Temporary sensory separation of lamb groups from ewes affects behaviors and serum levels of stress-related indicators of small-tailed Han lambs.

Under the current meat sheep breeding system, newborn lambs usually live with their mothers until weaning, and in daily management, they often need to be separated from their ewes for a short period due to dehorning, disease treatment, etc. Such short-term separation was considered to be a high-intensity stress for the lambs. This study aimed to explore the effects of 1 h sensory separations on behaviors and the concentration of stress-related indicators of small-tailed Han lambs. Lambs were assigned to four groups: auditory, visual, and tactile separation (AVT) group; visual and tactile separation (VT) group; tactile separation (T) group; and control (C) group. Then they were separated from their mothers for one hour on postnatal days 14, 21, 28, 35 and 42. Results showed the separated lambs (AVT, VT, and T groups) spent less time lying down relaxing and more time looking around, exploring, vocalizing, and attempting to escape (P < 0.05). Lambs separated by lack of tactile contact only exhibited the most escaping and moving behavior. Twin-born lambs showed less moving, escaping, and vocalizing than single-born lambs (P < 0.05). The separation also led to a rise in serum globulin levels and a decrease in tetraiodothyronine. In conclusion, this study showed that temporary 1 h ewe-lamb separations could affect behaviors and the serum levels of stress indicators of lambs. The behavioral responses were more obvious when lambs were separated by lack of tactile contact only, and in single-born lambs. It can conclude that indicated that when lambs need to be temporarily separated from ewes in daily management production, it would be better to let them stay together with their littermates, and make them avoid hearing or seeing the ewes, such management may partially reduce the separation stress, thereby improving the welfare and breeding efficiency of sheep.

Methyl gallate from Camellia nitidissima Chi flowers reduces quorum sensing related virulence and biofilm formation against Aeromonas hydrophila.

Aeromonas hydrophila, a Gram-negative zoonotic bacterium, causes high mortality in fish farming and immunocompromised patients. This study aimed to extract methyl gallate (MG) from the flowers of Camellia nitidissima Chi and evaluate its potential as a quorum sensing inhibitor (QSI) against Aeromonas hydrophila SHAe 115. MG reduced QS-associated virulence factors, including hemolysis, protease, and lipase, while impairing swimming motility and biofilm formation. Additionally, MG down-regulated positive regulatory genes (ahyR, fleQ) and up-regulated negative regulators (litR, fleN). This highlights MG's promise as a potent QSI for A. hydrophila SHAe 115, advancing strategies against infections in aquaculture and human health.

Anti-Biofilm and Antivirulence Activities of 1,2,6-tri-O-galloyl-ß-á´ -glucose against Proteus penneri.

AIMS: The present study investigated the anti-virulence and anti-biofilm effects of 1,2,6-tri-O-galloyl-ß-ᴅ-glucose (TGG), isolated from Camellia nitidissima Chi flowers, on Proteus penneri ALK 1200. METHODS AND RESULTS: TGG was isolated from C. nitidissima Chi flowers using various chromatographic techniques. The milk plate assay, azocasein assay, and exopolysaccharides (EPS) inhibition assay revealed that TGG effectively inhibited the production of crucial virulence factors, including protease and EPS, in P. penneri ALK 1200. Furthermore, fourier transform infrared spectroscopic (FT-IR) analysis indicated that TGG interfered with the composition of P. penneri ALK 1200's cellular component, potentially reducing the bacteria's pathogenicity. In addition, crystal violet assay, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM) analysis indicated a significant reduction in biofilm formation following TGG treatment. The swimming and swarming assays also showed that TGG reduced the motility of P. penneri ALK 1200. Furthermore, the qRT-PCR assay demonstrated that TGG down-regulated the expression of positive regulatory genes (hfq and flhD) responsible for motility and biofilm formation, while up-regulating the expression of the negative regulator of the quorum sensing system, bssS, in P. penneri ALK 1200. CONCLUSIONS: TGG displayed potent anti-QS and anti-biofilm activity towards P. penneri ALK 1200.

Effects of "audience effects"on animal mate choice: A review.

The information tranfered among individual animals can be shared by a network, which is consisted of the sender, the receiver, and the extra bystander of the communication signals. The bystanders can read and use the signal that is not sent directly to them and make use of it to interfere with the sender and the receiver, which is known as "audience effects" in the research area of animal behaviors. The processes of mate choice and mating of animals occur mainly in the network that is composed of the particular species. Increasing evidence show that the audience effects play an important role in regulating mating preference and mating strategy, resulting in changes in species evolution. Here, we review the role of audience effects on animal mate choice and evolution by clarifying the definition and functional explanations of audience effects, the factors contributing to audience effects, as well as the different impacts of audience effects on males and females. It would provide novel ideas to study the impacts of audience effects on mate choice and species evolution in the future.

Densely Connected Networks with Multiple Features for Classifying Sound Signals with Reverberation.

In indoor environments, reverberation can distort the signalseceived by active noise cancelation devices, posing a challenge to sound classification. Therefore, we combined three speech spectral features based on different frequency scales into a densely connected network (DenseNet) to accomplish sound classification with reverberation effects. We adopted the DenseNet structure to make the model lightweight A dataset was created based on experimental and simulation methods, andhe classification goal was to distinguish between music signals, song signals, and speech signals. Using this framework, effectivexperiments were conducted. It was shown that the classification accuracy of the approach based on DenseNet and fused features reached 95.90%, betterhan the results based on other convolutional neural networks (CNNs). The size of the optimized DenseNet model is only 3.09 MB, which is only 7.76% of the size before optimization. We migrated the model to the Android platform. The modified model can discriminate sound clips faster on Android thanhe network before the modification. This shows that the approach based on DenseNet and fused features can dealith sound classification tasks in different indoor scenes, and the lightweight model can be deployed on embedded devices.

Formation of oil-particle aggregates in the presence of marine algae.

After an oil spill, the formation of oil-particle aggregates (OPAs) is associated with the interaction between dispersed oil and marine particulate matter such as phytoplankton, bacteria and mineral particles. Until recently, the combined effect of minerals and marine algae in influencing oil dispersion and OPA formation has rarely been investigated in detail. In this paper, the impacts of a species of flagellate algae Heterosigma akashiwo on oil dispersion and aggregation with montmorillonite were investigated. This study has found that oil coalescence is inhibited due to the adhesion of algal cells on the droplet surface, causing fewer large droplets to be dispersed into the water column and small OPAs to form. Due to the role of biosurfactants in the algae and the inhibition of algae on the swelling of mineral particles, both the oil dispersion efficiency and oil sinking efficiency were improved, which reached 77.6% and 23.5%, respectively at an algal cell concentration (Ca) of 1.0 × 106 cells per mL and a mineral concentration of 300 mg L-1. The volumetric mean diameter of the OPAs decreased from 38.4 µm to 31.5 µm when Ca increased from 0 to 1.0 × 106 cells per mL. At higher turbulent energy, more oil tended to form larger OPAs. The findings may add knowledge about the fate and transport of spilled oil and provide fundamental data for oil spill migration modelling.

Evolution and phylogenetic diversity of the aquaporin gene family in arachnids.

Water flux across cells predominantly occurs through the pore formed by the aquaporin channels. Since water balance is one of the most important challenges to terrestrial animals, aquaporin evolution and diversity is known to play roles in animal terrestrialisation. Arachnids (Arthropoda: Chelicerata: Arachnida) are the second most diverse group and represent the pioneer land colonists in animals; however, there remains no thorough investigation on aquaporin evolution and diversity in this evolutionarily important lineage. Here we reported a phylogenetic study of aquaporin evolution and diversity using genomic data from 116 arachnid species covering almost all (15/16) extant orders. A previously unrecognised subfamily related to aquaporin-4 (i.e. Aqp4-like subfamily) via phylogenetic analysis was identified, suggesting certain underestimate of the arachnid aquaporin diversity in earlier studies probably due to limited taxonomic sampling. Further analysis indicates that this subfamily emerged deep within the life tree of arthropods. Gene tree of another Aqp4-like subfamily (PripL) shows an unexpected basal split between acariform mites (Acariformes) and other arachnids. A closer inspection demonstrated that the PripL evolved quickly and has been under differential selection pressure in acariform mites. Evidence is provided that the evolutionarily ancient Glp subfamily (i.e. aquaglyceroporin) is significantly expanded in terrestrial arachnids compared with their marine relatives. Finally, in spite of the phylogenetic diversity, there exists conservation of some exons in size, functional domain, and intron-insertion phase: an 81-bp and a 218-bp exon, respectively, in apq4-like and glp genes across Eumetazoa lineages including arachnids and human beings. Both exons encode the carboxyl-terminal NPA motif, implying the coding and splicing pressure during hundreds of million years of animal evolution. Hypotheses were tested to explore the possible link between these findings and arachnid terrestrialisation.

Effects of histone methylation modification on low temperature seed germination and growth of maize.

Low temperature is a limiting factor of seed germination and plant growth. Although there is a lot information on the response of maize to low temperatures, there is still poorly description of how histone methylation affects maize germination and growth development at low temperatures. In this study, the germination rate and physiological indexes of wild-type maize inbred lines B73 (WT), SDG102 silencing lines (AS), SDG102 overexpressed lines (OE) at germination stage and seedling stage were measured under low temperature stress (4 â„ƒ), and transcriptome sequencing was applied to analyze the differences of gene expression in panicle leaves among different materials. The results showed that the germination rate of WT and OE maize seeds at 4 â„ƒ was significantly lower than 25 â„ƒ. The content of MDA, SOD and POD of 4 â„ƒ seeding leaves higher than contrast. Transcriptome sequencing results showed that there were 409 different expression genes (DEGs) between WT and AS, and the DEGs were mainly up-regulated expression in starch and sucrose metabolism and phenylpropanoid biosynthesis. There were 887 DEGs between WT and OE, which were mainly up-regulated in the pathways of plant hormone signal transduction, porphyrin and chlorophyll metabolism. This result could provide a theoretical basis for analyzing the growth and development of maize from the perspective of histone methylation modification.

Flavonoids and other phenolics from Camellia nitidissima chi flowers.

From Camellia nitidissima Chi flowers four undescribed flavonoids, nitidissimol A, nitidissimol B, sexangularetin 3-O-(6''-trans-p-coumarolyglucopyranoside) and sexangularetin 3-O-(2''-trans-p-coumarolyglucopyranoside) (1-4), and two previously unreported phenolics, nitidissimol C, D (9, 10), were isolated first time along with ten known compounds, kaempferol 3-O-(6''-O-trans-p-coumaroyl)-ß-D- glucopyranoside (5), kaempferol-3-O-rhamnoside (6), Quercetin-3'-O-ß-D-glucoside (7), kaempferol-7-O-ß-D-glucoside (8), erythro-guaiacylglycerol-O-4'-coniferyl ether (11), threo-guaiacylglycerol-O-4'-coniferyl ether (12), protocatechuic acid (13), 1,2-Diethoxybenzene (14), ethyl shikimate (15), 2,4,6-trihydroxybenzoic acid 4-O-allopyranoside (16). The structures of the isolated compounds were elucidated by spectroscopic analysis of 1 D- and 2 D-NMR and MS data. Moreover, all isolated compounds (1-16) were tested for the antibacterial activity against Xanthomonas oryzae pv. oryzae and their quorum sensing inhibitory activity in Pseudomonas aeruginosa PAO1. However, no one showed signifcant inhibition of X. oryzae pv. oryzae (MIC90 > 0.1 mg/mL), nor did they significantly inhibit the pyocyanin synthesis, which is controlled by quorum sensing in PAO1.

A highly selective and rapid solvatochromic material for amide solvent detection.

The detection of amide solvents is essential to human health due to their carcinogenicity. A simple, solvatochromic luminescent material is presented for quick and accurate detection of amides. The luminescence color variation from blue to yellow is ascribed to the amide-induced phase transformation from Cs2ZnBr4:Cu to CsBr:Cu+/Cu2+.

Rapid and precise measurement of carbonate clumped isotopes using laser spectroscopy.

Carbonate clumped isotope abundance is an important paleothermometer, but measurement is difficult, slow, and subject to cardinal mass (m/z) interferences using isotope ratio mass spectrometry (IRMS). Here, we describe an optical spectroscopic measurement of carbonate clumped isotopes. We have adapted a tunable infrared laser differential absorption spectrometer (TILDAS) system to measure the abundances of four CO2 isotopologues used for clumped isotope thermometry. TILDAS achieves the same precision (0.01‰ SE) as IRMS measurements rapidly (∼50 min per carbonate analysis) and using small samples (<2 mg of calcite), without making assumptions about 17O abundance in the sample. A temperature calibration based on 406 analyses of CO2 produced by digestion of 51 synthetic carbonates equilibrated at 6° to 1100°C is consistent with results for natural carbonates and previous calibrations. Our system results were indistinguishable from IRMS systems after replicating the InterCarb interlaboratory calibration. Measurement by TILDAS could change the landscape for clumped isotope analysis.

Prioritization of cancer driver gene with prize-collecting steiner tree by introducing an edge weighted strategy in the personalized gene interaction network.

BACKGROUND: Cancer is a heterogeneous disease in which tumor genes cooperate as well as adapt and evolve to the changing conditions for individual patients. It is a meaningful task to discover the personalized cancer driver genes that can provide diagnosis and target drug for individual patients. However, most of existing methods mainly ranks potential personalized cancer driver genes by considering the patient-specific nodes information on the gene/protein interaction network. These methods ignore the personalized edge weight information in gene interaction network, leading to false positive results. RESULTS: In this work, we presented a novel algorithm (called PDGPCS) to predict the Personalized cancer Driver Genes based on the Prize-Collecting Steiner tree model by considering the personalized edge weight information. PDGPCS first constructs the personalized weighted gene interaction network by integrating the personalized gene expression data and prior known gene/protein interaction network knowledge. Then the gene mutation data and pathway data are integrated to quantify the impact of each mutant gene on every dysregulated pathway with the prize-collecting Steiner tree model. Finally, according to the mutant gene's aggregated impact score on all dysregulated pathways, the mutant genes are ranked for prioritizing the personalized cancer driver genes. Experimental results on four TCGA cancer datasets show that PDGPCS has better performance than other personalized driver gene prediction methods. In addition, we verified that the personalized edge weight of gene interaction network can improve the prediction performance. CONCLUSIONS: PDGPCS can more accurately identify the personalized driver genes and takes a step further toward personalized medicine and treatment. The source code of PDGPCS can be freely downloaded from https://github.com/NWPU-903PR/PDGPCS .

Ursolic Acid Enhances the Sensitivity of MCF-7 and MDA-MB-231 Cells to Epirubicin by Modulating the Autophagy Pathway.

Breast cancer is the leading cause of cancer death among women in the world, and its morbidity and mortality are increasing year by year. Epirubicin (EPI) is a commonly used drug for the treatment of breast cancer but unfortunately can cause cardiac toxicity in patients because of dose accumulation. Therefore, there is an urgent need for new therapies to enhance the sensitivity of breast cancer cells to EPI. In this study, we found ursolic acid (UA) can significantly improve the drug sensitivity of human breast cancer MCF-7/MDA-MB-231 cells to EPI. Next, we observed that the co-treatment of UA and EPI can up-regulate the expression of autophagy-related proteins Beclin-1, LC3-II/LC3-I, Atg5, and Atg7, and decrease the expression levels of PI3K and AKT, which indicates that the potential mechanism should be carried out by the regulating class III PI3K(VPS34)/Beclin-1 pathway and PI3K/AKT/mTOR pathway. Furthermore, we found the autophagy inhibitor 3-methyladenine (3-MA) could significantly reverse the inhibitory effect of co-treatment of UA and EPI on MCF-7 and MDA-MB-231 cells. These findings indicate that UA can dramatically enhance the sensitivity of MCF-7 and MDA-MB-231 cells to EPI by modulating the autophagy pathway. Our study may provide a new therapeutic strategy for combination therapy.

Selenium Attenuates S. aureus-Induced Inflammation by Regulation TLR2 Signaling Pathway and NLRP3 Inflammasome in RAW 264.7 Macrophages.

This study aimed to investigate the effects of selenium (Se) on the expression of Toll-like receptor (TLR) 2 and pyrin domain-containing protein (NLRP)3 inflammasome in macrophages infected by Staphylococcus aureus (S. aureus). RAW 264.7 macrophages were treated with 2 µmol/L Na2SeO3 for 12 h before infection with S. aureus for 2 h. Through Western blot, qRT-PCR, and ELISA analysis, the core molecules of TLR2 signaling pathway and NLRP3 inflammasome in RAW 264.7 macrophages were detected. Results showed that Se significantly reduced the elevated mRNA expression of TLR2, myeloid differentiation factor-88 (Myd88), NLRP3, Caspase-recruitment domain (ASC), and Caspase-1 induced by S. aureus. Furthermore, compared with I group, the protein expression of TLR2, Myd88, NLRP3, ASC, and Caspase-1 were suppressed in T group. In addition, the mRNA and protein expression of interleukin-1 beta (IL-1ß) induced by S. aureus were also decreased after Se treatment. In conclusion, Se inhibits S. aureus-induced inflammation by suppressing the activation of the TLR2 signaling pathway and NLRP3 inflammasome in RAW 264.7 macrophages.

Sesquiterpenoids and furan derivatives from the Orychophragmus violaceus (L.) O.E. Schulz endophytic fungus Irpex lacteus OV38.

Nine undescribed compounds, including six tremulane-type sesquiterpenoids, irpexolaceus A-F, one phenolic bisabolane-type sesquiterpenoid, irpexolaceus G, and two furan derivatives, irpexonjust A-B, as well as eight known analogs, were isolated from an endophytic fungus (Irpex lacteus OV38) of Orychophragmus violaceus (L.) O.E. Schulz, a Chinese medicinal and edible plant. The structures of these natural compounds were elucidated based on NMR, HRESIMS, single-crystal X-ray diffraction, and ECD spectroscopic data. Among the tested isolates (50 µg/mL), the inhibitory effects of irpexolaceus A, C, D, F, and G, irpexonjust B, and irpexlacte B against NO release from LPS-induced RAW 264.7 cells were higher than 45%, while irpexlacte C (42.6%), irpexolaceus B (39.6%), irpexonjust A (43.7%), and irpexolaceus E (33.6%) exhibited weaker inhibitory effects on the release of NO.

Selenium Ameliorates S. aureus-Induced Inflammation in Bovine Mammary Epithelial Cells by Regulating ROS-Induced NLRP3 Inflammasome.

In this study, the regulation effects of selenium (Se) on the expression of pyrin domain-containing protein (NLRP) 3 inflammasome and reactive oxygen species (ROS) in bovine mammary epithelial cells (bMECs) infected by Staphylococcus aureus (S. aureus) were detected. bMECs were treated with 8 µmol/L Na2SeO3 for 12 h before infection with S. aureus for 2 h. Through flow cytometry, Western blot, and qRT-PCR analysis, the expression of ROS and NLRP3 imflammasome was detected. Results showed Se significantly reduced the ROS level in bMECs; at the same time, the expressions of NLRP3, ASC, caspase-1, Pro-IL-1ß, and IL-1ß were also decreased. In conclusion, Se inhibits S. aureus-induced inflammation by suppressing the activation of NLRP3 inflammasome and ROS in bMECs.

Black Lycium barbarum polysaccharide attenuates LPS-induced intestine damage via regulation gut microbiota.

Lycium barbarums are traditionally used as a homology of medicinal plants in China with a potent role in metabolism and immunomodulation. The current study was performed to explore the attenuation effect and microbiota regulation of Lycium barbarum polysaccharide (BLBP) on lipopolysaccharide (LPS)-induced intestine damage in mice. A total of 70 mice were randomly divided into five groups; negative control (GA), LPS (GB), both treated with an equal volume of normal saline, and BLBP treatment groups GC (100 mg/kg), GD (200 mg/kg), and GE (400 mg/kg) via gavage for 19 days. On Day 19, mice in groups GB, GC, GD, and GE were treated with 10 mg/kg LPS for 24 h and euthanized to collect intestine samples for pathological examination and microbiota sequencing. The results showed a non-significant difference in body weight gain among the five mouse groups; however, mice in the GC and GE groups showed decreased weight gain. An H&E examination revealed that the integrity of intestinal villi was destroyed by LPS, while BLBP supplement alleviated intestinal damage with an increase in villus height and a decrease in crypt depth. A total of over 59,000, 40,000, 50,000, 45,000, and 55,000 raw sequences were found in groups GA, GB, GC, GD, and GE, respectively. LPS challenge decreased alpha diversity indexes significantly (p < 0.05), while a non-significant difference was found between different BLBP treatment groups and the GA group. A total of 8 phyla and 13 genera were found among five mouse groups, and BLBP partly restored the bacterial abundance in mice. LPS changed 282 metabolic pathways in KEGG L2, 77 metabolic pathways in KEGG L3, and 205 metabolic pathways in MetaCyc, respectively. The BLBP-supplemented groups, especially GE, showed reverse effects on those metabolic pathways. The current study revealed that BLBP can effectively decrease intestinal damage through the regulation of intestinal microbiota, which may provide new insights for the prevention of intestinal disease using food and medicine homologous of Lycium ruthenicum.

Effect of sexuality education for parents of preschoolers / 中国学校卫生

Objective@#To evaluate the effect of sexuality education for parents of preschoolers, to provide a basis for sexuality educational programs in rural settings.@*Methods@#This community intervention trial chose four kindergartens in Jiangyang District of Luzhou City as the intervention group and other four kindergartens in Jiangyou City of Mianyang City of Sichuan Province as the control group. Sexuality education for parents was carried out in the intervention group, and the control group received routine arrangement. The baseline survey was conducted from March to May 2019, and the final survey was conducted in December 2019. The investigators conducted a face to face or self filled questionnaires among each parent who agreed to participate in the survey by using the self designed questionnaire "Research on Current Situation and Countermeasures of Early Childhood Sexuality Education in Rural Areas (Parent)".@*Results@#Before the intervention, there was no significant difference in the basic information of parents and their children, and the situation of early childhood sexuality education between the intervention group and the control group( P > 0.05 ). After intervention, parents of the intervention group and the control group showed differences in correct rate of the knowledge regarding early childhood sexuality education (41.5%，32.1%), proportion of recognition of parental responsibility (90.7%， 81.3 %), sexuality education in the family (55.7%，45.9%), sexuality education in schools (70.2%，39.1%) and attitude behavior consistency (28.9%，16.3%) ( χ 2=4.05，8.05，4.17，42.48，9.59， P <0.05).@*Conclusion@#Sexuality education towards parents is effective through improving knowledge, responsibility, the implementation of sexuality education in the family, and attitude behavior consistency among parents.

Inhibition of Quorum Sensing and Biofilm Formation of Esculetin on Aeromonas Hydrophila.

Quorum sensing (QS) and biofilm formation inhibition activity of esculetin on Aeromonas hydrophila SHAe 115 were evaluated. Exposure to esculetin at 25, 50, and 100µg/ml significantly inhibited the production of protease and hemolysin, the formation of biofilms and attenuated the swarming motility of A. hydrophila SHAe 115. Biofilm forming inhibition was also observed through confocal laser scanning microscopy and scanning electron microscope. Quantitative real-time PCR analysis indicated that genes positively related to QS and biofilm formation were downregulated to varying degrees, while gene (litR) negatively related to biofilm formation was significantly upregulated. The phenotypic results were in good agreement with gene expression levels. These results indicated that esculetin would be a potential QS inhibitor for A. hydrophila.

Contents and yields of copper, iron, manganese and zinc would be affected by lucerne age and cut.

BACKGROUND: Lucerne is a perennial legume forage, which can produce multiple cuts in 1 year. Microelements play fundamental roles in the function, maintenance and adaptation to the environment for lucerne growth. However, the role of the accumulation of copper (Cu), iron (Fe), manganese (Mn) and Zinc (Zn), which vary with lucerne ages or cuts, has not been previously determined. Therefore, a hypothesis on the Cu, Fe, Mn and Zn in lucerne varying with age and cut was tested. METHODS: A total of 11, 8, 5, 4 and 1 year old lucerne (Medicago sativa Longdong) were selected as the material (until 2012 year), and samples were taken as three cuts at the cutting periods (early flowering stage) in 2012. Then, the contents and yields of Cu, Fe, Mn and Zn in lucerne were measured and calculated. RESULTS: The highest contents of Cu, Fe, Mn and Zn in lucerne were found in the 1 year old among the five ages, at the 3rd cut compared to the other two cuts, and in the leaf among the three organs. The highest yields of Cu, Fe, Mn and Zn were found in the older ages (11 and 8 years old), at the 3rd cut, and in the root among the three organs. The most positive correlations were found between contents, yields and biomass. CONCLUSIONS: The hypothesis was supported by the results. And the contents and yields of lucerne Cu, Fe, Mn and Zn were affected by the age, cut and organ. Furthermore, the yields of lucerne Cu, Fe, Mn and Zn were determined by their contents and lucerne biomass.