Establishment of methods to analyze the structural and functional integrity of the quail (Coturnix coturnix japonica) sperm plasma membrane.

1. The objectives of this study were to establish the use of the fluorophores Hoechst 33342 and propidium iodide for the evaluation of sperm plasma membrane integrity and to identify an adequate hypoosmotic solution for the evaluation of sperm membrane functionality in quails.2. Sperm samples were collected from the vas deferens of nine quails. After initial evaluation, the samples were subjected to a flash-frozen assay. Three treatments with the following proportions of fresh sperm and sperm subjected to flash freezing were prepared as follows: 100:0 (T100), 50:50 (T50), and 0:100 (T0). The hypoosmotic swelling test used distilled water (0 mOsm/l) and fructose solutions (50, 100, and 200 mOsm/l).3. Immediately after recovery, the samples showed 75.6 ± 5.0% motility with vigour of 3.7 ± 0.3 and 96.1 ± 0.5% of the sperm appeared normal. The membrane integrity test showed 62.2 ± 5.2% intact sperm at T100, 29.0 ± 4.1% at T50 and 0.1 ± 0.1% at T0. Moreover, a greater number of reactive sperm (74.7 ± 6.7%) were observed when incubated in distilled water (0 mOsm/l) in comparison to other solutions (P < 0.05).4. The association of fluorescent probes composed of Hoechst 33342 and propidium iodide provided an efficient assessment of the integrity of the plasmatic membrane of quail spermatozoa. However, the study identified that the hypoosmotic swelling test has little predictive value regarding sperm membrane functionality in this species.

Evaluation of the biofilm life cycle between Candida albicans and Candida tropicalis.

Candida tropicalis is an emergent pathogen with a high rate of mortality associated with its biofilm formation. Biofilm formation has important repercussions on the public health system. However, little is still known about its biofilm life cycle. The present study analyzed the biofilm life cycle of Candida albicans and C. tropicalis during various timepoints (24, 48, 72, and 96 h) through biomass assays, colony-forming unit (CFU) counting, and epifluorescence and scanning electron microscopies. Our results showed a significant difference between C. albicans and C. tropicalis biofilms in each biomass and viability assay. All-time samples in the biomass and viability assays confirmed statistical differences between the Candida species through pairwise Wilcoxon tests (p < 0.05). C. albicans demonstrated a lower biomass growth but reached nearly the same level of C. tropicalis biomass at 96 h, while the CFU counting assays exhibited a superior number of viable cells within the C. tropicalis biofilm. Statistical differences were also found between C. albicans and C. tropicalis biofilms from 48- and 72-h microscopies, demonstrating C. tropicalis with a higher number of total cells within biofilms and C. albicans cells with a superior cell area and higher matrix production. Therefore, the present study proved the higher biofilm production of C. tropicalis.

Fluorescence Microscopy Methods for the Analysis and Characterization of Lignin.

Lignin is one of the most studied and analyzed materials due to its importance in cell structure and in lignocellulosic biomass. Because lignin exhibits autofluorescence, methods have been developed that allow it to be analyzed and characterized directly in plant tissue and in samples of lignocellulose fibers. Compared to destructive and costly analytical techniques, fluorescence microscopy presents suitable alternatives for the analysis of lignin autofluorescence. Therefore, this review article analyzes the different methods that exist and that have focused specifically on the study of lignin because with the revised methods, lignin is characterized efficiently and in a short time. The existing qualitative methods are Epifluorescence and Confocal Laser Scanning Microscopy; however, other semi-qualitative methods have been developed that allow fluorescence measurements and to quantify the differences in the structural composition of lignin. The methods are fluorescence lifetime spectroscopy, two-photon microscopy, Föster resonance energy transfer, fluorescence recovery after photobleaching, total internal reflection fluorescence, and stimulated emission depletion. With these methods, it is possible to analyze the transport and polymerization of lignin monomers, distribution of lignin of the syringyl or guaiacyl type in the tissues of various plant species, and changes in the degradation of wood by pulping and biopulping treatments as well as identify the purity of cellulose nanofibers though lignocellulosic biomass.

Distribution and Chemical Composition of Lignin in Secondary Xylem of Cactaceae.

Cactaceae family has heterogeneity in the accumulation of lignocellulose due to the diversity of shapes and anatomy of the wood. Most studies focus on fibrous and dimorphic species; but the non-fibrous species are poorly studied. The aims of this work were to analyze the syringyl/guaiacyl ratio of lignin and its distribution in secondary xylem, especially in non-fibrous species. The syringyl/guaiacyl (S/G) ratio was quantified from 34 species of cacti by nitrobenzene oxidation of free-extractive wood. The distribution of lignocellulose in wood sections stained with safranin O/fast green was determined with epifluorescence microscopy. The S/G ratio was heterogeneous; most of the non-fibrous species had a higher percentage of syringyl, while the fibrous ones accumulate guaiacyl. Fluorescence emission showed that vessel elements and wide-band tracheids had similar tonalities. It is hypothesized that the presence of a higher percentage of syringyl in most cacti is part of the defense mechanism against pathogens, which together with the succulence of the stem represent adaptations that contribute to survival in their hostile environments.

Quorum Sensing Signaling Molecules Positively Regulate c-di-GMP Effector PelD Encoding Gene and PEL Exopolysaccharide Biosynthesis in Extremophile Bacterium Acidithiobacillus thiooxidans.

Acidithiobacillus species are fundamental players in biofilm formation by acidophile bioleaching communities. It has been previously reported that Acidithiobacillus ferrooxidans possesses a functional quorum sensing mediated by acyl-homoserine lactones (AHL), involved in biofilm formation, and AHLs naturally produced by Acidithiobacillus species also induce biofilm formation in Acidithiobacillus thiooxidans. A c-di-GMP pathway has been characterized in Acidithiobacillus species but it has been pointed out that the c-di-GMP effector PelD and pel-like operon are only present in the sulfur oxidizers such as A. thiooxidans. PEL exopolysaccharide has been recently involved in biofilm formation in this Acidithiobacillus species. Here, by comparing wild type and ΔpelD strains through mechanical analysis of biofilm-cells detachment, fluorescence microscopy and qPCR experiments, the structural role of PEL exopolysaccharide and the molecular network involved for its biosynthesis by A. thiooxidans were tackled. Besides, the effect of AHLs on PEL exopolysaccharide production was assessed. Mechanical resistance experiments indicated that the loss of PEL exopolysaccharide produces fragile A. thiooxidans biofilms. qRT-PCR analysis established that AHLs induce the transcription of pelA and pelD genes while epifluorescence microscopy studies revealed that PEL exopolysaccharide was required for the development of AHL-induced biofilms. Altogether these results reveal for the first time that AHLs positively regulate pel genes and participate in the molecular network for PEL exopolysaccharide biosynthesis by A. thiooxidans.

Biofilm characterization of Fusarium solani keratitis isolate: increased resistance to antifungals and UV light.

Fusarium solani has drawn phytopathogenic, biotechnological, and medical interest. In humans, it is associated with localized infections, such as onychomycosis and keratomycosis, as well as invasive infections in immunocompromised patients. One pathogenicity factor of filamentous fungi is biofilm formation. There is still only scarce information about the in vitro mechanism of the formation and composition of F. solani biofilm. In this work, we describe the biofilm formed by a clinical keratomycosis isolate in terms of its development, composition and susceptibility to different antifungals and ultraviolet light (UV) at different biofilm formation stages. We found five biofilm formation stages using scanning electron microscopy: adherence, germination, hyphal development, maturation, and cell detachment. Using epifluorescence microscopy with specific fluorochromes, it was elucidated that the extracellular matrix consists of carbohydrates, proteins, and extracellular DNA. Specific inhibitors for these molecules showed significant biofilm reductions. The antifungal susceptibility against natamycin, voriconazole, caspofungin, and amphotericin B was evaluated by metabolic activity and crystal violet assay, with the F. solani biofilm preformation to 24 h increased in resistance to natamycin, voriconazole, and caspofungin, while the biofilm preformation to 48 h increased in resistance to amphotericin B. The preformed biofilm at 24 h protected and reduced UV light mortality. F. solani isolate could produce a highly structured extra biofilm; its cellular matrix consists of carbohydrate polymers, proteins, and eDNA. Biofilm confers antifungal resistance and decreases its susceptibility to UV light. The fungal biofilm functions as a survival strategy against antifungals and environmental factors.

Comparison of three different extenders on Murrah buffaloes (Bubalus bubalis) semen freezability.

The use of frozen semen for artificial insemination is the main approach utilised for the genetic improvement of most domesticated species. The advantages include lower transportation costs, continuous availability of semen, fewer occurrences of sexually transmitted diseases and the incorporation of desirable genes in a relatively short amount of time. Nevertheless, the use of frozen semen in buffalo herds remains limited due to the loss of sperm quality when buffalo semen is frozen. So, the goal of this study was to evaluate the pre- and post-cryopreservation quality of buffalo semen diluted in three distinct freezing media: Tris-egg yolk, Botu-bov® (BB) and ACP-111®. Thirty-two ejaculates from four bulls were analysed in terms of kinetics, morphology and sperm viability by epifluorescence microscope. Thawed samples were also evaluated for capacitation-like damage, DNA fragmentation and plasma and acrosomal membrane integrity using flow cytometry. The Tris-egg yolk and BB® extenders yielded better results than the ACP-111® extender for kinetics parameter (total motility, progressive motility and percentage of rapid cells). However, semen samples were similar for parameters evaluated by flow cytometry. Taken together, the data indicate that in comparison with Tris-egg yolk and BB extender, ACP-111® can also be used as an extender for buffalo semen cryopreservation.

[Fungal biomass estimation in soils from southwestern Buenos Aires province (Argentina) using calcofluor white stain]. / Estimación de la biomasa fúngica en un suelo del sudoeste de la provincia de Buenos Aires (Argentina) con una tinción directa con blanco de calcoflúor.

Soil microorganisms are vital for ecosystem functioning because of the role they play in soil nutrient cycling. Agricultural practices and the intensification of land use have a negative effect on microbial activities and fungal biomass has been widely used as an indicator of soil health. The aim of this study was to analyze fungal biomass in soils from southwestern Buenos Aires province using direct fluorescent staining and to contribute to its use as an indicator of environmental changes in the ecosystem as well as to define its sensitivity to weather conditions. Soil samples were collected during two consecutive years. Soil smears were prepared and stained with two different concentrations of calcofluor, and the fungal biomass was estimated under an epifluorescence microscope. Soil fungal biomass varied between 2.23 and 26.89µg fungal C/g soil, being these values in the range expected for the studied soil type. The fungal biomass was positively related to temperature and precipitations. The methodology used was reliable, standardized and sensitive to weather conditions. The results of this study contribute information to evaluate fungal biomass in different soil types and support its use as an indicator of soil health for analyzing the impact of different agricultural practices.

Estimación de la biomasa fúngica en un suelo del sudoeste de la provincia de Buenos Aires (Argentina) con una tinción directa con blanco de calcoflúor / Fungal biomass estimation in soils from southwestern Buenos Aires province (Argentina) using calcofluor white stain

Los microorganismos del suelo son vitales para el correcto funcionamiento de los ecosistemas, principalmente por su papel en el ciclado de nutrientes. La intensificación del uso del suelo y las prácticas agrícolas alteran negativamente la actividad microbiana. La biomasa fúngica es uno de los parámetros más utilizados para estudiar el impacto de las actividades agrícolas en la estructura y el funcionamiento del suelo. El objetivo del presente trabajo fue estimar la biomasa fúngica en un suelo del sudoeste bonaerense con el fin de obtener valores de referencia que permitan usar este parámetro como un indicador de cambios en el ecosistema y, por otro lado, demostrar que la metodología empleada es sensible a las variaciones en las condiciones climáticas. Se colectaron muestras de suelos durante 2 años consecutivos. Se prepararon frotis de suelo y se tiñeron con soluciones de distintas concentraciones de blanco de calcoflúor y luego se estimó la biomasa fúngica observando los frotis con microscopio de epifluorescencia. Los valores de biomasa fúngica estimados variaron entre 2,23 y 26,89 μg Cfúngico/g de suelo y estuvieron dentro del rango esperable para el tipo de suelo estudiado. La biomasa fúngica mostró una relación positiva con la temperatura y las precipitaciones. La metodología empleada resultó ser confiable, repetible y sensible a cambios en las condiciones climáticas. Los resultados podrían usarse como valores de referencia para estudiar la biomasa fúngica de suelos bajo distintas condiciones y emplearse como indicadores del impacto de las distintas prácticas agrícolas sobre el ecosistema.

Soil microorganisms are vital for ecosystem functioning because of the role they play in soil nutrient cycling. Agricultural practices and the intensification of land use have a negative effect on microbial activities and fungal biomass has been widely used as an indicator of soil health. The aim of this study was to analyze fungal biomass in soils from southwestern Buenos Aires province using direct fluorescent staining and to contribute to its use as an indicator of environmental changes in the ecosystem as well as to define its sensitivity to weather conditions. Soil samples were collected during two consecutive years. Soil smears were prepared and stained with two different concentrations of calcofluor, and the fungal biomass was estimated under an epifluorescence microscope. Soil fungal biomass varied between 2.23 and 26.89 μg fungal C/g soil, being these values in the range expected for the studied soil type. The fungal biomass was positively related to temperature and precipitations. The methodology used was reliable, standardized and sensitive to weather conditions. The results of this study contribute information to evaluate fungal biomass in different soil types and support its use as an indicator of soil health for analyzing the impact of different agricultural practices.

Geometry and spatial orientation of a quadruple node of triple junctions in a europium-doped KI crystal as determined by epifluorescence microscopy.

The geometry and spatial orientation of a typical arrangement of four triple junctions and six grain boundaries sharing a common quadruple node in a Eu2+ -doped KI crystal are investigated by epifluorescence microscopy using the proper doping ion as a fluorochrome. To achieve this, an electronic three-dimensional reconstruction of the studied arrangement of crystal defects was built from microscopy images of different optical cross-sections of this arrangement. Previously, the doping ions were induced, by subjecting the crystal to a long annealing treatment, to form europium precipitates into the crystal grain boundaries. The optical properties of these precipitates were characterized by fluorescence spectrophotometry and used to tailor properly the microscope fluorescence mirror unit, whereas the single-crystal character of the microscope samples was tested by X-ray diffraction. By inspecting the reconstruction under handling, the dihedral angles between the grain boundaries that meet at a common triple junction as well as the angles between the triple junctions sharing the quadruple node were successfully measured at the quadruple node site. The measuring procedures are carefully described. The resulting values (132º, 109º, 119º, 125º, 111º, 124º, 124º, 111º, 125º, 129º, 109º and 122º ± 2º) for the dihedral angles depart for some few degrees from the characteristic angle (120º) of a 3-fold symmetry rotation, whereas the resulting values (104º, 111º, 117º, 103º, 100º and 121º ± 2º) for the triple junction angles are not far from the characteristic angle (109.47º) between the legs of a tetrahedron. These results, indicating that in the close neighbourhood of the quadruple node the studied arrangement of crystal defects deviates from a state of full structural stability, allow this arrangement to be fairly modelled in such a neighbourhood by a distorted tetrahedron. The angles between the studied triple junctions and the host lattice directions [11¯1], [111¯], [1¯11] and [1¯1¯1¯] were also measured at the quadruple node site, and the resulting values (8º, 7º, 6º and 8º ± 2º, respectively) indicate that a symmetry mismatching exists between the tetrahedral model of the studied Eu2+ -decorated arrangement of crystal defects and the KI matrix cubic crystal lattice. This symmetry mismatching is discussed to be responsible for the observed deviation from structural stability.

Patterns of microbial community biomass, composition and HPLC diagnostic pigments in the Costa Rica upwelling dome.

We investigated biomass, size-structure, composition, depth distributions and spatial variability of the phytoplankton community in the Costa Rica Dome (CRD) in June-July 2010. Euphotic zone profiles were sampled daily during Lagrangian experiments in and out of the dome region, and the community was analyzed using a combination of digital epifluorescence microscopy, flow cytometry and HPLC pigments. The mean depth-integrated biomass of phytoplankton ranged 2-fold, from 1089 to 1858 mg C m-2 (mean ± SE = 1378 ± 112 mg C m-2), among 4 water parcels tracked for 4 days. Corresponding mean (±SE) integrated values for total chlorophyll a (Chl a) and the ratio of autotrophic carbon to Chl a were 24.1 ± 1.5 mg Chl a m-2 and 57.5 ± 3.4, respectively. Absolute and relative contributions of picophytoplankton (∼60%), Synechococcus (>33%) and Prochlorococcus (17%) to phytoplankton community biomass were highest in the central dome region, while >20 µm phytoplankton accounted for ≤10%, and diatoms <2%, of biomass in all areas. Nonetheless, autotrophic flagellates, dominated by dinoflagellates, exceeded biomass contributions of Synechococcus at all locations. Order-of-magnitude discrepancies in the relative contributions of diatoms (overestimated) and dinoflagellates (underestimated) based on diagnostic pigments relative to microscopy highlight potential significant biases associated with making community inferences from pigments.

System for evaluation of oxidative stress on in-vitro-produced bovine embryos.

This study proposed a quantitative evaluation of oxidative status (OS) in bovine embryos. Sixteen-cell stage embryos, cultured under 5% O2, were treated with oxidative stress inducer menadione (0, 1, 2.5 and 5 µmol/l) for 24 h. Blastocyst rate (BLR) was recorded and expanded blastocysts were stained with CellROX®Green (CRG; OS evaluation) and evaluated under epifluorescence microscopy (ratio of pixel/blastomere). A significant effect of menadione was observed for BLR (P = 0.0039), number of blastomeres/embryo (P < 0.0001) and OS (P < 0.001). Strong negative correlations were found between BLR and the number of blastomeres with OS evaluation, demonstrating the efficacy of this analysis to evaluate OS levels of IVF bovine embryos.

Bacterias autótrofas y heterótrofas asociadas a nieve marina lodosa en arrecifes con escorrentía continental / Autotrophic and heterotrophic bacteria associated to muddy marine snow in reefs with terrestrial runoff / Bacterias autótrofas e heterotróficas associadas a neve marinila lodosa em recifes com escoamento continental

En agregados de nieve marina influenciada por escorrentía continental, la densidad de bacterias heterótrofas es mayor que la de autótrofas. Esta premisa fue puesta a prueba en cuatro zonas arrecifales localizadas a diferente distancia del Canal del Dique; principal fuente de aportes continentales a los arrecifes de coral del Archipiélago Nuestra Señora del Rosario, Cartagena, Caribe colombiano. Mediante epifluorescencia se determinó densidad promedio de microorganismos presentes en agregados de nieve marina. Los resultados mostraron mayor densidad de bacterias heterótrofas (3.63 x 10(4) ± 1.6 x 10(4) SE células mL-1) que de autótrofas (6 x 10³± 1.3 x 10³ SE células mL-1), principalmente en arrecifes cercanos a descarga de escorrentía continental (bacterias heterótrofas con densidad 8.9 x 10(4) células mL-1 y 3 x 10(4) células mL-1 para Isla Arena y Tesoro, respectivamente). La densidad de microorganismos encontrada es típica de zonas con alto contenido de materia orgánica particulada; por lo cual podría servir como potencial indicador de escorrentía continental. Estudios futuros deben enfocarse en determinar la composición de comunidad bacteriana asociada a nieve marina y posible virulencia sobre organismos arrecifales.

Em aglomerados de neve marinha influenciados por escoamento continental, a densidade de bacterias heterotróficas é maior que a de autótrofas. Esta premissa foi posta a prova em quatro zonas de recifes localizadas em diferentes distancias do Canal de Dique; principal fonte de contribuic.ao continental aos recifes do coral do Arquipélago Nossa Senhora do Rosàrio, Cartagena, Caribe colombiano. Mediante epifluorescencia determinou-se densidade de micro-organismos presentes em aglomerados de neve marinha. Os resultados mostraram maior densidade de bacterias heterotróficas (3.63 x 10(4) ± 1.6 x 10(4) SE células mL-1) que de autótrofas (6 x 10³ ± 1.3 x 10³ SE células mL-1), principalmente em recifes próximos a descarga de escoamento continental (bacterias heterotróficas com densidade 8.9 x 10(4) células mL-1 y 3 x 10(4) células mL-1 para a Ilha Arena e Tesoro, respetivamente). A densidade de micro-organismos é típica de zonas com elevado conteúdo de matéria orgànica particulada; pela qual pode servir como potencial indicador de escoamento continental. Estudos futuros devem enfocar-se em determinar a composicäo da comunidade bacteriana associada a neve marinha e possível virulencia sobre organismos recifais.

The density of heterotrophic bacteria is greater than autotrophic in marine snow aggregates influenced by continental runoff. Four coral reef areas at different distances from the Canal del Dique served to evaluate this premise; this canal is the main source of inland resources for the coral reefs of the Nuestra Señora del Rosario archipelago in Cartagena in the Colombian Caribbean. The average density of microorganisms in marine snow aggregates was determined using epifluorescence. The results showed higher density of heterotrophic bacteria (3.63 x 10(4) ± 1.6 x 10(4) SE cells mL-1) than autotrophic (6 x 10³± 1.3 x 10³ SE cells mL-1), mainly in reefs near continental runoff discharges (heterotrophic bacteria density of 8.9 x 10(4) cells mL-1 and 3 x 10(4) cells mL-1 Isla Arena and Tesoro, respectively). The density of microorganisms found is typical of high-particulate organic matter areas and, therefore, could be a potential indicator of continental runoff. Future studies should focus on determining the composition of the bacterial community associated with marine snow and its potential virulence on reef organisms.

Resin penetration in artificial enamel carious lesions after using sodium hypochlorite as a deproteinization agent.

OBJECTIVES: To study the degree of penetration of an adhesive resin in artificial enamel carious lesions after using sodium hypochlorite as deproteinization agent. STUDY DESIGN: Twenty included human third-molars, extracted for surgical indication, were used. Artificial lesions were created in the buccal and lingual sides of each specimen through a cycle of demineralization-remineralization. Samples were then incubated in human saliva for 7 days at 37 ° C. After surface cleaning, lesions and the peripheral sound enamel were etched with 37% orthophosphoric acid for 20 seconds. One lesion of each specimen was treated with 5.25% sodium hypochlorite (NaOCl) for one minute. The other lesion of each specimen was used as a control. Experimental and control lesions were sealed with a fluid resin marked with Rhodamine B. Lesions were sectioned for microscopic observation by epifluorescence and polarized light. The images obtained were analyzed morphometrically. The micrometer measurements were made with ImageJ ® software. The level of significance was assessed at p<0.05. RESULTS: The average sealant depth penetration in the control group was 94.9 ± 28.6 µm versus 122.8 ± 25.3 µm in the experimental group. This represents Δ 20.1% significantly greater penetration when using sodium hypochlorite (p<0.001). CONCLUSION: The results demonstrated a significant penetration of the sealing resin when the conventional technique is complemented with the application of 5.25% sodium hypochlorite for one minute in artificial enamel carious lesions.

Simultaneous assessment of plasmatic, acrosomal, and mitochondrial membranes in ram sperm by fluorescent probes / Avaliação simultânea das membranas plasmática, acrossomal e mitocondrial de espermatozoides de carneiros por sondas fluorescentes

In this experiment, it was defined a protocol of fluorescent probes combination: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and JC-1. For this purpose, four ejaculates from three different rams (n=12), all showing motility >80 percent and abnormal morphology <10 percent, were diluted in TALP medium and split into two aliquots. One of the aliquots was flash frozen and thawed in three continuous cycles, to induce damage in cellular membranes and to disturb mitochondrial function. Three treatments were prepared with the following fixed ratios of fresh semen:flash frozen semen: 0:100 (T0), 50:50 (T50), and 100:0 (T100). Samples were stained in the proposal protocol and evaluated by epifluorescence microscopy. For plasmatic membrane integrity, detected by PI probe, it was obtained the equation: v=1.09+0.86X (R²=0.98). The intact acrosome, verified by the FITC-PSA probe, produced the equation: v=2.76+0.92X (R²=0.98). The high mitochondrial membrane potential, marked in red-orange by JC-1, was estimated by the equation: v=1.90+0.90X (R²=0.98). The resulting linear equations demonstrate that this technique is efficient and practical for the simultaneous evaluations of the plasmatic, acrosomal, and mitochondrial membranes in ram spermatozoa.(AU)

Neste experimento, foi definida uma combinação de sondas fluorescentes: iodeto de propídio (PI), aglutinina de Pisum sativum conjugada ao isotiocionato de fluoresceína (FITC-PSA) e JC-1. Para esta proposta, quatro ejaculados de três carneiros (n=12), que apresentavam motilidade >80 por cento e alterações morfológicas <10 por cento, foram diluídos em meio TALP e divididos em duas alíquotas. Uma alíquota foi submetida a três ciclos de flash frozen e descongelação, para induzir danos nas membranas celulares e distúrbios na função mitocondrial. Três tratamentos foram preparados com as seguintes proporções preestabelecidas de sêmen fresco: sêmen submetido a flash frozen: 0:100 (T0), 50:50 (T50) e 100:0 (T100). As amostras foram coradas no protocolo proposto e avaliadas por microscopia de epifluorescência. Para integridade de membrana plasmática, detectada pela sonda PI, foi obtida a equação: v=1,09+0,86X (R²=0,98). O acrossomo intacto, verificado pela sonda FITC-PSA, produziu a equação: v=2,76+0,92X (R²=0,98). O alto potencial de membrana mitocondrial, marcada em vermelho-alaranjado pelo JC-1, foi estimado pela equação: v=1,90+0,90X (R²=0,98). As equações lineares resultantes demonstraram que a técnica é eficiente e prática para avaliação simultânea das membranas plasmática, acrossomal e mitocondrial em espermatozoides de carneiros.(AU)

Simultaneous assessment of plasmatic, acrosomal, and mitochondrial membranes in ram sperm by fluorescent probes / Avaliação simultânea das membranas plasmática, acrossomal e mitocondrial de espermatozoides de carneiros por sondas fluorescentes

In this experiment, it was defined a protocol of fluorescent probes combination: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and JC-1. For this purpose, four ejaculates from three different rams (n=12), all showing motility >80 percent and abnormal morphology <10 percent, were diluted in TALP medium and split into two aliquots. One of the aliquots was flash frozen and thawed in three continuous cycles, to induce damage in cellular membranes and to disturb mitochondrial function. Three treatments were prepared with the following fixed ratios of fresh semen:flash frozen semen: 0:100 (T0), 50:50 (T50), and 100:0 (T100). Samples were stained in the proposal protocol and evaluated by epifluorescence microscopy. For plasmatic membrane integrity, detected by PI probe, it was obtained the equation: v=1.09+0.86X (R²=0.98). The intact acrosome, verified by the FITC-PSA probe, produced the equation: v=2.76+0.92X (R²=0.98). The high mitochondrial membrane potential, marked in red-orange by JC-1, was estimated by the equation: v=1.90+0.90X (R²=0.98). The resulting linear equations demonstrate that this technique is efficient and practical for the simultaneous evaluations of the plasmatic, acrosomal, and mitochondrial membranes in ram spermatozoa.

Neste experimento, foi definida uma combinação de sondas fluorescentes: iodeto de propídio (PI), aglutinina de Pisum sativum conjugada ao isotiocionato de fluoresceína (FITC-PSA) e JC-1. Para esta proposta, quatro ejaculados de três carneiros (n=12), que apresentavam motilidade >80 por cento e alterações morfológicas <10 por cento, foram diluídos em meio TALP e divididos em duas alíquotas. Uma alíquota foi submetida a três ciclos de flash frozen e descongelação, para induzir danos nas membranas celulares e distúrbios na função mitocondrial. Três tratamentos foram preparados com as seguintes proporções preestabelecidas de sêmen fresco: sêmen submetido a flash frozen: 0:100 (T0), 50:50 (T50) e 100:0 (T100). As amostras foram coradas no protocolo proposto e avaliadas por microscopia de epifluorescência. Para integridade de membrana plasmática, detectada pela sonda PI, foi obtida a equação: v=1,09+0,86X (R²=0,98). O acrossomo intacto, verificado pela sonda FITC-PSA, produziu a equação: v=2,76+0,92X (R²=0,98). O alto potencial de membrana mitocondrial, marcada em vermelho-alaranjado pelo JC-1, foi estimado pela equação: v=1,90+0,90X (R²=0,98). As equações lineares resultantes demonstraram que a técnica é eficiente e prática para avaliação simultânea das membranas plasmática, acrossomal e mitocondrial em espermatozoides de carneiros.

Enumeração de bactérias em leite cru, utilizando a técnica de contagem direta por microscopia de epifluorescent (Direct Epifluorescent Filter Technique - DEFT) / Enumeration of bacteria in raw milk, utilizing the straight technique of tally by microscopy of epifluorescência (Direct Epifluorescent Filter Technique - DEFT)

Avaliou-se o método rápido de análise quantitativa de bactérias por microscopia de epifluorescência (DEFT) no leite cru tipo C recebido pelo Laticínios Escola da Universidade Federal de Viçosa, Viçosa-MG. Um total de 190 amostras foram quantificadas pelo método DEFT e pelo método oficial de contagem padrão de mesófilos aeróbios em placa (CPP). Pôde-se observar a distinguir células coradas de alaranjado (consideradas viáveis) e células coradas de verde ( consideradas mortas), sendo que apenas as alaranjadas foram enumeradas. O resultado da análise de regressão línea apresentou um coeficiente de correção r = 0,83, sendo que a equação de regressão ficou Y = 0,68X + 1,96; onde Y = log10 UFC/mL (CPP). O limite de detecção apresentado pelo método DEFT ficou em 5,3 x 10³ CME/mL. O método DEFT apresentou um CV de 1,6 por cento. Baseando-se nas análises comparativas, pode-se inferir que o método DEFT tem um grande potencial para ser usado no Brasil para avaliar o nível de contaminação do leite cru tipo C. Além da possibilidade da sua aplicação em outros estudos na área de alimentos.(AU)

Quick and reliable methods to count bacterial contamination in foods are required for the industrial quality control. The Direct Epifluorescent Filter Technique (OEFT) can analyse samples in less than 30 minutes, with the advantage 10 differentiate viable and dead cells. A total of 190 samples of grade C raw milk were sampled at a dairy processing plant and analyzed for the Standard Plate Count and DEFT to compare the two methods. The time required to obtainli results by the DEFT method was 20 minutes. 1t IO as possible to distinguish viable cells, fluorescing orange, and dead bacteria, fluorescing green. The correlation coefficient (r) was 0.83. The precision measured by the coefficient of variation was good (CV = 1.62%). The results confirmed that the DEFT is method that gives accurate results rapidly, needs small place, and has a relatively low cost. (AU)