Chlorophyll a fluorescence and leaf temperature are early indicators of oil palm diseases

Bud rot (BR) caused by Phytophthora palmivora and lethal wilt (LW) whose causal agent is unknown, are two diseases currently posing a threat to the oil palm ( Elaeis guineensis . Jacq) industry. BR, first reported in 1964, has destroyed more than 85,000 ha. LW, first reported in 1994 in the Llanos Orientales in Colombia, has destroyed more than 5,000 ha. Chlorophyll a fluorescence is useful as a provider of information about the efficiency of the photosynthetic process when plants are subjected to biotic or abiotic stresses. Oil palms affected by BR and LW showed anomalies in the photosynthetic system, manifested by reductions in Fv / F M and PSII. Changes in PSII, variable fluorescence yield ( Fv ) and maximum fluorescence in light-adapted leaves ( F M ) were observed from the start of BR infection. The most sensitive and early indicators of LW disease were leaf temperature and basal fluorescence ( F 0 ). Fv/F 0 significantly changed in diseased palms, indicating problems with movement of electrons through the electron transport chain. Leaf temperature changed in response to both diseases, but variation was greater in LW. We concluded that damage to the photochemical system caused by the diseases affected the processes by which the plant captures and transports energy, causing a physiological imbalance in the plant reflected in the observed variations in chlorophyll a fluorescence and leaf temperature. The two parameters began to change early in the onset of BR and before visual symptoms appeared in LW, which is very important to the management of both diseases, the foundation of which is early detection.

Chlorophyll a fluorescence and leaf temperature are early indicators of oil palm diseases

Bud rot (BR) caused by Phytophthora palmivora and lethal wilt (LW) whose causal agent is unknown, are two diseases currently posing a threat to the oil palm ( Elaeis guineensis . Jacq) industry. BR, first reported in 1964, has destroyed more than 85,000 ha. LW, first reported in 1994 in the Llanos Orientales in Colombia, has destroyed more than 5,000 ha. Chlorophyll a fluorescence is useful as a provider of information about the efficiency of the photosynthetic process when plants are subjected to biotic or abiotic stresses. Oil palms affected by BR and LW showed anomalies in the photosynthetic system, manifested by reductions in Fv / F M and PSII. Changes in PSII, variable fluorescence yield ( Fv ) and maximum fluorescence in light-adapted leaves ( F M ) were observed from the start of BR infection. The most sensitive and early indicators of LW disease were leaf temperature and basal fluorescence ( F 0 ). Fv/F 0 significantly changed in diseased palms, indicating problems with movement of electrons through the electron transport chain. Leaf temperature changed in response to both diseases, but variation was greater in LW. We concluded that damage to the photochemical system caused by the diseases affected the processes by which the plant captures and transports energy, causing a physiological imbalance in the plant reflected in the observed variations in chlorophyll a fluorescence and leaf temperature. The two parameters began to change early in the onset of BR and before visual symptoms appeared in LW, which is very important to the management of both diseases, the foundation of which is early detection.(AU)

Fluorescent test accurately predicts leak of ischemic colon anastomosis in rats

Purpose: To investigate if fluorescein fluorescent test can predict dehiscence in a model of ischemic colonic anastomosis in rats. Methods: This experimental controlled trial randomly assigned 55 rats to four groups. Anastomoses were performed in non-ischemic colon segments (control group) and in ischemic colon segments measuring 1, 2 or 3 cm long (groups 1, 2 and 3, respectively). Fluorescein was injected and the tissues were examined under ultraviolet light. Seven days later, a second-look surgery was performed to check for the presence or absence of anastomosis dehiscence. Results: Twenty-four rats presented anastomotic dehiscence during the second-look surgery. Reticular and nonfluorescent patterns were significantly associated with the occurrence of anastomotic dehiscence. Fluorescein fluorescence had a sensitivity of 95.8%, specificity of 89.2%, positive predictive value of 88.4%, negative predictive value of 96.2%, and accuracy of 92.3% to predict anastomotic dehiscence. Conclusion: Fluorescein fluorescent test can accurately predict leak in a model of ischemic colonic anastomosis in rats.(AU)

Use of green fluorescent protein labeled non-tuberculous mycobacteria to evaluate the activity quaternary ammonium compound disinfectants and antibiotics

Although infections with NonTuberculous Mycobacteria have become less common in AIDS patients, they are important opportunistic infections after surgical procedures, likely because they are ubiquitous and not efficiently killed by many commonly used disinfectants. In Venezuela there have recently been many non-tuberculous mycobacteria soft tissue infections after minor surgical procedures, some apparently related to the use of a commercial disinfectant based on a Quaternary Ammonium Compound. We studied the activity of this and other quaternary ammonium compounds on different non-tuberculous mycobacteria by transforming the mycobacteria with a dnaA-gfp fusion and then monitoring fluorescence to gauge the capacity of different quaternary ammonium compounds to inhibit bacterial growth. The minimum inhibitory concentration varied for the different quaternary ammonium compounds, but M. chelonae and M. abscessus were consistently more resistant than M. smegmatis, and M. terrae more resistant than M. bovis BCG.(AU)

Advances in flow cytometry in basic and applied equine andrology

The aim of this review is to present the current probes available that assess different compartments and functions of stallion spermatozoa, including assays to investigate the functionality of the membranes, nucleus and mitochondria, and to study cell signaling in this particular cell. New multi-parametric protocols for the assessment of stallion sperm, recently developed in the laboratory of the authors, will also be presented. The potential clinical applicability of diagnostic tests based on flow cytometry will also be discussed.

Advances in flow cytometry in basic and applied equine andrology

The aim of this review is to present the current probes available that assess different compartments and functions of stallion spermatozoa, including assays to investigate the functionality of the membranes, nucleus and mitochondria, and to study cell signaling in this particular cell. New multi-parametric protocols for the assessment of stallion sperm, recently developed in the laboratory of the authors, will also be presented. The potential clinical applicability of diagnostic tests based on flow cytometry will also be discussed.(AU)

Validation of the CellRox Deep Red ® fluorescent probe to oxidative stress assessment in equine spermatozoa

Considering the importance of ROS influence on sperm functionality and some limitations in sperm oxidative stress assessment methods, a field to studies of new techniques are still open. In this sense, the aim of this study is to validate the ROS detection technique through the CellRox Deep Red Reagent®probe in stallion sperm. Four stallions were used and the analyses were conducted on four replicates of semen samples from each of stallion (n = 16). The results of the polynomial regression presented a quadratic effect, high determination coefficient value (R2 = 0.88) and high significant P value (P < 0.0001). The CellRox Deep Red® fluorescent probe is able to detect reactive oxygen species in equine sperm, indicating accurately the occurrence of oxidative stress in stallion semen.

Validation of the CellRox Deep Red ® fluorescent probe to oxidative stress assessment in equine spermatozoa

Considering the importance of ROS influence on sperm functionality and some limitations in sperm oxidative stress assessment methods, a field to studies of new techniques are still open. In this sense, the aim of this study is to validate the ROS detection technique through the CellRox Deep Red Reagent®probe in stallion sperm. Four stallions were used and the analyses were conducted on four replicates of semen samples from each of stallion (n = 16). The results of the polynomial regression presented a quadratic effect, high determination coefficient value (R2 = 0.88) and high significant P value (P < 0.0001). The CellRox Deep Red® fluorescent probe is able to detect reactive oxygen species in equine sperm, indicating accurately the occurrence of oxidative stress in stallion semen.(AU)

The dynamic structure of Spitzenkörpers of Trichosporon asahii examined by the fluorescent probe FM4-64

The Spitzenkörper is a dynamic and specialized multicomponent cell complex present in the tips of hyphal cells. The amphiphilic styryl dye FM4-64 was found to be ideal for imaging the dynamic changes of the apical vesicle cluster within growing hyphal tips. It is widely used as a marker of endocytosis and to visualize vacuolar membranes. Here we performed uptake experiments using FM4-64 to study the dynamic of the Spitzenkörper in Trichosporon asahii. We observed that Spitzenkörpers were present at the tip of the budding site of the spore, blastospore, and the germ tube of T. asahii. We also found that Spitzenkörpers were present at the tip of the hyphae as well as the subapical regions. Cytochalasin D, an inhibitor of actin polymerization, leads to abnormal Spitzenkörper formation and loss of cell polarity.(AU)

Caracterização física e estrutural de espermatozoides no ejaculado de Caprinos Azul, Canindé, Moxotó e Repartido / Physical and structural characterization of sperm in the ejaculate from Azul, Caninde, Moxotó and Repartido goat races

This study aimed to evaluate physically and structurally ejaculates from locally adapted goats in therainy season. Semen pool formed by the four goats of each breed was evaluated for physical, diluted in Tris-eggyolk and frozen. After thawing, it was diluted in Tris and incubated with carboxyfluorescein diacetate andpropidium iodide, to evaluate the integrity of the plasma membrane and JC1 to assess mitochondrial activity.The percentage of cells with damaged membrane and mitochondrial activity in spermatozoa between breed werecompared, every hour for three hours. There was no significant difference (P > 0,05) in relation to thepercentage of cells with damaged plasma membrane, as well as between the incubation times. The Azul breedhad lower mitochondrial activity (P < 0,05). The evaluated semen of all breeds showed no significant damage tothe plasma membrane in the rainy season, but the Azul breed had lower mitochondrial activity in sperm cells.(AU)

Análise da integridade de membrana de espermatozoides extraídos de fragmentos frescos de testículos de cães / Sperm membrane integrity analysis from dog fresh testis

To evaluate testicular tissue before cryopreservation is important to quantify and qualify losses ofsperm during the freezing/thawing of testicular tissue. The aim of this study was to analyze the sperm plasmamembrane structural and functional integrity on post-pubertal dogs (n = 7) fresh testis. The sperm fromtesticular tissue were submitted to plasma membrane structural and functional integrity analysis by fluorescentmicroscopy using SYBR 14 and Propidium Iodide and the hiposmotic test, respectively. For statistical analysis,it was used normality test (Shapiro Wilk) and Wilcoxon test (R 3.3.1; P > 0.05). It was observed 94.9% and91.8% spermatozoa presenting structural and functional integrity, respectively. Thus, analysis of the membraneintegrity is an important tool to analyze spermatozoa extracted from testicles. Despite the fluorescence be apractical method, it has a high cost.(AU)

Caracterização física e estrutural de espermatozoides no ejaculado de Caprinos Azul, Canindé, Moxotó e Repartido / Physical and structural characterization of sperm in the ejaculate from Azul, Caninde, Moxotó and Repartido goat races

This study aimed to evaluate physically and structurally ejaculates from locally adapted goats in therainy season. Semen pool formed by the four goats of each breed was evaluated for physical, diluted in Tris-eggyolk and frozen. After thawing, it was diluted in Tris and incubated with carboxyfluorescein diacetate andpropidium iodide, to evaluate the integrity of the plasma membrane and JC1 to assess mitochondrial activity.The percentage of cells with damaged membrane and mitochondrial activity in spermatozoa between breed werecompared, every hour for three hours. There was no significant difference (P > 0,05) in relation to thepercentage of cells with damaged plasma membrane, as well as between the incubation times. The Azul breedhad lower mitochondrial activity (P < 0,05). The evaluated semen of all breeds showed no significant damage tothe plasma membrane in the rainy season, but the Azul breed had lower mitochondrial activity in sperm cells.

Análise da integridade de membrana de espermatozoides extraídos de fragmentos frescos de testículos de cães / Sperm membrane integrity analysis from dog fresh testis

To evaluate testicular tissue before cryopreservation is important to quantify and qualify losses ofsperm during the freezing/thawing of testicular tissue. The aim of this study was to analyze the sperm plasmamembrane structural and functional integrity on post-pubertal dogs (n = 7) fresh testis. The sperm fromtesticular tissue were submitted to plasma membrane structural and functional integrity analysis by fluorescentmicroscopy using SYBR 14 and Propidium Iodide and the hiposmotic test, respectively. For statistical analysis,it was used normality test (Shapiro Wilk) and Wilcoxon test (R 3.3.1; P > 0.05). It was observed 94.9% and91.8% spermatozoa presenting structural and functional integrity, respectively. Thus, analysis of the membraneintegrity is an important tool to analyze spermatozoa extracted from testicles. Despite the fluorescence be apractical method, it has a high cost.

Facial nerve identification with fluorescent dye in rats

PURPOSEThe parotidectomy technique still has an elevated paresis and paralysis index, lowering patient life's quality. The correct identification of the facial nerve can prevent nerve damage. Fluorescent dye identifies nerves in experimental studies but only few articles focused its use on facial nerve study in parotidectomies. We aimed to stain the rat facial nerve with fluorescent dye to facilitate visualization and dissection in order to prevent injuries.METHODSForty adult male Wistar rats were submitted to facial injection of saline solution (Gsf-control group, 10) or fluorescent dye solution (Gdye group, 30) followed by parotidectomy preserving the facial nerve, measuring the time for localization and facility of localization (LocTime and LFN). Nerve function was assessed using the Vibrissae Movements (PMV) and Eyelid Closure Motion (PFP) scores.RESULTSNerve localization was faster in Gdye group, with 83% Easy LFN rate. The Gdye group presented with low nerve injury degree and better PMV and PFP scores, with high sensitivity and accuracy.CONCLUSIONSThis experimental method of facial nerve fluorescence was effective for intraoperative nerve visualization, identification and preservation. The technique may be used in future facial nerve studies, translated to humans, contributing to the optimization of parotid surgery in the near future.(AU)

Produção in vitro de embriões utilizando-se sêmen sexado de touros 5/8 Girolando / Production of in vitro embryo using sexed sperm of 5/8 Girolando bulls

We evaluated the "in vitro" blastocyst rate production using bovine sexed semen. Semen from three bulls was used to verify the individual"s semen variation, cleavage rates and embryo production. In this study, we employed reproductive biotechnologies, computer analysis of post-thawed semen and fluorescent probes for sperm cells integrity analysis (plasma membrane, acrosome membrane and mitochondrial potential). A total of 959 oocysts went through in vitro maturation steps for in vitro fertilization and cultivation, being 473 with sexed semen and 486 with conventional semen. The cleavage rate was observed in blastocysts on D2 and D7. Data were analyzed by SPSS 16.0 software using analysis of variance (ANOVA), Student"s t-test was used to detect differences between groups, and chi-square analysis for in vitro production results (P 0.05 ). The results differed between conventional (31.06%) and sexed semen (21.10%) in the obtainment of blastocyst. When the blastocyst production was individually compared in sexed semen samples (27.69%, 17.93% and 25.56%, bulls 1, 2 and 3, respectively) we verified T2 T1 and T1 = T3 and T2 = T3. [...]

Avaliou-se a taxa de produção de blastocisto in vitro utilizando-se o sêmen bovino sexado. Foram utilizados três reprodutores para verificar a variação individual do sêmen, taxas de clivagem e produção embrionária. O trabalho utilizou-se de biotécnicas reprodutivas, análise computadorizada do sêmen pós-descongelação e sondas fluorescentes para análises de integridade da célula espermática (membrana plasmática, membrana acrossomal e potencial mitocondrial). Um total de 959 oócitos passou por etapas de maturação in vitro, fertilização in vitro (sexado, n= 473; convencional, n = 486) e cultivo in vitro. A taxa de clivagem foi observada no D2 e a de blastocistos no D7. Os dados foram analisados pelo programa SPSS 16.0 empregando-se a análise de variância (ANOVA), sendo o teste t-Student usado para detectar diferenças entre os grupos e o Qui-quadrado  para análise dos resultados da produção in vitro (P 0,05). Os resultados diferiram entre o sêmen convencional (31,06%) e sexado (21,10%) para produção de blastocisto. Quando comparada a produção de blastocisto individualmente nas amostras de sêmen sexado (27,69%; 17,93% e 25,56%, touros 1, 2 e 3, respectivamente), percebeu-se que T2 T1 e T1=T3 e T2=T3. Quanto às análises de cinética espermática, as amostras de sêmen sexado mostraram diferençasentre os touros nas variáveis velocidade curvilínea, velocidade linear e velocidade do trajeto em que o T1(117,7±1,6 µm/s; 60,0±0,3 µm/s; 73,6±0,4 µm/s, respectivamente) quando comparado aostouros T2 (80,2±2,3 µm/s; 47,0±2,0 µm/s; 57,7±0,9 µm/s, respectivamente) e T3 (86,4±5,7 µm/s; 46,2±2,7 µm/s; 53,8±2,8 µm/s, respectivamente) obteve valores mais elevados. As análises daintegridade da célula espermática não diferiram entre as amostras de sêmen convencional, já no sêmen sexado a integridade de membrana foi a variável que diferiu estatisticamente entre os touros, em que o T1 (38 ±2,7) diferiu do T3(53,8± 1,8) (P=0,009), mas não divergiu do T2 (44,1±4,4).[...]

Produção in vitro de embriões utilizando-se sêmen sexado de touros 5/8 Girolando / Production of in vitro embryo using sexed sperm of 5/8 Girolando bulls

We evaluated the "in vitro" blastocyst rate production using bovine sexed semen. Semen from three bulls was used to verify the individual"s semen variation, cleavage rates and embryo production. In this study, we employed reproductive biotechnologies, computer analysis of post-thawed semen and fluorescent probes for sperm cells integrity analysis (plasma membrane, acrosome membrane and mitochondrial potential). A total of 959 oocysts went through in vitro maturation steps for in vitro fertilization and cultivation, being 473 with sexed semen and 486 with conventional semen. The cleavage rate was observed in blastocysts on D2 and D7. Data were analyzed by SPSS 16.0 software using analysis of variance (ANOVA), Student"s t-test was used to detect differences between groups, and chi-square analysis for in vitro production results (P 0.05 ). The results differed between conventional (31.06%) and sexed semen (21.10%) in the obtainment of blastocyst. When the blastocyst production was individually compared in sexed semen samples (27.69%, 17.93% and 25.56%, bulls 1, 2 and 3, respectively) we verified T2 T1 and T1 = T3 and T2 = T3. [...](AU)

Avaliou-se a taxa de produção de blastocisto in vitro utilizando-se o sêmen bovino sexado. Foram utilizados três reprodutores para verificar a variação individual do sêmen, taxas de clivagem e produção embrionária. O trabalho utilizou-se de biotécnicas reprodutivas, análise computadorizada do sêmen pós-descongelação e sondas fluorescentes para análises de integridade da célula espermática (membrana plasmática, membrana acrossomal e potencial mitocondrial). Um total de 959 oócitos passou por etapas de maturação in vitro, fertilização in vitro (sexado, n= 473; convencional, n = 486) e cultivo in vitro. A taxa de clivagem foi observada no D2 e a de blastocistos no D7. Os dados foram analisados pelo programa SPSS 16.0 empregando-se a análise de variância (ANOVA), sendo o teste t-Student usado para detectar diferenças entre os grupos e o Qui-quadrado  para análise dos resultados da produção in vitro (P 0,05). Os resultados diferiram entre o sêmen convencional (31,06%) e sexado (21,10%) para produção de blastocisto. Quando comparada a produção de blastocisto individualmente nas amostras de sêmen sexado (27,69%; 17,93% e 25,56%, touros 1, 2 e 3, respectivamente), percebeu-se que T2 T1 e T1=T3 e T2=T3. Quanto às análises de cinética espermática, as amostras de sêmen sexado mostraram diferençasentre os touros nas variáveis velocidade curvilínea, velocidade linear e velocidade do trajeto em que o T1(117,7±1,6 µm/s; 60,0±0,3 µm/s; 73,6±0,4 µm/s, respectivamente) quando comparado aostouros T2 (80,2±2,3 µm/s; 47,0±2,0 µm/s; 57,7±0,9 µm/s, respectivamente) e T3 (86,4±5,7 µm/s; 46,2±2,7 µm/s; 53,8±2,8 µm/s, respectivamente) obteve valores mais elevados. As análises daintegridade da célula espermática não diferiram entre as amostras de sêmen convencional, já no sêmen sexado a integridade de membrana foi a variável que diferiu estatisticamente entre os touros, em que o T1 (38 ±2,7) diferiu do T3(53,8± 1,8) (P=0,009), mas não divergiu do T2 (44,1±4,4).[...](AU)

Corantes comumente empregados na citogenética vegetal / Dyes usually employed in plant cytogenetics

O emprego dos corantes, na citogenética vegetal, data de muitos anos, uma vez que as pesquisas nas áreas da citologia e histologia vêm sendo desenvolvidas constantemente desde os primeiros estudos celulares no século XIX. Inicialmente, eram extraídos de fontes vegetais ou animais, sendo atualmente produzidos sinteticamente em escala comercial. Os corantes são classificados em não fluorescentes e fluorescentes, conforme suas propriedades químicas e a escolha de uso é de acordo com o tipo de estrutura celular ou grupo celular a ser analisado. A diversidade de tipos e compostos químicos existentes nos diferentes corantes proporciona sua aplicação em estudos avançados na citogenética clássica e molecular. Uma revisão de suas propriedades químicas e emprego é apresentada para os corantes não fluorescentes orceína, hematoxilina, Giemsa, carmin; e para os fluorescentes 4',6-diamidino-2-fenilindol (DAPI), cromomicina A (CMA), fluoresceína e rodamina.(AU)

The use of dyes in plant cytogenetics goes back many years, as research in the fields of cytology and histology has been constantly developed since the first cellular studies in the 19(th) century. Initially they were taken from plant or animal sources, and now they are produced synthetically on a commercial scale. These dyes are classified in fluorescent and non-fluorescent, according to their chemical properties and the choice of use is based on the type of cell structure or cell group to be analyzed. The diversity of types and chemical compounds available in different dyes provides their application in advanced and classical cytogenetics studies. A review of their chemical properties and use is presented for the non-fluorescent dyes orcein, hematoxylin, Giemsa, and carmine; and for the fluorescent dyes 4',6-diamidino-2-phenylindole (DAPI), chromomycin A (CMA), fluorescein, and rhodamine.(AU)

Dyes usually employed in plant cytogenetics / Corantes comumente empregados na citogenética vegetal

The use of dyes in plant cytogenetics goes back many years, as research in the fields of cytology and histology has been constantly developed since the first cellular studies in the 19th century. Initially they were taken from plant or animal sources, and now they are produced synthetically on a commercial scale. These dyes are classified in fluorescent  and non-fluorescent, according to their chemical properties and the choice of use is based on the type of cell structure or cell group to be analyzed. The diversity of types and chemical compounds available in different dyes provides their application in advanced and classical cytogenetics studies. A review of their chemical properties and use is presented for the non-fluorescent dyes orcein, hematoxylin, Giemsa, and carmine; and for the fluorescent dyes 4,6-diamidino-2-phenylindole (DAPI), chromomycin A (CMA), fluorescein, and rhodamine.

O emprego dos corantes, na citogenética vegetal, data de muitos anos, uma vez que as pesquisas nas áreas da citologia e histologia vêm sendo desenvolvidas constantemente desde os primeiros estudos celulares no século XIX. Inicialmente, eram extraídos de fontes vegetais ou animais, sendo atualmente produzidos sinteticamente em escala comercial. Os corantes são classificados em não fluorescentes e fluorescentes, conforme suas propriedades químicas e a escolha de uso é de acordo com o tipo de estrutura celular ou grupo celular a ser analisado. A diversidade de tipos e compostos químicos existentes nos diferentes corantes proporciona sua aplicação em estudos avançados na citogenética clássica e molecular. Uma revisão de suas propriedades químicas e emprego é apresentada para os corantes não fluorescentes orceína, hematoxilina, Giemsa, carmin; e para os fluorescentes 4,6-diamidino-2-fenilindol (DAPI), cromomicina A (CMA), fluoresceína e rodamina.

Dyes usually employed in plant cytogenetics / Corantes comumente empregados na citogenética vegetal

The use of dyes in plant cytogenetics goes back many years, as research in the fields of cytology and histology has been constantly developed since the first cellular studies in the 19th century. Initially they were taken from plant or animal sources, and now they are produced synthetically on a commercial scale. These dyes are classified in fluorescent  and non-fluorescent, according to their chemical properties and the choice of use is based on the type of cell structure or cell group to be analyzed. The diversity of types and chemical compounds available in different dyes provides their application in advanced and classical cytogenetics studies. A review of their chemical properties and use is presented for the non-fluorescent dyes orcein, hematoxylin, Giemsa, and carmine; and for the fluorescent dyes 4,6-diamidino-2-phenylindole (DAPI), chromomycin A (CMA), fluorescein, and rhodamine.(AU)

O emprego dos corantes, na citogenética vegetal, data de muitos anos, uma vez que as pesquisas nas áreas da citologia e histologia vêm sendo desenvolvidas constantemente desde os primeiros estudos celulares no século XIX. Inicialmente, eram extraídos de fontes vegetais ou animais, sendo atualmente produzidos sinteticamente em escala comercial. Os corantes são classificados em não fluorescentes e fluorescentes, conforme suas propriedades químicas e a escolha de uso é de acordo com o tipo de estrutura celular ou grupo celular a ser analisado. A diversidade de tipos e compostos químicos existentes nos diferentes corantes proporciona sua aplicação em estudos avançados na citogenética clássica e molecular. Uma revisão de suas propriedades químicas e emprego é apresentada para os corantes não fluorescentes orceína, hematoxilina, Giemsa, carmin; e para os fluorescentes 4,6-diamidino-2-fenilindol (DAPI), cromomicina A (CMA), fluoresceína e rodamina.(AU)

Techniques for sperm evaluation using fluorescent probes / Técnicas para avaliação espermática utilizando sondas fluorescentes

A variety of laboratory tests were developed to obtain more reliable results of sperm evaluation and increase the accuracy of sperm fertility predictions. These tests detected damage of sperm specific compartments or organelles, which cannot be detected in routine sperm analysis. The use of fluorescent probes and detection using fluorescent microscopy or flow cytometry is an important tool but a more precise and accurate laboratory test is needed. Propidium iodide and 6-carboxyfluorescein diacetate are used for evaluations of plasmatic membrane integrity. Fluorescein isothiocyanate, associated with conjugated lecithin Psium sativum or Arachis hypogaea, are used for evaluations of acrosome integrity. Two probes, MitoTracker or Rhodamine123, are generally used to measure the absence or presence of mitochondrial potential. However, a better option is 5,5; 6,6 - tetrachloro - 1,1; 3,3 -tetraetilbenzimidazolil-carbocyanine (JC-1) dye, which assesses not only the presence of mitochondrial potential and distinguished spermatozoa with poorly and highly functional mitochondria. Two techniques, TUNEL or COMETA, and the Acridine Orange Test (AOT) dye are used to evaluate chromatin integrity. A fluorescence technique based on chlortetracycline (CTC) or Merocyanine 540 is used to estimate whether sperm pass by or through the capacitation process. This review focuses on the fluorescent probes that are most widely used to evaluate plasma membrane integrity, capacitation, acrosome integrity, chromatin integrity and mitochondrial potential.(AU)

Diversos testes laboratoriais têm sido desenvolvidos com o intuito de obter resultados confiáveis nas avaliações espermáticas, aumentando a qualidade e a confiabilidade do sêmen utilizado em técnicas de reprodução assistida. Esses testes têm permitido detectar danos em compartimentos e organelas específicas da célula espermática, que não são detectados nas análises de rotina. Dentre esses testes, o uso de sondas fluorescentes e sua detecção em microscópio de epifluorescência ou citometria de fluxo se tornaram ferramenta importante quando uma avaliação mais acurada é necessária. Para a avaliação da integridade de membrana plasmática, pode ser utilizado o iodeto de propídio, associado ao diacetato de 6-carboxifluoresceína. As avaliações de integridade acrossomal podem ser feitas através do isotiocianato de fluoresceína, associado às lecitinas conjugadas, como a Psium sativum ou Arachis hypogaea. O potencial mitocondrial pode ser avaliado quanto à ausência ou presença através da sonda Mitotracker ou Rodamina 123. Outra opção ainda melhor pode ser observada utilizando o corante iodeto de 5,5; 6,6 tetracloro 1,1; 3,3 tetraetilbenzimidazolil-carbocianina (JC-1), este corante avalia a presença do potencial mitocondrial e avalia quanto à classificação do grau. Para avaliar a integridade de cromatina, pode ser empregado o uso de duas técnicas conhecidas como TUNEL ou COMETA, ou pode ser utilizado o corante Acridine Orange Test (AOT). Para avaliar se o espermatozoide está passando ou passou pelo processo de capacitação, pode ser utilizada a técnica de fluorescência a base de clortetraciclina, o CTC, quelante do cálcio, ou a Merocianina 540. Esta revisão enfatiza as principais sondas fluorescentes disponíveis para avaliar a integridade de membrana plasmática, capacitação, integridade acrossomal, integridade da cromatina, e potencial mitocondrial.(AU)