Ruminal fermentation and enteric methane production of legumes containing condensed tannins fed in continuous culture.

A continuous-culture fermentor study was conducted to assess nutrient digestibilities, volatile fatty acid (VFA) concentrations, microbial protein synthesis, bacterial nitrogen (N) efficiency, and enteric methane (CH4) production of four 50:50 grass-legume diets, randomly assigned in a 4 × 4 Latin square design. Four legumes with different concentrations of condensed tannins (CT) were tested: alfalfa [ALF; Medicago sativa L., non-CT legume]; birdsfoot trefoil [BFT; Lotus corniculatus L., low-CT legume]; crown vetch [CV; Securigera varia (L.) Lassen, moderate-CT legume]; and sericea lespedeza [SL; Lespedeza cuneata (Dum. Cours.) G. Don, high-CT legume]. Orchardgrass (Dactylis glomerata L.) was the common forage used in all diets. Four fermentors were evaluated over four 10-d periods by feeding 82 g of dry matter (DM)/d in 4 equal feedings. Methane output was recorded every 10 min. Effluent samples were collected during the last 3 d of the experiment, composited by fermentor and period, and analyzed for pH and VFA, as well as DM, organic matter, crude protein, neutral detergent fiber, and acid detergent fiber for determination of apparent and true nutrient digestibilities. Microbial protein synthesis and bacterial efficiency were estimated by analysis of N flows and purines. The CT concentrations were 3, 21, 38 and 76 g/kg of DM for ALF, BFT, CV, and SL diets, respectively. The SL diet had decreased fiber digestibilities and total VFA concentrations compared with the other diets. This resulted in the least total CH4 production in the SL diet. Bacterial N efficiency per kilogram of organic matter truly digested was lower in the SL diet than in the BFT and CV diets. The lowest CH4 production per unit of digestible nutrients was also found in the SL diet. Further work should be conducted to find optimal diets (by testing other legumes, rations, and sources of CT) for reducing CH4 emissions without negatively affecting ruminal digestion to maintain or improve productivity.

Exposure of heavy metals in coal gangue soil, in and outside the mining area using BCR conventional and vortex assisted and single step extraction methods. Impact on orchard grass.

It was investigated that toxic metals (cadmium and lead) enhanced in coal gangue soil used for the reclamation of soil, creates adverse impacts on atmosphere. Presently the chemical fractionation of toxic metals in coal, inner and outer coal gangue soil samples of Lakhra coalfield were studied along with the impact on the orchard grass grown on coal gangue soil in a subside land of the coal mining area. The BCR sequential extraction scheme (BCR-SES) was carried out to determine the different chemical profiles of heavy metals in coal and coal gangue soil samples. For comparative purpose time saving conventional single step extraction (CSE) and vortex assisted single step extraction (VSE) schemes based on same working setting used BCR extraction scheme. The all three procedures were validated by a certified soil sample (BCR 483) and standard addition method in real samples. The total Cd and Pb in coal, soil and grass samples were determined prior to oxidize by acid mixture. The separation of each fraction of Cd, and Pb in all types of environmental samples by VSE could be completed in 30-120 s. The extracted Cd and Pb concentrations in reducible fractions by CSE and VSE extraction procedure were 2.5-5% higher than those values gained through BCR-SES. About 71 and 50% of Cd and Pb in coal samples were observed in acid soluble, reducible and oxidizable fraction, respectively, whilst rest of Cd and Pb (29 and 50%) were found in residual phase. The orchard grass (Dactylis glomerata) grown on land reclaimed with OSG was analysed, the bioaccumulate Cd and Pb may create adverse impacts on grazing cattle.

Infection Rates and Alkaloid Patterns of Different Grass Species with Systemic Epichloë Endophytes.

Symbiotic Epichloë species are fungal endophytes of cool-season grasses that can produce alkaloids with toxicity to vertebrates and/or invertebrates. Monitoring infections and presence of alkaloids in grasses infected with Epichloë species can provide an estimate of possible intoxication risks for livestock. We sampled 3,046 individuals of 13 different grass species in three regions on 150 study sites in Germany. We determined infection rates and used PCR to identify Epichloë species diversity based on the presence of different alkaloid biosynthesis genes, then confirmed the possible chemotypes with high-performance liquid chromatography (HPLC)/ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) measurements. Infections of Epichloë spp. were found in Festuca pratensis Huds. (81%), Festuca ovina L. aggregate (agg.) (73%), Lolium perenne L. (15%), Festuca rubra L. (15%) and Dactylis glomerata L. (8%). The other eight grass species did not appear to be infected. For the majority of Epichloë-infected L. perenne samples (98%), the alkaloids lolitrem B and peramine were present, but ergovaline was not detected, which was consistent with the genetic evaluation, as dmaW, the gene encoding the first step of the ergot alkaloid biosynthesis pathway, was absent. Epichloë uncinata in F. pratensis produced anti-insect loline compounds. The Epichloë spp. observed in the F. ovina agg. samples showed the greatest level of diversity, and different intermediates of the indole-diterpene pathway could be detected. Epichloë infection rates alone are insufficient to estimate intoxication risks for livestock, as other factors, like the ability of the endophyte to produce the alkaloids, also need to be assessed.IMPORTANCE Severe problems of livestock intoxication from Epichloë-infected forage grasses have been reported from New Zealand, Australia, and the United States, but much less frequently from Europe, and particularly not from Germany. Nevertheless, it is important to monitor infection rates and alkaloids of grasses with Epichloë fungi to estimate possible intoxication risks. Most studies focus on agricultural grass species like Lolium perenne and Festuca arundinacea, but other cool-season grass species can also be infected. We show that in Germany, infection rates and alkaloids differ between grass species and that some of the alkaloids can be toxic to livestock. Changes in grassland management due to changing climate, especially with a shift toward grasslands dominated with Epichloë-infected species such as Lolium perenne, may result in greater numbers of intoxicated livestock in the near future. We therefore suggest regular monitoring of grass species for infections and alkaloids and call for maintaining heterogenous grasslands for livestock.

High-concentrate diets based on forages harvested at different maturity stages affect ruminal synthesis of B vitamins in lactating dairy cows.

Effects of plant maturity on apparent ruminal synthesis and post-ruminal supply of B vitamins were evaluated in two feeding trials. Diets containing alfalfa (Trial 1) or orchardgrass (Trial 2) silages harvested either (1) early cut, less mature (EC) or (2) late cut, more mature (LC) as the sole forage were offered to ruminally and duodenally cannulated lactating Holstein cows in crossover design experiments. In Trial 1, conducted with 16 cows (569±43 kg of empty BW (ruminal content removed) and 43.7±8.6 kg/day of 3.5% fat-corrected milk yield; mean±SD) in two 17-day treatment periods, both diets provided ~22% forage NDF and 27% total NDF, and the forage-to-concentrate ratios were 53 : 47 and 42 : 58 for EC and LC, respectively. In Trial 2, conducted with 13 cows (588±55 kg of empty BW and 43.7±7.7 kg/day of 3.5% fat-corrected milk yield; mean±SD) in two 18-day treatment periods, both diets provided ~25% forage NDF and 31% total NDF; the forage-to-concentrate ratios were 58 : 42 and 46 : 54 for EC and LC, respectively. Thiamin, riboflavin, niacin, vitamin B6, folates and vitamin B12 were measured in feed and duodenal content. Apparent ruminal synthesis was calculated as the duodenal flow minus the intake. Diets based on EC alfalfa decreased the amounts of thiamin, niacin and folates reaching the duodenum, whereas diets based on EC orchardgrass increased riboflavin duodenal flow. Daily apparent ruminal synthesis of thiamin, riboflavin, niacin and vitamin B6 were correlated negatively with their intake, suggesting a microbial regulation of their concentration in the rumen. Vitamin B12 apparent ruminal synthesis was correlated negatively with total volatile fatty acids concentration, but positively with ruminal pH and microbial N duodenal flow.

Effect of starchy or fibrous carbohydrate supplementation of orchardgrass on ruminal fermentation and methane output in continuous culture.

A 4-unit dual-flow continuous culture fermentor system was used to assess the effects of supplementing orchardgrass (Dactylis glomerata L.) with 2 levels [5 and 10% of total dry matter (DM) fed] of starchy (barley grain, BAR) or fibrous (beet pulp, BP) carbohydrate sources on nutrient digestibility, volatile fatty acid (VFA) production, bacterial protein synthesis, and CH4 output. Treatments were randomly assigned to fermentors in a 4×4 Latin square design with a 2×2 factorial arrangement using 7 d for microbial adaptation and 3 d for sample collection. Treatments included (1) 57g of DM orchardgrass + 3g of DM BAR, (2) 54g of DM orchardgrass + 6g of DM BAR, (3) 57g of DM orchardgrass + 3g of DM BP, or (4) 54g of DM orchardgrass + 6g of DM BP. Feedings occurred at 0900, 1030, 1400, and 1900h throughout four 10-d periods. Gas samples for CH4 analysis were collected 6 times daily at 0725, 0900, 1000, 1355, 1530, and 1630h. Fermentor samples for pH, NH3-N, and VFA analysis were taken on d 8, 9, and 10. Samples were also analyzed for DM, organic matter, crude protein, purines, neutral detergent fiber, and acid detergent fiber to determine nutrient digestibilities and estimation of bacterial protein synthesis. Apparent and true DM and organic matter digestibilities were not affected by supplement source. Apparent neutral and acid detergent fiber digestibilities were greater for BAR than BP. Conversely, apparent crude protein digestibility was greater for BP than BAR. Mean and maximum pH tended to be greatest for BAR than BP. Minimum pH was greater at the lower level (5% of diet DM) of supplementation. Barley produced greater concentrations of total VFA and acetate, whereas BP had greater daily outputs of CH4. Significant supplement type × level interactions were found for bacterial N flow and efficiency. Overall, supplementing orchardgrass with BP improved crude protein digestibility, reduced fiber digestibility and total VFA concentration, but increased CH4 output compared with BAR.

Effect of water-soluble carbohydrate content in orchardgrass pasture on grazing time and rumen fermentation in dairy cows.

Two experiments were conducted to clarify the effect of water-soluble carbohydrate (WSC) content in orchardgrass pasture on the diurnal distribution of grazing time. Six ruminally cannulated, non-lactating dairy cows were grazed on either of two pastures with different orchardgrass cultivars containing low WSC (LWSC; cultivar: 'Hokkai 28') or high WSC (HWSC; cultivar: 'Harunemidori'). The cows were grazed in morning and evening sessions in experiment 1, whereas the cows were grazed throughout the day in experiment 2. In experiment 1, grazing time of the cows on HWSC was longer than that of the cows on LWSC (P < 0.01). This difference was larger in the morning session than in the evening session (pasture × grazing session: P < 0.05). Effects on herbage intake were similar to those on grazing time. In experiment 2, daily total grazing time was longer for the cows on HWSC than for those on LWSC (P < 0.05). The cows on HWSC spent a longer time grazing than those on LWSC in the morning between 03.00 and 09.00 hours (P < 0.01). The results indicated that prolonged grazing time in the period between dawn and early morning could increase daily herbage intake in cows grazed on pastures of orchardgrass cultivars with high-WSC content.

Divergent utilization patterns of grass fructan, inulin, and other nonfiber carbohydrates by ruminal microbes.

Fructans are an important nonfiber carbohydrate in cool season grasses. Their fermentation by ruminal microbes is not well described, though such information is needed to understand their nutritional value to ruminants. Our objective was to compare kinetics and product formation of orchardgrass fructan (phlein; PHL) to other nonfiber carbohydrates when fermented in vitro with mixed or pure culture ruminal microbes. Studies were carried out as randomized complete block designs. All rates given are first-order rate constants. With mixed ruminal microbes, rate of substrate disappearance tended to be greater for glucose (GLC) than for PHL and chicory fructan (inulin; INU), which tended to differ from each other (0.74, 0.62, and 0.33 h(-1), respectively). Disappearance of GLC had almost no lag time (0.04 h), whereas the fructans had lags of 1.4h. The maximum microbial N accumulation, a proxy for cell growth, tended to be 20% greater for PHL and INU than for GLC. The N accumulation rate for GLC (1.31h(-1)) was greater than for PHL (0.75 h(-1)) and INU (0.26 h(-1)), which also differed. More microbial glycogen (+57%) was accumulated from GLC than from PHL, though accumulation rates did not differ (1.95 and 1.44 h(-1), respectively); little glycogen accumulated from INU. Rates of organic acid formation were 0.80, 0.28, and 0.80 h(-1) for GLC, INU, and PHL, respectively, with PHL tending to be greater than INU. Lactic acid production was more than 7-fold greater for GLC than for the fructans. The ratio of microbial cell carbon to organic acid carbon tended to be greater for PHL (0.90) and INU (0.86) than for GLC (0.69), indicating a greater yield of cell mass per amount of substrate fermented with fructans. Reduced microbial yield for GLC may relate to the greater glycogen production that requires ATP, and lactate production that yields less ATP; together, these processes could have reduced ATP available for cell growth. Acetate molar proportion was less for GLC than for fructans, and less for PHL than for INU. In studies with pure cultures, all microbes evaluated showed differences in specific growth rate constants (µ) for GLC, fructose, sucrose, maltose, and PHL. Selenomonas ruminantium and Streptococcus bovis showed the highest µ for PHL (0.55 and 0.67 h(-1), respectively), which were 50 to 60% of the µ achieved for GLC. The 10 other species tested had µ between 0.01 and 0.11h(-1) with PHL. Ruminal microbes use PHL differently than they do GLC or INU.

The effect of high polyphenol oxidase grass silage on metabolism of polyunsaturated fatty acids and nitrogen across the rumen of beef steers.

Polyphenol oxidase (PPO) activity in red clover (Trifolium pratense) has been reported to reduce both proteolysis and lipolysis, resulting in greater N use efficiency and protection of PUFA across the rumen. Although high levels of PPO have been reported in grasses such as cocksfoot (orchard grass; Dactylis glomerata), no in vivo research has determined whether grass PPO elicits the same response as red clover PPO. To test the hypothesis that silage ensiled from grass with high levels of PPO protects N and PUFA across the rumen, 6 steers with ruminal and duodenal cannulas were offered cocksfoot silage (CO; high-PPO grass), perennial ryegrass silage (PR; Lolium perenne; low-PPO grass), or red clover silage (RC; high-PPO control) at 16 g DM/kg BW daily with the experiment consisting of two 3 × 3 Latin squares with 21-d periods, consisting of 12 d of diet adaptation, 6 d of duodenal marker infusion, 2 d of duodenal sampling, and 1 d of ruminal sampling. All silages were well preserved, with DM of 34.4, 55.3, and 45.4% for CO, PR, and RC. Activity of PPO in silages was low due to deactivation but was greater in CO than either PR or RC (0.15 vs. 0.05 and 0.08 µkatal/g DM). Protein-bound phenol (mg/g DM) as a measure of the degree of oxidation and an indication of PPO protection was greatest for RC (15.9) but comparable for PR (10.1) and CO (12.2). Biohydrogenation of C18 PUFA was significantly lower on RC compared to the 2 grass silages with CO greater than PR. Despite lower levels of total fatty acid intake and subsequent duodenal flow, CO resulted in greater levels of phytanic acid and total branched and odd chain fatty acids in duodenal digesta than RC or PR. Ruminal ammonia concentration was greatest for RC, with no difference between the grasses. Duodenal flow of microbial N and efficiency of microbial protein synthesis were lowest for CO and comparable for RC and PR. The CO (high-grass PPO) did not result in elevated levels of C18 PUFA escaping the rumen or improve efficiency of total N transfer through the rumen compared to PR. The RC resulted in a greater flow of N and nonmicrobial N to the duodenum than the 2 grasses with PR greater than CO.

Soluble phenolic compounds in fresh and ensiled orchard grass (Dactylis glomerata L.), a common species in permanent pastures with potential as a biomass feedstock.

High-value coproducts can greatly improve the feasibility of utilizing plant feedstocks for biorefining and biofuel production. Plant polyphenolics have potential application in the pharmaceutical and cosmetic industries. Orchard grass varieties have been noted for accumulation of polyphenolic compounds, and the current study determined the soluble phenol profile and content in the orchard grass variety 'Abertop'. Hydroxycinnamates and flavonoids were monitored during the transition from vegetative to flowering stage at maximum crop yield. Caffeic acid derivatives, related to bioactives in the Asian medicinal herb Salvia miltiorrhiza , and novel hydroxycinnamate-flavone conjugates were also identified in extracts. Harvest yields of hydroxycinnamates and flavonoids ranged from 2.6 to 4.0 kg/ha and from 2.1 to 5.1 kg/ha, respectively. Abundant compounds showed high levels of antioxidant activity comparable with that of trolox. Minimal changes in soluble phenol content and composition were observed after ensiling with the exception of increases in caffeic acid, a caffeic acid derivative, and a caffeic acid breakdown product, dihydroxystyrene.

Effect of hay steaming on forage nutritive values and dry matter intake by horses.

Management strategies for horses with respiratory disease include soaking hay before feeding. Hay steaming is an alternative to this practice; however, little is known about its impact on forage nutritive values or intake. The objective was to determine the effect of steaming on forage nutritive value and intake by horses. Two alfalfa (Medicago sativa L.)-orchardgrass (Dactylis glomerata L.) mixed hays were evaluated: a low moldy (NM) and moderately moldy (MM) hay. Six mature horses were used in a 10 d crossover design. Three horses were assigned to each hay type and treatments were switched on d 6. Each day, one bale of each hay was sampled (pre- and poststeaming) and steamed for 90 min using a commercial hay steamer. Two flakes of steamed or unsteamed NM or MM hay were weighed and offered simultaneously to each horse in individual hay nets. Horses were allowed access to hay for 2 h, orts were collected, and 2 h DMI was calculated. Six additional bales of NM and MM were used to evaluate the effect of steaming on total suspended particulate (TSP). Flakes of unsteamed or steamed hay were agitated in an electric cement mixer, and TSP were recorded every min for 30 min using a tapered element oscillating microbalance sampler. Paired t tests and PROC MIXED of SAS (SAS Inst. Inc., Cary, NC) were used to compare steamed and unsteamed hay nutritive values, mold concentration, TSP, and 2 h DMI. Steaming increased hay moisture and therefore reduced DM to 77 and 81% for NM and MM, respectively (P < 0.001). In NM and MM hay, steaming reduced P content by 16 and 17%, respectively (P ≤ 0.007). Steaming reduced water-soluble carbohydrates (WSC) and ethanol-soluble carbohydrates (ESC) by 13% (P = 0.001) and 27% (P = 0.003), respectively, for MM but had no effect on NM (P > 0.05). Steaming reduced mold concentrations in both hays by ≥ 91% (P < 0.001). Total suspended particulate of MM hay was reduced by 55% (P = 0.043), but TSP in NM hay was not affected by steaming (P = 0.445). Dry matter intake of NM was increased by steaming; horses ingested 0.64 kg of unsteamed and 2.02 kg of steamed hay (P < 0.001). Dry matter intake of MM was not affected by steaming (P > 0.05). For NM hay, steaming decreased P and mold concentrations and increased DMI of the hay but had no effect on TSP. In MM hay, steaming reduced P, WSC, ESC, mold concentrations, and TSP but did not affect DMI. Steaming represents a strategy for reducing TSP and mold concentrations and increasing DMI in some hays but can result in leaching of essential nutrients.

Functional characterization of orchardgrass cytosolic Hsp70 (DgHsp70) and the negative regulation by Ca2+/AtCaM2 binding.

When plants are exposed to extreme temperature, stress-inducible proteins are highly induced and involved in subcellular defence mechanisms. Hsp70, one of stress-inducible proteins, functions as an ATP-dependent molecular chaperone in broad organisms to process such as the inhibition of protein denaturation, promotion of protein folding, and renaturation of denatured proteins. In this study, we isolated a heat-inducible orchardgrass Hsp70 (DgHsp70) that is a homolog of cytosolic Hsp70 that possesses a CaM-binding domain. Purified DgHsp70 protein displayed dose-dependent ATPase, holdase, and ATP-dependent foldase activities. To investigate functional roles of DgHsp70 by the association of Arabidopsis calmodulin-2 (AtCaM2), showing heat-sensitive reduction on transcription, we first characterized the binding activity by gel-overlay assay. DgHsp70 binds to AtCaM2 in the presence of Ca(2+) via a conserved CaM-binding domain. Ca(2+)/AtCaM2 binding decreased ATPase activity of DgHsp70, and concomitantly, reduced foldase activity. Based on the protein structure of bovine Hsc70, which is the closest structural homolog of DgHsp70, a CaM-binding domain is located near the ATP-binding site and CaM may span the ATP-binding pocket of Hsp70. Its decreased functional foldase activity may be caused by blocking ATP hydrolysis after Ca(2+)/AtCaM2 binding. It may associate with inhibition of functional activity of DgHsp70 in the absence of stress and/or de novo protein synthesis of DgHsp70 in the presence of thermal stress condition.

Detection of plant volatiles after leaf wounding and darkening by proton transfer reaction "time-of-flight" mass spectrometry (PTR-TOF).

Proton transfer reaction-time of flight (PTR-TOF) mass spectrometry was used to improve detection of biogenic volatiles organic compounds (BVOCs) induced by leaf wounding and darkening. PTR-TOF measurements unambiguously captured the kinetic of the large emissions of green leaf volatiles (GLVs) and acetaldehyde after wounding and darkening. GLVs emission correlated with the extent of wounding, thus confirming to be an excellent indicator of mechanical damage. Transient emissions of methanol, C5 compounds and isoprene from plant species that do not emit isoprene constitutively were also detected after wounding. In the strong isoprene-emitter Populus alba, light-dependent isoprene emission was sustained and even enhanced for hours after photosynthesis inhibition due to leaf cutting. Thus isoprene emission can uncouple from photosynthesis and may occur even after cutting leaves or branches, e.g., by agricultural practices or because of abiotic and biotic stresses. This observation may have important implications for assessments of isoprene sources and budget in the atmosphere, and consequences for tropospheric chemistry.

Occurrence of associative effects between grasses and legumes in binary mixtures on in vitro rumen fermentation characteristics.

When animals are fed a grass and legume mixture, digestive interactions can occur in the rumen between the substrates contained in the different plants, and the response of the animal to the combination of forages can differ from the balanced median values of their components considered individually. Our objective was to assess the associative effects between temperate forages in 8 grass-legume binary combinations on in vitro rumen fermentation characteristics to highlight synergies or antagonisms in terms of nutritional and environmental impacts. Two grasses (cocksfoot, CF; ryegrass, RG) and 4 legume species (alfalfa, A; white clover, WC; red clover, RC; sainfoin, SAN) were incubated alone and in grass-legume mixture (1:1, wt/wt) in batches containing buffered rumen fluid during 3.5 and 24 h. For each substrate and each incubation time, parameters describing the degradation of the energetic and nitrogenous compounds and their partition into fermentation end products were measured. Data were subjected to ANOVA using a mixed procedure to test quadratic contrasts. At 3.5 h of incubation, many quadratic effects were observed. The presence of A, WC, or RC in mixtures quadratically increased the NH(3)-N production (up to +28% when compared with the calculated value, P < 0.001), whereas the presence of condensed tannin (CT) activity in SAN considerably decreased it (up to -67%, quadratic effect, P < 0.001) and the N disappearance. To a lesser extent, the presence of SAN reduced the proportion of methane in the gas produced (up to 7%, quadratic effect, P = 0.018). Generally, the degradation of OM and NDF was not stimulated by mixing grass and legume, except for SAN. However, the presence of SAN severely impaired the NDF digestion at the early phase of fermentation. At 24 h of incubation, few associative effects were observed in comparison with those observed at 3.5 h of incubation, but the effect of CT of SAN on N metabolism was still clearly present. This study shows that, among the grass-legume mixtures tested in vitro, only the presence of SAN can interact with the grasses to reduce the degradation of proteins and the production of methane but with a transitory negative effect on fiber digestion.

Effects of spontaneous heating on estimates of total digestible nutrients for alfalfa-orchardgrass hays packaged in large round bales.

Large round or large square hay packages are more likely to heat spontaneously during storage than hay packaged in conventional (45 kg) bales, and the effects of this phenomenon on the associated energy estimates for these hays can be severe. Our objectives for this project were to assess the relationship between estimates of total digestible nutrients (TDN) and spontaneous heating and to describe any important differences in energy estimates that may result specifically from 2 methods of estimating truly digestible fiber (TD-Fiber). Using the summative approach to estimate TDN, TD-Fiber can be estimated from inputs of protein-corrected neutral detergent fiber (NDFn) and acid detergent lignin (TD-FiberLIG) or from NDFn and 48-h neutral detergent fiber digestibility (TD-FiberNDFD). Throughout 2006 and 2007, mixed alfalfa (Medicago sativa L.)-orchardgrass (Dactylis glomerata L.) hays from 3 individual harvests were obtained from the same 8.2-ha research site near Stratford, Wisconsin. Both options for estimating TD-Fiber (TD-FiberLIG or TD-FiberNDFD) were then used independently via the summative approach to estimate the total TDN concentrations (TDN-LIG or TDN-NDFD, respectively) within these hays. Estimates of both TDN-LIG and TDN-NDFD then were related to heating degree days >30 degrees C accumulated during storage by various regression techniques. Changes (poststorage - prestorage) in TDN-LIG that occurred during storage (DeltaTDN-LIG) were best fitted with a nonlinear decay model in which the independent variable was squared [Y=(11.7 x e(-0.0000033xxxx)) - 11.6; R(2)=0.928]. For changes in TDN-NDFD (DeltaTDN-NDFD), a quadratic regression model provided the best fit (Y=0.0000027x(2) - 0.010x+0.4; R(2)=0.861). Generally, DeltaTDN-LIG estimates were 2.0 to 4.0 percentage units lower than DeltaTDN-NDFD estimates when heating exceeded 500 HDD. For regressions on maximum internal bale temperature, both DeltaTDN-LIG (Y=-0.38x+16.3; R(2)=0.954) and DeltaTDN-NDFD (Y=-0.25x+10.2; R(2)=0.848) were best fitted by linear models with heterogeneous (P<0.001) slopes and intercepts. In both cases, coefficients of determination were high, suggesting that simple measures of spontaneous heating are excellent predictors of energy losses in heated forages. Regardless of method, reductions in TDN were associated primarily with losses of nonfiber carbohydrate, which is known to occur via oxidation of sugars during spontaneous heating. For heated forages, some discrepancy between TDN-LIG and TDN-NDFD existed because the relationship between NDFD and spontaneous heating was shown previously to be very poor, resulting in minimal changes for estimates of TD-FiberNDFD as a consequence of heating. In contrast, TD-FiberLIG declined in close association with heating, largely because TD-FiberLIG was sensitive to changes in concentrations of both NDFn and acid detergent lignin. Discrepancies between TDN-LIG and TDN-NDFD were exacerbated further when neutral detergent fiber rather than NDFn was used to estimate TD-FiberNDFD. Estimates of TDN declined by as much as 13.0 percentage units within severely heated hays, and this is a serious consequence of spontaneous heating.

Effects of spontaneous heating on forage protein fractions and in situ disappearance kinetics of crude protein for alfalfa-orchardgrass hays packaged in large round bales.

During 2006 and 2007, forages from 3 individual hay harvests were used to assess the effects of spontaneous heating on concentrations of crude protein (CP), neutral detergent insoluble CP (NDICP), acid detergent insoluble CP (ADICP), and in situ disappearance kinetics of CP and NDICP for large round bales of mixed alfalfa (Medicago sativa L.) and orchardgrass (Dactylis glomerata L.). Over the 3 harvests, 96 large round bales were made at preset bale diameters of 0.9, 1.2, or 1.5m and at moisture concentrations ranging from 9.3 to 46.6%. Internal bale temperatures were monitored daily during an outdoor storage period. The change in concentrations of NDICP (poststorage - prestorage) increased with heating degree days (HDD) >30 degrees C in a relationship best explained with a nonlinear model {Y=24.9 - [22.7 x (e(-0.000010 x x x x))]; R(2)=0.892} that became asymptotic at +24.9 percentage units of CP, thereby indicating that NDICP increases rapidly within bales that heat spontaneously. When maximum internal bale temperature (MAX) was used as the independent variable, the best regression model was quadratic and the coefficient of determination was still relatively high (R(2)=0.716). The change in concentrations of ADICP (poststorage - prestorage; DeltaADICP) also increased with HDD and was best fitted to a nonlinear model {Y=14.9 - [15.7 x (e(-0.0000019 x x x x))]} with a very high coefficient of determination (R(2)=0.934). A similar quartic response was observed for the regression of DeltaADICP on MAX (R(2)=0.975). Increases in DeltaADICP as a result of heating (HDD or MAX) were paralleled by concurrent increases in hemicellulose at relatively low increments of heating, but the inverse relationship was observed as hemicelluloses likely became reactive and concentrations decreased in more severely heated hays. Changes in ruminal disappearance rate of CP were best fitted to cubic models for regressions on both HDD (R(2)=0.939) and MAX (R(2)=0.876); these changes represented an approximate 50% rate reduction in severely heated hays relative to prestorage controls. Within ranges of heating most commonly encountered under field conditions, changes in rumen-degradable protein decreased in a primarily linear relationship with HDD or MAX. However, the mean change in rumen-degradable protein for the 4 most severely heated hays was only -2.6 percentage units of CP, which represents a minimal reduction from prestorage controls and is far less than the maximum of -7.9 percentage units of CP observed with less-severe heating. Interpretation of these results was complicated by poor recovery of NDICP from our most severely heated hays following machine rinsing of 0-h Dacron bags; theoretically, and by definition, this unrecovered pool of NDICP is assumed to be entirely degradable in the rumen. It remains unclear whether these responses could be corroborated in vivo or by other analytical techniques, or whether the magnitude of HDD or MAX for our most severely heated hays exceeds the reliable limits for estimating RDP via in situ methodology.

Extraction, structural characterisation and evaluation of hydroxycinnamate esters of orchard grass (Dactylis glomerata) as substrates for polyphenol oxidase.

Polyphenol oxidase (PPO) activity has been reported in orchard grass (Dactylis glomerata); however, to date, no endogenous substrates have been identified. In the present study, we report the isolation and structural elucidation of PPO substrates in this species. The free phenol fraction was extracted, separated by reverse-phase chromatography and six potential substrates, including two hydroxycinnamate esters, were identified by UV spectrometry, electrospray ionisation-tandem mass spectrometry (LC-ESI-MS(n)) and 1D and 2D NMR analyses ((1)H NMR, (13)C NMR, DEPT, COSY, HMQC and HMBC). Furthermore, three caffeoylquinic acids (3-CQA, 4-CQA and 5-CQA) were identified by comparison of their spectral data (ESI-MS) with those of known compounds and literature data. Five of these compounds were demonstrated to be substrates for orchard grass PPO.

Characterization of peptidic and carbohydrate cross-reactive determinants in pollen polysensitization.

BACKGROUND: Cross-reactivity may be due to protein sequence or domain homologies and/or the existence of cross-reactive carbohydrate determinants (CCDs). The clinical relevance of peptidic cross-reactivities is well known, whereas that of CCDs is still a question of debate. The aim of this study is to characterize the IgE specificity of various patients suffering from pollen polysensitization to identify both peptidic and carbohydrate cross-reactive determinants. MATERIAL AND METHODS: Rapeseed, grass and Arabidopsis proteins were separated by isoelectric focusing, followed by SDS-PAGE, and transferred to a nitrocellulose sheet. The sheets were incubated either with an individual serum from a birch+grass-sensitive patient, followed by anti-human IgE, or with labelled Concanavalin A (ConA). Binding inhibition was tested by incubation of the sera with a mixture of sugar residues. RESULTS: The results showed two different patterns of cross-reacting sera: a pattern that implies few proteins, not always glycosylated and known as allergens, and a pattern that implies numerous proteins with molecular masses over 30 kDa. This second pattern was very close to the ConA -binding pattern. The IgE binding was abolished by pre-incubation with sugar residues only in the case of the second pattern. DISCUSSION: This study shows that multiple pollen sensitizations could result from multiple sensitizations to specific proteins or from a cross-sensitization to a wide range of glycoproteins. Two-D blots allow to characterize a cross-sensitization due to carbohydrate determinants, and thus to improve the diagnosis of allergy and its medical treatment.

Effective rumen degradation of dry matter, crude protein and neutral detergent fibre in forage determined by near infrared reflectance spectroscopy.

The objective of the present study was to examine if near infrared reflectance spectroscopy (NIRS) could be used to predict degradation parameters and effective degradation from scans of original forage samples. Degradability of dry matter (DM), crude protein (CP) and neutral detergent fibre (NDF) of 61 samples of perennial ryegrass (Lolium perenne L.) and orchardgrass (Dactylis glomerata L.) was tested by using the in situ technique. The grass samples were harvested at three different stages, early vegetative growth, early reproductive growth and late reproductive growth. Degradability was described in terms of immediately rumen soluble fraction (a fraction, for DM and CP only as NDF does not contain a soluble fraction), the degradable but not soluble faction (b fraction) and the rate of degradation of the b fraction (c value). Overall effective degradability of DM, CP and NDF was also calculated. Near infrared reflectance spectroscopy was examined for its ability to predict degradation parameters and to make a direct prediction of effective degradation from scans of the original samples of perennial ryegrass and orchardgrass. Prediction of effective degradation of the different feed fractions showed different accuracy. The coefficients of determination (R(2)) from regressions of predicted vs. measured effective degradation, using a cross-validation method, were 0.92 for DM, 0.78 for CP and 0.61 for NDF. The attempt to predict the degradation parameters (a, b and c) by NIRS was less successful as the coefficients of determination for the degradation parameters were low. Concentrations of CP and NDF in the original samples were predicted by using NIRS and the validated R(2) value was 0.98 for CP and 0.92 for NDF. It is concluded that using NIRS predictions from scans of original samples is a promising method to obtain values for the effective degradation of DM, CP and NDF in ruminant feeds, but that larger calibration sets are necessary for obtaining improved accuracy.

Fractional and structural characterization of hemicelluloses from perennial ryegrass (Lolium perenne) and cocksfoot grass (Dactylis glomerata).

Sequential three-stage treatments with 80% EtOH containing 0.2% NaOH, 2.5% H2O2-0.2% EDTA containing 1.5% NaOH and 2.5% H2O2-0.2% TAED containing 1.0% NaOH at 75 degrees C for 3h released 8.0% and 10.4%, 79.1% and 77.0% and 12.9% and 12.5% of the original hemicelluloses from perennial grass and cocksfoot grass, respectively. It was found that the four alkaline peroxide-soluble hemicellulosic fractions contained higher amounts of xylose (33.4-38.2%), uronic acids (9.3-15.3%) and rhamnose (3.0-3.9%), but were lower in glucose (25.1-28.3%), galactose (13.3-15.3%) and mannose (0.4-1.5%) than those of the two alkaline EtOH-soluble hemicellulosic fractions in which glucose (32.9-36.0%), xylose (20.1-22.6%), arabinose (14.1-21.4%), galactose (16.6-19.9%), mannose (4.1-9.9%) and uronic acids (3.4-7.4%) were the major sugar components. 13C NMR spectroscopy confirmed that all the six hemicellulosic fractions were composed of galactoarabinoxylans, 4-O-methylglucuronoarabinoxylans and beta-glucan. In addition, the studies showed that the four alkaline peroxide-soluble hemicellulosic fractions were more linear and acidic and had larger molecular weights (Mw, 28,400-38,650 g mol(-1)) than those of the two alkaline EtOH-soluble hemicellulosic fractions (Mw, 16,460-17,420 g mol(-1)).

Aerobic stability of wheat and orchardgrass round-bale silages during winter.

Using recently developed technology, balage is often stored in large (1.2 x 1.2 m) round bales that are wrapped in plastic film with an in-line wrapper. The aerobic stability of this fermented forage is important, particularly during winter months when it is fed to livestock or sold as a cash crop. Two types of forage, orchardgrass [Dactylis glomerata L.; 54.4% dry matter (DM)] and wheat (Triticum aestivum L.; 62.4% DM), were packaged in large round bales and wrapped with an in-line wrapper during May 2002. Twenty-one bales of each balage type were unwrapped and exposed to air on Dec. 10, 2002 for 0, 2, 4, 8, 16, 24, or 32 d (ambient temperature range = 0.6 to 19.4 degrees C) to evaluate aerobic stability. For both orchardgrass and wheat balage, final bale weight, concentration of DM, and pH were not affected by exposure time. Across both balage types, DM recoveries were > or = 97% for all bales, indicating that both balage types were very stable when exposed to air. For orchardgrass balage, exposure time had no effect on concentrations of NDF, ADF, hemicellulose, cellulose, or lignin, thereby indicating that little deterioration occurred. Similarly, no contrast relating any fiber component with exposure time was significant for wheat balage. Concentrations of crude protein (CP) were not affected by exposure time for wheat balage, but there was a tendency for exposed orchardgrass bales to have greater concentrations of CP than bales sampled on d 0. Exposure time had no effect on 48-h in situ digestibility of DM for wheat balage, but there was a tendency for a linear increase with exposure time for orchardgrass balage. However, the overall range (78.2 to 80.5%) over the 32-d exposure period was very narrow, and this response is probably of limited biological significance. Generally, concentrations of fermentation acids were low, primarily because of the high concentration of DM within these balages, and only minimal changes in these acids were observed over the exposure interval. These results suggest that the balage evaluated in this trial during winter conditions was very stable after exposure to air for up to 32 d. This should allow for considerable flexibility with respect to feeding, transport, and marketing of balage during winter months without significant aerobic deterioration.