were found in milk from cows offered the RC–G silage. The G-30 diet resulted in higher concentration of 18:2n-6 and the same concentration of 18:3n-3 in the milk as the other grass silages, despite lower intake levels of these FAs. The apparent recoveries of 18:3n-3 from feed to milk were 5.74%, 4.27%, 4.10% and 5.31% for G-30, G-90, G-120 and RC–G, respectively. A higher recovery when red clover is included in the diet confirms previous reports. The higher apparent recovery of 18:3n-3 on the G-30 treatment may be related to the lower silage DM intake, which led to a higher relative proportion of ingested FAs originating from concentrates compared with the G-90 and G-120 diets. With the rates and types of concentrates used in this study, the achieved differences in FA composition among the silages were not enough to influence the concentrations of unsaturated FAs in milk.     

Effect of grain and forage fractions of corn silage on milk production and composition in dairy cows

Corn silage (CS) is associated with a reduction in milk fat content. The fact that CS is constituted of a grain and a forage fraction could explain this effect. This experiment evaluated the effect of grain fraction of CS on rumen fermentation, production performance and milk composition. Earless CS (ECS) was harvested after manually removing corn ears from the plant. Whole CS (WCS) was harvested from the same field on the same day. Eight (four ruminally fistulated) multiparous Holstein cows (84 days in milk) were utilized in a double 4 × 4 Latin square with 21-day periods. Treatments were (dry matter (DM) basis) (1) 23.0% WCS; (2) 12.4% ECS plus 10.6% high moisture corn (HMC) to obtain reconstituted CS (RCS); (3) 23.0% ECS; and (4) 23.0% timothy silage (TS). Diets were formulated to be isonitrogenous and were fed as total mixed ration once a day. DM intake (DMI), milk yield, 4.0% fat-corrected milk (FCM), as well as protein concentration and yield were higher for WCS than ECS. Compared with WCS, cows tended to eat less with RCS, and produced less milk and milk protein. However, yield of FCM was similar between WCS and RCS. Milk fat concentration and yield, as well as the specific ratio of t11 18:1 to t10 18:1 in milk fat did not differ among diets. Milk urea-N tended to be higher for ECS than WCS and TS, whereas ruminal NH₃-N was higher with ECS than TS. Rumen pH decreased linearly with time after feeding but was not different between treatments. Higher acetate and lower propionate concentration resulted in greater acetate to propionate ratio with ECS compared with WCS. In conclusion, removing grain fraction from CS decreased milk production and modified rumen fermentation without affecting milk fat concentration and yield. Moreover, despite some differences in DMI and total ruminal volatile fatty acid concentration between WCS and RCS, the restoration of FCM yield, using HMC in RCS diets, to a level of production similar to WCS highlights the importance of energy and nutrients supplied by the grain fraction of CS to support milk yield.     

Influence of damaging and wilting red clover on lipid metabolism during ensiling and in vitro rumen incubation

This paper describes the relationship between protein-bound phenols in red clover, induced by different degrees of damaging before wilting and varying wilting duration, and in silo lipid metabolism. The ultimate effect of these changes on rumen biohydrogenation is the second focus of this paper. For this experiment, red clover, damaged to different degrees (not damaged (ND), crushing or frozen/thawing (FT)) before wilting (4 or 24 h) was ensiled. Different degrees of damaging and wilting duration lead to differences in polyphenol oxidase (PPO) activity, measured as increase in protein-bound phenols. Treatment effects on fatty acid (FA) content and composition, lipid fractions (free FAs, membrane lipids (ML) and neutral fraction) and lipolysis were further studied in the silage. In FT, red clover lipolysis was markedly lower in the first days after ensiling, but this largely disappeared after 60 days of ensiling, regardless of wilting duration. This suggests an inhibition of plant lipases in FT silages. After 60 days of ensiling no differences in lipid fractions could be found between any of the treatments and differences in lipolysis were caused by reduced FA proportions in ML of wilted FT red clover. Fresh, wilted (24 h) after damaging (ND or FT) and ensiled (4 or 60 days; wilted 24 h; ND or FT) red clover were also incubated in rumen fluid to study the biohydrogenation of C18:3n-3 and C18:2n-6 in vitro. Silages (both 60 days and to a lower degree 4 days) showed a lower biohydrogenation compared with fresh and wilted forages, regardless of damaging. This suggests that lipids in ensiled red clover were more protected, but this protection was not enhanced by a higher amount of protein-bound phenols in wilted FT compared with ND red clover. The reduction of rumen microbial biohydrogenation with duration of red clover ensiling seems in contrast to what is expected, namely a higher biohydrogenation when a higher amount of FFA is present. This merits further investigation in relation to strategies to activate PPO toward the embedding of lipids in phenol-protein complexes.     

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     

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.     

Predicting duodenal flows and absorption of fatty acids from dietary characteristics in ovine and bovine species: a meta-analysis approach

Dietary and ruminal factors modify the ruminal biohydrogenation (RBH) of polyunsaturated fatty acids (FA), with duodenal FA flows being quantitatively and qualitatively different from FA intake. Using a meta-analysis approach from a database on duodenal flows of FA in ruminants, this study aimed to determine predictive equations for duodenal and absorbed flows of saturated FA, C18:1, C18:2 and C18:3 isomers, odd- and branched-chain FA (OBCFA), C20:5n-3, C22:5n-3 and C22:6n-3 and to quantify the effects of dietary and digestive factors on those equations. The database was divided into four subsets: forage, seed, vegetable oils or animal fats (oil/fat), and fish products (fish) subsets. Models of duodenal and absorbed FA flows were obtained through variance–covariance analysis. Effects of potential interfering factors (conservation mode and botanical families of forages, lipid source, technological processing of lipid supplements, diet composition and animal characteristics) were analysed. We obtained 83 models for duodenal FA flows as a function of FA intake for saturated FA (C14:0, C16:0 and C18:0), C18:1, C18:2 and C18:3 isomers and seven other models for OBCFA. For the seed/oil/fat subset, intakes of total C18:3, C18:2 and starch content increased the duodenal t11-C18:1 flow with 0.08, 0.16 and 0.005 g/kg of dry matter intake (DMI), respectively, whereas intake level [(DMI×100)/BW] decreased it. The c9c12c15-C18:3 RBH was higher for oil/fat than seed (96.7% v. 94.8%) and a protective effect of Leguminosae v. Gramineae against RBH for that FA appeared in the forage subset. The duodenal C17:0 flow increased with starch content and decreased with ruminal pH, respectively, whereas duodenal iso-C16:0 flow decreased with dietary NDF content for the seed/oil/fat subset. The duodenal C20:5n-3, C22:5n-3 and C22:6n-3 flows depended on their respective intake and the inhibitory effect of C22:6n-3 on duodenal C18:0 flow was quantified. Thirteen models of absorbed FA flows were performed depending on their respective duodenal flows. This study determined the effects of different qualitative and quantitative dietary and digestive factors, allowing for improved predictions of duodenal and absorbed FA flows.     

Nitrogen and fatty acid rumen metabolism in cattle offered high or low polyphenol oxidase red clover silage

Polyphenol oxidase (PPO) in red clover (RC) has been shown to reduce both lipolysis and proteolysis in silo and implicated (in vitro) in the rumen. However, all in vivo comparisons have compared RC with other forages, typically with lower levels of PPO, which brings in other confounding factors as to the cause for the greater protection of dietary nitrogen (N) and C18 polyunsaturated fatty acids (PUFA) on RC silage. This study compared two RC silages which when ensiled had contrasting PPO activities (RC+ and RC−) against a control of perennial ryegrass silage (PRG) to ascertain the effect of PPO activity on dietary N digestibility and PUFA biohydrogenation. Two studies were performed the first to investigate rumen and duodenal flow with six Hereford×Friesian steers, prepared with rumen and duodenal cannulae, and the second investigating whole tract N balance using six Holstein-Friesian non-lactating dairy cows. All diets were offered at a restricted level based on animal live weight with each experiment consisting of two 3×3 Latin squares using big bale silages ensiled in 2010 and 2011, respectively. For the first experiment digesta flow at the duodenum was estimated using a dual-phase marker system with ytterbium acetate and chromium ethylenediaminetetraacetic acid as particulate and liquid phase markers, respectively. Total N intake was higher on the RC silages in both experiments and higher on RC− than RC+. Rumen ammonia-N reflected intake with ammonia-N per unit of N intake lower on RC+ than RC−. Microbial N duodenal flow was comparable across all silage diets with non-microbial N higher on RC than the PRG with no difference between RC+ and RC−, even when reported on a N intake basis. C18 PUFA biohydrogenation was lower on RC silage diets than PRG but with no difference between RC+ and RC−. The N balance trial showed a greater retention of N on RC+ over RC−; however, this response is likely related to the difference in N intake over any PPO driven protection. The lack of difference between RC silages, despite contrasting levels of PPO, may reflect a similar level of protein-bound-phenol complexing determined in each RC silage. Previously this complexing has been associated with PPOs protection mechanism; however, this study has shown that protection is not related to total PPO activity.     

Effects of conservation method on fatty acid composition of silage

This experiment was conducted to investigate effects of wilting and additives on the fatty acid (FA) composition of grass silage. The crop used was timothy (Phelum pratense L., cv. Grindstad), and the additives were Proens? (formic acid and propionic acid, 60–66 g/100 g and 25–30 g/100 g, respectively), the bacterial inoculant Siloferm® Plus (Pediococcus acidilactici and Lactobacillus plantarum) and water (control). The wilted material reached a dry matter (DM) content of 336 g/kg at the first cut and 350 g/kg at the second cut. Neither wilting nor the additives had any major effect on the FA proportions, with the only differences in the concentrations of C16:0 and C18:3. Silage treated with bacterial inoculant contained a higher proportion of C16:0 (P<0.05) than silage treated with acid, and a lower (P<0.05) concentration of C18:3 than silage treated with either of the other two additives. In the silages, there were lower (P<0.05) proportions of C16:0, C18:0, C18:1 and C18:3, and higher (P<0.05) proportions of C16:1, C18:2 and other identified FAs, than in the fresh material. A wilting process shorter than 24 h, to a DM content of 330–350 g/kg, did not have any effect on the proportions of FAs in P. pratense L., cv. Grindstad. Since the different additives and wilting strategies tested in this study did not affect the proportions of FAs in silage to a major extent, the results indicate that such a process offers a robust means to avoid losses of FAs that can occur during wilting, while retaining the positive effects of wilting, such as reduced losses of nutrients through effluents.     

Effect of replacing grass silage with maize silage in the diet on bovine milk fatty acid composition

Even though extensive research has examined the role of nutrition on milk fat composition, there is less information on the impact of forages on milk fatty acid (FA) composition. In the current study, the effect of replacing grass silage (GS) with maize silage (MS) as part of a total mixed ration on animal performance and milk FA composition was examined using eight multiparous mid-lactation cows in a replicated 4 × 4 Latin square with 28-day experimental periods. Four treatments comprised the stepwise replacement of GS with MS (0, 160, 334 and 500 g/kg dry matter (DM)) in diets containing a 54 : 46 forage : concentrate ratio on a DM basis. Replacing GS with MS increased (P < 0.001) the DM intake, milk yield and milk protein content. Incremental replacement of GS with MS in the diet enhanced linearly (P < 0.001) the proportions of 6:0-14:0, decreased (P < 0.01) the 16:0 concentrations, but had no effect on the total milk fat saturated fatty acid content. Inclusion of MS altered the distribution of trans-18:1 isomers and enhanced (P < 0.05) total trans monounsaturated fatty acid and total conjugated linoleic acid content. Milk total n-3 polyunsaturated fatty acid (PUFA) content decreased with higher amounts of MS in the diet and n-6 PUFA concentration increased, leading to an elevated n-6 : n-3 PUFA ratio. Despite some beneficial changes associated with the replacement of GS with MS, the overall effects on milk FA composition would not be expected to substantially improve long-term human health. However, the role of forages on milk fat composition must also be balanced against the increases in total milk and protein yield on diets containing higher proportions of MS.     

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.     

Effect of chestnut and quebracho tannins on fatty acid profile in rumen liquid- and solid-associated bacteria: an in vitro study

Tannins are phenolic compounds that interfere with biohydrogenation (BH) of polyunsaturated fatty acids (FAs). The aim of the present in vitro study was to investigate the effects of two different sources of tannins on FA profiles of rumen bacteria, with particular reference to rumenic and vaccenic acid. A control diet (C; composed of 300 g/kg of wheat straw, 132 g/kg of soyabean meal, 96 g/kg of barley meal, 152 g/kg of maize meal, 300 g/kg of maize gluten and 20 g/kg of mineral vitamin premix, all expressed on dry matter (DM)) and four diets, obtained by adding to C two different types of tannins from chestnut (TC) and from quebracho (TQ) at two concentration levels (49 and 82 g/kg DM), were compared. The content of the main unsaturated FAs (C18:1 cis9, C18:1 trans11, C18:2 cis9, cis12 and C18:3 cis9, cis12, cis15) from solid-associated bacteria (SAB) and liquid-associated bacteria (LAB) was affected by the presence of tannins in the diets. In particular, C18:1 trans11 content was significantly increased, especially with TC1, whereas the decreasing of C18:1 cis9 was unaffected, regardless of the presence or the kind of tannins added to feeds. SAB contained higher amounts of intermediates of polyunsaturated FA BH (as C18:1 trans11 and C18:2 cis9, trans11) than LAB that were characterized by a higher amount of C18:0. In the concentration range adopted in this study, the effect of TC and TQ on changes of bacterial FA profile was comparable. Tannins seem to be a good means to modulate the FA profile of rumen bacteria, favouring the accumulation of C18:1 trans11 during in vitro rumen fermentation.     

Effects of starch-rich or lipid-supplemented diets that induce milk fat depression on rumen biohydrogenation of fatty acids and methanogenesis in lactating dairy cows

Optimizing milk production efficiency implies diets allowing low methane (CH₄) emissions and high dairy performance. We hypothesize that nature of energy (starch v. lipids) and lipid supplement types (monounsaturated fatty acid (MUFA) v. polyunsaturated fatty acid (PUFA) mitigate CH₄ emissions and can induce low milk fat content via different pathways. The main objective of this experiment was to study the effects of starch-rich or lipid-supplemented diets that induce milk fat depression (MFD) on rumen biohydrogenation (RBH) of unsaturated fatty acids (FA) and enteric CH₄ emissions in dairy cows. Four multiparous lactating Holstein cows (days in milk=61±11 days) were used in a 4×4 Latin square design with four periods of 28 days. Four dietary treatments, three of which are likely to induce MFD, were based (dry matter basis) on 56% maize silage, 4% hay and 40% concentrates rich in: (1) saturated fatty acid (SFA) from Ca salts of palm oil (PALM); (2) starch from maize grain and wheat (MFD-Starch); (3) MUFA (cis-9 C18:1) from extruded rapeseeds (MFD-RS); and (4) PUFA (C18:2n-6) from extruded sunflower seeds (MFD-SF). Intake and milk production were measured daily. Milk composition and FA profile, CH₄ emissions and total-tract digestibility were measured simultaneously when animals were in open-circuit respiration chambers. Fermentation parameters were analysed from rumen fluid samples taken before feeding. Dry matter intake, milk production, fat and protein contents, and CH₄ emissions were similar among the four diets. We observed a higher milk SFA concentration with PALM and MFD-Starch, and lower milk MUFA and trans-10 C18:1 concentrations in comparison to MFD-RS and MFD-SF diets, while trans-11 C18:1 remained unchanged among diets. Milk total trans FA concentration was greater for MFD-SF than for PALM and MFD-Starch, with the value for MFD-RS being intermediate. Milk C18:3n-3 content was higher for MFD-RS than MFD-SF. The MFD seems more severe with MFD-SF and MFD-RS than PALM and MFD-Starch diets, because of a decrease in milk SFA concentration and a stronger shift from trans-11 C18:1 to trans-10 C18:1 in milk. The MFD-SF diet increased milk trans FA (+60%), trans-10 C18:1 (+31%), trans-10,cis-12 CLA (+27%) and PUFA (+36%) concentrations more than MFD-RS, which explains the numerically lowest milk fat yield and indicates that RBH pathways of PUFA differ between these two diets. Maize silage-based diets rich in starch or different unsaturated FA induced MFD with changes in milk FA profiles, but did not modify CH₄ emissions.     

Effects of capric acid on rumen methanogenesis and biohydrogenation of linoleic and α-linolenic acid

Capric acid (C10:0), a medium chain fatty acid, was evaluated for its anti-methanogenic activity and its potential to modify the rumen biohydrogenation of linoleic (C18:2n-6) and α-linolenic acids (C18:3n-3). A standard dairy concentrate (0.5 g), supplemented with sunflower oil (10 mg) and linseed oil (10 mg) and increasing doses of capric acid (0, 10, 20 and 30 mg), was incubated with mixed rumen contents and buffer (1 : 4 v/v) for 24 h. The methane inhibitory effect of capric acid was more pronounced at the highest (30 mg) dose compared to the medium (20 mg) (-85% v. -34%), whereas the lower dose (10 mg) did not reduce rumen methanogenesis. A 23% decrease in total short-chain fatty acid (SCFA) production was observed, accompanied by shifts towards increased butyrate at 20 mg and increased propionate at 30 mg of capric acid (P < 0.001). Capric acid linearly decreased the extent of biohydrogenation of C18:2n-6 and C18:3n-3, by up to 60% and 86%, respectively. This reduction was partially due to a lower extent of lipolysis when capric acid was supplemented. Capric acid at 20 and 30 mg completely inhibited the production of C18:0 (P < 0.001), resulting in an accumulation of biohydrogenation intermediates, mainly C18:1t10 + t11 and C18:2t11c15. In contrast to effects on rumen fermentation (methane production and proportions of SCFA), 30 mg of capric acid did not induce major changes in rumen biohydrogenation as compared to the medium (20 mg) dose. This study revealed the dual action of capric acid, being inhibitory to both methane production and biohydrogenation of C18:2n-6 and C18:3n-3.     

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.