Effect of incubation and analysis temperatures on sperm kinematics and morphometrics during human semen analysis / Efecto de las temperaturas de incubación y análisis sobre la cinemática y la morfometría de los espermatozoides durante el análisis de semen humano

Introduction: Human semen analysis must be performed after the liquefaction of the ejaculate. This takes place about 30min after ejaculation and samples must be maintained in the lab during this time. The temperatures for this incubation and the final analysis of motility are crucial but seldom taken into account. This study aims to examine the effect of these temperatures on various sperm parameters both manually (sperm count, motility, morphology, viability, chromatin condensation and maturation and DNA fragmentation) and CASA (kinematics and morphometrics, using an ISAS®v1 CASA-Mot and CASA-Morph systems, respectively) analyzed. Methods: Seminal samples from thirteen donors were incubated for 10min at 37°C followed by additional 20min at either room temperature (RT, 23°C) or 37°C and then examined following WHO 2010 criteria. Results: The data obtained show that there were no significant differences (P>0.05) in the subjective sperm quality parameters with incubation temperature. On the other hand, the head sperm morphometric parameters were significantly higher after room temperature incubation showing, in addition, lower ellipticity (P<0.05). Furthermore, kinematic parameters were evaluated both at RT and 37°C for the two incubation temperatures. In general, the four temperature combinations showed that kinematic parameters followed this order: RT-RT Conclusions: Our results showed that temperature control during both incubation and analysis is needed for accurate semen analysis, recommending the use of 37°C during the entire process. (AU)

Introducción: El análisis de semen humano debe realizarse después de la licuefacción del eyaculado. Esto ocurre aproximadamente a los 30minutos después de la eyaculación. Las temperaturas para esta incubación y el análisis final de la motilidad son cruciales, pero rara vez se tienen en cuenta. Este estudio tiene como objetivo examinar el efecto de estas temperaturas en varios parámetros de los espermatozoides tanto de forma manual (recuento de espermatozoides, motilidad, morfología, viabilidad, condensación y maduración de la cromatina y fragmentación del ADN) como CASA (cinemática y morfometría, utilizando un CASA-Mot ISAS®v1 y Sistemas CASA-Morph, respectivamente) analizados. Métodos: Las muestras seminales de 13 donantes se incubaron durante 10minutos a 37°C, seguidas de 20minutos adicionales a temperatura ambiente (TA, 23°C) o a 37°C y luego se examinaron siguiendo los criterios de la OMS 2010. Resultados: Los datos obtenidos muestran que no hubo diferencias significativas (p>0,05) en los parámetros subjetivos de calidad del esperma con la temperatura de incubación. Por otro lado, los parámetros morfométricos de la cabeza de los espermatozoides fueron significativamente más altos después de la incubación a temperatura ambiente, mostrando, además, una elipticidad más baja (p<0,05). Además, los parámetros cinemáticos se evaluaron tanto a temperatura ambiente como a 37°C para las dos temperaturas de incubación. En general, las cuatro combinaciones de temperatura mostraron que los parámetros cinemáticos siguieron este orden: RT-RT < RT-37 < 37-37 < 37-RT (temperaturas de incubación y análisis, respectivamente). Conclusiones: Nuestros resultados mostraron que el control de la temperatura durante la incubación y el análisis es necesario para un análisis de semen preciso, recomendando el uso de 37°C durante todo el proceso. (AU)

Effect of incubation and analysis temperatures on sperm kinematics and morphometrics during human semen analysis.

INTRODUCTION: Human semen analysis must be performed after the liquefaction of the ejaculate. This takes place about 30min after ejaculation and samples must be maintained in the lab during this time. The temperatures for this incubation and the final analysis of motility are crucial but seldom taken into account. This study aims to examine the effect of these temperatures on various sperm parameters both manually (sperm count, motility, morphology, viability, chromatin condensation and maturation and DNA fragmentation) and CASA (kinematics and morphometrics, using an ISAS®v1 CASA-Mot and CASA-Morph systems, respectively) analyzed. METHODS: Seminal samples from thirteen donors were incubated for 10min at 37°C followed by additional 20min at either room temperature (RT, 23°C) or 37°C and then examined following WHO 2010 criteria. RESULTS: The data obtained show that there were no significant differences (P>0.05) in the subjective sperm quality parameters with incubation temperature. On the other hand, the head sperm morphometric parameters were significantly higher after room temperature incubation showing, in addition, lower ellipticity (P<0.05). Furthermore, kinematic parameters were evaluated both at RT and 37°C for the two incubation temperatures. In general, the four temperature combinations showed that kinematic parameters followed this order: RT-RT

Human kinematic and morphometric sperm subpopulation analysis using CASA technology: A new approach to spermatozoa classification / Análisis cinemático y morfométrico de subpoblaciones de espermatozoides humanos utilizando tecnología CASA: un nuevo enfoque para la clasificación de los espermatozoides

Introduction: Semen analysis is a clinical method aimed at determining the fertility of a male individual. The traditional subjective method lacks the reliability that can be achieved by computer-assisted sperm analysis (CASA) technology. Unfortunately, this technology has only been used when taking into consideration individually different sperm characteristics. The aim of this work is to present an integrative mathematical approach that considers different seminal variables to establish human sperm subpopulations. Methods: Samples were obtained from thirteen volunteers via masturbation and were analyzed by the routine subjective method and two objective systems, CASA Motility (CASA-Mot) and CASA Morphology (CASA-Morph). Results: Seminogram variables were reduced to three principal components (PC) showing two subpopulations. Kinematics and morphometric variables each rendered three PCs for four subpopulations. Conclusions: These results lay the foundations for future studies including different geographical, social, ethnic and age range conditions with the aim of achieving a definitive view of the human semen picture. (AU)

Introducción: El análisis de semen es el método clínico para determinar la fertilidad masculina. El método subjetivo tradicional carece de la fiabilidad, que se puede obtener con el uso de la tecnología del análisis de semen asistido por ordenador (CASA). Desafortunadamente, esta tecnología se ha venido utilizando únicamente teniendo en cuenta de forma independiente las diversas características de los espermatozoides. El objetivo del presente estudio es presentar una aproximación matemática que incluye diversas variables seminales para definir las posibles subpoblaciones espermáticas. Métodos: Las muestras se obtuvieron por masturbación de 13 voluntarios, que se analizaron de forma subjetiva, así como con 2 sistemas objetivos, para el análisis de la movilidad (CASA-Mot) y la morfología (CASA-Morph). Resultados: Tanto las variables cinemáticas como las morfométricas rindieron 3 componentes principales y 4 subpoblaciones. Conclusión: Estos resultados sientan las bases para estudios futuros que incluyan diferencias geográficas, sociales, étnicas o de rango de edad con el ánimo de obtener una definición concluyente sobre las características seminales de la especie humana. (AU)

Human kinematic and morphometric sperm subpopulation analysis using CASA technology: A new approach to spermatozoa classification.

INTRODUCTION: Semen analysis is a clinical method aimed at determining the fertility of a male individual. The traditional subjective method lacks the reliability that can be achieved by computer-assisted sperm analysis (CASA) technology. Unfortunately, this technology has only been used when taking into consideration individually different sperm characteristics. The aim of this work is to present an integrative mathematical approach that considers different seminal variables to establish human sperm subpopulations. METHODS: Samples were obtained from thirteen volunteers via masturbation and were analyzed by the routine subjective method and two objective systems, CASA Motility (CASA-Mot) and CASA Morphology (CASA-Morph). RESULTS: Seminogram variables were reduced to three principal components (PC) showing two subpopulations. Kinematics and morphometric variables each rendered three PCs for four subpopulations. CONCLUSIONS: These results lay the foundations for future studies including different geographical, social, ethnic and age range conditions with the aim of achieving a definitive view of the human semen picture.

Sperm morphological and morphometric evaluation in captive collared peccaries (Pecari tajacu) / Avaliação morfológica e morfométrica de espermatozoides de catetos (Pecari tajacu)

The aim of this study was to compare different staining methods for the evaluation of sperm morphology by light microscopy and also to describe the morphometry of the entire sperm in collared peccaries (Pecari tajacu). Semen from 10 males was obtained by electroejaculation and evaluated for sperm motility, vigor, and concentration. Semen smears were prepared through three different staining methods: Bengal rose, brome-phenol blue, and eosin-nigrosin. Smears were evaluated under light microscopy and sperm morphologic alterations were determined in percentage. In addition, sperm morphometric analysis was conducted by light microscopy coupled to image analyzer software. The smears stained with Bengal Rose provide the best results for the visualization of the sperm tail, midpiece, and head. The use of eosin-nigrosin stain did not allow an adequate impregnation, and some sperm presented a few contrasts with the background. A higher incidence of bent coiled tails was verified in the use of brome-phenol blue staining (P<0.05). Through morphometric evaluation, it was observed that the tail occupies the greatest proportion (89%) of the sperm which presents a discretely elongated head. According to the results, the use of the Bengal Rose stain is recommended for the morphologic evaluation of the collared peccary sperm.

O objetivo deste estudo foi comparar diferentes métodos de coloração para avaliação da morfologia espermática por microscopia de luz e também descrever a morfometria completa de espermatozoides de catetos (Pecari tajacu). Sêmen de 10 machos foi obtido por eletroejaculação e avaliado quanto à motilidade espermática, vigor e concentração. Foram preparados por três diferentes métodos de coloração: Rosa de Bengala, Azul de Bromofenol e Eosina-Nigrosina. Os esfregaços foram avaliados por microscopia de luz, e determinado o percentual das alterações morfológicas. Ainda, a análise da morfometria espermática foi realizada por microscópio de luz acoplado a um softwere de análise de imagens. Os esfregaços corados com Rosa de Bengala apresentaram melhores resultados de visualização da cauda, peça intermediária e cabeça dos espermatozoides. O uso do corante Eosina-Nigrosina não permitiu uma adequada impregnação e alguns dos espermatozoides apresentaram pouco contraste com o fundo da lâmina. Uma maior incidência de cauda fortemente enrolada foi verificada com o uso do corante Azul de Bromofenol (P<0.05). Através da avaliação morfométrica foi observada que a cauda ocupa a maior proporção (89%) do espermatozoide, e a cabeça apresenta-se discretamente alongada. De acordo com os resultados, o uso do corante Rosa de Bengala é recomendado para a avaliação morfológica de espermatozoides de catetos.

Avaliação computacional da compactação da cromatina e de características morfométricas da cabeça de espermatozoides de coelho (Oryctolagus cuniculus) / Computational evaluation of chromatin condensation and morphometric characteristics of rabbit (Oryctolagus cuniculus) sperm head

Investigou-se a correlação entre a morfometria da cabeça e a intensidade da condensação e heterogeneidade da cromatina em espermatozoides de coelho (Oryctolagus cuniculus). Para tal, utilizaram-se 35 esfregaços de sêmen de coelhos da raça Nova Zelândia, corados com azul de toluidina e avaliados por análise de imagem computacional. As imagens foram obtidas digitalmente em tons de cinza e avaliadas por algoritmos desenvolvidos em ambiente de programação Scilab. As mensurações obtidas da cabeça dos espermatozoides foram área, perímetro, comprimento, largura, relação comprimento largura, elipsidade, fator de forma, descritores Fourier e simetria lateral e anteroposterior. Também foram avaliadas a intensidade da compactação e a heterogeneidade da cromatina espermática. Os espermatozoides de coelho apresentaram compactação e heterogeneidade cromatínica mais intensas do que os de touro e observou-se correlação significativa entre características morfométricas da cabeça e compactação e heterogeneidade cromatínica. Conclui-se que a cromatina é importante para a constituição morfológica da cabeça de espermatozoides de coelho e que a cromatina espermática de coelho é naturalmente mais heterogênea e menos compactada que a de touro.

The correlation between the head morphometry and the intensity of condensation and heterogeneity of sperm chromatin were investigated in rabbit (Oryctolagus cuniculus). To this, 35 semen smears from New Zealand rabbits were stained with toluidine blue and evaluated by computer image analysis. The images were obtained in digital grayscale and analyzed by algorithms developed in the Scilab programming environment. The measurements obtained from the sperm heads were area, perimeter, length, width, length:width ratio, ellipticity, shape factor, lateral and anterior-posterior symmetries, and Fourier descriptors. The intensity and heterogeneity of the compaction of sperm chromatin was also evaluated. The rabbit spermatozoa showed chromatin heterogeneity and condensation more intense than the bull spermatozoa, and it was observed correlation between morphometric characteristics of the head and chromatin compaction and heterogeneity. The results suggest that chromatin is important for the morphological constitution of the head morphology spermatozoa head, as well as, rabbit sperm chromatin is inherently more heterogeneous and less condensed than the bull sperm chromatin.