Clover Species Research Articles

Optimizing polyphenol extraction and UPLC-MS/MS analysis from red clover (Trifolium pratense L.) species using response surface methodology

Polyphenols were extracted from red clover (Trifolium pratense L.) plants following conventional extraction. The combined factors of ethanol concentration (60–80%), extraction time (15–45 min) and extraction temperature (40–80°C) were evaluated. Central composite design software was used to conduct Response Surface Methodology (RSM) modelling and optimize the polyphenol yield from the red clover. Total phenolic content, antioxidant activity and ultra-performance liquid chromatography mass-spectrometry (UPLC-MS/MS) analysis were used to quantify polyphenols. The RSM model identified ethanolic composition having greatest influence (p < 0.05) on the polyphenol yield with extraction yields increasing with percentage ethanol. The optimal extraction conditions were identified as 80% ethanol for 45 minutes at 40°C. When compared with an exhaustive polyphenol extraction method (70% ethanol for 21 h at room temperature), the optimal extraction resulted in a 25.6% increase in TPC yield. UPLC-MS/MS was performed to quantify the individual polyphenols present within the sample, in which biochanin A and formononetin were present in abundance. The outcomes of this modelling and optimization study demonstrate that polyphenols from red clover can be extracted efficiently with minimal environmental and energy cost and thus had the capability to serve as a source of valuable nutraceutical and pharmaceutical products.

Grasslands and flood mitigation – Contrasting forages improve surface water infiltration rates

Grasslands globally deliver many ecosystem services, including water management to alleviate flood risk reduction. Two replicated field experiments were conducted to study how agricultural forage species with diverse rooting systems, sown as single species, affected rooting, soil structure and earthworm populations, and consequently water infiltration to understand how they each might influence flood risk from grasslands. Experiment One showed soils under red clover (Trifolium pratense), white clover (Trifolium repens) and chicory (Cichorium intybus) had higher infiltration rates three years after establishment, compared to perennial ryegrass (Lolium perenne). Higher red clover and chicory root biomass or increased earthworm abundance under white clover may have caused these effects. Experiment Two monitored infiltration at intervals over several years post establishment to understand the timeframe for changes in rates; plantain (Plantago lanceolata) was sown as an additional forage. Infiltration declined post establishment, the timing and extent of decline varying with forages; forage effects were significant after 27 months (P < 0.05). Infiltration rates were higher under red and white clover compared to ryegrass, with chicory and plantain intermediate (P < 0.05). Forages again differed in likely mechanisms delivering higher water infiltration, notably between the two clover species. White clover had higher earthworm biomass (P < 0.05), whereas red clover had a higher average root diameter compared to the other forages (P < 0.05). Drivers of intermediate benefits of chicory and plantain also differed: chicory had higher earthworm abundance (month 38) compared to plantain, which had higher average root diameter compared to ryegrass (month 41); 30 months post-establishment soil bulk density was lower under both forages compared to ryegrass and red clover, with white clover intermediate (P < 0.05); bulk density and penetration resistance did not relate to infiltration. Findings demonstrate that a shift from perennial ryegrass-dominated pastures to swards with more contrasting forages provides an ecohydrological approach to mitigating flood risk and climate adaptation.

De novo genome assembly of white clover (Trifolium repens L.) reveals the role of copy number variation in rapid environmental adaptation

BackgroundWhite clover (Trifolium repens) is a globally important perennial forage legume. This species also serves as an eco-evolutionary model system for studying within-species chemical defense variation; it features a well-studied polymorphism for cyanogenesis (HCN release following tissue damage), with higher frequencies of cyanogenic plants favored in warmer locations worldwide. Using a newly generated haplotype-resolved genome and two other long-read assemblies, we tested the hypothesis that copy number variants (CNVs) at cyanogenesis genes play a role in the ability of white clover to rapidly adapt to local environments. We also examined questions on subgenome evolution in this recently evolved allotetraploid species and on chromosomal rearrangements in the broader IRLC legume clade.ResultsIntegration of PacBio HiFi, Omni-C, Illumina, and linkage map data yielded a completely de novo genome assembly for white clover (created without a priori sequence assignment to subgenomes). We find that white clover has undergone extensive transposon diversification since its origin but otherwise shows highly conserved genome organization and composition with its diploid progenitors. Unlike some other clover species, its chromosomal structure is conserved with other IRLC legumes. We further find extensive evidence of CNVs at the major cyanogenesis loci; these contribute to quantitative variation in the cyanogenic phenotype and to local adaptation across wild North American populations.ConclusionsThis work provides a case study documenting the role of CNVs in local adaptation in a plant species, and it highlights the value of pan-genome data for identifying contributions of structural variants to adaptation in nature.

Tolerance of plantain (Plantago lanceolata) to translocated herbicides wiped on flower stems

ABSTRACT Growing plantain (Plantago lanceolata) within pastures reduces nitrate leaching following grazing. Plantain is usually grown with grass and clover species, making selective weed control challenging. One strategy to selectively remove tall weeds in pastures with mixed species is to apply herbicides using weed wipers. However, for many months of the year, plantain has upright leafless flower stems that will intercept herbicide applied using wipers. To determine if this means plantain might be damaged by wipers used in such swards, two glasshouse experiments tested the tolerance of mature plantain plants to the application of herbicides suitable for use in weed wipers (glyphosate, clopyralid, aminopyralid, dicamba, metsulfuron, picloram and triclopyr) to the flower stems. Aminopyralid (0.075% ai solution) and a low rate of glyphosate (1.8% ai) caused the least decrease in plantain biomass and should be safe for weed wiping within swards containing plantain. A higher rate of glyphosate (7.2% ai) and a glyphosate/metsulfuron mix (0.9% + 0.01% ai, respectively) caused the most damage to plantain, particularly following simulated rainfall after application. Thus, applying some herbicides using wipers should be possible for selective weed control within plantain swards even if rain falls soon afterwards, though field trials are required to fine-tune recommendations.

Clover in vineyards, a potential trap plant for the mealybug Pseudococcus calceolariae—a vector of GLRaV-3 to grapevines but not clover species

Clover in vineyards, a potential trap plant for the mealybug Pseudococcus calceolariae—a vector of GLRaV-3 to grapevines but not clover species

Clover (Trifolium spp.) Inclusion in Kentucky Bluegrass (Poa pratensis) Lawns

Historically, white clover (Trifolium repens) seed was included in turfgrass seed mixtures to provide biodiversity and nitrogen (N) to lawns. White clover dicultures have been studied recently for inclusion in both warm- and cool-season turfgrasses, with the goals of reducing fertilizer applications and providing pollinator forage in lawns; however, other clovers have not been as widely researched in turfgrass. The objectives of this study were to evaluate 1) if white, strawberry (T. fragiferum), crimson (T. incarnatum), and rose (T. hirtum) clovers can persist in dicultures with Kentucky bluegrass (Poa pratensis); 2) if clover inclusion in dicultures impacts broadleaf weed cover; and 3) if low levels of N fertilization impact clover persistence or quality of clover–bluegrass dicultures. Kentucky bluegrass was grown as a monoculture or as a diculture with each of the four clover species. Each mono- or diculture was then treated with a low rate of N fertilizer (48.8 kg⋅ha–1 N) or no N fertilizer to determine quality and percentage of grass, clover, or weed and bare-soil cover. Dicultures contained similar or less weed and bare-soil cover, and maintained similar or greater quality compared with bluegrass monocultures, indicating clover and Kentucky bluegrass dicultures are suitable alternatives to Kentucky bluegrass monoculture lawns, and can potentially lead to reduced fertilizer and pesticide requirements. Fertilizer generally had no effect on cover, likely because of the low rates of N applied.

Estrogenic Isoflavones in Clover Plants, Flower Nectar, Unripe Honeys and Mature Honeys: A Natural Biochemical Transformation of Isoflavones by Honeybees.

This study is the first to report on the presence of oestrogenic compounds in different clover flower nectar samples, in bee-deposited nectars collected from hive combs (unripe honey) and in mature honeys harvested from the same hives. The clover species investigated were two red clover (Trifolium pratense) cultivars, bred specifically for high isoflavone content, alongside a sainfoin (Onobrychis viciifolia) and a purple clover (T. purpureum) cultivar. A total of eight isoflavones, four of them non-glycosidic (biochanin A, formononetin, genistein and daidzein) the others glycosidic (sissotrin, ononin, genistin and daidzin), were targeted for identification and quantification in this study using high-performance thin-layer chromatography (HPTLC). Leaves and flower bracts of the clover samples were also investigated. Different isoflavone profiles were found across the four clover species and also in the different samples collected from each species indicating that, most likely due to the activity of honeybee (Apis mellifera) salivary enzymes, biochemical conversions take place when these bioactive compounds transition from flower nectar into ripe honey. Among the four investigated clover species, the two red clover cultivars, including their honeys, were found to contain higher levels of estrogenic compounds compared to other two cultivars.

Productive response of two species of chickens (Mexican native and Sasso), in confinement and grazing white clover (Trifolium repens L.)

Objective: To determine the productive behavior and forage consumption (Trifolium repens L.) of Mexican native and Sasso chickens, under two rearing systems (confinement or grazing).&#x0D; Design/Methodology/Approach: One-hundred twenty-eight chickens (64 Mexican native (MN) and 64 Sasso (S)) of 35 d of age were randomly distributed in two production systems to obtain four repetitions (eight chickens per repetition) of each of the following genotype × system combinations: Mexican native in grazing, Mexican native in confinement, Sasso in grazing, and Sasso in confinement. A completely randomized experimental design with a 2×2 factorial arrangement was used, with genotype and production system as its main factors. The following variables were evaluated: feed consumption, weight gain, and feed conversion. Additionally, forage consumption in grazing birds was determined.&#x0D; Results: The productive performance variables were not affected by the production system factors or by its interaction with the bird genotype. However, the genotype did influence the variables considered: the Sasso birds recorded better values (p ≤ 0.05) than Mexican native specimens. Regarding forage consumption, no differences were observed (p ≤ 0.05) between bird genotypes and, in both cases, the accumulated consumption at the 49 d of study was close to 60 g of DM.&#x0D; Study Limitations/Implications: It is necessary to carry out a socioeconomic study as well as a defoliation level analysis with the aim of improving the use of the resource.&#x0D; Findings/Conclusions: Mexican native birds had a lower productive performance with a similar forage consumption.

Physiological and Biochemical Responses of Melilotus albus to Saline and Alkaline Stresses

Sweet clover (Melilotus albus) is a high-quality leguminous forage grass with salinity tolerance, drought tolerance, and cold hardiness. We selected four varieties of sweet clover with different sensitivities (061898, 061930, No. 55 white flower, and Ningxia white flower) and analyzed the effects of different concentrations of three sodium salts (Na2CO3, NaHCO3, and NaCl) on their physiology and biochemistry responses. Growth and development indexes (such as germination rate, root length, shoot length), chlorophyll content, osmotic regulators (proline, soluble sugar), malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were determined under saline–alkali stress. Seed germination and seedling growth of all four clover species were significantly inhibited under saline–alkali stress. During germination, seed germination rate, root length, and shoot length decreased with increasing saline and alkaline concentration. Under saline–alkali stress, chlorophyll content tended to increase and then decrease, cell damage and death increased, and malondialdehyde, soluble sugar, and proline content tended to increase and then decrease. Moreover, the activities of SOD, POD, and CAT all increased and then decreased. Under Na2CO3 stress, the decrease in chlorophyll content of the resistant variety 061898 was less than in the sensitive Ningxia white flower variety. As the concentrations of Na2CO3, NaHCO3, and NaCl increased, the maximum photochemical efficiency of PSII was significantly affected. The resistant 061898 is capable of maintaining higher photosynthetic efficiency. Furthermore, under treatments with the three kinds of saline–alkali solutions, cell damage and death for Ningxia white flower were greater than in 061898. For 061898, the increases in soluble sugar and proline content were greater and the increase in malondialdehyde content was less, while the antioxidant enzyme activities were higher than those in Ningxia white flower. All four sweet clover varieties had higher stress resistance with neutral than with alkaline salts. When stressed by medium to high saline–alkali concentrations, sweet clover seedlings had increased osmotic substance content, enhanced antioxidant enzyme activity, and regulated physiological metabolism. Additionally, sweet clovers regulated the expression of WRKY33, GH3, CYCD3, OXI1, MKK2, MYC2, JAZ, COI1, PYL, PP2C, TGA, and MPK3 to adapt to the saline–alkali environment and improve saline–alkali tolerance. Our analysis of the sweet clover salinity tolerance mechanism contributes to its further use and is of significant importance for addressing land salinization and promoting sustainable agricultural and pastoral practices in China.

Deep N acquisition in cultivated grasslands: Uptake of slow-release 15N-labeled ammonium in hemiboreal monospecific leys

AimsTo develop a methodology to study uptake and redistribution by plants of NH4+ from deep soil, applying it to investigate deep root N uptake by cultivated grassland species.MethodsA slow-release 15NH4+ label adsorbed to clinoptilolite was placed into soil (depth 42 cm) well below the densest root zone in well-established monospecific stands of five grass and two clover species. Species showing a variety of deep rooting patterns, N acquisition strategy, forage qualities, and persistence in hemiboreal conditions were chosen. The label was placed in early spring and tracked throughout one or two growing seasons in two repeated experiments.ResultsAfter two growing seasons ~ 90% of the label was tracked in the soil and harvested herbage of grasses, less in clovers. Deep N uptake was limited in spring, increased during mid-season, and was strongest in autumn in all species, despite lower herbage yield in autumn. Species differed in ability to recover and maintain 15N in the soil–plant system. In one growing season, Lolium perenne L., Phleum pratense L., Schedonorus pratensis (Huds.) P.Beauv. and Schedonorus arundinaceus (Schreb.) Dumort herbage recovered ~ 65% of the label, Poa pratensis L. 54%, and Trifolium pratense L. and Trifolium repens L. 36–48%. Label transport to topsoil was observed, mainly attributable to plant nutrient redistribution rather than physical diffusion.ConclusionsThe innovative slow-release 15N label enabled tracing species differences and seasonal changes in uptake of NH4+ from deep soil. Among the tall-growing grasses, growth vigor appeared as important for deep N uptake as expected root depth.

ASSESSMENT OF HEAVY METALS ACCUMULATION IN PERENNIAL LEGUMES (WHITE CLOVER AND RED CLOVER) IN THE COPȘA MICĂ AREA, ROMANIA

The impact of heavy metals on perennial legumes, just like on other plant species, can be detrimental and has several ecological and agricultural implications. The ability of perennial legumes to fix atmospheric nitrogen through symbiotic interactions with nitrogen-fixing bacteria in their root nodules makes them a significant group of plants. The present study aims to evaluate the total concentration of heavy metals (cadmium, lead, copper, and zinc) in red and white clover plants harvested from grasslands located in heavily polluted areas (Copșa Mică, Axente Sever, Valea Viilor, Micăsasa and Târnava). White clover (Trifolium repens L.) recorded Cd content values between 0.06 - 3.53 mg/kg dry weight, Cu had values between 4.09 - 33.4 mg/kg dry weight, Pb content was between 0.18 - 4.77 mg/kg dry weight and Zn had values between 38 - 186 mg/kg dry weight. Regarding red clover (Trifolium pratense L.) the heavy metal content values were as follows: for Cd (0.08 - 3.38 mg/ kg d.w), for Cu (6.03 - 13.5 mg/kg d.w), for Pb (0.13 – 2.34 mg/kg d.w), for Zn (33.1 - 169 mg/kg d.w). Our results show that clover species harvested from grasslands in these geographical areas have high levels of heavy metals due to metallurgical activities. Grazing in these areas affected by heavy metal pollution can pose serious health problems for animals and later for humans through consumption of their products.

Seed dormancy and germination ecology of several clover species

This research was carried out to evaluate the effect of (1) different methods of breaking seed dormancy (physical and chemical scarification, high temperature, hot water and after-ripening), and (2) environmental factors (temperature, salinity and water stress) on seed germination of Trifolium angustifolium, T. arvense, T. fragiferum, T. fucatum, T. repens, T. subterraneum and Trifolium sp. All experiments were conducted in the Seed Research Laboratory of Gorgan University of Agricultural Sciences and Natural Resources, Iran, in 2021. All the examined clover species were dormant after harvest, with little germination. Mechanical scarification with sandpaper or sulphuric acid resulted in more than 88 and 52% germination, respectively. Seeds were able to germinate between 5 and 35°C, but the highest germination (≥ 98% on average) occurred at 20°C. Fluctuating temperature regimes were more efficient in improving the germination than constant temperatures. Seeds of clover species, in addition to having a hard seed coat, have a non-deep physiological dormancy. Fresh seeds showed a high sensitivity to water stress compared with after-ripened seeds. However, relative resistance to salinity stress was observed. The results obtained in this research can be used to develop effective sowing strategies and support the successful establishment of these clover species.

Бобовият компонент на горски садинови ливади в условията на „детелинова година“

A study of the species composition, biological, morphological and productive characteristics of the legume component of forest Chrysopogon gryllus meadows a year with high spring moisture availability was conducted. A very high species diversity of leguminous grasses was found - 17 species from 7 genera. Until the mowing of the meadows, a distinct turnover of species and an almost equal total productivity of dry mass from the legume component per ten-day period was observed. Thus, the productivity of fresh and dry mass of the legume component and, accordingly, the nitrogen fixation levels in the orchard meadows have a stable high level until mowing. The annual clovers can be identified as the legume with the strongest response to increasing moisture availability. A high productivity of mesophytic perennial clover species has also been reported, which significantly increase the fodder value of the grasslands. The species Dorycnium herbaceum and Medicago falcata have the highest productivity of fresh and dry mass when mowing the Chrysopogon gryllus meadows and have the greatest contribution to the forage value of the harvested hay. According to the morphological analysis, the legume component is characterized by a very high variability, both in plant height and in the relative share of leaves and flowers in the formed biomass, which determines its importance for the functional and biological diversity in the studied meadow type. The results are indicative of a great self-recovery potential of the Chrysopogon gryllus meadows in the the foothills of the Central Balkan, as well as a lack of degradation processes under their current mode of use.

Effects of multispecies and monoculture forages on nutrient digestibility and fermentation responses using an in vitro rumen simulation technique (RUSITEC)

Enteric methane (CH4) emissions are a major contributor to global greenhouse gas emissions. A number of different CH4 mitigation strategies have been proposed, and the inclusion of alternative forages into grazing systems could be a practical and feasible option. Multispecies swards comprise a mixture of forage species and can increase grassland productivity, reduce nitrogen fertiliser requirements, and reduce fertiliser-associated nitrous oxide emissions. This study investigated the potential benefit of multispecies swards in reducing ruminal CH4 production. Using the in vitro rumen simulation technique, we compared the effects of different forages (perennial ryegrass, timothy, red clover, white clover, chicory, ribwort plantain and their equi-proportional mixture) on CH4 and gas production, ruminal fermentation parameters and nutrient digestibility. We also compared these responses to perennial ryegrass produced with a higher nitrogen application rate (300 N). The experiment was conducted over 21 d, with the initial 14 d allowed for dietary adaptation. Over the subsequent 7 d, there were significant effects of forage type on CH4 production, ruminal fermentation and digestibility. Ribwort plantain, chicory and white clover showed lower CH4 production; with chicory producing 73% and 57% less CH4 (mmol d−1) compared to 300 N perennial ryegrass and 150 N perennial ryegrass incubation, respectively. Chicory had 72% lower CH4 produced per gram of organic matter digested (mmol g−1) than that of 300 N perennial ryegrass. Chicory and 300 N perennial ryegrass had greater (P < 0.01) organic matter and nutrient digestibility (DM, CP, NDF, and ADF) than other forages. Greater ammonia nitrogen concentration was observed in chicory and both clover species, compared to the other forage species (P < 0.01). In general, the response of the six-species mixture was not significantly different to the average response of the six component monocultures. Thus, from these data, the performance of multispecies swards for these responses (in general) was predictable from the performance of the component monocultures. These results showed that different grassland forages, especially chicory and white clover, have the potential to reduce ruminal CH4 emissions and could be a promising anti-methanogenic alternative to chemical CH4 inhibitors and feed additives. More generally, the relatively wide variation in CH4 abatement potential across a small sample of plant species suggests the merit of wider screening to identify grassland species with high CH4 abatement potential in vitro.

An assessment of resistance of two plantain (Plantago lanceolata) cultivars to phenoxy herbicides

ABSTRACT Plantain (Plantago lanceolata L.) is becoming widely adopted in New Zealand livestock systems due to its productivity, nutritional values and its role in reducing nitrate leaching from grazed pastures. Although sometimes grown alone, plantain is generally used in mixtures with grass and clover species; however, this makes it more difficult to selectively control weeds within these swards using herbicides. There has been some selection for phenoxy herbicide resistance in a cultivar of plantain, Agritonic, in New Zealand. Its level of resistance to MCPB, MCPA, an MCPB/MCPA mix, 2,4-D and 2,4-DB was compared to that of a more susceptible cultivar, Tonic, in dose-response experiments. The results showed that the resistance ranged between 1.3 and 3.5-fold compared to Tonic, and it was only significant for 2,4-D and MCPB/MCPA mix in the first and second experiments, respectively. Among all the tested herbicides, rates of MCPB and the MCPB/MCPA mix that are currently used in clover-based pastures were less damaging to Agritonic plants than the other herbicides tested. However, variability in resistance of the Agritonic plants resulted in some surviving the recommended rates of MCPA, 2,4-DB and 2,4-D, suggesting there is potential to select for higher levels of resistance to phenoxy herbicides.

Differences in the Ripening of Two Clover Species and the Effect of Pre-harvest Desiccation

Experiments were focused on differences in the ripening of crimson clover (Trifoliumincarnatum L.) and red clover (Trifolium pratense L.) and on the influence of the pre-harvest treatment with desiccants. The drying of leaves, stems and heads during ripening, regrowing, breakdown of heads and yield parameters including seed were compared. The crimson clover exhibited the natural drying of leaves, stems and heads faster than the red clover. In crimson clover, the drying of leaves, stems and heads before the harvest was 80 - 100%, 50– 95%, and 100 %, respectively. In red clover, the drying of leaves, stems and heads was 25 - 84 %, 20–72% and 45–99%, respectively. There were also differences in the regrowing and in the breakdown of heads. In the pre-harvest treatments of crimson clover and red clover, the effect of diquat was compared with the efficiency of other herbicide substances (pyraflufen-ethyl, carfentrazone-ethyl and pelargonic acid) and with the efficiency of a high dose of DAM fertilizer (liquid fertilizer, urea-ammonium nitrate). Compared with diquat, the drying was slower with the other active substances. Significant effects of tested active substances on yield, WTS (weight of thousand seeds) and germination were not recorded.

A phytochemical study of the clover growing in Kuzbass

In addition to studying bioactive organic compounds in plants, it is increasingly important to determine the biological role of elements in plants growing in environmentally unfavorable areas. One of such regions in Russia is Kuzbass with its intensively developing chemical, metallurgical, and coal mining sectors. In this study, we assessed the plant materials of red clover (Trifolium pratense L.), alsike clover (Trifolium hybridum L.), and white clover (Trifolium repens L.) collected from their natural populations in Kuzbass.&#x0D; The qualitative and quantitative composition of heavy metals in the clover samples was determined voltammetrically. The contents of molybdenum and phosphorus were measured by the photocolorimetric method. Total nitrogen and protein were determined by the Kjeldahl method. Nickel, cobalt, and chromium were quantified by spectrophotometry.&#x0D; We analyzed the plant materials of the clover samples for heavy metals and found that the content of lead was the least in red clover and the highest in alsike clover. Copper varied in a larger range and was minimal in red clover compared to that in alsike and white clover. Zinc was found at higher concentrations of in white and red clover compared to that in alsike clover. The levels of cadmium exceeded the maximum permissible concentrations in all the clover samples. We also revealed that the clover samples contained different amounts of various amino acids, including arginine, valine, lysine, glycine, aspartic acid, and alanine.&#x0D; The plant materials of the clover species growing in Kuzbass can be used to improve the fertility of soil and nitrogen regime. However, the clover species should not be used in bulk feed for farm animals because of high concentrations of cadmium.

Mapping groundcover of clover species in hill pastures in Wairarapa

Tokaroa Farm (608 ha) with sheep and beef in the Wairarapa has predominantly sunny, north-facing slopes. Average annual rainfall is 810 mm with three months summer-dry. High quality pasture is needed in late winter/early spring for lactating stock and in spring to finish stock early. The survey mapped the distribution of resident subterranean (sub) and white clovers across four land classes based on slope and aspect. ArcGIS identified 60 GPS points that were visually assessed in December 2019 and November 2021. At the majority of points in both years, both white and sub clover were present. In 2019, white clover groundcover was 18.1% and higher (P&lt;0.05) than sub clover at 12.6%. Management class had no effect. In 2021 there was no species effect (both ~11%) but there was a trend (P=0.062) for less white clovergroundcover in the ‘shady’ class (6.7%) than the ‘flat’ class at 17.5%. Pasture management was focused on promoting early season sub clover, but the unexpectedly high groundcover of white clover provided feed during wetter than average summers. Understanding the continuum from summer-dry annual sub clover areas to summer-wet perennial white clover dominance across New Zealand pastures provides tools for the selection and management of appropriate legumes.

Methane emission, nutrient digestibility, and rumen microbiota in Holstein heifers fed 14 different grass or clover silages as the sole feed

This experiment investigated the variation in enteric methane production and associated gas exchange parameters, nutrient digestibility, rumen fermentation, and rumen microbiome when a range of silages based on different forage types (grass or clover), and different species within the 2 types, were fed as the sole feed to heifers. Three grass species (perennial ryegrass, festulolium, and tall fescue) and 2 clover species (red clover and white clover) were included. Perennial ryegrass was harvested at 2 maturity stages in the primary growth, white clover was harvested once in the primary growth, and 4 cuts of festulolium and tall fescue and 3 cuts of red clover were harvested during the growing season, giving 14 different silage batches in total. Sixteen Holstein heifers 16 to 21 mo old and 2 to 5 mo in pregnancy were fed the silages ad libitum as the sole feed in an incomplete crossover design. Each silage was fed to 4 heifers, except for the 2 perennial ryegrass silages, which were fed to 8 heifers; in total 64 observations. The CH4 production was measured for 3 d in respiration chambers. Heifers fed clover silages had higher dry matter intake (DMI) compared with heifers fed grass silages, and heifers fed tall fescue silages had the numerically the lowest DMI. Compared with grass silages, feeding clover silages led to higher crude protein digestibility but lower neutral detergent fiber (NDF) digestibility. Rumen pH was higher in heifers fed clover silages compared with those fed grass silages. Based on composition analysis, the rumen microbiota of the heifers clustered clearly according to forage type and species. More specifically, 7 of the 34 dominating rumen bacterial genus-level groups showed higher relative abundances for the clover silages, whereas 7 genus-level groups showed higher abundances for the grass silages. Methane yield was higher for heifers fed grass silages than for those fed clover silages when methane production was related to dry matter and digestible organic matter intake, whereas the opposite was seen when related to NDF digestion. The gross energy lost as methane (CH4 conversion factor, %) reduced from 7.5% to 6.7%, equivalent to an 11% reduction. The present study gives the outlines for choosing the optimal forage type and forage species with respect to nutrient digestibility and enteric methane emission in ruminants.

Rumen fermentation and forage degradability in dairy cows offered perennial ryegrass, perennial ryegrass and white clover, or a multispecies forage

An experiment was conducted to compare the rumen fermentation and degradability of three forage treatments. A perennial ryegrass (PRG; Lolium perenne), perennial ryegrass and white clover (Trifolium pratense) (PRGWC) and multispecies forage (MULTI), containing two grass species; perennial ryegrass and timothy (Phleum pratense); two clover species; white clover and red clover (Trifolium repens); and two herb species; chicory (Cichorium intybus) and plantain (Plantago lanceolata), were compared. Six rumen fistulated lactating dairy cows were used in a replicated 3 × 3 Latin square experiment consisting of 3 periods lasting 21-days. Cows were allowed ad libitum access to fresh forage and offered 2.71 kg DM concentrate each day. During days 1 – 14 of each period, cows were acclimatized to dietary treatments before entering a 7 – day sampling period. Indwelling rumen pH probes were inserted into the rumen of each cow on day 15 and 16. Rumen fluid samples were taken on day 18 every 2 h for rumen volatile fatty acid and ammonia nitrogen (NH3N) analysis. Forage dry matter (DM) and crude protein (CP) degradability was assessed via in sacco incubation beginning on day 19 of each period. Treatment had no effect on dry matter intake (DMI) or milk yield. There was no difference in mean rumen pH between treatments. The rumen pH of cows offered MULTI was below pH 6.0 for a higher percentage of time than the other treatments, however the concentration of acetic acid in the rumen of cows was similar between treatments. Effective degradability (ED) of DM was higher (P = 0.003) for MULTI (0.77) compared to PRG (0.68) and PRGWC (0.66). The soluble CP fraction of MULTI and PRGWC was significantly lower than that of PRG (P = 0.007), though PRGWC had the highest rumen ammonia concentration of all treatments (P = 0.002). MULTI had the highest ED and rate of DM degradation in the rumen, and lowest soluble CP of all treatments. Multispecies forages can support milk production in lactating dairy cows; however, care must be taken to ensure adequate dietary NDF due to the low fiber concentration and highly degradable nature of the forage.