Antifungal Mechanism of Ruta graveolens Essential Oil: A Colombian Traditional Alternative against Anthracnose Caused by Colletotrichum gloeosporioides.

Here, we report for the first time on the mechanisms of action of the essential oil of Ruta graveolens (REO) against the plant pathogen Colletotrichum gloeosporioides. In particular, the presence of REO drastically affected the morphology of hyphae by inducing changes in the cytoplasmic membrane, such as depolarization and changes in the fatty acid profile where straight-chain fatty acids (SCFAs) increased by up to 92.1%. In addition, REO induced changes in fungal metabolism and triggered apoptosis-like responses to cell death, such as DNA fragmentation and the accumulation of reactive oxygen species (ROS). The production of essential enzymes involved in fungal metabolism, such as acid phosphatase, ß-galactosidase, ß-glucosidase, and N-acetyl-ß-glucosaminidase, was significantly reduced in the presence of REO. In addition, C. gloeosporioides activated naphthol-As-BI phosphohydrolase as a mechanism of response to REO stress. The data obtained here have shown that the essential oil of Ruta graveolens has a strong antifungal effect on C. gloeosporioides. Therefore, it has the potential to be used as a surface disinfectant and as a viable replacement for fungicides commonly used to treat anthracnose in the postharvest testing phase.

Antimicrobial activity of phenyllactic acid against Klebsiella pneumoniae and its effect on cell wall membrane and genomic DNA.

As Klebsiella pneumoniae (KP) has acquired high levels of resistance to multiple antibiotics, it is considered a worldwide pathogen of concern, and substitutes for traditional antibiotics are urgently needed. 3-Phenyllactic acid (PLA) has been reported to have antimicrobial activity against food-borne bacteria. However, there was no experiment evidence for the exact antibacterial effect and mechanism of PLA kills pathogenic KP. In this study, the Oxford cup method indicated that PLA is effective to KP with a minimum inhibitory concentration of 2.5 mg/mL. Furthermore, PLA inhibited the growth and biofilm formation of in a time- and concentration-dependent manner. In vivo, PLA could significantly increase the survival rate of infected mice and reduce the pathological tissue damage. The antibacterial mode of PLA against KP was further explored. Firstly, scanning electron microscopy illustrated the disruption of cellular ultrastructure caused by PLA. Secondly, measurement of leaked alkaline phosphatase demonstrated that PLA disrupted the cell wall integrity of KP and flow cytometry analysis with propidium iodide staining suggested that PLA damaged the cell membrane integrity. Finally, the results of fluorescence spectroscopy and agarose gel electrophoresis demonstrated that PLA bound to genomic DNA and initiated its degradation. The anti-KP mode of action of PLA was attributed to the destruction of the cell wall, membrane, and genomic DNA binding. These findings suggest that PLA has great potential applications as antibiotic substitutes in feed additives against KP infection in animals.

Effect of Detergents Based on Sodium Dodecyl Sulfate on Functional Metrics of Frozen-Thawed Collared Peccary (Pecari tajacu) Semen.

We evaluated the effects of detergents based on sodium dodecyl sulfoxide (SDS) on the functional parameters of collared peccary frozen-thawed sperm. Semen aliquots from ten individuals were diluted in a Tris-egg yolk-glycerol extender alone or with 0.5% Equex STM® paste or SDS (at 0.1%, 0.3% or 0.5% (v/v) concentration). Samples were fast frozen in liquid nitrogen with a post-thaw evaluation of motility, membrane functionality and integrity, mitochondrial activity, sperm binding ability and thermal resistance. The treatments without SDS (41.8 ± 3.5%) and those containing Equex (41.8 ± 4.4%) or 0.1% SDS (41.2 ± 5.5%) provided greater sperm motility (p < 0.05) than those containing SDS 0.3% (30.5 ± 4.7%) and 0.5% (31.2 ± 6.3%). Immediately after thawing, only treatments containing 0.1% SDS effectively preserved sperm straightness (STR) when compared to the negative control. All treatments preserved the amplitude of lateral head (ALH) and straightness (STR) during a thermal resistance test (p > 0.05), but SDS 0.5% impaired the membrane functionality and mitochondrial activity after thawing (p < 0.05). All treatments provided a similar recovery of sperm binding ability after thawing (p < 0.05). Our results showed that the addition of 0.1% SDS to the Tris-yolk-glycerol extender optimized the freeze-thaw recovery of peccary semen.

Seed Priming with Glass Waste Microparticles and Red Light Irradiation Mitigates Thermal and Water Stresses in Seedlings of Moringa oleifera.

The association between population increase and the exploitation of natural resources and climate change influences the demand for food, especially in semi-arid regions, highlighting the need for technologies that could provide cultivated species with better adaptation to agroecosystems. Additionally, developing cultivation technologies that employ waste materials is highly desirable for sustainable development. From this perspective, this study aimed to evaluate whether seed priming with glass waste microparticles used as a silicon source under red light irradiation mitigates the effects of thermal and water stress on seedlings of Moringa oleifera. The experimental design was set up in randomized blocks using a 2 × 2 × 2 factorial arrangement consisting of seed priming (NSP-no seed priming, and SPSi-seed priming with glass microparticles under red light irradiation), soil water replenishment (W50-50%, and W100-100% of crop evapotranspiration-ETc), and temperature change (TC30°-30 °C day/25 °C night and TC40°-40 °C day/35 °C night). Seed priming with glass microparticles under red light irradiation mitigated the effects of thermal and water stress on seedlings of Moringa oleifera seedlings through the homeostasis of gas exchange, leaf water status, osmotic adjustment, and the antioxidant mechanism.

Paraoxonase 1 activity in the sperm-rich portion of boar ejaculates is positively associated with sperm quality.

Associations of the activity of the paraoxonase 1 (PON1) enzyme with boar sperm quality still needs to be characterized, since boar ejaculates present distinct portions with differences in sperm concentration and quality. This study evaluated PON1 activity in the serum, in the distinct portions of boar ejaculates and estimated correlations with sperm quality parameters. Ejaculates and blood samples were collected from six boars for three weeks (two per week per boar; n = 36). Serum and post-spermatic portion PON1 activities were positively correlated (P = 0.01) but were both uncorrelated with the PON1 activity in the sperm-rich portion and in the whole ejaculate (P > 0.05). Differences in PON1 activity among boars were only observed in the sperm-rich portion of the ejaculate (P < 0.05). The PON1 activity in the serum and in the post-spermatic portion was generally negatively correlated with parameters of spermatozoa kinetics (P < 0.05). In the sperm-rich portion, PON1 activity was positively correlated with sperm concentration (P < 0.0001), curvilinear distance and velocity (both P < 0.05) and DNA integrity (P < 0.05), but negatively correlated with straightness and linearity (P < 0.05). Thus, boar ejaculates with increased PON1 activity in the sperm-rich portion may present increased concentration and spermatozoa with acceptable curvilinear velocity and distance and DNA integrity, which suggests that PON1 activity may be a biomarker for potential fertility.

Adverse Effects of Single Neutrophil Extracellular Trap-Derived Components on Bovine Sperm Function.

Neutrophil extracellular traps (NETs) play a key role in fertilisation by eliminating microorganisms and entrapping spermatozoa in the female reproductive tract (FRT). The deleterious effects of NETs on spermatozoa have been previously described; however, individual exposure to NET-derived components in bull spermatozoa has not been explored. The aim of this study was to evaluate the effects of the main NET-derived proteins, histone 2A (H2A), neutrophil elastase (ELA), myeloperoxidase (MPO), pentraxin 3 (PTX), cathepsin G (Cat-G), and cathelicidin LL37 (LL-37), at concentrations of 1, 10, and 30 µg/mL, on sperm parameters. Sperm were selected and incubated with different NET-derived proteins for 4 h. Membrane and acrosome integrity, lipoperoxidation, and membrane phospholipid disorders were also evaluated. Bovine polymorphonuclear neutrophil (PMN)/sperm co-cultures were evaluated by scanning electron microscopy and immunofluorescence. All NET-derived proteins/enzymes resulted in a reduction in membrane integrity, acrosome integrity, and lipoperoxidation at a concentration of 30 µg/mL. Bovine PMN/sperm co-cultures showed marked NET formation in the second hour. In conclusion, all NET-derived proteins/enzymes exerted cytotoxic effects on bull sperm, and this effect should be considered in future investigations on the uterine microenvironment and the advancement of spermatozoa in the FRT.

The in vitro effect of delta-9-tetrahydrocannabinol and cannabidiol on whole blood viscosity, elasticity and membrane integrity.

BACKGROUND: The main biological activities of cannabis are due to the presence of several compounds known as cannabinoids. Delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are two of the main cannabinoids. Studies have shown that the effects of THC can be modulated by CBD. OBJECTIVE: This study aims to look at the effect of different concentrations of THC and CBD separately and in combination, on blood viscosity, elasticity and membrane integrity. METHODS: Blood samples were collected from twenty-four healthy adult non-smokers. Blood viscosity and elasticity were determined using the Vilastic Scientific Bioprofiler for different concentrations (0, 2.5, 25, 50 and 100 ng/ml) of CBD and THC respectively, as well as in extracts with combinations of CBD and THC in 4:1 and 1:1 ratios respectively. Repeated measures analysis of variance (ANOVA) was used to determine the difference between the means of the groups. RESULTS: Blood viscosity increased significantly with increasing concentrations of both THC and CBD from 25 ng/ml up to 100 ng/ml ranging from 6.45 ± 0.36 mPa·s to 11.60 ± 1.12 mPa·s for THC and ranging from 5.46 ± 0.24 mPa·s to 9.91 ± 1.10 mPa·s for CBD respectively, being more pronounced in the extracts at 21.33 ± 2.17 mPa·s for the 4THC:1CBD extract and 21.76 ± 1.88 mPa·s for the 1THC:1CBD extract. There was no significant increase in elasticity for THC and CBD separately. However, a significant increase in elasticity was observed in the extracts. THC and CBD affected red cell morphology resulting in complete disintegration at the highest concentrations. CONCLUSIONS: THC and CBD increased red blood cell viscosity and elasticity separately and in combination. They also adversely affected membrane integrity.

Paraoxonase 1 activity in the sperm-rich portion of boar ejaculates is positively associated with sperm quality

Associations of the activity of the paraoxonase 1 (PON1) enzyme with boar sperm quality still needs to be characterized, since boar ejaculates present distinct portions with differences in sperm concentration and quality. This study evaluated PON1 activity in the serum, in the distinct portions of boar ejaculates and estimated correlations with sperm quality parameters. Ejaculates and blood samples were collected from six boars for three weeks (two per week per boar; n = 36). Serum and post-spermatic portion PON1 activities were positively correlated (P = 0.01) but were both uncorrelated with the PON1 activity in the sperm-rich portion and in the whole ejaculate (P > 0.05). Differences in PON1 activity among boars were only observed in the sperm-rich portion of the ejaculate (P < 0.05). The PON1 activity in the serum and in the post-spermatic portion was generally negatively correlated with parameters of spermatozoa kinetics (P < 0.05). In the sperm-rich portion, PON1 activity was positively correlated with sperm concentration (P < 0.0001), curvilinear distance and velocity (both P < 0.05) and DNA integrity (P < 0.05), but negatively correlated with straightness and linearity (P < 0.05). Thus, boar ejaculates with increased PON1 activity in the sperm-rich portion may present increased concentration and spermatozoa with acceptable curvilinear velocity and distance and DNA integrity, which suggests that PON1 activity may be a biomarker for potential fertility.(AU)

Citral and geraniol induce necrotic and apoptotic cell death on Saccharomyces cerevisiae.

Essential oils and their main components, monoterpenes, have been proven to be important alternatives for the control of pathogenic and spoiling microorganisms, but the mode of action of these compounds is poorly understood. This work aimed to determine the mode of action of citral and geraniol on the model yeast Saccharomyces cerevisiae using a flow cytometry approach. Exponentially growing yeast cells were treated with different concentrations of citral and geraniol for 3 h, and evaluated for cell wall susceptibility to glucanase, membrane integrity, reactive oxygen species (ROS) accumulation, mitochondrial membrane potential, and metacaspase activity. Results provide strong evidence that citral and geraniol acute fungicidal activity against Saccharomyces cells involves the loss of membrane and cell wall integrity resulting in a dose-dependent apoptotic/necrotic cell death. However, yeast cells that escape this first cell membrane disruption, particularly evident on sub-lethal concentration, die by metacaspase-mediated apoptosis induced by the accumulation of intracellular ROS. The deleted mutant on the yca1 gene showed high tolerance to citral and geraniol.

Overtime expression of plasma membrane and mitochondrial function markers associated with cell death in human spermatozoa exposed to nonphysiological levels of reactive oxygen species.

In many cell types, the potential of reactive oxygen species to induce death processes has been largely demonstrated. Studies in spermatozoa have associated the imbalance of reactive oxygen species and phosphatidylserine externalisation as an apoptosis marker. However, the lack of consensus about time effect in the joint expression of these and other death markers has made it difficult to understand the set of mechanisms influenced beyond the concentration effect of reactive oxygen species to stimulate cell death. Here, the plasma membrane permeability and integrity, phosphatidylserine externalisation and mitochondrial membrane potential were jointly evaluated as death markers in human spermatozoa stimulated with H2 O2 . The results showed a profound and sustained effect of dissipation in the mitochondrial membrane potential and an increased phosphatidylserine externalisation in human spermatozoa exposed to 3 mmol-1 of H2 O2 at 30 min. This was followed by an increased membrane permeability after 45 min. The last observed event was the loss of cell membrane integrity at 60 min. In conclusion, mitochondria are rapidly affected in human spermatozoa exposed to reactive oxygen species, with the barely detectable mitochondrial membrane potential coexisting with the high phosphatidylserine externalisation in cells with normal membrane permeability.

Synthetic Peptide ΔM4-Induced Cell Death Associated with Cytoplasmic Membrane Disruption, Mitochondrial Dysfunction and Cell Cycle Arrest in Human Melanoma Cells.

Melanoma is the most dangerous and lethal form of skin cancer, due to its ability to spread to different organs if it is not treated at an early stage. Conventional chemotherapeutics are failing as a result of drug resistance and weak tumor selectivity. Therefore, efforts to evaluate novel molecules for the treatment of skin cancer are necessary. Antimicrobial peptides have become attractive anticancer agents because they execute their biological activity with features such as a high potency of action, a wide range of targets, and high target specificity and selectivity. In the present study, the antiproliferative activity of the synthetic peptide ΔM4 on A375 human melanoma cells and spontaneously immortalized HaCaT human keratinocytes was investigated. The cytotoxic effect of ΔM4 treatment was evaluated through propidium iodide uptake by flow cytometry. The results indicated selective toxicity in A375 cells and, in order to further investigate the mode of action, assays were carried out to evaluate morphological changes, mitochondrial function, and cell cycle progression. The findings indicated that ΔM4 exerts its antitumoral effects by multitarget action, causing cell membrane disruption, a change in the mitochondrial transmembrane potential, an increase of reactive oxygen species, and cell cycle accumulation in S-phase. Further exploration of the peptide may be helpful in the design of novel anticancer peptides.

Exploratory Study of Fatty Acid Profile in Two Filmy Ferns with Contrasting Desiccation Tolerance Reveal the Production of Very Long Chain Polyunsaturated Omega-3 Fatty Acids.

Lipids are fundamental components of cell membranes and play a significant role in their integrity and fluidity. Alteration in lipid composition of membranes has been reported to be a major response to abiotic environmental stresses. This work was focused on the characterization of frond lipid composition and membrane integrity during a desiccation-rehydration cycle of two filmy fern species with contrasting desiccation tolerance: Hymenophyllum caudiculatum (less tolerant) and Hymenophyllum plicatum (more tolerant). The relative water content decreased without differences between species when both filmy ferns were subjected to desiccation. However, H. plicatum reached a higher relative water content than H. caudiculatum after rehydration. Fatty acids profiles showed the presence of a very long chain polyunsaturated fatty acid during the desiccation-rehydration cycle, with eicosatrienoic acid being the most abundant. Additionally, propidium iodide permeation staining and confocal microscopy demonstrated that, following the desiccation-rehydration cycle, H. plicatum exhibited a greater membrane integrity than H. caudiculatum. The lack of some very long chain fatty acids such as C22:1n9 and C24:1n9 in this species contrasting with H. plicatum may be associated with its lower membrane stability during the desiccation-rehydration cycle. This report provides the first insight into the fatty acid composition and dynamics of the membrane integrity of filmy ferns during a desiccation-rehydration cycle. This could potentially play a role in determining the different levels of desiccation tolerance and microhabitat preferences exhibited by Hymenophyllaceae species.

Assessment of the antibacterial, antivirulence, and action mechanism of Copaifera pubiflora oleoresin and isolated compounds against oral bacteria.

The microorganisms that constitute the oral microbiome can cause oral diseases, including dental caries and endodontic infections. The use of natural products could help to overcome bacterial resistance to the antimicrobials that are currently employed in clinical therapy. This study assessed the antimicrobial activity of the Copaifera pubiflora oleoresin and of the compounds isolated from this resin against oral bacteria. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays provided values ranging from 6.25 to > 400 µg/mL for the C. pubiflora oleoresin and its isolated compounds. The fractional inhibitory concentration index (FICI) assay showed that the oleoresin and chlorhexidine did not act synergistically. All the tested bacterial strains formed biofilms. MICB50 determination revealed inhibitory action: values varied from 3.12-25 µg/mL for the oleoresin, and from 0.78 to 25 µg/mL for the ent-hardwickiic acid. Concerning biofilm eradication, the C. pubiflora oleoresin and hardwickiic acid eradicated 99.9 % of some bacterial biofilms. Acid resistance determination showed that S. mutans was resistant to acid in the presence of the oleoresin and ent-hardwickiic acid at pH 4.0, 4.5, and 5.0 at all the tested concentrations. Analysis of DNA/RNA and protein release by the cell membrane demonstrated that the oleoresin and hardwiickic acid damaged the bacterial membrane irreversibly, which affected membrane integrity. Therefore, the C. pubiflora oleoresin and ent-hardwickiic acid have potential antibacterial effect and can be used as new therapeutic alternatives to treat oral diseases such as dental caries and endodontic infections.

Can short-term exposure to copper and atrazine be cytotoxic to microalgae?

Aquatic environments can be easily contaminated due to anthropogenic activities that may affect local biota. Microalgae are abundant and have an important role on the food chain. Consequently, they stand out as promising models for studies of contaminants. This study investigated the cytotoxic effects of atrazine and copper (separate and mixture) exposure in microalgae Desmodesmus communis, as well as its cellular defense due to ABC (ATP-binding cassette) proteins activity against the xenobiotics. We analyzed two different ABC proteins activity pathways: P-gp, which is responsible for nonspecific substance efflux, and MRP that is associated with metals efflux. It was observed that the microalgae exposure to atrazine (90 nM) and copper (141 nM) has been considered cytotoxic. When contaminants were mixed, only the combination of both highest concentrations tested was cytotoxic. The P-gp blocker, verapamil, demonstrated that the contaminants tested caused proteins inhibition. However, the MK-571 (MRP blocker) did not block pump activity. There was an inverse relationship between ABC protein activity and cytotoxicity; non-cytotoxic conditions suggest increased activity of microalgae defense proteins.

A closer look into the physical interactions between lipid membranes and layered double hydroxide nanoparticles.

Layered double hydroxide nanoparticles (LDH-NPs) constitute promising nanocarriers for drug and gene delivery. Although their cell internalization has been studied, the interaction between LDH-NPs and biological membrane models, such as giant unilamellar vesicles (GUVs), remains unexplored. These vesicles are widely-used membrane models that allow minimizing the complexity and uncertainty associated with biological systems to study the physical interactions in the absence of cell metabolism effects. With such an approach the physicochemical properties of the membrane can be differentiated from the biological functionalities involved in cell internalization and the membrane-mediated internalization can be directly understood. In this work, we describe for the first time the interaction of LDH-NPs with freestanding negatively charged POPC:POPS GUVs by fluorescence microscopy. The experiments were performed with fluorescein labeled LDH-NPs of about 100 nm together with different fluorophores in order to evaluate the NPs interactions with the vesicles as well as their impact on the membrane morphology and permeability. Positively charged LDH-NPs are electrostatically accumulated at the GUVs membrane, altering its lateral phospholipid distribution and increasing the stiffness and permeability of the membrane. The adsorption of albumin (LDH@ALB) or polyacrylic acid (LDH@PA) passivates the surface of LDH-NPs eliminating long-range electrostatic attraction. The absence of membrane-mediated internalization of either LDH@ALB or LDH@PA, represents an advantage in the use of LDH-NPs as drug or nucleic acids nanocarriers, because suitable functionalization will allow an optimal cell targeting.

Sperm characterization of the Amazonian freshwater cururu stingray Potamotrygon wallacei (Potamotryogonidae): basic knowledge for reproduction and conservation plans.

This study aimed to describe the morphology and sperm quality of free-living adult males of cururu stingray Potamotrygon wallacei, endemic from the Rio Negro basin, Brazilian Amazon. The sperm was collected in loco from the seminal vesicle region and fixed in buffered saline formaldehyde solution for further evaluation of morphometry, sperm plasma membrane integrity and sperm concentration. The spermatozoa presented a total length of 138.25 ± 1.82 µm with a helical shape and a long head. A high percentage of cells with intact membrane (98 ± 2%) and normal spermatozoa (92 ± 1%) were observed. The cell concentration was 0.34 ± 0.05 × 1010 spermatozoa/ml of semen. These observations are unprecedented for potamotrygonid species and will serve as a basis for future management and conservation strategies.

Association of L-arginine with heparin on the sperm capacitation improves in vitro embryo production in bovine.

We aimed to evaluate the effects of L-arginine (L-arg) in the quality of in vitro heparin-induced capacitation of cryopreserved bovine spermatozoa and its effects on IVP. The experimental groups were: Control 0 hour without pre-capacitation, and groups of sperm capacitated for 30 min in the absence of COC with heparin (Control 30 min), with 1 mM L-arg and with 1 mM L-arg + heparin. The capacitation pattern was evaluated by chlortetracycline assay and the integrity of the plasma membrane (PM) and acrosome membrane (AM) by the association of Hoescht 33342 and propidium iodide. Further, we assess the sperm quality by the rate of in vitro blastocysts production. Treatment with 1 mM L-arg + heparin increased the percentage of capacitated sperm when compared to Control 0 hour and the treatment with heparin (61.1 vs. 18.2 and 47.0%, respectively, P<0.05). The addition of 1 mM L-arg to the medium has capacitated the spermatozoa (26.2 ± 3.8) but was less effective than heparin (47.0 ± 4.0) (P<0.05). There was no difference in the percentage of sperm with intact PM between treatments when compared to Control 0 hour (P>0.05). The group capacitated with 1 mM L-arg + heparin for 30 min increased the blastocyst rate compared to Control IVF (53.7 vs. 40.8%, P<0.05). We conclude that the addition of L-arg with heparin increases the number of capacitated spermatozoa in vitro with 30 min of pre-incubation in the absence of COC not altering the integrity of plasma and acrosomal membrane. This treatment in the absence of COC was the most effective method for blastocysts production, and the method of pre-incubation could be used to assess the role of other substances in the sperm capacitation and its effect on IVP.

The ideal holding time for boar semen is 24 h at 17 °C prior to short-cryopreservation protocols.

Boar semen cannot be immediately cryopreserved, it need be hold at 17 °C prior to cryopreservation, holding time has been used to improve cryopreserved boar semen, since holding time allows a prolonged interaction between spermatozoa and seminal plasma components. However, until now only few periods of holding time have been studied, and boar semen had been held at 17 °C for 24 h to facilitate its manufacture. Thus, this experiment aims to study the effect several holding time (0, 4, 8, 12, 24, 28 and 32 h) on boar spermatozoa post-thawed (PT) characteristics. Fifteen sperm-rich fractions of ejaculate were extended in Beltsville Thawing Solution and storage at 17 °C. After each holding time (0, 4, 8, 12, 24, 28 and 32 h), a sample was centrifuged, and sperm pellet was diluted in an extender composed of sugars, amino acids, buffers, 20% egg yolk (v/v), antibiotics, 2% glycerol as a cryoprotectant, and 2% methylformamide (v/v). Cryopreservation was performed with an automatic cryopreservation system. Cryopreserved boar semen was evaluated to spermatozoa kinetics, plasma and acrosomal membranes integrity, mitochondrial membrane potential, detection of superoxide anion, plasma membrane fluidity, and peroxidation. Twenty-four hours of holding increase total and progressive motility, rapid spermatozoa, and integrity of plasma and acrosome membranes. To mitochondrial membrane potential, 32 h is needed. However, holding time was not able to control the superoxide anion amount neither membrane lipid peroxidation, and had no effects on membrane fluidity. Thus, to reach the best results of PT boar semen the ideal holding time is 24 h.

Electrical conductivity as an indicator of damage due to low temperatures in beans leaves / Condutividade elétrica como indicador de danos por temperaturas baixas em folhas de feijão

The electrical conductivity test indirectly evaluates cell membrane disorganization by quantifying the electrolytes released into the water after tissue imbibing. The objective of this work was to evaluate methodological variations in the electrical conductivity test, for it to serve as an indicator of low temperature-induced damages and estimate the cold tolerance of bean plants. Cultivar IPR Uirapuru plants were subjected to minimum temperatures of 4°C, 2°C, 0°C, -1°C, -2°C, -3°C, and -4°C for 1 h in a growth chamber under controlled conditions. After the treatment period, the response of plants to cold stress was evaluated by determination of the total protein content, and catalase (CAT) and ascorbate peroxidase (APX) enzymatic activities, and evaluation of photosystem II (Fm/Fv) efficiency and leaf anatomy. These results were compared with those obtained in the electrical conductivity test, which was performed in plants under cold stress as well as under a non-stress environment, with 2, 4, 6, and 8 leaf discs immersed in 30 mL of distilled water for 24 h in BOD, at temperatures of 25°C, 30°C, and 35°C. Analysis of variance was performed using a completely randomized design, and for electrical conductivity, a number of discs × cold stress temperature combinations were used for each soak temperature. The averages were compared using the Turkey’s test at 5% and 10% probability. Pearson correlation coefficient (r) between the conductivity averages and other cold stress evaluation data was also performed. The results showed a marked reduction in the ratio (Fv/Fm) only in the treatments at -3°C and -4°C, which indicated tissue death. At temperatures below 0°C, there was a collapse of the leaf blade tissues, and it was not possible to differentiate the palisade parenchyma from the spongy parenchyma in the treatments at -2°C, -3°C, and -4°C. There was an increase in the protein content since the temperature -3°C...

O teste de condutividade elétrica avalia de forma indireta a desorganização da membrana celular, pela quantificação dos eletrólitos liberados na água após a embebição dos tecidos. Assim, o objetivo do trabalho foi avaliar as variações de metodologias no teste de condutividade elétrica como indicador de danos por temperaturas baixas para estimar a tolerância ao frio de plantas de feijão preto. Dessa forma, as plantas da cultivar IPR Uirapuru foram submetidas às temperaturas mínimas de 4°C, 2°C, 0°C, -1°C, -2°C, -3°C e -4°C, por uma hora, em câmara de crescimento sob condições controladas. Após esse período, a resposta das plantas ao estresse por frio foi avaliada pela determinação do teor de proteínas totais, atividade enzimática da catalase (CAT) e da ascorbato peroxidase (APX), além da avaliação da eficiência do fotossistema II (Fv/Fm) e da anatomia foliar. Esses resultados foram comparados com os obtidos no teste de condutividade elétrica o qual foi realizado nas plantas após o estresse frio e em plantas não estressadas (ambiente) com 2, 4, 6 e 8 discos foliares imersos em 30 ml de água destilada, mantidos por 24 horas em B.O.D nas temperaturas de 25°C, 30°C e 35°C. A análise de variância foi realizada em delineamento inteiramente casualizado, e para condutividade elétrica utilizou-se fatorial número de discos x temperatura de estresse ao frio para cada temperatura de embebição separadamente. As médias foram comparadas pelo teste Tukey a 5 e 10% de probabilidade. Realizou-se a correlação de Pearson (r) entre as médias de condutividade e os demais testes de avaliação do estresse por frio. Nos resultados observou-se acentuada redução da relação (Fv/Fm) apenas nos tratamentos -3°C e -4°C indicando morte dos tecidos. Para temperaturas inferiores a 0°C houve colapso dos tecidos do limbo foliar, não sendo possível diferenciar o parênquima paliçádico do parênquima esponjoso nos tratamentos -2, -3 e -4°C...

Electrical conductivity as an indicator of damage due to low temperatures in beans leaves / Condutividade elétrica como indicador de danos por temperaturas baixas em folhas de feijão

The electrical conductivity test indirectly evaluates cell membrane disorganization by quantifying the electrolytes released into the water after tissue imbibing. The objective of this work was to evaluate methodological variations in the electrical conductivity test, for it to serve as an indicator of low temperature-induced damages and estimate the cold tolerance of bean plants. Cultivar IPR Uirapuru plants were subjected to minimum temperatures of 4°C, 2°C, 0°C, -1°C, -2°C, -3°C, and -4°C for 1 h in a growth chamber under controlled conditions. After the treatment period, the response of plants to cold stress was evaluated by determination of the total protein content, and catalase (CAT) and ascorbate peroxidase (APX) enzymatic activities, and evaluation of photosystem II (Fm/Fv) efficiency and leaf anatomy. These results were compared with those obtained in the electrical conductivity test, which was performed in plants under cold stress as well as under a non-stress environment, with 2, 4, 6, and 8 leaf discs immersed in 30 mL of distilled water for 24 h in BOD, at temperatures of 25°C, 30°C, and 35°C. Analysis of variance was performed using a completely randomized design, and for electrical conductivity, a number of discs × cold stress temperature combinations were used for each soak temperature. The averages were compared using the Turkeys test at 5% and 10% probability. Pearson correlation coefficient (r) between the conductivity averages and other cold stress evaluation data was also performed. The results showed a marked reduction in the ratio (Fv/Fm) only in the treatments at -3°C and -4°C, which indicated tissue death. At temperatures below 0°C, there was a collapse of the leaf blade tissues, and it was not possible to differentiate the palisade parenchyma from the spongy parenchyma in the treatments at -2°C, -3°C, and -4°C. There was an increase in the protein content since the temperature -3°C...(AU)

O teste de condutividade elétrica avalia de forma indireta a desorganização da membrana celular, pela quantificação dos eletrólitos liberados na água após a embebição dos tecidos. Assim, o objetivo do trabalho foi avaliar as variações de metodologias no teste de condutividade elétrica como indicador de danos por temperaturas baixas para estimar a tolerância ao frio de plantas de feijão preto. Dessa forma, as plantas da cultivar IPR Uirapuru foram submetidas às temperaturas mínimas de 4°C, 2°C, 0°C, -1°C, -2°C, -3°C e -4°C, por uma hora, em câmara de crescimento sob condições controladas. Após esse período, a resposta das plantas ao estresse por frio foi avaliada pela determinação do teor de proteínas totais, atividade enzimática da catalase (CAT) e da ascorbato peroxidase (APX), além da avaliação da eficiência do fotossistema II (Fv/Fm) e da anatomia foliar. Esses resultados foram comparados com os obtidos no teste de condutividade elétrica o qual foi realizado nas plantas após o estresse frio e em plantas não estressadas (ambiente) com 2, 4, 6 e 8 discos foliares imersos em 30 ml de água destilada, mantidos por 24 horas em B.O.D nas temperaturas de 25°C, 30°C e 35°C. A análise de variância foi realizada em delineamento inteiramente casualizado, e para condutividade elétrica utilizou-se fatorial número de discos x temperatura de estresse ao frio para cada temperatura de embebição separadamente. As médias foram comparadas pelo teste Tukey a 5 e 10% de probabilidade. Realizou-se a correlação de Pearson (r) entre as médias de condutividade e os demais testes de avaliação do estresse por frio. Nos resultados observou-se acentuada redução da relação (Fv/Fm) apenas nos tratamentos -3°C e -4°C indicando morte dos tecidos. Para temperaturas inferiores a 0°C houve colapso dos tecidos do limbo foliar, não sendo possível diferenciar o parênquima paliçádico do parênquima esponjoso nos tratamentos -2, -3 e -4°C...(AU)