Microscale Spatiotemporal Variation and Generation Mechanisms of Reactive Oxygen Species in the Rhizosphere of Ryegrass: Coupled Biotic-Abiotic Processes.

Reactive oxygen species (ROS) play key roles in soil biogeochemical processes, yet the occurrence and accumulation of ROS in the rhizosphere are poorly documented. Herein, we first developed a ROS-trapping membrane to in situ determine ROS in the ryegrass rhizosphere and then quantified the temporal and spatial variations of representative ROS (i.e., O2•─, H2O2, and •OH). Fluorescence imaging clearly visualized the production of ROS in the rhizosphere. Both O2•─ and H2O2 content increased first and then declined throughout the life cycle of ryegrass, while •OH concentration decreased continuously. Spatially, ROS contents remained at a relatively high level at 0-5 mm and then descended with increasing distance. The concentrations of ROS in different soils followed the order of black soil > latosol soil > yellow-brown soil > tier soil ∼ red soil. Analysis of soil properties suggested that both biotic factors (microbial community) and abiotic factors (Fe(II) and water-soluble phenols) played critical roles in ROS production. The combined processes, including Fe(II) and water-soluble phenol-mediated electron transfer, microbial community-driven extracellular O2•─ release, and Fe(II)/Fe(III) cycling, may be responsible for ROS production. These findings provide insights into ROS-associated rhizosphere effects and inspiration for the phytoremediation of pollutants and element cycling.

Biostimulating effect of l-tryptophan on the yield and chemical and microbiological quality of perennial ryegrass (Lolium perenne) herbage and silage for ruminant.

BACKGROUND: This study aimed to evaluate the effect of l-tryptophan (l-TRP) used in the cultivation of Lolium perenne on the yield, and chemical and microbiological quality of its herbage and silage. l-Tryptophan was applied in doses of 5, 0.5, 0.05 kg ha-1 . The experiment was conducted with a control group (C) and a comparative control group (C+) with higher nitrogen fertilization. RESULTS: The dose of 5 kg ha-1 had a significant effect on herbage yield, which increased by 15% compared to group C and approximated the value achieved in group C+. The treatment with l-TRP caused a significant increase in water-soluble carbohydrate (WSC) content only in Tr5 (165 g kg-1 DM), which was reflected in a more beneficial course of fermentation, lower pH (4.59), and a higher sum of fermentation acids, including lactic acid (94.7 g kg-1 DM). Ryegrass treatment with a high l-TRP dose effectively reduced the loads of Clostridium spp. and fungi, and increased the count of Bacillus spp. The l-TRP significantly reduced N-NH3 content in Tr05 (98.6 g kg-1 TN) compared with C+ (123 g kg-1 TN) and butyric acid content in Tr05 (from 1.35 g kg-1 DM in the C to 0.38 g kg-1 DM). CONCLUSION: The most effective dose turned out to be a dose of 5 kg ha-1 , which allowed a higher yield and a better fermentation course to be achieved. This work presents the feasibility of using l-TRP to optimize nutrient consumption by Lolium perenne and ultimately to affect the quality of its silage as a feedstuff. © 2020 Society of Chemical Industry.

Toxic metal phytoextraction potential and health-risk parameters of some cultivated plants when grown in metal-contaminated river sediment of Danube, near an industrial town.

Toxic metal phytoextraction potential of some higher plants, the white mustard (Sinapis alba L.), perennial rye grass (Lolium perenne L.) and also two cultivated plants, as green pea (Pisum sativum L. var. Rajnai törpe), radish (Raphanus sativus L. var. Szentesi óriás vaj), was studied in a field experiment, along the river Danube in close vicinity of an industrial town, Dunaújváros, Hungary. Soil/sediment and the various plant organs (leaves, stems and roots) were assessed for the contamination with some potentially toxic elements (PTE), such as the cadmium (Cd), nickel (Ni), copper (Cu), and zinc (Zn). It was found that Cd and Ni concentration was below, while the Cu and Zn elements were above the Hungarian permissible limits in each of the studied soil/sediment samples. Bioconcentration factor (BAF) was less than 1 in the shoot biomass of test plant samples and followed the order of Cu > Zn > Cd and Ni. Phytoremediation potential of selected test plants was found to be rather limited. The translocation factor (TF) was more than 1 for Cu and Zn elements, at each test plants. Cadmium was translocated into the leaves in case of the radish, only. Considering of the potential human daily intake of metals (DIM), it was less than 1 both for the adults and for the children. Health risk index (HRI) values of children, however, were higher than 1 for the Cd in case of radish, and for Zn and Cu in case of the pea. Results suggest that consumption of these plants grown in gardens of contaminated sediments can result in some risks for citizens in the industrial town of Dunaújváros. Further studies are required to identify appropriate plants with greater toxic metal phytoextraction potential.

Determination of Arginine δ15N Values in Plant and Animal Proteins by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry.

Nitrogen (N) stable isotope techniques are widely used in ecology, archaeology, and forensic science to explore trophic relationships and provenances of organisms and materials, most widely using bulk δ15N values of whole organisms, tissues, or other materials. However, compound-specific isotope values can provide more diagnostic isotope "fingerprints" and specific information about metabolic processes. Existing techniques for nitrogen isotope analysis allow the determination of δ15N values of 14 amino acids (AAs), accounting for ca. 75% of plant protein and collagen N. The majority of remaining N is from arginine, comprising 16 and 14% of collagen and plant protein N, respectively. We therefore aimed to develop a method to detect arginine and determine its δ15N value (δ15NArg) by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), to further contribute to the understanding of the metabolic routing of this important AA. We demonstrate that arginine, as its N-acetyl isopropyl ester, is amenable to GC analysis using a 15 m midpolarity DB-35 column, eluting with baseline resolution from other AAs. The recorded δ15N value by GC-C-IRMS was within the error of that of the underivatized compound determined by elemental analyzer-isotope ratio mass spectrometry (EA-IRMS). The newly developed GC-C-IRMS method was applied to modern plant protein and cattle collagen, enabling their δ15NArg values to be related to AA biosynthesis. Determination of archaeological cattle collagen δ15NArg values confirmed the suitability of this method to provide further insights into past diets and ecosystems. Bulk collagen δ15N value reconstruction including δ15NArg values better reflect the measured bulk values, as the isotopic ratio of 91% of collagen N can now be determined at the compound-specific level.

Host Range Expansion of an Endemic Insect Herbivore is Associated With High Nitrogen and Low Fibre Content in Exotic Pasture Plants.

Endemic moth species of the genus Wiseana spp. (Hepialidae) have become serious pests of introduced pasture plants in New Zealand. The original native host plants of these moths have not been confirmed. This study investigated the performance (survival, development time, weight gain) of three Wiseana species on seven putative host plants: five native and two exotic species. The aim was to identify native hosts for the three Wiseana species and to compare their performance on native plants and exotic pasture plants. The chemical composition of the seven putative host plants was investigated to compare native and exotic plant chemistries, and to test for associations between plant characteristics and performance of selected Wiseana species. Carbon, nitrogen, silica and fibre contents were measured for each plant species; primary metabolite composition was determined by gas chromatography-mass spectrometry. For the three moth species, increased survival and weight gain were significantly associated with high nitrogen and low fibre contents in one exotic host plant, white clover (Trifolium repens), although one species, W. umbraculata, did not complete development to adult on any of the plants tested, including clover. Two exotic plants (T. repens, Lolium perenne × Lolium multiflorum), and two native plants (Aciphylla squarrosa and Festuca actae) supported W. copularis development to the adult stage, but only one exotic (T. repens) and one native (F. actae) species supported complete development of W. cervinata. Exotic and native plant species had distinct metabolite profiles, but there was no significant association between metabolite composition and Wiseana performance. We conclude that W. copularis and W. cervinata, but not W. umbraculata, have expanded their host range, because of their ability to use both native and new hosts. No evidence was found for a host shift, i.e., a loss of performance on the ancestral host compared with the new host.

Contrasting Effects of Grass - Endophyte Chemotypes on a Tri-Trophic Cascade.

Systemic grass-endophytes of the genus Epichloë symbiotically infect the above-ground plant parts of many grass species, where they produce alkaloids in a grass- and endophyte-specific manner that are toxic or deterrent to herbivores. An increasing number of studies show cascading negative effects of endophyte-derived alkaloids that extend to higher trophic levels, harming beneficial insects, including those that control aphid populations. Lacewings are one of the major biological aphid controls, and are especially resistant to insecticides and pollutants, but their susceptibility to endophyte infection in the food chain has never been studied. Our study found variability in aphid population growth depending on the endophyte-grass chemotype, where aphid population growth was lowest on chemotypes known for producing high amounts of loline alkaloids. We also showed that larval and pupal development and mortality of the Common Green Lacewing (Chrysoperla carnea) was, in a non-choice experiment, not affected by endophyte infection in the food chain. This is a first indication that lacewings might be resistant to endophyte-derived alkaloids and could be robust biocontrol agents when applied together with endophyte-infected grass, possibly replacing chemical pesticides.

Metal immobilization and nitrate reduction in a contaminated soil amended with zero-valent iron (Fe0).

Technologies based on zero-valent iron (Fe0) are increasingly being used to immobilize metals in soils and remove metals and nitrate from waters. However, the impact of nitrate reduction on metal immobilization in metal contaminated soils has been poorly investigated so far. Here, different concentrations of Fe0 filings (1%, 2% and 5%; wt%) were applied to a metal contaminated soil. The resulting nitrate reduction and metal (Cd and Zn) immobilization was investigated using a column leaching experiment for 12 weeks. Corrosion of Fe0 filings and precipitation of Fe oxyhydroxydes (FeOOH) on the surfaces of the filings were observed using SEM-EDS and EMPA-WDS at the end of the experiment. Compared to the untreated soil, total nitrate amounts released were lowered by 47%, 59% and 87% in the presence of 1%, 2% and 5% of Fe0, respectively. Concomitantly with nitrate reduction, Cd and Zn concentrations in leachates were strongly alleviated in the presence of Fe0, which was partly attributed to the rise of soil pH subsequent to nitrate reduction. More importantly, biotests with Lupinus albus L. revealed that the mechanisms involved in metal immobilization are stable to root-induced acidification. However, Fe0 was not efficient to reduce Cd concentration in Lolium multiflorum Lam., indicating that root processes other than acidification may re-mobilize metals.

Comparison of cadmium uptake and transcriptional responses in roots reveal key transcripts from high and low-cadmium tolerance ryegrass cultivars.

Cadmium (Cd), which seriously affects plant growth and crop production, is harmful to humans. Previous studies revealed ryegrass (Lolium multiflorum Lam.) exhibits Cd tolerance, and may be useful as a potential hyperaccumulator because of its wide distribution. In this study, the physiological and transcriptional responses of two ryegrass cultivars [i.e., high (LmHC) and low (LmLC) Cd tolerance] to Cd stress were investigated and compared. The Cd tolerance of LmHC was greater than that of LmLC at various Cd concentrations. The uptake of Evans blue dye revealed that Cd-induced root cell mortality was higher in LmLC than in LmHC after a 12-h Cd treatment. Furthermore, the content and influx rate of Cd in LmLC roots were greater than in LmHC roots under Cd stress conditions. The RNA sequencing and quantitative real-time PCR data indicated that the Cd transport regulatory genes (ABCG37, ABCB4, NRAMP4, and HMA5) were differentially expressed between the LmLC and LmHC roots. This expression-level diversity may contribute to the differences in the Cd accumulation and translocation between LmLC and LmHC. These findings may help clarify the physiological and molecular mechanisms underlying ryegrass responses to Cd toxicity. Additionally, ryegrass may be able to hyperaccumulate toxic heavy metals during the phytoremediation of contaminated soil.

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.

The effect of by-product inclusion and concentrate feeding rate on milk production and composition, pasture dry matter intake, and nitrogen excretion of mid-late lactation spring-calving cows grazing a perennial ryegrass-based pasture.

Interest is growing in the use of by-products as economical sources of nutrients that complement grazed grass, particularly at times when grass supply is insufficient to meet the nutritional demands of lactating dairy cattle. The objective of this research was to assess the effect of the amount of by-product inclusion and concentrate feeding rate on pasture dry matter intake, milk production and composition, and N excretion from spring-calving cows grazing summer pasture during mid-late lactation. Forty-eight Holstein Friesian dairy cows were randomly assigned to 1 of 4 dietary treatments in a 2 × 2 factorial design. Cows were grazed in one group on a perennial ryegrass-based sward, with pelleted concentrates offered twice daily during milking over a 63-d experimental period. The dietary treatments were 3 kg of concentrate containing 35% by-products; 6 kg of concentrate containing 35% by-products; 3 kg of concentrate containing 95% by-products; and 6 kg of concentrate containing 95% by-products on a fresh matter basis. The by-products used were soybean hulls, palm kernel expeller, and maize dried distillers grains with solubles, included in equal proportions on a dry matter basis. Pasture dry matter intake (14.5 kg/d) was not affected by the amount of by-product inclusion or feeding rate. By-product inclusion had no effect on milk yield (27.1 kg/d) or milk solids (MS) yield (2.0 kg/d). Cows offered 6 kg of concentrate had a greater milk (+1.6 kg/d) and MS (+0.13 kg/d) yield, consumed more N (+0.08 kg/d), and excreted a lower proportion of N in the milk (0.25 vs. 0.27) and feces (0.39 vs. 0.41) and a higher proportion in the urine (0.39 vs. 0.32) compared with cows offered 3 kg of by-product-based concentrate. In conclusion, by-products can be included at up to 95% of the concentrate fed to cows grazing pasture without affecting pasture dry matter intake, milk production or composition, or N excretion. Cows offered 6 kg of concentrates produced more milk and MS than cows offered 3 kg but had higher urinary N excretion. Economics of this yield response will depend on milk and concentrate prices.

Exploration of Chlorophyll a Fluorescence and Plant Gas Exchange Parameters as Indicators of Drought Tolerance in Perennial Ryegrass.

Perennial ryegrass (Lolium perenne L.) belongs to the common cultivated grass species in Central and Western Europe. Despite being considered to be susceptible to drought, it is frequently used for forming the turf in urban green areas. In such areas, the water deficit in soil is recognized as one of the most important environmental factors, which can limit plant growth. The basic aim of this work was to explore the mechanisms standing behind the changes in the photosynthetic apparatus performance of two perennial ryegrass turf varieties grown under drought stress using comprehensive in vivo chlorophyll fluorescence signal analyses and plant gas exchange measurements. Drought was applied after eight weeks of sowing by controlling the humidity of the roots ground medium at the levels of 30, 50, and 70% of the field water capacity. Measurements were carried out at four times: 0, 120, and 240 h after drought application and after recovery (refilling water to 70%). We found that the difference between the two tested varieties' response resulted from a particular re-reduction of P700+ (reaction certer of PSI) that was caused by slower electron donation from P680. The difference in the rate of electron flow from Photosystem II (PSII) to PSI was also detected. The application of the combined tools (plants' photosynthetic efficiency analysis and plant gas exchange measurements) allowed exploring and explaining the specific variety response to drought stress.

Effectiveness of pig sludge as organic amendment of different textural class mine tailings with different periods of amendment-contact time.

The present study assesses the effect of tailing texture (loamy sand (LT) and sandy loam (ST)), dose of pig sludge (0, 50, 100 and 200 t ha-1) and amendment-contact time (14, 28 and 42 days) on physicochemical quality of amended substrate using Lolium perenne var Nui as a bioindicator. The main properties of LT differed of ST in levels of total organic carbon (0.19 and 0.58%), in pH (4.6 and 8.5), total Cu (202 and 1647 mg kg-1) and Zn content (31 and 137 mg kg-1). Soil pore water of experimental substrates was characterized for pH, electrical conductivity (EC) and Cu2+ ion activity (pCu2+) while ammonium nitrogen (NH4+-N), infiltration rate and general physicochemical characteristics were measured in substrates. Shoot biomass (SB), root biomass (RB) and the RB:SB ratio was calculated for L. perenne. The results showed there to be a significant interaction (p < 0.05) between tailing texture, sludge dose and amendment-contact time for pCu2+, infiltration rate, SB, RB, and RB: SB ratio, but not for pH, EC, or NH4+-N. However, sludge dose and amendment-contact time significantly affected all variables. By increasing dosages of pig sludge, pore water pH increased, and this was associated with decreases in pCu2+ and the infiltration rate. High doses of pig sludge (100 and 200 t ha-1) impaired growth of L. perenne irrespective of tailing texture and amendment-contact time, likely because of the rise of EC (up to 14 mS cm-1). For both tailing textures, the highest biomass was obtained after incorporation of 50 t ha-1 of pig sludge, with increasing values as amendment-contact time rose. In conclusion, effective management of pig sludge for tailing reclamation should guarantee doses <50 t ha-1 and amendment-contact time >28 days, irrespective of tailing texture.

Stability of pyrrolizidine alkaloids from Senecio vernalis in grass silage under different ensilage conditions.

BACKGROUND: This study evaluated the degradation of pyrrolizidine alkaloids (PAs) from eastern groundsel (Senecio vernalis) in grass silage prepared with different inoculants. Silages were produced from ryegrass with 230 g kg-1 dry matter (DM) content and mixed with eastern groundsel (9:1; w/w fresh matter basis) containing 5.5 g kg-1 DM PA. Treatments were: CON (untreated control), LP (3.0 × 105 cfu g-1 Lactobacillus plantarum DSMZ 8862/8866) or LBLC (7.3 × 104 cfu g-1 Lactobacillus buchneri LN40177 / Lactobacillus casei LC32909), and each of the treatments in combination with 30 g kg-1 molasses. Silages were prepared in glass jars and opened after 3, 10, and 90 days. Fermentation characteristics were determined and the PAs analyzed. RESULTS: Although the levels of fermentation acids differed between treatments, results indicated good quality of all silages during 90 days. Significant time (P < 0.001) and treatment (P < 0.001) effects were observed for PAs. Concentrations of senecionine and seneciphylline decreased with molasses, declined over time, and were negatively correlated with lactic, propionic, and butyric acid, or with lactic and butyric acid in case of seneciphylline. In all silages, seneciphylline and senecionine N-oxides were undetectable after 3 days, whereas senkirkine, the most abundant PA, remained stable. CONCLUSIONS: Silage prepared from grass contaminated with eastern groundsel still contained high PA levels, and was hence a potential health hazard. Molasses supplementation reduced concentrations of senecionine and seneciphylline, while the bacterial inoculants had no effect. Other potentially toxic PA metabolites were not analyzed in the present study and further research is needed. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Meta-analysis of the effect of white clover inclusion in perennial ryegrass swards on milk production.

There is increased demand for dairy products worldwide, which is coupled with the realization that consumers want dairy products that are produced in a sustainable and environmentally benign manner. Forage legumes, and white clover (Trifolium repens L.; WC) in particular, have the potential to positively influence the sustainability of pasture-based ruminant production systems. Therefore, there is increased interest in the use of forage legumes because they offer opportunities for sustainable pasture-based production systems. A meta-analysis was undertaken to quantify the milk production response associated with the introduction of WC into perennial ryegrass swards and to investigate the optimal WC content of dairy pastures to increase milk production. Two separate databases were created. In the grass-WC database, papers were selected if they compared milk production of lactating dairy cows grazing perennial ryegrass-WC (GC) swards with that of cows grazing perennial ryegrass-only swards (GO). In the WC-only database, papers were selected if they contained milk production from lactating dairy cows grazing on GC swards with varying levels of WC content. Data from both databases were analyzed using mixed models (PROC MIXED) in SAS (SAS Institute, Cary, NC). Within the grass-WC database, where mean sward WC content was 31.6%, mean daily milk and milk solids yield per cow were increased by 1.4 and 0.12 kg, respectively, whereas milk and milk solids yield per hectare were unaffected when cows grazed GC compared with GO swards. Stocking rate and nitrogen fertilizer application were reduced by 0.25 cows/ha and 81 kg/ha, respectively, on GC swards compared with GO swards. These results highlight the potential of GC production systems to achieve similar levels of production to GO systems but with reduced fertilizer nitrogen inputs, which is beneficial from both an economic and environmental point of view. In the context of increased demand for dairy products, there may be potential to increase the productivity of GC systems by increasing fertilizer nitrogen use to increase stocking rate and carrying capacity while also retaining the benefit of WC inclusion on milk production per cow.

Nanoscale zero-valent iron assisted phytoremediation of Pb in sediment: Impacts on metal accumulation and antioxidative system of Lolium perenne.

Lead (Pb) is a highly toxic environmental pollutant, and could result in toxic effects on living organisms. The effects of 0, 100, 200, 500, 1000 and 2000 mg/kg of nZVI on plant growth, Pb accumulation and antioxidative responses of Lolium perenne were investigated. Results showed that the total Pb contents in L. perenne with the treatment of low concentrations of nZVI (100, 200 and 500 mg/kg) were higher than those in the non-nZVI treatments, and the highest Pb accumulation capacity of 1175.40 µg per pot was observed in L. perenne with the treatment of 100 mg/kg nZVI. However, the total Pb contents in L. perenne decreased at high concentrations of nZVI (1000 and 2000 mg/kg). This might be resulted from the decrease of photosynthetic chlorophyll content and the aggravated oxidative stress induced by the high concentration of nZVI, which caused the decrease of plant biomass and metal accumulation capacity in plant. Moreover, the sequential extraction experiments results showed that the lowest acid soluble fraction of Pb in the sediments was found in the treatment with 100 mg/kg of nZVI, indicating that 100 mg/kg was the optimum concentration for nZVI to assist the phytoremediation of Pb-polluted sediment. To conclude, these findings provide a promising method to remediate Pb-polluted sediment by nZVI assisted phytoremediation.

Effect of substituting fresh-cut perennial ryegrass with fresh-cut white clover on bovine milk fatty acid profile.

BACKGROUND: Including forage legumes in dairy systems can help address increasing environmental/economic concerns about perennial ryegrass monoculture pastures. This work investigated the effect of substituting fresh-cut grass with increasing quantities of fresh-cut white clover (WC) on milk fatty acid (FA) profile and transfer efficiency of dietary linoleic (LA) and α-linolenic (ALNA) acids to milk fat. Three groups of three crossbred dairy cows were used in a 3 × 3 crossover design. Dietary treatments were 0 g kg-1 WC + 600 g kg-1 grass, 200 g kg-1 WC + 400 g kg-1 grass, and 400 g kg-1 WC + 200 g kg-1 grass. All treatments were supplemented with 400 g kg-1 concentrates on a dry matter basis. Cows had a 19-day adaptation period to the experimental diet before a 6-day measurement period in individual tie stalls. RESULTS: Increasing dietary WC did not affect dry matter intake, milk yield or milk concentrations of fat, protein or lactose. Milk polyunsaturated FA concentrations (total n-3, total n-6, LA and ALNA) and transfer efficiency of LA and ALNA were increased with increasing dietary WC supply. CONCLUSION: Inclusion of WC in pastures may increase concentrations of nutritionally beneficial FA, without influencing milk yield and basic composition, but any implications on human health cannot be drawn. © 2018 The Authors. Journal of the Science of Food and Agriculture published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry.

Pollensomes as Natural Vehicles for Pollen Allergens.

Olive (Olea europaea) pollen constitutes one of the most important allergen sources in the Mediterranean countries and some areas of the United States, South Africa, and Australia. Recently, we provided evidence that olive pollen releases nanovesicles of respirable size, named generically pollensomes, during in vitro germination. Olive pollensomes contain allergens, such as Ole e 1, Ole e 11, and Ole e 12, suggesting a possible role in allergy. The aim of this study was to assess the contribution of pollensomes to the allergic reaction. We show that pollensomes exhibit allergenic activity in terms of patients' IgE-binding capacity, human basophil activation, and positive skin reaction in sensitized patients. Furthermore, allergen-containing pollensomes have been isolated from three clinically relevant nonphylogenetically related species: birch (Betula verrucosa), pine (Pinus sylvestris), and ryegrass (Lolium perenne). Most interesting, pollensomes were isolated from aerobiological samples collected with an eight-stage cascade impactor collector, indicating that pollensomes secretion is a naturally occurring phenomenon. Our findings indicate that pollensomes may represent widespread vehicles for pollen allergens, with potential implications in the allergic reaction.

Milk production and composition, nitrogen utilization, and grazing behavior of late-lactation dairy cows as affected by time of allocation of a fresh strip of pasture.

Eighty late-lactation dairy cows were used to examine the effects of allocating a new pasture strip of a sward based on ryegrass (Lolium perenne L.) in the morning (a.m.; ∼0730 h) or in the afternoon (p.m.; ∼1530 h) on milk production and composition, nitrogen (N) utilization, and grazing behavior. Cows grazed the same pasture strips for 24 h and were offered the same daily herbage allowance. Herbage composition differed among treatments; p.m. herbage had greater dry matter (DM; 22.7 vs. 19.9%), organic matter (OM; 89.5 vs. 88.9%), and water-soluble carbohydrate (10.9 vs. 7.6%) concentrations and lesser crude protein (20.5 vs. 22.2%) and neutral detergent fiber (48.8 vs. 50.4%) concentrations compared with a.m. herbage. Total fatty acids (FA), α-linolenic acid, and polyunsaturated FA (PUFA) were greater in a.m. herbage, whereas monounsaturated FA were greater in p.m. herbage. Estimates of herbage DM intake did not differ among treatments. Daily milk yields and milk fat and milk protein concentrations were similar among treatments, whereas milk fat (684 vs. 627 g/cow), milk protein (545 vs. 505 g/cow), and milk solids (milk fat + milk protein) yields (1,228 vs. 1,132 g/cow) tended to be greater for cows on p.m. herbage. Rumenic acid and total PUFA in milk were greater for cows on a.m. herbage, whereas oleic acid was greater for cows on p.m. herbage. Estimates of urinary N excretion (g/d) did not differ among treatments, but urinary N concentrations were greater for cows on a.m. herbage (5.85 vs. 5.36 g/L). Initial herbage mass (HM) available (kg of DM/ha) and instantaneous HM disappearance rates (kg of DM/ha and kg of DM/h) did not differ, but fractional disappearance rates (0.56 vs. 0.74 per hour for a.m. vs. p.m., respectively) differed. Under the current conditions, timing of pasture strip allocation altered the herbage nutrient supply to cows; allocating a fresh strip of pasture later in the day resulted in moderate increases in milk and milk solids yields in late-lactation dairy cows. Conversely, a greater concentration of precursor FA in a.m. herbage resulted in a greater concentration of beneficial FA in milk, compared with cows on p.m. herbage.

The effects of high-sugar ryegrass/red clover silage diets on intake, production, digestibility, and N utilization in dairy cows, as measured in vivo and predicted by the NorFor model.

Grass silage-based diets often result in poor nitrogen utilization when fed to dairy cows. Perennial ryegrass cultivars with high concentrations of water-soluble carbohydrates (WSC) have proven potential for correcting this imbalance when fed fresh, and have also been shown to increase feed intake, milk production, and N utilization. The possibility of achieving corresponding effects with silage-based diets was investigated in change-over experiments in an incomplete block design with 16 (yr 1) or 12 (yr 2) Swedish Red dairy cows in mid lactation. Measurements on N excretion and rumen parameters were performed on subgroups of 8 and 4 cows, respectively. In yr 1, 2 ryegrass cultivars (standard = Fennema; high-WSC = Aberdart) and 2 cuts (first and second) were compared. In all treatments, ryegrass silage was mixed 75/25 on a dry matter (DM) basis, with red clover silage before feeding out. In yr 2, 1 basic mixture from the different cuts of these 2 cultivars was used and experimental factors were red clover silage inclusion (25 or 50%) and sucrose addition (0 or 10%) on a silage DM basis. Differences in WSC concentration in the silage mixtures in yr 1 were minor, whereas the differences between cuts were more substantial: 100 compared with 111 g/kg of DM for first-cut silage and 39 compared with 47 g/kg of DM for second-cut silage. The silages fed in yr 2 had a WSC concentration of 115 or 102 g/kg of DM (25 or 50% red clover, respectively), but when sucrose was added WSC concentration reached 198 and 189 g/kg of DM, respectively. Milk production (kg/d) did not differ between treatments in either year. Red clover inclusion to 50% of silage DM increased milk protein. Nitrogen efficiency (milk N/feed N) increased from 0.231 to 0.254 with sucrose inclusion in yr 2 (average for the 2 red clover levels). Overall rumen pH was 5.99 and increased sucrose level did not affect pH level or daily pH pattern. Sucrose addition reduced neutral detergent fiber digestibility, particularly at higher inclusion rates of clover. Rumen pool of total purines did not differ between treatments, nor did protein production assessed from urinary allantoin. The NorFor feed evaluation model overestimated digestibility of neutral detergent fiber and N, but underestimated N excretion in feces. We concluded that addition of WSC to dairy cow diets at levels up to 3 kg of WSC per day (>14% of DM) does not dramatically affect cow performance.

Cryopreservation Of Nili-Ravi Buffalo Bull Sperm in Cryodiluent Supplemented with Lolium perenne Protein Preparations.

BACKGROUND: Semen from the Nili-Ravi buffalo bull, Bubalus bubalis, shows poor survival after freeze storage compared to bovine (Bos taurus and Bos indicus) semen. Freeze-susceptibility distinctions in these two genera have been attributed to differences in sperm membranes. MATERIALS AND METHODS: We measured the impact of protein preparations derived from a frost-resistant perennial grass, Lolium perenne, with ice recrystallization inhibition activity on the low temperature storage of B. bubalis semen. RESULTS: When the L. perenne preparations (0.1, 1, 10 µg/mL) were added to buffalo semen [2 ejaculates per bull (N=3) per replicate (r=3)] in Tris-citrate extender (50×106sperm mL-1), there was no impact on semen quality, as measured by sperm motility and plasma membrane integrity, after storage at 4 degree C (P>0.05). However, when semen supplemented with the grass proteins (0.1 and 1 µg mL-1) was evaluated after freezing and storage in liquid nitrogen for 24 h, post-thaw sperm progressive motility and plasma membrane integrity was higher (P<0.05) than in control samples. Post-thaw sperm viability and sperm acrosome integrity was similar (P > 0.05) to controls. CONCLUSION: The improvement in cryopreserved buffalo sperm progressive motility and plasma membrane integrity suggests that the use of these easily-made preparations may improve fertility after cryopreservation and offers the prospect of improved conception rates after artificial insemination with cryopreservation.