Exogenous silicon induces aluminum tolerance in white clover (Trifolium repens) by reducing aluminum uptake and enhancing organic acid secretion.

Excessive aluminum (Al) in acidic soils is a primary factor that hinders plant growth. The objective of the present study was to investigate the effect and physiological mechanism of exogenous silicon (Si) in alleviating aluminum toxicity. Under hydroponic conditions, 4 mM Al significantly impeded the growth of white clover; however, pretreatments with 1 mM Si mitigated this inhibition, as evidenced by notable changes in growth indicators and physiological parameters. Exogenous silicon notably increased both shoot and root length of white clover and significantly decreased electrolyte leakage (EL) and malondialdehyde (MDA) content compared to aluminum treatments. This positive effect was particularly evident in the roots. Further analysis involving hematoxylin staining, scanning electron microscopy (SEM), and examination of organic acids (OAs) demonstrated that silicon relieved the accumulation of bioactive aluminum and ameliorated damage to root tissues in aluminum-stressed plants. Additionally, energy-dispersive X-ray (EDX) analysis revealed that additional silicon was primarily distributed in the root epidermal and cortical layers, effectively reducing the transport of aluminum and maintaining the balance of exchangeable cations absorption. These findings suggest that gradual silicon deposition in root tissues effectively prevents the absorption of biologically active aluminum, thereby reducing the risk of mineral nutrient deficiencies induced by aluminum stress, promoting organic acids exudation, and compartmentalizing aluminum in the outer layer of root tissues. This mechanism helps white clover alleviate the damage caused by aluminum toxicity.

Allelopathic effect of the methanol extract of the weed species-red sorrel (Rumex acetosella L.) on the growth, phytohormone content and antioxidant activity of the cover crop - white clover (Trifolium repens L.).

Allelopathy is a biological process in which one organism releases biochemicals that affect the growth and development of other organisms. The current investigation sought to determine the allelopathic effect of Rumex acetosella on white clover (Trifolium repens) growth and development by using its shoot extract (lower IC50 value) as a foliar treatment. Here, different concentrations (25, 50, 100, and 200 g/L) of shoot extract from Rumex acetosella were used as treatments. With increasing concentrations of shoot extract, the plant growth parameters, chlorophyll and total protein content of Trifolium repens decreased. On the other hand, ROS, such as O2.- and H2O2, and antioxidant enzymes, including SOD, CAT, and POD, increased with increasing shoot extract concentration. A phytohormonal study indicated that increased treatment concentrations increased ABA and SA levels while JA levels were reduced. For the identification of allelochemicals, liquid‒liquid extraction, thin-layer chromatography, and open-column chromatography were conducted using R. acetosella shoot extracts, followed by a seed bioassay on the separated layer. A lower IC50 value was obtained through GC/MS analysis. gammaSitosterol was identified as the most abundant component. The shoot extract of Rumex acetosella has strong allelochemical properties that may significantly impede the growth and development of Trifolium repens. This approach could help to understand the competitive abilities of this weed species and in further research provide an alternate weed management strategy.

Chemical Basis for Determining the Allelopathic Potential of Invasive Plant Wall Barley (Hordeum murinum L. subsp. murinum).

The study investigated compounds present in the invasive grass Hordeum murinum L. subsp. murinum and tested the allelopathic potential of this plant against common meadow species Festuca rubra L. and Trifolium repens L. Gas chromatography-mass spectrometry (GC-MS) performed separately on the ears and stalks with leaves of wall barley revealed 32 compounds, including secondary metabolites, that may play an important role in allelopathy. Two compounds, N-butylbenzenesulfonamide (NBBS) and diphenylsulfone (DDS), were described for the first time for wall barley and the Poaceae family. The presence of 6,10,14-trimethylpentadecan-2-one (TMP) has also been documented. Aqueous extracts of H. murinum organs (ears and stalks with leaves) at concentrations of 2.5%, 5%, and 7.5% were used to evaluate its allelopathic potential. Compared to the control, all extracts inhibited germination and early growth stages of meadow species. The inhibitory effect was strongest at the highest concentration for both the underground and aboveground parts of the seedlings of the meadow species tested. Comparing the allelopathic effect, Trifolium repens proved to be more sensitive. In light of the results of the study, the removal of wall barley biomass appears to be important for the restoration of habitats where this species occurs due to its allelopathic potential.

Winter survival in red clover: experimental evidence for interactions among stresses.

BACKGROUND: There is a lack of knowledge on the combined effects of different stresses on plants, in particular different stresses that occur during winter in temperate climates. Perennial herbaceous plants in temperate regions are exposed to many different stresses during winter, but except for the fact that cold temperatures induce resistance to a number of them, very little is known about their interaction effects. Knowledge about stress interactions is needed in order to predict effects of climate change on both agricultural production and natural ecosystems, and to develop adaptation strategies, e.g., through plant breeding. Here, we conducted a series of experiments under controlled conditions to study the interactions between cold (low positive temperature), clover rot infection (caused by Sclerotinia trifoliorum) and freezing, in red clover (Trifolium pratense) accessions. We also compared our results with winter survival in field experiments and studied associations between stress and shoot growth. RESULTS: Exposure to low positive temperatures (cold acclimation) induced resistance to clover rot. There was a clear negative interaction effect between freezing stress and clover rot infection, resulting in up to 37% lower survival rate compared to what would have been expected from the additive effect of freezing and infection alone. Freezing tolerance could continue to improve during incubation under artificial snow cover at 3 °C in spite of darkness, and we observed compensatory shoot growth following freezing after prolonged incubation. At the accession level, resistance to clover rot was negatively correlated with growth in the field during the previous year at a Norwegian location. It was also negatively correlated with the shoot regrowth of control plants after incubation. Clover rot resistance tests under controlled conditions showed limited correlation with clover rot resistance observed in the field, suggesting that they may reveal variation in more specific resistance mechanisms. CONCLUSIONS: We here demonstrate, for the first time, a strong negative interaction between freezing and infection with a winter pathogen. We also characterize the effects of cold acclimation and incubation in darkness at different temperatures on winter stress tolerance, and present data that support the notion that annual cycles of growth and stress resistance are associated at the genetic level.

Epidural Hematoma Associated with Red Clover Use After Epidural Injection: A Case Report.

An epidural hematoma is a rare but devastating complication after interventional pain procedures. The etiology is multifactorial, including anatomical variations, inherited coagulation disorders, and consumption of anticoagulants or antiplatelet substances. Specifically, in regard to platelet aggregation, the consumption of herbal medicine is often forgotten as a potential cause for coagulation profile disorders, potentially leading to an epidural hematoma. We present the case of a patient who developed an epidural hematoma after a cervical epidural block, most likely associated with daily "red clover" consumption.

Water Stress Influences Phytoestrogen Levels in Red Clover (Trifolium pratense) but Not Kura Clover (T. ambiguum).

Some forage legumes synthesize phytoestrogens. We conducted a glasshouse study to investigate how water stress (drought and waterlogging) influences phytoestrogen accumulation in red clover and kura clover. Compared to the red clover control, the 20 day drought resulted in an over 100% increase in the phytoestrogens formononetin and biochanin A, which together accounted for 91-96% of the total phytoestrogens measured. Waterlogging resulted in elevated concentrations of daidzein, genistein, and prunetin but not formononetin or biochanin A. Concentrations of phytoestrogens in kura clover were low or undetectable, regardless of water stress treatment. Leaf water potential was the most explanatory single-predictor of the variation in concentrations of formononetin, biochanin A, and total phytoestrogens in red clover. These results suggest that drought-stressed red clover may have higher potential to lead to estrogenic effects in ruminant livestock and that kura clover is a promising alternative low- or no-phytoestrogen perennial forage legume.

Neurotherapeutic effects of quercetin-loaded nanoparticles and Biochanin-A extracted from Trifolium alexandrinum on PI3K/Akt/GSK-3ß signaling in the cerebral cortex of male diabetic rats.

Diabetes mellitus (DM) is a severe metabolic disease that can have significant consequences for cognitive health. Bioflavonoids such as Trifolium alexandrinum (TA), quercetin (Q), and Biochanin-A (BCA) are known to exert a wide range of pharmacological functions including antihyperglycemic activity. This study aimed to investigate the neurotherapeutic effects of quercetin-loaded nanoparticles (Q-LNP) and BCA extracted from TA against diabetes-induced cerebral cortical damage through modulation of PI3K/Akt/GSK-3ß and AMPK signaling pathways. Adult male Wistar albino rats (N = 25) were randomly assigned to one of five groups: control, diabetics fed a high-fat diet (HFD) for 2 weeks and intraperitoneally (i.p.) injected with STZ (40 mg/kg), and diabetics treated with Q-LNP (50 mg/kg BW/day), BCA (10 mg/kg BW/day), or TA extract (200 mg/kg BW/day). Treatments were applied by oral gavage once daily for 35 days. Diabetic rats treated with Q-LNP, BCA, and TA extract showed improvement in cognitive performance, cortical oxidative metabolism, antioxidant parameters, and levels of glucose, insulin, triglyceride, and total cholesterol. In addition, these treatments improved neurochemical levels, including acetylcholine, dopamine, and serotonin levels as well acetylcholinesterase and monoamine oxidase activities. Furthermore, these treatments lowered proinflammatory cytokine production for TNF-α and NF-κB; downregulated the levels of IL-1ß, iNOS, APP, and PPAR-γ; and attenuated the expressions of PSEN2, BACE, IR, PI3K, FOXO 1, AKT, AMPK, GSK-3ß, and GFAP. The histopathological examinations of the cerebral cortical tissues confirmed the biochemical results. Overall, the present findings suggest the potential therapeutic effects of TA bioflavonoids in modulating diabetes-induced cerebral cortical damage.

Root uptake of cereal benzoxazinoids grants resistance to root-knot nematode invasion in white clover.

Plants synthesize a plethora of chemical defence compounds, which vary between evolutionary lineages. We hypothesize that plants evolved the ability to utilize defence compounds synthesized and released by neighbouring heterospecific plants. In two experiments, we incubated clover (Trifolium repens L.) seedlings with individual benzoxazinoid (BX) compounds (2,4-dihydroxy-1,4-benzoxazin-3-one, 2-hydroxy-1,4-benzoxazin-3-one, benzoxazolinone, and 6-methoxy- benzoxazolin-2-one), a group of bioactive compounds produced by cereals, to allow clover BX uptake. Subsequently, we transplanted the seedlings into soil and quantified BX root and shoot content and invasion of root-knot nematodes in clover roots up to 8 weeks after transplantation. We show that clover root uptake of BXs substantially enhanced clover's resistance against the root-knot nematode Meloidogyne incognita. This effect lasted up to 6 weeks after the clover roots were exposed to the BXs. BXs were absorbed by clover roots, and then translocated to the shoots. As a result of clover metabolization, we detected the parent BXs and a range of their transformation products in the roots and shoots. Based on these novel findings, we envisage that co-cultivation of crop species with complementary and transferable chemical defence systems can add to plant protection.

Genome-wide identification of Aux/IAA gene family in white clover (Trifolium repens L.) and functional verification of TrIAA18 under different abiotic stress.

BACKGROUND: White clover (Trifolium repens L.) is an excellent leguminous cool-season forage with a high protein content and strong nitrogen-fixing ability. Despite these advantages, its growth and development are markedly sensitive to environmental factors. Indole-3-acetic acid (IAA) is the major growth hormone in plants, regulating plant growth, development, and response to adversity. Nevertheless, the specific regulatory functions of Aux/IAA genes in response to abiotic stresses in white clover remain largely unexplored. RESULTS: In this study, we identified 47 Aux/IAA genes in the white clover genome, which were categorized into five groups based on phylogenetic analysis. The TrIAAs promoter region co-existed with different cis-regulatory elements involved in developmental and hormonal regulation, and stress responses, which may be closely related to their diverse regulatory roles. Collinearity analysis showed that the amplification of the TrIAA gene family was mainly carried out by segmental duplication. White clover Aux/IAA genes showed different expression patterns in different tissues and under different stress treatments. In addition, we performed a yeast two-hybrid analysis to investigate the interaction between white clover Aux/IAA and ARF proteins. Heterologous expression indicated that TrIAA18 could enhance stress tolerance in both yeast and transgenic Arabidopsis thaliana. CONCLUSION: These findings provide new scientific insights into the molecular mechanisms of growth hormone signaling in white clover and its functional characteristics in response to environmental stress.

The inclusion of companion forages in the diet alongside perennial ryegrass increased dry matter intake and organic matter digestibility in sheep.

The inclusion of companion forages in the diet of ruminant animals is gaining popularity in temperate regions due to observed improvements in animal performance. The aim of this study was to assess the effect of diet type on DM intake (DMI) and organic matter digestibility (OMD) in sheep. Furthermore, the effect of sward type on diet nutritive quality was investigated. Five dietary treatments were investigated using a 5 × 5 Latin square design experiment: Perennial ryegrass (Lolium perenne L.; PRG) only or PRG plus white clover (Trifolium repens L.;PRG + WC), red clover (Trifolium pratense L.; PRG + RC), chicory (Chicorium intybus L.; PRG + Chic) or plantain (Plantago lanceolata L.; PRG + Plan) at a ratio of 75% PRG and 25% of the respective companion forage and 100% PRG for the grass only treatment on a DM basis. Twenty Belclare castrated male (wether) sheep were housed in metabolism crates across five feeding periods. Individual DMI and faecal output were recorded daily and digestibility parameters were subsequently calculated. Results show that the inclusion of any companion forage increased DMI (kg/day DM) which ranged from 1.55 ± 0.038 (PRG) to 1.76 ± 0.038 (PRG + Chic) (P < 0.001). The PRG + WC (825 ± 1.1), PRG + RC (823 ± 1.1) and PRG + Chic (826 ± 1.1) diets had a greater in vitro OMD (g/kg DM) when compared to PRG (819 ± 1.1) or PRG + Plan (816 ± 1.1) (P < 0.001). Furthermore, the PRG + Chic (830 ± 2.9) diet had a greater in vivo OMD (g/kg DM) (P < 0.01) when compared to the PRG, PRG + RC, and PRG + Plan diets. Regression analysis showed that in vitro estimates moderately reflected in vivo measurements (r2 = 0.61). The inclusion of any companion forage increased dietary CP content and reduced the proportion of NDF in the diet. Crude protein concentration increased by an average of 16.5 g/kg DM and NDF content was reduced by 25.3 g/kg DM, on average, with companion forage inclusion (P < 0.001). Results suggest that binary sward mixtures benefit pasture-based sheep production systems, boosting sward quality, aiding increased DM intakes of a more digestible diet in the summer period.

Evaluation Health Risks and Sorption of Hexavalent Chromium (Cr(VI) by Biochar and Iron Doped Zinc Oxide Modified Biochar (Fe-ZnO@BC) Using Trifolium: A Green Synthesis Technique.

Contamination of aquatic and terrestrial environment with hexavalent chromium Cr(VI) is one of the major hazards worldwide due its carcinogenicity, persistency and immobility. Different research techniques have been adopted for Cr(VI) remediation present in terrestrial and aquatic media, while adsorption being the most advance, low cost, environmentally friendly and common method. The present study discussed the mechanisms of Parthenium hysterophorus derived biochar, iron-doped zinc oxide nanoparticles (nFe-ZnO) and Fe-ZnO modified biochar (Fe-ZnO@BC) involved in Cr(VI) mobility and bioavailability. Pot experiments were conducted to study the effect of Parthenium hysterophorus derived biochar, nFe-ZnO and Fe-ZnO@BC application rates (2%, 2 mg/kg, 10 mg/kg, respectively). The results indicated that the addition of soil amendments reduced Cr(VI) mobility. The findings revealed that the reduction in chromium mobility was observed by P. hysterophorus BC, and Fe-ZnO@BC but nFe-ZnO application significantly (p = 0.05) reduced Cr(VI) and CrT uptake as compared to the control treatments. The results of SEM coupled with EDS showed a high micropores and channel, smooth surface which helped in adsorption, and may enhance soil conditions. The concentration index (CI) by different amendments in trifolium plant was followed the descending order as: nFe-ZnO > Fe-ZnO@BC > P. hysterophorus BC after 30, 60 and 90 days of harvesting, respectively. In addition, human health risk index was found less than one (H1 < 1.0) in amended soils as compared to control treatments.

Determination of Physicochemical Characteristics, Phytochemical Profile and Antioxidant Activity of Various Clover Honeys.

This paper reports on some physicochemical and phytochemical characteristics (i. e. pH, electrical conductivity, colour, moisture content, total phenolic content, sugar profile) and in vitro antioxidant activity of honeys harvested from five legume species, red clover (Trifolium pratense), balansa clover (T. michelianum), Persian clover (T. resupinatum), purple clover (T. purpureum) and sanfoin, also known as holy clover (Onobrychis viciifolia), that were grown in enclosed shade houses to ensure that the honeys' characteristics are reflective of a truly monofloral honey. Glucose and fructose, determined via High-Performance Thin-Layer Chromatography (HPTLC) analysis, were found as the main sugars in all investigated honeys with the ratio of fructose to glucose ranging from 1 : 1.2 to 1 : 1.6. The honeys' pH values ranged from 3.9 to 4.6 which met Codes Alimentarius (CA) requirements. The moisture content was found to be between 17.6 and 22.2 % which in some cases was slightly higher than CA requirements (≤20 %). The honeys' colour values, prior and after filtration, were between 825.5-1149.5 mAU and 532.4-824.8 mAU respectively, illustrating golden yellow to deep yellow hues. The total phenolic content (TPC) of the honeys was determined using a modified Folin-Ciocalteu assay. Their antioxidant activity was captured by the Ferric Reducing-Antioxidant Power (FRAP) assay as well as HPTLC analysis coupled with 2,2-diphenyl-1-picrylhydrazyl (DPPH) derivatisation. The highest total phenolic content was found in red clover honey (45.4 mg GAE/100 g) whereas purple clover honey showed the highest level of activity in the FRAP assay (7.3 mmol Fe2+/kg). HPTLC-DPPH analysis of the honeys' organic extracts demonstrated the presence of various bioactive compounds that contribute to their overall antioxidant activity. This study developed a methodology for producing monofloral clover honeys in a space limited, enclosed production system, which allowed to collate important baseline data for these honeys that can serve as the foundation for their potential future development into commercial honeys, including honeys that can be used for medicinal purposes.

Symbiotic efficiency of Rhizobium leguminosarum sv. trifolii strains originating from the subpolar and temperate climate regions.

Red clover (Trifolium pratense L.) is a forage legume cultivated worldwide. This plant is capable of establishing a nitrogen-fixing symbiosis with Rhizobium leguminosarum symbiovar trifolii strains. To date, no comparative analysis of the symbiotic properties and heterogeneity of T. pratense microsymbionts derived from two distinct geographic regions has been performed. In this study, the symbiotic properties of strains originating from the subpolar and temperate climate zones in a wide range of temperatures (10-25 °C) have been characterized. Our results indicate that all the studied T. pratense microsymbionts from two geographic regions were highly efficient in host plant nodulation and nitrogen fixation in a wide range of temperatures. However, some differences between the populations and between the strains within the individual population examined were observed. Based on the nodC and nifH sequences, the symbiotic diversity of the strains was estimated. In general, 13 alleles for nodC and for nifH were identified. Moreover, 21 and 61 polymorphic sites in the nodC and nifH sequences were found, respectively, indicating that the latter gene shows higher heterogeneity than the former one. Among the nodC and nifH alleles, three genotypes (I-III) were the most frequent, whereas the other alleles (IV-XIII) proved to be unique for the individual strains. Based on the nodC and nifH allele types, 20 nodC-nifH genotypes were identified. Among them, the most frequent were three genotypes marked as A (6 strains), B (5 strains), and C (3 strains). Type A was exclusively found in the temperate strains, whereas types B and C were identified in the subpolar strains. The remaining 17 genotypes were found in single strains. In conclusion, our data indicate that R. leguminosarum sv. trifolii strains derived from two climatic zones show a high diversity with respect to the symbiotic efficiency and heterogeneity. However, some of the R. leguminosarum sv. trifolii strains exhibit very good symbiotic potential in the wide range of the temperatures tested; hence, they may be used in the future for improvement of legume crop production.

Yield of white clover and orchard grass cultivated in mining passives adding black earth and compost as a substrate.

Mining environmental liabilities generate environmental pollution. The objective of the present study was to determine the yield of white clover (Trifolium repens) and orchard grass (Dactylis glomerata) cultivated in mining environmental passives adding black earth and compost as a substrate in the Buenaventura Julcani Huancavelica Company. The treatments were the combinations: 4: 3.1: 3.1: 3:1.1 kg of RP: RP, TN: RP, C: RP, TN, C respectively. They were distributed in four treatments with twelve experimental units for each species of leguminous and gramineous grass, we worked according to the completely randomized design (DCA) with a 2 x 4 factorial arrangement, the experimental unit being a treatment with twelve repetitions. The variables evaluated were: germination percentage (TG) and stem survival percentage (TST). For the statistical analysis, the SPSS software was used.

Screening of heavy metal-resistant rhizobial and non-rhizobial microflora isolated from Trifolium sp. growing in mining areas.

Plant growth-promoting rhizobacteria (PGPR) can promote plant growth and development with several beneficial effects, especially in challenging environmental conditions, such as the presence of toxic contaminants. In this study, 49 isolates obtained from Trifolium sp. nodules growing on a Pb/Zn mine site were characterized for PGP traits including siderophores production, phosphate solubilization, extracellular enzymes production, and antifungal activity. The isolates were also screened for their ability to grow at increasing concentrations of NaCl and heavy metals, including lead, zinc, cobalt, copper, nickel, cadmium, and chromium. The findings of our study indicated that isolates Cupriavidus paucula RSCup01-RSCup08, Providencia rettgeri RSPro01, Pseudomonas putida RSPs01, Pseudomonas thivervalensis RSPs03-RSPs09, and Acinetobacter beijerinckii RSAci01 showed several key traits crucial for promoting plant growth, thus demonstrating the greatest potential. Most isolates displayed resistance to salt and heavy metals. Notably, Staphylococcus xylosus RSSta01, Pseudomonas sp. RSPs02, Micrococcus yunnanensis RSMicc01, and Kocuria dechangensis RSKoc01 demonstrated a significant capacity to grow at salt concentrations ranging from 10 to 20%, and isolates including Cupravidus paucula RSCup01-RSCup08 exhibited resistance to high levels of heavy metals, up to 1300 mg/L Pb++, 1200 mg/L Zn++, 1000 mg/L Ni++, 1000 mg/L Cd++, 500 mg/L Cu++, 400 mg/L Co++, and 50 mg/L CrVI+. Additionally, the analysis revealed that metal-resistant genes pbrA, czcD, and nccA were exclusively detected in the Cupriavidus paucula RSCup01 strain. The results of this study provide insights into the potential of plant growth-promoting rhizobacteria strains that might be used as inoculants to improve phytoremediation in heavy metal-contaminated soils.

Pantoea trifolii sp. nov., a novel bacterium isolated from Trifolium rubens root nodules.

A novel bacterium, designated strain MMK2T, was isolated from a surface-sterilised root nodule of a Trifolium rubens plant growing in south-eastern Poland. Cells were Gram negative, non-spore forming and rod shaped. The strain had the highest 16S rRNA gene sequence similarity with P. endophytica (99.4%), P. leporis (99.4%) P. rwandensis (98.8%) and P. rodasii (98.45%). Phylogenomic analysis clearly showed that strain MMK2T and an additional strain, MMK3, should reside in the genus Pantoea and that they were most closely related to P. endophytica and P. leporis. Genome comparisons showed that the novel strain shared 82.96-93.50% average nucleotide identity and 26.2-53. 2% digital DNA:DNA hybridization with closely related species. Both strains produced siderophores and were able to solubilise phosphates. The MMK2T strain was also able to produce indole-3-acetic acid. The tested strains differed in their antimicrobial activity, but both were able to inhibit the growth of Sclerotinia sclerotiorum 10Ss01. Based on the results of the phenotypic, phylogenomic, genomic and chemotaxonomic analyses, strains MMK2T and MMK3 belong to a novel species in the genus Pantoea for which the name Pantoea trifolii sp. nov. is proposed with the type strain MMK2T (= DSM 115063T = LMG 33049T).

Enrichment of milk antioxidant activity by dietary supplementation of red clover isoflavone in cows and its improvement on mice intestinal health.

Red clover (Trifolium pratense) isoflavone was supplemented to dairy cows, and antioxidant capacity of milk was assessed. Treated cows increased the activities of antioxidant enzymes, reduced production of oxidation products, and enhanced the concentrations of vitamin E and vitamin C. Moreover, milk fatty acid profile was positive influenced by 8 g/kg red clover isoflavone, with changes in the lower saturated and higher unsaturated fatty acids. We further demonstrated the efficacy of antioxidant capacity of milk in mice, found that milk from cows feeding red clover isoflavone increased the expressions of antioxidant enzymes, and alleviated lipopolysaccharide (LPS)-stimulated tissue damage of duodenum and jejunum, which was related to upregulated metabolism pathways of carbohydrate, lipid, and amino acid, as well as downregulated inflammatory related pathways. Together, dietary supplementation of red clover isoflavone is an effective way to improve milk antioxidant capacity, providing a natural strategy for developing functional foods.

Efficacy of plant-derived dietary supplements in improving overall menopausal symptoms in women: An updated systematic review and meta-analysis.

This updated systematic review and meta-analysis aims to confirm the effectiveness of plant-based supplements in improving overall menopausal symptoms and vasomotor symptoms. A systematic review of the literature was conducted by searching the PubMed/MEDLINE, Web of Science, EMBASE, and CENTRAL databases up to June 2022. Randomized placebo-controlled clinical trials that evaluated the effects of dietary supplements on menopausal symptoms were included. Outcome measures included daily hot flash frequency, Kupperman's index, Menopause Rating Scale, and Greene Climacteric Scale. Pooled data were analyzed using a fixed-effects model and expressed as a weighted mean difference with a 95% confidence interval for continuous outcomes. For qualitative assessment, 67 studies were selected. For quantitative assessment, 54 reports were obtained from 61 studies. The study participants were peri- or postmenopausal women aged 38-85, most of whom experienced hot flashes as a menopausal symptom. The investigational products included 28 soy-derived, 6 red clover-derived, and 28 other plant-derived supplements. Qualitative assessment revealed that approximately 76% of the studies were generally of fair or good quality, whereas 24% were of low quality. Meta-analysis results indicated significant improvements in all questionnaire scores, including hot flash frequency, in the dietary supplement group compared with the placebo group. Comprehensive evaluation using different questionnaire tools showed that the various plant-derived dietary supplements can significantly alleviate menopausal symptoms. However, further rigorous studies are needed to determine the association of plant-derived dietary supplements with menopausal health because of the general suboptimal quality and heterogeneous nature of current evidence.

Reducing dietary protein and supplementation with starch or rumen-protected methionine and its effect on performance and nitrogen efficiency in dairy cows fed a red clover and grass silage-based diet.

The increasing cost of milk production, in association with tighter manure N application regulations and challenges associated with ammonia emissions in many countries, has increased interest in feeding lower crude protein (CP) diets based on legume silages. Most studies have focused on alfalfa silage, and little information is available on low-CP diets based on red clover silage. Our objectives were to examine the effects of dietary CP content and supplementing a low-CP diet with dietary starch or rumen-protected Met (RPMet) on the performance, metabolism, and nitrogen use efficiency (NUE; milk N output/N intake) in dairy cows fed a red clover and grass silage-based diet. A total of 56 Holstein-Friesian dairy cows were blocked and randomly allocated to 1 of 4 diets over a 14-wk feeding period. Diets were based on red clover and grass silages at a ratio of 50:50 on a dry matter (DM) basis and were fed as a total mixed ration, with a 53:47 ratio of forage to concentrate (DM basis). The diets were formulated to supply a similar metabolizable protein (MP) content, and had a CP concentration of either 175 g/kg DM (control [CON]) or 150 g/kg DM (low-protein [LP]), or LP supplemented with either additional barley as a source of starch (LPSt; +64 g/kg DM) or RPMet (LPM; +0.3 g/100 g MP). At the end of the 14-wk feeding period, 20 cows (5 per treatment) continued to be fed the same diets for a further 6 d, and total urine output and fecal samples were collected. We observed that dietary treatment did not affect DM intake, with a mean of 21.5 kg/d; however, we also observed an interaction between diet and week with intake being highest in cows fed LPSt in wk 4 and CON in wk 9 and 14. Mean milk yield, 4% fat-corrected milk, and energy-corrected milk were not altered by treatment. Similarly, we found no effect of dietary treatment on milk fat, protein, or lactose content. In contrast, milk and plasma urea concentrations were highest in cows fed CON. The concentration of blood plasma ß-hydroxybutyrate was highest in cows receiving LPM and lowest in LPSt. Apparent NUE was 28.6% in cows fed CON and was higher in cows fed any of the low-protein diets (LP, LPSt, or LPM), with a mean value of 34.2%. The sum of milk fatty acids with a chain length below C16:0 was also highest in cows fed CON. We observed that dietary treatment did not affect the apparent whole-tract nutrient digestibility of organic matter, N, neutral detergent fiber, and acid detergent fiber, with mean values of 0.785, 0.659, 0.660, and 0.651 kg/kg respectively, but urinary N excretion was approximately 60 g/d lower in cows fed the low-CP diets compared with CON. We conclude that reducing the CP content of red clover and grass silage-based diets from 175 to 150 g/kg DM while maintaining MP supply did not affect performance, but reduced the urinary N excretion and improved NUE, and that supplementing additional starch or RPMet had little further effect.

Genome-wide identification and characterization of NAC transcription factor family members in Trifolium pratense and expression analysis under lead stress.

BACKGROUND: The NAC TF family is widely involved in plant responses to various types of stress. Red clover (Trifolium pratense) is a high-quality legume, and the study of NAC genes in red clover has not been comprehensive. The aim of this study was to analyze the NAC gene family of red clover at the whole-genome level and explore its potential role in the Pb stress response. RESULTS: In this study, 72 TpNAC genes were identified from red clover; collinearity analysis showed that there were 5 pairs of large fragment replicators of TpNAC genes, and red clover was found to be closely related to Medicago truncatula. Interestingly, the TpNAC genes have more homologs in Arabidopsis thaliana than in soybean (Glycine max). There are many elements in the TpNAC genes promoters that respond to stress. Gene expression analysis showed that all the TpNAC genes responded to Pb stress. qRT-PCR showed that the expression levels of TpNAC29 and TpNAC42 were significantly decreased after Pb stress. Protein interaction network analysis showed that 21 TpNACs and 23 other genes participated in the interaction. In addition, the TpNAC proteins had three possible 3D structures, and the secondary structure of these proteins were mainly of other types. These results indicated that most TpNAC members were involved in the regulation of Pb stress in red clover. CONCLUSION: These results suggest that most TpNAC members are involved in the regulation of Pb stress in red clover. TpNAC members play an important role in the response of red clover to Pb stress.