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.     

Red clover polyphenol oxidase and lipid metabolism

Increasing the polyunsaturated fatty acid (PUFA) composition of milk is acknowledged to be of benefit to consumer health. Despite the high PUFA content of forages, milk fat contains only about 3% of PUFA and only about 0.5% of n-3 fatty acids. This is mainly due to intensive lipid metabolism in the rumen (lipolysis and biohydrogenation) and during conservation (lipolysis and oxidation) such as drying (hay) and ensiling (silage). In red clover, polyphenol oxidase (PPO) has been suggested to protect lipids against degradation, both in the silage as well as in the rumen, leading to a higher output of PUFA in ruminant products (meat and milk). PPO mediates the oxidation of phenols and diphenols to quinones, which will readily react with nucleophilic binding sites. Such binding sites can be found on proteins, resulting in the formation of protein-bound phenols. This review summarizes the different methods that have been used to assess PPO activity in red clover, and an overview on the current understanding of PPO activity and activation in red clover. Knowledge on these aspects is of major importance to fully harness PPO's lipid-protecting role. Furthermore, we review the studies that evidence PPO-mediated lipid protection and discuss its possible importance in lab-scale silages and further in an in vitro rumen system. It is demonstrated that high (induction of) PPO activity can lead to lower lipolysis in the silage and lower biohydrogenation in the rumen. There are three hypotheses on its working mechanism: (i) protein-bound phenols could directly bind to enzymes (e.g. lipases) as such inhibiting them; (ii) binding of quinones in and between proteins embedded in a lipid membrane (e.g. in the chloroplast) could lead to encapsulation of the lipids; (iii) direct binding of quinones to nucleophilic sites in polar lipids also could lead to protection. There is no exclusive evidence on which mechanism is most important, although there are strong indications that only lipid encapsulation in protein-phenol complexes would lead to an effective protection of lipids against ruminal biohydrogenation. From several studies it has also become apparent that the degree of PPO activation could influence the mode and degree of protection. In conclusion, this review demonstrates that protein-bound phenols and encapsulation in protein-phenol complexes, induced by PPO-mediated diphenol oxidation, could be of interest when aiming to protect lipids against pre-ruminal and ruminal degradation.     

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.     

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.     

Effects of mixtures of red clover and maize silages on the partitioning of dietary nitrogen between milk and urine by dairy cows

Eight multiparous lactating Holstein-Friesian cows were used to evaluate the partitioning of dietary nitrogen (N) from diets based on mixtures of red clover and maize silages in comparison with diets based on ryegrass silage. All cows received 4 kg/day of a standard dairy concentrate with one of four forage treatments in an incomplete changeover design with three 4-week periods. Three treatments were based on mixtures of red clover and maize silage. N intake was altered both by varying the ratio of these silages (40/60 and 25/75 on a dry matter (DM) basis) and by an additional treatment for which the DM intake of the 40/60 mixture was restricted to the level achieved with grass silage. Rumen passage rates were estimated from faecal excretion curves following a pulse oral dose of Dysprosium-labeled silage and urinary excretion of purine derivatives (PD) was used as an index of rumen microbial protein synthesis. Red clover silage mixtures led to significantly increased feed intake (21.5, 20.7 and 15.2 kg DM/day for 40/60 and 25/75 red clover/maize silage mixtures and grass silage, respectively), milk production (25.8, 27.8 and 20.0 kg/day for the same treatments, respectively) and milk component yields, but were without effect on milk fat and protein concentrations. The large increase in the yield of milk (24.5 kg/day) and milk components for the restricted red clover/maize silage treatment, in comparison with the grass silage treatment, was proportionately greater than the increase in DM intake (16.6 kg DM/day). There were no significant treatment effects on diet digestibility, while the higher intakes of red clover silage mixtures were associated with higher rumen passage rates (5.82%, 6.24% and 4.55%/h, respectively). There were significant effects of both N intake and forage source on the partitioning of dietary N between milk and urine. When dietary protein was diluted by the inclusion of maize silage, red clover silage led to increased milk N and reduced urinary N in comparison with grass silage. Improvements in N utilisation may be related to increased dietary starch and/or rumen passage rates leading to increased microbial protein synthesis for these treatments. Urinary excretion of PD was significantly higher for all diets based on mixtures of red clover and maize silages, in comparison with grass silage. Urinary N output was close to literature predictions based on N intake for the diet based on ryegrass silage, but 40 to 80 g/day (25% to 30%) less than predicted for the diets based on the mixtures of red clover and maize silages.     

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.     

Preference of dairy cows for ryegrass, white clover and red clover, and its effects on nutrient supply and milk quality

Two experiments were conducted with 30 dairy cows each, to study the preference for fresh (Experiment 1) and ensiled (Experiment 2) ryegrass, white and red clover. Both experiments consisted of three choice diets with white or red clover or both, offered with ryegrass, and two diets with ryegrass mixed with white or red clover (40% clover). Cows consumed diets with 37.7% fresh white and 45.9% red clover, and no preference was observed when the cows were offered all three forages. By contrast, cows preferred white and red clover silage (73.0 and 69.2%, respectively) over ryegrass silage (of lower nutritive quality). When offered three forages, cows preferred white (59.8%) over red clover (17.5%) and ryegrass (22.7%). Choice diets resulted in diets similar (fresh forages) or higher in nutrient content and digestibility (silages). Treatments did not affect feed intake and performance. Choices compared to mixed diets with red clover silage were preferable regarding the fatty acid composition of the milk fat. Obviously, only large differences in nutrient and energy concentration facilitate preferences for clovers over ryegrass, which could, depending on clover type, be beneficial in terms of the milk's fatty acid composition.     

The Impact of Using Alternative Forages on the Nutrient Value within Slurry and Its Implications for Forage Productivity in Agricultural Systems

Alternative forages can be used to provide valuable home-grown feed for ruminant livestock. Utilising these different forages could affect the manure value and the implications of incorporating these forages into farming systems, needs to be better understood. An experiment tested the hypothesis that applying slurries from ruminants, fed ensiled red clover (Trifolium pratense), lucerne (Medicago sativa) or kale (Brassica oleracea) would improve the yield of hybrid ryegrass (Lolium hybridicum), compared with applying slurries from ruminants fed ensiled hybrid ryegrass, or applying inorganic N alone. Slurries from sheep offered one of four silages were applied to ryegrass plots (at 35 t ha⁻¹) with 100 kg N ha⁻¹ inorganic fertiliser; dry matter (DM) yield was compared to plots only receiving ammonium nitrate at rates of 0, 100 and 250 kg N ha⁻¹ year⁻¹. The DM yield of plots treated with 250 kg N, lucerne or red clover slurry was significantly higher than other treatments (P<0.001). The estimated relative fertiliser N equivalence (FNE) (fertiliser-N needed to produce same yield as slurry N), was greatest for lucerne (114 kg) >red clover (81 kg) >kale (44 kg) >ryegrass (26 kg ha⁻¹ yr⁻¹). These FNE values represent relative efficiencies of 22% (ryegrass), 52% (kale), 47% (red clover) and 60% for lucerne slurry, with the ryegrass slurry efficiency being lowest (P = 0.005). Soil magnesium levels in plots treated with legume slurry were higher than other treatments (P<0.001). Overall, slurries from ruminants fed alternative ensiled forages increased soil nutrient status, forage productivity and better N efficiency than slurries from ruminants fed ryegrass silage. The efficiency of fertiliser use is one of the major factors influencing the sustainability of farming systems, these findings highlight the cascade in benefits from feeding ruminants alternative forages, and the need to ensure their value is effectively captured to reduce environmental risks.     

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.     

Effects of ensiled forage legumes on performance of store finishing lambs

Male Beulah speckled face lambs (initial live weight (LW) 28.8 ± 0.31 kg) were allocated to three dietary treatments to evaluate the performance of store lambs of a hill breed when offered ensiled lucerne (Medicago sativa), red clover (Trifolium pratense) or ryegrass. Second-cut silage bales (wilted and inoculated) were prepared from 3-year old lucerne and red clover stands and a 1-year old ryegrass sward. All the lambs were group-housed and offered ad libitum ryegrass silage during a 3-week co-variate period. This was followed by a week of dietary changeover period, after which the lambs were housed individually and offered their treatment diet ad libitum. All the lambs received a flat rate supplement of pelleted molassed sugarbeet (250 g fresh weight/day). Individual intakes were determined daily, and weekly measurements of LW and body condition score (CS) were made. Additional measurements were taken by scanning the lambs for depth of Longissimus dorsi (LD) muscle and subcutaneous fat. Over an experimental period of 7 weeks, the lambs offered red clover silage had a higher voluntary silage dry matter (DM) intake, total DM intake and metabolisable energy (ME) intake (P < 0.001) than lambs offered either lucerne or ryegrass silage. This resulted in a faster (P < 0.001) growth rate and increase (P < 0.001) in CS, with no difference between lucerne and ryegrass silages. The feed conversion efficiency (FCE) was 8.0 ± 0.61 kg feed/kg gain for lambs fed red clover silage, compared with 16.6 ± 2.82 and 10.6 ± 1.94 kg feed/kg gain for lucerne and ryegrass silage, respectively. The CP intake was higher (P < 0.001) for lambs fed the lucerne and red clover silages than for the ryegrass silage treatment. The concentration of plasma total protein (TP) was higher (P < 0.05) for lambs offered ryegrass silage versus lucerne and red clover silage. Urea concentrations were highest for lambs fed lucerne silage and lowest for those fed ryegrass silage (P < 0.05). The glucose concentration was higher (P < 0.05) for lambs offered red clover silage, whereas non-esterified fatty acids (NEFA) concentration was higher (P < 0.05) for lambs offered lucerne silage. Substituting ryegrass silage with red clover silage has the potential to improve the performance of finishing store lambs.     

Effects of polyphenol oxidase on lipolysis and proteolysis of red clover silage with and without a silage inoculant (Lactobacillus plantarum L54)

Polyphenol oxidase (PPO) has been shown to reduce proteolysis and lipolysis in red clover through deactivation of proteolytic and lipolytic enzymes and/or through formation of protein–phenol–lipid complexes. This experiment investigated the time course of both lipolysis and proteolysis in two red clover lines with different PPO activities with and without addition of a silage inoculant to help understand the action of PPO in the silo, and its potential effects on protein and glycerol-based lipid conservation, and to determine effects of a more rapid pH reduction with inoculation on PPO activity. Four silages were prepared from high or low PPO precision chopped red clover in 60 test-tube-silos, each containing 120 g fresh weight: (a) high PPO red clover without inoculation (H−), (b) low PPO red clover without inoculation (L−), (c) high PPO red clover with inoculation (H+), and (d) low PPO red clover with inoculation (L+). Each treatment had three replicates for each time point of 1, 2, 4, 8 and 90 days. The inoculant used was Lactobacillus plantarum strain L54 applied at a rate of 10⁶ CFU/g fresh weight. Silage pH was reduced (P < 0.001) by inoculation with no effect of PPO. Inoculation had no effect on either lipolysis or free amino acid release, although more (P < 0.01) soluble protein and less (P < 0.01) ammonia-N was in inoculated silages. H silages had a lower level of both proteolysis (release of free amino acids, P < 0.05) and lipolysis (loss of membrane lipid, P < 0.01) than L red clover silages. Results indicate that PPO reduced proteolysis and lipolysis in the silo and that inoculation had no adverse effects on PPO activity.     

Ruminal large and small particle kinetics in dairy cows fed red clover and grass silages harvested at two stages of growth

Passage, comminution and digestion rates of large and small particles were estimated using a rumen evacuation technique and total faecal collection with five lactating dairy cows in a 5 × 5 Latin square experiment. Two grass and two red clover silages harvested at early and late primary growth stages and a 1:1 mixture of late harvest grass and early harvest red clover were the dietary treatments. Cows received 9.0 kg supplementary concentrate per day. Ruminal contents and faeces were divided into large (>1.25 mm) and small (1.25–0.038 mm) particles by wet sieving. Indigestible neutral detergent fibre (iNDF) was determined by 12 days ruminal in situ incubation followed by neutral detergent extraction. Plant species did not affect ruminal particle size distribution, whereas advancing forage maturity decreased the proportion of large particles for both grass and red clover silage diets. Ruminal pool size of iNDF was higher (P<0.001) with red clover compared to grass silage diets. Ruminal passage rates of iNDF and potentially digestible NDF (pdNDF) increased with decreasing particle size (P<0.01). Passage rate of iNDF for small particles was slower (P<0.01) when red clover compared to grass silage diets were fed. Particle comminution rate in the rumen was slower (P<0.001) with red clover compared to grass silage diets and it increased (P<0.01) with advancing forage maturity. The contribution of particle comminution to ruminal mean retention time of iNDF in the ruminal large particle pool was smaller (P<0.01) in red clover compared to grass silage diets and it increased (P<0.05) with the mixed silage compared to the separate silages. Passage rate of pdNDF for both large and small particles was not affected by dietary treatments. Digestion rate of pdNDF for large particles was faster (P<0.001) with red clover compared to grass silage diets. Differences in ruminal passage and digestion rates of the large and small particles, in addition to differences in the passage and digestion rates of red clover compared to grass silage diets, emphasize the need to consider particle size and forage type in metabolic models predicting feed intake and fibre digestibility in ruminants.     

Effect of harvest time of red and white clover silage on chewing activity and particle size distribution in boli,rumen content and faeces in cows

The study examined the effects of harvest time of red and white clover silage on eating and ruminating activity and particle size distribution in feed boli, rumen content and faeces in cows. The clover crops were harvested at two stages of growth and ensiled in bales. Red clover crops had 36% and 45% NDF in dry matter (DM) at early (ER) and late (LR) harvest, respectively, and the white clover crops had 19% and 29% NDF in DM at the early (EW) and late (LW) harvest, respectively. The silages were fed restrictively (80% of ad libitum intake) twice daily to four rumen cannulated non-lactating Jersey cows (588 ± 52 kg) in a 4 × 4 Latin square design. Jaw movements (JM) were recorded for 96 h continuously. Swallowed boli, rumen mat, rumen fluid and faeces samples were collected, washed in nylon bags (0.01 mm pore size) and freeze-dried before dry sieving through 4.750, 2.360, 1.000, 0.500, 0.212 and 0.106 mm into seven fractions. The length (PL) and width (PW) values of rumen and faeces particles within each fraction were measured by use of image analysis. The eating activity (min/kg DM intake; P < 0.05) was higher in LR compared with the other treatments. The eating activity (min/kg NDF intake; P < 0.05) was affected by clover type with highest values for white clover silage. The mean ruminating time (min/kg DM), daily ruminating cycles (P < 0.001) and JM during ruminating (P < 0.05) were affected by treatment with increasing values at later harvest time. The proportion of washed particle DM of total DM in boli (P < 0.001), rumen mat (P < 0.001), rumen fluid (P < 0.01) and faeces was (P < 0.001) highest by feeding LR. There were identified two peaks (modes 1 and 2) on the probability density distribution (PDF) of PW values of rumen mat and faeces, but only one peak (mode 1) for PL values. There was no difference in the mean and mode 1 PW and PL value in rumen mat between the four treatments. The mean PL, mode PL, mode 2 PW and mean PW in faeces were highest for LR (P < 0.05). The mean particle size in boli measured by sieving was higher at white clover compared with red clover treatments (P < 0.001) and the highest value in faeces was found in LR (P < 0.01). The two peaks on PDF for width values of rumen mat and faeces particles are most likely related to the leaves and the stems/petioles. In conclusion, the mean total chewing activity per kg DM was lowest for the white clover silage and increased for both silages due to later harvest time. The mean particle size in boli was smallest for LR, whereas the mean PL and PW in faeces were highest for the LR.     

Effect of combined ensiling of sorghum and soybean with or without molasses and lactobacilli on silage quality and in vitro rumen fermentation

Two sorghum (Sorghum bicolor (L.) Moench) varieties (SG₁ & SG₂), with the former showing higher grain and total DM yield, but also increased tannin contents compared to the latter, and one soybean (Glycine max (L.) Merr.) variety (INCASOY-35) were sown, harvested (at pasty grain state), chopped and ensiled (CIAP, Cuba). Silages were made in following combinations: either SG₁ or SG₂ combined with soybean in two proportions (0.4 and 0.6). All silages were prepared with or without molasses (3.5% of fresh material) and Lactobacillus sp. as inoculant (3 × 10⁵ colony forming units/g). Silage quality parameters included pH, ammonia, lactate, acetate, butyrate and water soluble carbohydrates content. Further, both fresh and ensiled materials were incubated in vitro with buffered rumen fluid to study the fermentation characteristics. Silage of a good quality could be produced with both sorghum varieties alone, but combined silages showed improved quality compared to soybean silage (p<0.05 for all quality characteristics). Addition of molasses and bacterial inoculant further improved silage quality (p<0.05 for all quality characteristics). In vitro incubation (24 h) of ensiled material resulted in lower acetate and higher propionate proportion compared to fresh forages. However, ensiling without molasses and inoculant reduced in vitro short chain fatty acid production and hence the apparent rumen degradability of organic mater as well as the fermentation rate. As expected, a higher proportion of sorghum increased the molar propionate proportion and the fractional fermentation rate, whereas ammonia (mmol/L) concentrations were reduced. SG₁ silages produced higher molar propionate proportions, lower acetate proportions and ammonia concentrations.     

Differences in rate of ruminal hydrogenation of C18 fatty acids in clover and ryegrass

Biohydrogenation of C18 fatty acids in the rumen of cows, from polyunsaturated and monounsaturated to saturated fatty acids, is lower on clover than on grass-based diets, which might result in increased levels of polyunsaturated fatty acids in the milk from clover-based diets affecting its nutritional properties. The effect of forage type on ruminal hydrogenation was investigated by in vitro incubation of feed samples in rumen fluid. Silages of red clover, white clover and perennial ryegrass harvested in spring growth and in third regrowth were used, resulting in six silages. Fatty acid content was analysed after 0, 2, 4, 6, 8 and 24 h of incubation to study the rate of hydrogenation of unsaturated C18 fatty acids. A dynamic mechanistic model was constructed and used to estimate the rate constants (k, h) of the hydrogenation assuming mass action-driven fluxes between the following pools of C18 fatty acids: C18:3 (linolenic acid), C18:2 (linoleic acid), C18:1 (mainly vaccenic acid) and C18:0 (stearic acid) as the end point. For k_C18:1,C18:2 the estimated rate constants were 0.0685 (red clover), 0.0706 (white clover) and 0.0868 (ryegrass), and for k_C18:1,C18:3 it was 0.0805 (red clover), 0.0765 (white clover) and 0.1022 (ryegrass). Type of forage had a significant effect on k_C18:1,C18:2 (P < 0.05) and a tendency to effect k_C18:1,C18:3 (P < 0.10), whereas growth had no effect on k_C18:1,C18:2 or k_C18:1,C18:3 (P > 0.10). Neither forage nor growth significantly affected k_C18:0,C18:1, which was estimated to be 0.0504. Similar, but slightly higher, results were observed when calculating the rate of disappearance for linolenic and linoleic acid. This effect persists regardless of the harvest time and may be because of the presence of plant secondary metabolites that are able to inhibit lipolysis, which is required before hydrogenation of polyunsaturated fatty acids can begin.     

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.     

In vitro ruminal fermentation characteristics and utilisable CP supply of sainfoin and birdsfoot trefoil silages and their mixtures with other legumes

The extensive protein degradation occurring during ensiling decreases the nutritive value of silages, but this might be counteracted by tannins. Therefore, silages from two legume species containing condensed tannins (CT) – sainfoin (SF) and birdsfoot trefoil (two cultivars: birdsfoot trefoil, cv. Bull (BTB) and birdsfoot trefoil, cv. Polom) – were compared for their in vitro ruminal fermentation characteristics. The effect of combining them with two CT-free legume silages (lucerne (LU) and red clover (RC)) was also determined. The supply of duodenally utilisable CP (uCP) in the forages was emphasised. The legumes were each harvested from three field sites. After 24 h of wilting on the field, the legumes were ensiled in laboratory silos for 86 days. Proximate constituents, silage fermentation characteristics, CT content and CP fractions were determined. Subsequently, silage samples and 1 : 1 mixtures of the CT-containing and CT-free silages were incubated for 24 h in batch cultures using ruminal fluid and buffer (1 : 2, v/v). Each treatment was replicated six times in six runs. The effects on pH, ammonia and volatile fatty acid concentrations, protozoal counts, and total gas and methane production were determined. uCP content was calculated by considering the CP in the silage and the ammonia in the incubation fluid from treatments and blanks. Statistical evaluation compared data from single plants alone and together with that from the mixtures. Among treatments, SF silage contained the least CP and the most CT. The non-protein nitrogen content was lower, favouring neutral detergent soluble and insoluble protein fractions, in the SF and RC silages. Absolute uCP content was lowest in SF and SF mixtures, although the ratio to total CP was the highest. In comparison with LU, the ammonia concentration of the incubation fluid was lower for SF, RC and BTB and for the mixture of SF with LU. The total gas and methane production was similar among the treatments, and the total volatile fatty acid production was decreased with the CT-containing legumes. Protozoal count was increased with the mixtures containing LU and either SF or BTB compared with single LU. In conclusion, compared with the other legumes, SF and RC have similar advantages as they show limited proteolysis during ensiling. In addition, SF supplies more uCP relative to total CP. The CT-containing legumes also differed in their effect on ruminal fermentation and ammonia formation, probably because of their different CT contents. Thus, SF and its mixtures appear promising for improving the protein utilisation of ruminants.     

Protein degradation in the rumen of red clover forage at various stages of growth and conserved as silage or wrapped big bales

In order to study the extent to which rumen soluble nitrogen can contribute to the intestinal flow, a study was carried out to simultaneously assess the dynamics of protein disappearance from dacron bags placed in the rumen and the amount of various N products in the rumen fluid (total nitrogen (tN), ammonia nitrogen (NH3-N), non-ammonia nitrogen (NAN)). The measurements were carried out on 4 sheep fed successively various red clover forages. These forages included the initial growth of fresh red clover (50% bud, first flower, and full flower). In addition, one silage and one wrapped big bale at the first flower stage and two wrapped big bales (harvested at 51% and 71% dry matter) at the full flower stage were given. The effective degradability of nitrogen (DegN) for a fresh forage estimated from the nylon bag procedure did not vary (p > 0.05) with the vegetation stage (0.727 for the bud stage, 0.694 at the first flower, 0.706 at the full flower). The DegN of the silage was higher (p < 0.05; 0.735) and the DegN of the wrapped big bale was markedly lower (p < 0.05; 0.660), than the original fresh forage at the first flower. The DegN of the wrapped big bales made at 51 and 71% DM, respectively, were 0.625 and 0.604 against 0.706 for fresh forage at the full flower stage. The concentrations of tN and NAN in the rumen fluid were low, highest 1 h to 2 h after feeding, and then decreasing up to 7 h after feeding whatever the growth stage and conservation mode. A part of the solubilised nitrogen remained as protein 1 h after feeding for fresh red clover harvested at various growth stages, while minimal protein could be seen in the rumen fluid after the sheep were fed silage or wrapped big bales. The part of NAN escaping rumen degradation and transiting with the rumen fluid was between 7 and 13% of the nitrogen disappearing from the nylon bags (NAN/CP x DegN) placed in the rumen. There was only a small difference for forages at different stages of growth, or modes of conservation. This fraction was higher for wrapped big bales and particularly for the late stage forage (wrapped big bale, 71% DM, harvested at the full flower stage).     

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.     

Degradability characteristics of dry matter and crude protein of forages in ruminants

Twelve corn silages, 22 grass silages and 14 grass hays, obtained from various farms located in the lower Fraser Valley region of British Columbia, and 16 alfalfa hays, grown primarily in the Columbia basin of central Washington State, were evaluated using both the rumen and the mobile nylon bag in situ techniques. Nylon bags containing each forage were incubated in duplicate for 0, 2, 4, 8, 12, 24, 48, 72, or 96 h in two of six non-lactating Holstein cows fitted with rumen and duodenal cannulae. All forage types were evaluated in terms of the following dry matter (DM) and crude protein (CP) digestion characteristics: soluble fraction A, degradable fraction B, degradation rate, lag phase, and effective degradability. The mobile nylon bag technique was used to determine intestinal disappearance of DM and CP from the forages following pre-incubation in the rumen for 12 h. Significant (P < 0.05) differences in degradation characteristics occurred within all forages with regard to the soluble and potentially degradable DM and CP fractions. Soluble CP content in the rumen varied from 44.08 to 75.37% and from 18.74 to 65.38% in the corn and grass silages, respectively, and from 48.27 to 75.43% and from 30.13 to 65.95% in the alfalfa and grass hays, respectively. Significant differences within each forage type were also observed for the degradable CP in fraction B: 10.89 to 45.28% for corn silage, 20.72 to 82.77% for grass silage, 16.67 to 44.88% for grass hay and 25.44 to 62.93% for alfalfa hays. Significant differences (P > 0.05) were observed in fractional rates of ruminal DM degradation of the grass hays and corn silages. Significant differences did exist in the fractional rates of ruminal CP degradation within all forage types with the exception of alfalfa hays. Effective degradabilities of DM and CP were also significantly different between samples of a particular forage type. The mobile nylon bag data indicated that approximately 20% of the original CP in the grass silage, grass hay and alfalfa hay samples disappeared in the intestine and that there was significant variation between individual samples. On average, in the corn silage samples more than 10% of the original nitrogenous material disappeared in the intestine. The results presented in this study clearly demonstrate that the use of tabulated values for describing individual batches of forages in terms of their degradability characteristics is inaccurate since they may not reflect the particular forage being used in the ration and thus may lead to errors in diet formulation.