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.     

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.     

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.     

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.     

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.     

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.     

Dietary preference of dairy heifers grazing ryegrass and white clover,with and without an anti-bloat treatment

The effect of an anti-bloat treatment on the dietary preference of non-pregnant Holstein×Friesian heifers for ryegrass and white clover was studied. Eight groups of two heifers grazed 0.51 ha plots containing adjacent monocultures of grass alongside clover with either 25 or 75% clover (by ground area) and either with or without a monensin intra-ruminal anti-bloat device. Preference was measured by recording the time spent grazing grass or clover between dawn and dusk by manual scan sampling at 2 min intervals over a 48 h period. The observers also recorded a visual assessment of bloat (on a scale of 0–5). The heifers selected 51.2% clover when offered 25% clover and 76.6% clover when offered 75% clover. Analysis showed an overall partial preference for clover of 63.9% (mean value across all treatments), although the relative abundance of clover did influence preference. There was a diurnal pattern to preference, with a stronger preference for clover in the morning, and the preference for grass increasing towards the evening. These results are very similar to those previously reported for sheep, suggesting that there may be a common biological basis for partial preference for grass and clover in sheep and cattle. None of the animals showed any significant signs of bloat, and the anti-bloat treatment had no effect on the proportion of clover selected. This indicates that avoidance of moderate or severe bloat is unlikely to be the mechanism for partial preference in cattle. However, it is possible that mild, sub-clinical bloat could still play a role in diet selection.     

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.     

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.     

Characteristics of boli formed by dairy cows upon ingestion of fresh ryegrass,lucerne or chicory

This study examined the comminution of fresh herbage, subsequent nutrient release, and the characteristics of swallowed boli from three physically and chemically contrasting forages during ingestive mastication by dairy cows. The extent and pattern of nutrient release will determine their availability to rumen microflora, and potentially influence their efficiency of use. The forages evaluated were perennial ryegrass (ryegrass, Lolium perenne L., cv Alto AR37), lucerne (Medicago sativa L., cv Torlesse) and chicory (Cichorium intybus L., cv Choice). Experimental design was a 3×3 cross-over with three forages and three consecutive 1-day measurement periods, conducted twice. Six non-lactating, pregnant, multiparous Holstein-Friesian×Jersey cows (Bos taurus) were used, with the first cross-over applied to three mature (10.1±0.61 years old; BW 631±64 kg) cows, and the second to three young (4.8±0.02 years; BW 505±19 kg) cows. Fresh cut forage was offered to the cows following partial rumen evacuation. Swallowed boli were collected directly at the cardia at the commencement, middle and end of the first feeding bout of the first meal of the day. Forage species did not affect the fresh weight of ingested boli (mean 169 g, P=0.605) but the proportion of saliva in boli varied between forage. Boli of chicory contained the greatest amount of herbage material and least amount of saliva, whereas ryegrass boli were the opposite. Boli fresh weight tended to increase as time in the meal progressed, but the age of the cow was not shown to affect any boli characteristics or nutrient release. Particle size reduction was affected by forage, with 31%, 38% and 35% of chicory, lucerne and ryegrass herbage reduced to <2 mm. There was little evidence of relationship between comminution and any physical or chemical characteristic of the forage, except in ryegrass where extent of comminution was moderately correlated with herbage strength. Proportional release of herbage soluble carbohydrate exceeded that of N during mastication. Differences in loss of N were moderately correlated with the amount of N in the herbage (R²=0.53) but herbage comminution was not strongly correlated with release of either N or carbohydrate. These findings illustrate the complex animal×forage interactions that occur during mastication, and that it is not possible to infer nutrient loss from herbage based on herbage characteristics as the driver for this differ between species.     

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).     

Phyto-oestrogens in herbage and milk from cows grazing white clover, red clover, lucerne or chicory-rich pastures

A grazing experiment was carried out to study the concentration of phyto-oestrogens in herbage for cattle and in milk during two periods (May and June). Forty-eight Danish Holstein cows were divided into four groups with four treatment diets; white clover, red clover, lucerne and chicory-rich pastures. Each experimental period lasted 15 days. Herbage samples from the first day and individual milk samples from the last day of the experimental period were analysed for phyto-oestrogens using LC-MS technique. The total concentration of phyto-oestrogens was 21 399 mg/kg dry matter (DM) for red clover and 238 to 466 mg/kg DM for the other three herbages mainly due to a much higher concentration of biochanin A, formononetin and glycitein in red clover. In the milk, the total concentration of phyto-oestrogens was 253 to 397 μg/l for red clover milk and 56 to 91 μg/l in the milk from the other three treatments. This was especially due to a higher concentration of equol, daidzein and formononetin in the red clover milk. The concentration of biochanin A was significantly higher in milk from the red clover treatment in May while no differences were observed in June. Enterodiol was similar across treatments while the concentration of enterolactone was significantly lower for red clover milk compared with the other treatments. Of the tested pastures, red clover appears to have the highest concentration and to be the best source of phyto-oestrogens, especially equol, in bovine milk.     

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.     

Evaluation of physical structure value in spring-harvested grass/clover silage and hay fed to heifers

The physical structure value of conserved grass/clover forages of spring harvest was evaluated by assessing effects of harvest time, conservation method, iNDF/NDF ratio and NDF intake (NDFI) per kg BW on chewing activity and fecal particle size in dairy heifers. A mixed sward consisting of ryegrass (Lolium perenne), red clover (Trifolium pratense) and white clover (Trifolium repens) was harvested in 2009 on May 9 (early) and 25 (late), and both cuts were conserved as silage and hay. The early silage, early hay, late silage and late hay contained dry matter (DM) of 454, 842, 250 and 828 g/kg, and NDF of 315, 436, 414 and 503 g/kg DM, respectively. Forages were fed as sole feed to four Jersey heifers of 435±30 kg BW in a 4×4 Latin square experiment. Feeding level was 90% of individual ad libitum intake, divided equally across two daily meals offered at 0800 and 1530 h. Chewing activity was estimated from recorded jaw movements (JM) oscillations continuously logged for 96 h and summarized per 24 h as mean effective rumination time and eating time. Eating behavior was further observed during four 20-min test meals. Weight proportion of large feces particles (>1.0 mm) and geometric mean fecal particle size (GPS) were calculated. Potentially indigestible NDF (iNDF) was estimated by incubation for 288 h in situ. The daily DM intake (DMI) decreased with progressing maturity at harvest (P<0.001) while daily NDFI was unaffected by harvest time (P>0.05). Earlier harvest led to less rumination per kg NDFI (P<0.01), similar eating time per kg NDFI (P>0.05) and similar proportion of large particles (P>0.01) compared with later harvest. Rumination time per kg NDFI decreased with higher NDFI per kg BW (P<0.001) and with lower iNDF/NDF ratio (P<0.01). Content and potential digestibility of NDF was greater in hay than in silage from the same harvest probably due to field loss and therefore confounded effects of conservation method. This study of high digestibility grass/clover silage and hay showed that NDF content and NDFI per kg BW affect fecal particle size and rumination time per kg NDF, and suggests implementation of NDFI per kg BW in systems evaluating physical structure in diets.     

Interaction between Sitona lepidus and red clover lines selected for formononetin content

Adult clover root weevil Sitona lepidus show a feeding preference for white clover Trifolium repens over red clover Trifolium pratense. The effects on S. lepidus of three red clover T. pratense lines, selected for high, medium, or low levels of the isoflavone formononetin in foliage, were compared in three experiments using white clover as a control. In a no‐choice slant board experiment, weevil larval weights were greater for larvae feeding on white clover roots than those feeding on roots of the red clovers. The effect of larval root herbivory on plant growth was similar for all four clovers. Following root herbivory, a large increase in root and shoot formononetin levels was observed in the high‐formononetin selection of red clover but little change in the low‐formononetin red clover. In a no‐choice experiment with sexually mature female adult weevils feeding on foliage of the four clovers, all the red clovers had increased weevil mortality. Female weevils eating the high‐formononetin red clover laid fewer eggs than weevils eating white clover. The red clover diet caused a large accumulation of abdominal fat and/or oil in the weevils, whereas weevils feeding on white clover did not accumulate fat/oil. When sexually immature adult weevils were given a choice of foliage from all four clovers, white clover was eaten preferentially, and the low‐formononetin red clover was preferred to the high‐formononetin red clover. The results suggest that formononetin and associated metabolites in red clover may act as chemical defences against adult S. lepidus and that distribution in forage legumes can be manipulated by plant breeding to improve root health.     

Feed intake and milk production in dairy cows fed different grass and legume species: a meta-analysis

The aim of this meta-analysis was to compare feed intake, milk production, milk composition and organic matter (OM) digestibility in dairy cows fed different grass and legume species. Data from the literature was collected and different data sets were made to compare families (grasses v. legumes, Data set 1), different legume species and grass family (Data set 2), and different grass and legume species (Data set 3+4). The first three data sets included diets where single species or family were fed as the sole forage, whereas the approach in the last data set differed by taking the proportion of single species in the forage part into account allowing diets consisting of both grasses and legumes to be included. The grass species included were perennial ryegrass, annual ryegrass, orchardgrass, timothy, meadow fescue, tall fescue and festulolium, and the legume species included were white clover, red clover, lucerne and birdsfoot trefoil. Overall, dry matter intake (DMI) and milk production were 1.3 and 1.6 kg/day higher, respectively, whereas milk protein and milk fat concentration were 0.5 and 1.4 g/kg lower, respectively, for legume-based diets compared with grass-based diets. When comparing individual legume species with grasses, only red clover resulted in a lower milk protein concentration than grasses. Cows fed white clover and birdsfoot trefoil yielded more milk than cows fed red clover and lucerne, probably caused by a higher OM digestibility of white clover and activity of condensed tannins in birdsfoot trefoil. None of the included grass species differed in DMI, milk production, milk composition or OM digestibility, indicating that different grass species have the same value for milk production, if OM digestibility is comparable. However, the comparison of different grass species relied on few observations, indicating that knowledge regarding feed intake and milk production potential of different grass species is scarce in the literature. In conclusion, different species within family similar in OM digestibility resulted in comparable DMI and milk production, but legumes increased both DMI and milk yield compared with grasses.     

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 offering dairy cows diets differing in phosphorus concentration over four successive lactations: 2. Health, fertility, bone phosphorus reserves and nutrient utilisation

This experiment examined the long-term effects of offering diets containing low levels of dietary phosphorus (P) on dairy cow health, fertility and bone composition, and the effect of dietary P level on nutrient utilisation. One hundred winter-calving Holstein-Friesian dairy cows were offered diets containing either 'high' or 'low' levels of dietary P over a 4-year period. Rations offered during the winter included grass silage, maize silage (70 : 30 dry matter (DM) basis, approximately) and concentrates (10.0 to 12.0 kg/cow per day). During the summer periods in years 1 and 2, half of the cows grazed both day and night, while the remaining cows grazed by day, and were housed by night and offered grass silage. During years 3 and 4, all cows grazed both day and night during the summer period. Concentrate feed levels during the summer periods were 3.0 to 4.0 kg/cow per day. Different dietary P levels were achieved by offering concentrates containing either high or low P levels during the winter period (approximately 7.0 or 4.4 g P/kg DM, respectively) and during the summer period (approximately 6.8 or 3.6 g P/kg DM, respectively). Total ration P levels averaged 4.9 and 3.6 g P/kg DM for the high and low P winter diets, respectively, and 4.2 and 3.6 g P/kg DM for the high and low P summer diets, respectively. A total of 95, 70, 50 and 22 cows completed each of lactations from 1 to 4, respectively. Neither the incidence of lameness or mastitis, or milk somatic cell count, were affected by dietary P level (P > 0.05), while none of the fertility parameters recorded in any of lactations from 1 to 4 was affected by the dietary P level (P > 0.05). Dietary P level had no effect on the specific gravity, ash or calcium content of rib cortical bone cores (n = 78 cows), while the P content of cortical bone (g/kg fresh, g/kg DM and mg/ml fresh bone) was lower with cows offered low P diets (P < 0.05). Dietary P level had no significant effect on the digestibility of either the DM, nitrogen, energy or acid detergent fibre fraction of the diet (P > 0.05), while faecal P excretions were reduced by a mean of 27 g/cow per day with cows offered the low P diets during the winter period. The results of this study indicate that dietary P levels can be reduced to proportionately 0.8 (approximately) of current UK feeding standards (Agricultural and Food Research Council, 1991), with no detrimental effect on dairy cow health or fertility, while having only minor effects on bone composition.     

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.     

Effects of feed intake on enteric methane emissions from sheep fed fresh white clover (Trifolium repens) and perennial ryegrass (Lolium perenne) forages

Published analyses of enteric methane (CH₄) emissions from sheep and cattle show an inverse relationship between feed intake and CH₄ yield (g CH₄/kg dry matter (DM) intake), which suggests opportunities for reducing CH₄ emissions from feed eaten and per unit of animal production. Most relationships between feed intake and CH₄ yield have been based on animals fed conserved feeds, especially silages and grains. Our research is a series of experiments with fresh white clover (Trifolium repens) and perennial ryegrass (Lolium perenne; ryegrass) forages fed to sheep at a range of feed intake levels. This study was comprised of four experiments where good quality freshly harvested white clover or ryegrass were fed to sheep over a three-fold range in DM intake, and CH₄ emissions were measured in respiration chambers for two consecutive days in each experiment. Measurements were made from 16 sheep in Experiment 1 (fed at 1.6 × metabolizable energy requirements for maintenance; ME_m), 28 sheep in Experiment 2 (at 0.8 and 2.0 × ME_m), eight sheep and two measurement periods in Experiment 3 (at 1.6 × ME_m), and 30 sheep in Experiment 4 (fed at 0.8, 1.2, 1.6, 2.0 and 2.5 × ME_m). Prior to each experiment, sheep had a 10 d acclimatization period to diets. Apparent digestibility was measured over 7 d from sheep in Experiments 1, 3 and 4, along with collection of rumen digesta for volatile fatty acid (VFA) determination. Although CH₄ yields differed when sheep were fed white clover or ryegrass at similar intakes, the differences were inconsistent and mean values similar across all experiments. This, and a similar structure of all experiments, enabled combined analysis of data from all four experiments using the restricted maximum likelihood (REML) procedure to estimate effects of feed intake level on digestibility, digestible nutrient intake, gas emissions, and VFA concentrations in the rumen. The REML analysis showed that when DM intake increased from 0.40 to 1.60 kg/d, the predicted responses were an increase in CH₄ production (g/d) of 187% (12.4–35.6 g/d; P<0.001), and a decline in CH₄ yield of 21% (25.6–20.2 g/kg DM intake; P<0.001). High feed intake levels were associated with increased molar proportions (mM of total VFA) of propionate from 0.17 to 0.21 (P=0.038). Single and multiple regressions were completed on the data from all experiments, with organic matter (OM) intake predicting 0.87 of the variation in CH₄ production, and molar proportion of propionate predicting 0.60 of the variation in CH₄ yield. Increasing feed intakes by 1 kg/d of DM reduced CH₄ yield by 4.5 g/kg DM intake. Plant chemical composition was weakly related to CH₄ yield. High intakes of fresh forages will lower CH₄ yield from fermentation, but effects of feed composition on CH₄ emissions were minor. The interaction between effects of feed intake and rumen function requires further investigation to understand relationships with CH₄ emissions.