Isoflavone profiles of red clovers and their distribution in different parts harvested at different growing stages

The isoflavone compositions and concentrations in the leaf, flower, petiole, and stem of 13 red clover cultivars were studied using high-performance liquid chromatography coupled with a diode array and a mass spectrometric detector with negative electrospray ionization. Different cultivars showed significantly different concentrations of individual and total isoflavones. The leaf contained the highest overall concentration, followed by the stem, petiole, and flower. Biochanin A and formononetin were the predominant isoflavones in all cultivars and all parts, along with eight other minor aglycones, daidzein, genistein, glycitein, irilone, orobol, pratensein, pseudobaptigenin, and prunetin, and four minor malonylglycosides, genistein-7-glucoside-6' '-malonate, orobol-7-glucoside-6' '-malonate, formononetin-7-glucoside-6' '-malonate, and biochanin A-7-glucoside-6' '-malonate. The isoflavone compositions and concentrations were also found to be different between red clover parts harvested at the early bud stage and the late flowering stage. Sample storage and handling prior to analysis were also found to be important. Samples in this study were kept at -5 degrees C for a few days before being freeze-dried and were found to contain mainly the aglycones of isoflavones. This may actually be an advantage in that "natural" and more bioactive isoflavones can be obtained without using chemical hydrolysis. Findings in this study therefore provide important information for developing isoflavone-rich red clovers and for optimizing harvest timing and choosing the right part of the red clover plant.     

Changes of isoflavones during the growth cycle of Lupinus albus

The objective of this study was to monitor the changes in isoflavone content in different plant organs (leaves, stems, roots) during the crop growth stage of three cultivars of Lupinus albus (white lupin) under field conditions, taking into account sowing time effects (autumn and early spring) and cultivar effects. Three sampling dates (from late vegetative to late grain growth stages) were evaluated. Seven isoflavones and four flavonoids were identified by LC-ESI-MS analysis. The isoflavone content was higher in leaves than in stems, and it was highest before flowering, whereas it decreased during maturity. Autumn-sown plants showed higher isoflavone content than early spring-sown plants, especially in late vegetative and early reproductive stages. Genistein 7- O-glucoside was the main isoflavone of leaves and stems in the late vegetative stages of early spring sowing, whereas genistein was the main isoflavone under autumn sowing. Variation among cultivars affected only marginally the total isoflavone content. No isoflavones were detected in seeds.     

Extraction of isoflavone malonylglucosides from Trifolium pratense L

Extraction of isoflavone malonylglucosides from red clover (Trifolium pratense L.) is a complicated procedure. This is due to the relatively unstable character of the thermolabile glucoside malonates as well as by action of native beta-glucosidases, resulting in a rapid degradation of malonylated glucosides into their corresponding aglucones. In this study, Tris was identified as a suitable beta-glucosidase inhibitor in red clover extracts, optimized at 350 mM Tris in 80% ethanol at pH 7.2. Extraction of fresh red clover leaves using Tris increased the concentration of malonate conjugated isoflavones approximately 13 to 24 times as opposed to extraction without Tris. A comparison of isoflavone profiles obtained after extraction with and without Tris of different plant organs of red clover and several species within the family Fabaceae suggests that the amount and/or activity of the degenerative beta-glucosidase enzymes vary for the different plant parts of red clover and among the species studied. Therefore, the use of standard extraction methods may well result in overestimation of the concentration of aglucones and consequently underestimation of the malonylglucoside isoflavones concentration depending on the plant species and plant part studied.     

Correlations of oil and protein with isoflavone concentration in soybean [Glycine max (L.) Merr.

Twelve isoflavones were detected by high-performance liquid chromatography in seeds of 17 soybean [Glycine max (L.) Merrill] cultivars grown at three locations. 6' '-O-Malonyldaidzin and 6' '-O-malonylgenistin together constituted 71-81% of total isoflavones, which ranged in concentration from 2038 to 9514 microg/g and averaged 5644 microg/g across locations and cultivars. The total as well as several individual isoflavones had a moderate negative correlation with oil across locations and cultivars. Six cultivars had a moderate or strong negative correlation of total isoflavones with oil. Five cultivars had a moderate or strong positive correlation of total isoflavones with protein. These results suggest that judicious selection of germplasm for soybean breeding may facilitate development of soybean lines with desirable isoflavone concentrations.     

Identification of urinary metabolites of the red clover isoflavones formononetin and biochanin A in human subjects

Dietary supplements manufactured from red clover are widely marketed to provide the beneficial health effects of isoflavones without changing the original diet. In this study the metabolism of formononetin and biochanin A, the principal isoflavones of red clover, was studied in human subjects. Seven women ingested four red clover dietary supplements, and the metabolites of the isoflavones were identified in their urine samples. The structures of trimethylsilyl derivatives of the metabolites were established by GC-MS. New reduced metabolites of formononetin (dihydroformononetin and angolensin) and biochanin A (dihydrobiochanin A and 6'-hydroxyangolensin) were identified in urine samples using authentic reference compounds. Possible metabolic pathways are presented for the red clover isoflavones formononetin and biochanin A.     

Retention and changes of soy isoflavones and carotenoids in immature soybean seeds (Edamame) during processing

Isoflavones and carotenoids in four experimental genotypes and Hutcheson cultivar soybeans were evaluated as a function of processing treatments and maturity. Total isoflavone and carotenoid contents were affected by genotypes and maturity stages (p < 0.0001). Total isoflavones ranged from 472 microg/g (in NTCPR93-40) to 2280 microg/g (in Hutcheson). Lutein contents ranged from 895 (in NTCPR93-286) to 2119 (in Honey Brown), and beta-carotene ranged from 291 (in Hutcheson) to 491 (in NICPR92-40) microg/100 g. Mean total isoflavone retention percentages in immature Hutcheson soybeans were 46% (boiling), 53% (freezing), and 40% (freeze-drying). Mean retentions of lutein and beta-carotene, respectively, were 92 and 73% in frozen, 62 and 62% in boiled, and 34 and 27% in freeze-dried soybeans. Boiling caused a substantial increase in daidzin, genistin, and genistein. The results show that post-harvest changes in total isoflavones and carotenoids in soybeans are influenced by processing methods, but genotype has an effect on isoflavone and carotenoid profiles during seed development.     

Comparison of isoflavone concentrations in soybean (Glycine max (L.) Merrill) sprouts grown under two different light conditions

We determined and compared the composition and content of isoflavones in the cotyledon, hypocotyl, and root of 17 soybean sprout varieties grown under dark and light conditions. The total average isoflavone concentrations in 17 soybean sprout varieties were 2167 microg g(-1) (green sprout) and 2538 microg g(-1) (yellow sprout) in cotyledons, 1169 microg g(-1) (green sprout) and 1132 microg g(-1) (yellow sprout) in hypocotyls, and 2399 microg g(-1) (green sprout) and 2852 microg g(-1) (yellow sprout) in roots. There were no significant differences in total isoflavone concentrations between the green and yellow sprouts. However, significant differences in total isoflavone amounts were observed among the three organs, with roots exhibiting the highest total isoflavone concentrations followed by cotyledons and hypocotyls. Total daidzin concentrations of green (775 microg g(-1)) and yellow (897 microg g(-1)) sprouts increased to more than 4 times that in seeds (187 microg g(-1)). Yellow sprouts contained the highest (1122 microg g(-1)) total genistin concentrations, and green (155 microg g(-1)) and yellow (155 microg g(-1)) sprouts had more total glycitin concentrations than seeds. In cotyledons of green and yellow sprouts, genistin, daidzen, and glycitin constituted more than 67%, more than 28%, and less than 4% of the total isoflavone contents, respectively. In hypocotyls, total daidzin represented more than 45% of the total isoflavones, and total glycitin was higher than in cotyledons and roots. Malonylglycoside concentrations were highest in cotyledons, whereas glycoside concentrations were highest in hypocotyls and roots. The high accumulation of isoflavones in roots is consistent with isoflavones serving as signal molecules in the induction of microbial genes involved in soybean (Glycine max) nodulation.     

Seasonal variation of red clover (Trifolium pratense L., Fabaceae) isoflavones and estrogenic activity

Red clover (Trifolium pratense L., Fabaceae) dietary supplements are currently used to treat menopausal symptoms because of their high content of the mildly estrogenic isoflavones daidzein, genistein, formononetin, and biochanin A. These compounds are estrogenic in vitro and in vivo, but little information exists on the best time to harvest red clover fields to maximize content of the isoflavones and thus make an optimal product. Samples of cultivated red clover above-ground parts and flower heads were collected in parallel over one growing season in northeastern Illinois. Generally, autohydrolytic extracts of above-ground parts contained more isoflavones and had more estrogenic activity in Ishikawa endometrial cells as compared with extracts of flower heads. Daidzein and genistein contents peaked around June to July, while formononetin and biochanin A contents peaked in early September. Flower head and total above-ground parts extracts exhibited differential estrogenic activity in an Ishikawa (endometrial) cell-based alkaline phosphatase induction assay, whereas nondifferential activity was observed for most extracts tested in an MCF-7 (breast) cell proliferation assay when tested at the same final concentrations. Ishikawa assay results could be mapped onto the extracts' content of individual isoflavones, but MCF-7 results did not show such a pattern. These results suggest that significant metabolism of isoflavones may occur in MCF-7 cells but not in Ishikawa cells; therefore, caution is advised in the choice of bioassay used for the biological standardization of botanical dietary supplements.     

Isoflavone composition, phenol content, and antioxidant activity of soybean seeds from India and Bulgaria

Isoflavone levels and isoflavone chemical composition in 11 cultivars of soybean, including 4 Indian and 7 genotypes of soybean grown in Bulgaria, were analyzed as determined by C 18 reversed phase high-performance liquid chromatography coupled with a photodiode array detector. Antioxidant activity of soybean extracts was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, and total phenolic compounds (TPC) were determined by using Folin-Ciocalteu reagent. The range of total isoflavones (TI) was 558.2-1048.6 microg g (-1) of soy in Indian cultivars, and it was 627.9-1716.9 microg g (-1) of soy in the case of Bulgarian cultivars. The highest and lowest total isoflavone contents were observed for Maus-2 (1048.6 microg g (-1) of soy) and Hardee (558.2 microg g (-1) of soy), respectively, for the Indian cultivars, and they were observed for Boryara (1716.9 microg g (-1) of soy) and Line 5 (627.9 microg g (-1) of soy) for the Bulgarian genotypes. DPPH radical scavenging activity did not differ significantly among the cultivars and did not correlate with TI, whereas TPC correlated well with TI and weakly with DPPH. Malonylglucoside of all the aglycones, total genistein (TGin), and total daidzein (TDin) showed strong correlation with total isoflavones, whereas acetylglucoside and aglycone levels did not significantly correlate with total isoflavone. Profiling of soybean isoflavone is helpful in understanding the regulation of isoflavone biosynthesis for greater improved resistance of crops to disease and greater health benefits for humans. This comparative study of soybean cultivars grown in India and Bulgaria throws light on their composition and nutraceutical value.     

Growth,water use efficiency,and sodium and potassium acquisition by tomato cultivars grown under salt stress

Three cultivars of tomato (Lycopersicon esculentum Mill., cvs. Sera, 898, Rohaba) were grown under different levels of NaCl in nutrient solution to determine effects of salt stress on shoot and root dry matter (DM), plant height, water use efficiency (WUE, g DM kg^‐1 water evapotranspired), shoot sodium (Na) and potassium (K) concentrations, and K versus Na selectivity (S_K,Na). Increasing NaCl concentration in nutrient solution adversely affected shoot and root DM, plant height, WUE, K concentration, and K/Na ratio of all cultivars. Shoot Na concentrations increased with increasing NaCl concentration in the nutrient solution. Although increasing salt concentration in the solution adversely affected growth of all cultivars, the cultivar Sera had the highest shoot and root DM than the other two cultivars (898 and Rohaba). Shoot and root DM of cultivar 898 was most affected by salt, while cultivar Rohaba had an intermediate salt sensitivity. The cultivar Sera generally had higher WUE values, shoot K concentrations, and S_K,Na, but had lower shoot Na concentrations than the other two cultivars when plants were grown under different salt levels. Greater Na exclusion, higher K uptake and shoot S_K,Na are suggested as being plant strategies for salt tolerance.     

LC-ESI-MS study of the flavonoid glycoside malonates of red clover (Trifolium pratense)

High-performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) was applied to the analysis of the flavonoids and their glycoside malonates of the flowers and leaves of red clover (Trifolium pratense). Through LC-MS comparative studies on the plant extracts and their malonate-free extracts, approximately 20 flavonoid glycoside malonates were detected in the flower extract. Eight were identified as genistin 6' '-O-malonate (39), formononetin 7-O-beta-D-glucoside 6' '-O-malonate (40), biochanin A 7-O-beta-D-glucoside 6' '-O-malonate (41), trifoside 6' '-O-malonate (42), irilone 4'-O-beta-D-glucoside 6' '-O-malonate (43), pratensein 7-O-beta-D-glucoside 6' '-O-malonate (44), isoquercitrin 6' '-O-malonate (45), and 3-methylquercetin 7-O-beta-D-glucoside 6' '-O-malonate (46). About 15 other flavonoids and clovamides were proved to be present in this extract. The study also found that the flowers contained flavones as the major flavonoids, whereas the leaves had isoflavones as the major flavonoids. This is the first detection of the six malonates (39 and 42-46) in the extracts of red clover, and among them, 42, 43, and 46 are new compounds.     

Red clover Trifolium pratense L. phytoestrogens: UV-B radiation increases isoflavone yield, and postharvest drying methods change the glucoside conjugate profiles

Isoflavone extracts of red clover Trifolium pratense L. (cv. Pawera) with dissimilar glucoside conjugate profiles were obtained by employing different postharvest drying methods. The most prominent isoflavones found were formononetin and biochanin A and their corresponding glucosides and malonyl glucoside esters. Postharvest freeze drying inhibited the conversion of the glycosides to the aglycones, while vacuum drying allowed for maximum conversion of the glycosides to their corresponding aglycones. Air drying produced a low level of the aglycones formononetin and biochanin A, and oven drying promoted decarboxylation of the malonyl glucosides to the acetyl glucosides. Exposure to enhanced UV-B radiation resulted in an increase in total formononetin and biochanin A isoflavone levels, indicating that harvest during a period of high ambient UV-B radiation may be appropriate for maximum yield. The levels of caffeic acid and flavonols also increased by about 40 and 250%, respectively, on exposure to enhanced UV-B radiation.     

Bioactive polyphenols in leaves, stems, and berries of Saskatoon (Amelanchier alnifolia Nutt.) cultivars

The Saskatoon berry is currently cultivated in many parts of the world for its suitability for various food products and due to its high content of nutrients and polyphenols. To determine the phytochemical profile of a Saskatoon plant, polyphenols from leaves, stems, and berries were screened from four cultivars grown in Finland using HPLC-DAD and HPLC-ESI/MS. The phenolic composition and concentrations varied among plant parts and cultivars. The main berry components were cyanidin-based anthocyanins (63% of the phenols), quercetin-derived flavonol glycosides, and hydroxycinnamic acids. The total anthocyanin content varied between 258.7 and 517.9 mg/100 fresh weight among cultivars. Protocatechuic acid was found for the first time in Saskatoon berries. The leaves consisted of quercetin- and kaempferol-derived glycosides (41% of the phenols), hydroxycinnamic acids (36%), catechins, and some neolignans. Quercetin 3-galactoside and 3-glucoside, (-)-epicatechin, and chlorogenic acid were the main phenolics in the leaves of all cultivars. The stem components were flavanone and flavonol glycosides (55% of the phenols), catechins (38%), and hydroxybenzoic acids. Concentrations of the main compound, eriodictyol 7-glucoside, varied among cultivars from 3.3 to 6.5 mg/g of stem dry weight. Very high proanthocyanidin contents were found in stems and leaves (10-14% of dry biomass), whereas berries contained a low amount of proanthocyanidins (3% of dry biomass). The findings reveal that leaves and stems of Saskatoon cultivars possess high amounts of various phenolic compounds that may offer new functional raw materials for a wide range of food and health products.     

FACE对杂交籼稻汕优63干物质生产与分配的影响

2005--2006年利用我国惟一的农田开放式空气CO2浓度增高（FACE）研究平台,以三系杂交籼稻汕优63为供试材料,设计比大气CO2浓度（对照,370umol．mol^-1）高200umol．mol^-1的FACE处理（570umol．mol^-1）和施N量为125kg·hm^-2（LN）、250kg·hm^-2（NN）处理,研究其对汕优63物质生产与分配的影响。结果表明：（1）两季平均,FACE处理使汕优63移栽-分蘖中期、分蘖中期-拔节期、拔节-抽穗期和抽穗-成熟期干物质生产量分别比对照增加了39％、20％、32％和41％,结果使成熟期生物产量显著增加（＋33％）;（2）与干物质生产量相比,汕优63平均叶面积指数（LAI）和净同化率（NAR）对CO2的响应有相似的季节性变化趋势,但LAI的响应值明显大于NAR;（3）FACE处理使汕优63不同生育时期叶片和稻穗占地上部干重的比例下降,而使茎鞘占地上部干重的比例明显增加;（4）三系杂交籼稻汕优63成熟期生物产量以及不同生育阶段平均LAI、NAR和干物质生产量对FACE的响应与粳稻品种存在明显差异．而干物质分配差异较小。     

Quantification of five isoflavones and coumestrol in various solid agroenvironmental matrices using ¹³c₃-labeled internal standards

We developed and validated three different sample preparation and extraction methods followed by HPLC-MS/MS (negative electrospray ionization) analysis for the quantification of estrogenic isoflavones (formononetin, daidzein, equol, biochanin A, and genistein) and coumestrol in red clover, soil, and manure. Plant and manure samples were solid-liquid extracted, whereas soil was extracted with accelerated solvent extraction. Absolute recoveries were between 80 and 93%, 20 and 30%, and 14 and 91% for plant, soil, and manure samples, respectively. Relative recoveries ranged from 75 to 105% for all matrices, indicating that isotope-labeled internal standards (13C?-formononetin, 13C?-daidzein, 13C?-equol, 13C?-biochanin A, and 13C?-genistein) were capable to compensate for losses during analysis. The limits of detection in red clover, soil, and manure were 3-9 μg/g(dryweight(dw)), 0.6-8.2 ng/g(dw), and 34.2 ng/g(dw) to 17.0 μg/g(dw), respectively. Formononetin was the most dominant compound in red clover plants (up to 12.5 mg/g(dw)) and soil (up to 3.3 μg/g(dw)), whereas equol prevailed in manure (up to 387 μg/g(dw)).     

Boron,molybdenum and selenium status in different plant parts in forage legumes and vegetable crops 1

This study was conducted in Prince Edward Island (P.E.I.), Canada to characterize the levels of B, Mo, and Se in the various plant parts of alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.), broccoli (Brassica oleracea var. italica Plenck.), Brussels sprouts (Brassica oleracea var. gemmifera Zenker), cauliflower (Brassica oleracea var. botrytis L.), and rutabaga (Brassica napobrassica Mill). The three micronutrient anions were consistently highest in the leaves and lowest in the stems. The lower halves of the vegetable crops usually contained close to the highest amounts of B and Mo. In forage legumes, the B concentration was higher in the bottom than in the upper leaves, but B was lower in the bottom than upper stems. Since leaves consistently contained the highest amounts of B and Mo, it is recommended that most recently matured leaves in the Brassica species, and young leaves in forage legumes be sampled to characterize the B and Mo status of these crops. Selenium is of significance to livestock, but not in plant nutrition; therefore, only the parts fed to animals, e.g. the whole vegetative tops of forage legumes, should be used to determine the crop Se status.     

Phytoestrogen content of foods of animal origin: dairy products, eggs, meat, fish, and seafood

Dietary phytoestrogens may be involved in the occurrence of chronic diseases. Reliable information on the phytoestrogen content in foods is required to assess dietary exposure and disease risk in epidemiological studies. However, existing analyses have focused on only one class of these compounds in plant-based foods, and there is only little information on foods of animal origin, leading to an underestimation of intake. This is the first comprehensive study of phytoestrogen content in animal food. We have determined the phytoestrogen content (isoflavones: biochanin A, daidzein, formononetin, genistein, and glycitein; lignans: secoisolariciresinol and matairesinol; coumestrol; equol; enterolactone; and enterodiol) in 115 foods of animal origin (including milk and milk-products, eggs, meat, fish, and seafood) and vegetarian substitutes using liquid chromatography-mass spectrometry (LC-MS) with (13)C-labeled internal standards. Phytoestrogens were detected in all foods analyzed; the average content was 20 microg/100 g of wet weight (isoflavones, 6 microg/100 g; lignans, 6 microg/100 g; equol, 3 microg/100 g; and enterolignans, 6 microg/100 g). In infant soy formula, 19 221 microg/100 g phytoestrogens were detected (compared to 59 microg/100 g in non-soy formula). Our study shows that all foods analyzed contained phytoestrogens and most foods (except for fish, seafood, and butter) contained mammalian phytoestrogens (enterolignans and equol). This is the first comprehensive study of phytoestrogen content of foods of animal origin and will allow for a more accurate estimation of exposure to dietary phytoestrogens.     

Potassium,Phosphorus, Sulfur,and Boron Fertilization Effects on Soybean Isoflavone Content and Other Seed Characteristics

ABSTRACT

Soybean [Glycine max (L.) Merr.] seeds contain isoflavones that have positive impacts on human health. The objective of this study was to determine the impact of pre-plant mineral fertilization on isoflavone, oil and crude protein concentrations, and seed yield of field-grown soybean. The effects of potassium (0, 50, 100, and 150 kg K ha^?1), phosphorus (0, 25, 50, and 75 kg P ha^?1), sulfur (0, 15, 30, 45 kg S ha^?1), and boron (0, 1.5, 3.0, and 4.5 kg B ha^?1) were tested separately, each with two 00 soybean cultivars (‘Golden’ and ‘Grand Prix’) grown in replicated trials at Sainte-Anne-de-Bellevue, Québec, Canada in 2002/3. Seed total and individual isoflavone concentrations were determined by high-performance liquid chromatography. Seed yield, 100-seed weight, and oil and crude protein (CP) contents were determined concurrently. Across years and cultivars, no fertilizer treatments effects were observed for most variables. This overall lack of response to fertilizers was attributed to the relatively high initial fertility of the sandy loam and sandy clay loam soils used. However, total and individual isoflavone concentrations were significantly affected by year and cultivar. Across experiments, total isoflavone concentration was 33% greater on average in 2003 than in 2002, which was characterized by above-average temperatures and severe drought. Cultivar with the greatest isoflavone concentration varied depending on the year. Fertilization does not appear to be a viable strategy to increase isoflavone concentration of soybean seeds on medium-to high-fertility soils.     

Response of two pomegranate (Punica granatum L.) cultivars to six boron concentrations: Growth performance,nutrient status,gas exchange parameters,chlorophyll fluorescence,and proline and carbohydrate content

One-year-old, own-rooted pomegranate cultivars “Ermioni” and “Wonderful” plants were irrigated for 75 days with modified Hoagland nutrient solutions containing 0–10 mg L^?1 boron (B). At the end of the experiment, the control plants of “Ermioni” presented better growth performance than those of “Wonderful.” However, there were no differences in the treatments with high B concentration (5.0 or 10 mg L^?1). Control “Wonderful” plants had higher fresh and dry matter than control “Ermioni” plants. Moreover, the highest B concentrations in nutrient solution led to a significant increase in chlorophyll and carbohydrate content in the leaves of cultivar “Ermioni.” Furthermore, leaf proline concentration, gas exchange, chlorophyll fluorescence, and micro–macronutrients of both cultivars were not affected by any of the tested B treatments. B concentration in plant parts was linearly correlated to B supply. The highest B concentrations were observed in roots followed by stems and apical and basal leaves.     

Yield and chemical composition of five common grassland species in response to nitrogen fertiliser application and phenological growth stage

Abstract

The interaction of grassland management factors such as plant species, rate of nitrogen (N) fertiliser application and stage of maturity at harvest, will determine the optimal balance of herbage yield, nutritive quality and ensilability for ruminant nutrition and/or industrial applications. This study investigated the effects of N fertiliser input and harvest date on the yield and chemical composition of five common grass species, and made comparisons with red clover. Perennial ryegrass (PRG), Italian ryegrass (IRG), tall fescue, cocksfoot, timothy and red clover were grown under two inorganic N fertiliser inputs (0 kg N ha^?1 and 125 kg N ha^?1; except red clover) and harvested at five dates (fortnightly from 12 May to 7 July) in the primary growth. Regression analysis of these data allowed comparison of the yield and chemical composition of each grass species at common growth stages, without the confounding effects of variation in maturity between grass species at common harvest dates. Of the grass species investigated, timothy was most productive in terms of dry matter (DM) yield and thus has the potential to provide a cheaper feed per unit DM. However, the most digestible grass species was PRG, with timothy being the lowest, and this could impact on both animal and bioenergy production potential. The most suitable grass species for ensiling was IRG (particularly when grown without fertiliser N) due to its higher water soluble carbohydrate (WSC) concentration and lower buffering capacity (BC) compared to all other grass species. In comparison to the grasses receiving inorganic N fertiliser, red clover had a numerically lower DM yield, but a higher mean DM digestibility and crude protein concentration. The lower WSC concentration and higher BC of the red clover may result in a greater preservation challenge during ensiling.