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.

Optimal frame rate when there were stallion sperm motility evaluations and determinations for kinematic variables using CASA-Mot analysis in different counting chambers.

This study was conducted to determine optimum image capture frame rates (FRO) when there was evaluation of different types of counting chambers used for CASA-Mot determinations of stallion sperm motility. Sperm VCL was determined at frame rates of 25-250 f/s in: 1) Spermtrack® (Spk) 10 and 20 chambers (drop displacement-type chambers 10 and 20 µm-deep respectively; and 2) ISAS®D4C10, ISAS®D4C20 (10 and 20 µm-deep respectively) and ISAS®D4C20 L (20 µm-deep) capillary loaded chambers. Values for different sperm kinematic variables were determined using each chamber at 250 f/s, which is the maximum frame rate that the software can be used for analyses. With evaluation of Spk chambers, there was a greater curvilinear velocity (VCL), average path velocity (VAP), straight line velocity (STR), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) values (P < 0.05) than with capillary loaded chambers, with there being greatest values with 20 µm-deep chambers. With the Spk10 chamber, VCL and ALH were greater at the chamber centre than periphery. There were no such differences for the Spk20 chamber. With evaluation of the D4C10 chamber, VSL and STR were less when there was a sperm deposition point towards the chamber end, while there were the opposite for the D4C20 chamber. When there was evaluation of the D4C20 chamber, there were also greater VCL, WOB and BCF values in distal areas. With use of most of these chambers, data should be collected from different fields and means determined, however, this is not necessary with Spk20 chambers.

Combined effects of type and depth of counting chamber, and rate of image frame capture, on bull sperm motility and kinematics.

Semen quality assessment requires accurate, reliable and objective methods for examination of sperm variables including sperm motility. For preparation of semen samples for artificial insemination, as a genetic resource, samples that are used for insemination need to have the capacity to result in a highly acceptable fertility rate. Several methods have been developed for evaluation of bull sperm in laboratory conditions and for preparation of doses for artificial insemination. Computer-assisted semen analyses can provide objective information on various sperm variables. Nevertheless, this equipment requires fine-calibrations considering differences among species, breeds and conditions for sample evaluation and data analyses. In the present study, there was examination of the interaction between factors such as image frame rate and type and depth of counting chamber in which sperm were evaluated, together with differences between bulls of four breeds. The use of the Spermtrack® reusable 10 µm-depth chamber provided more reliable results than results obtained using disposable chambers (10 and 20 µm depth). A capture rate of at least 90 fps is required for assessment of sperm motility percentage, whereas a rate of 250 fps is needed for obtaining consistent kinematic data. Differences among breeds in the present study indicate conditions for sperm analyses should include specific equipment calibrations for each breed. These results contribute to development of more precise conditions for assessments of bull sperm quality taking into account breed differences and the requirement each breed has for the adequate evaluation and preparation of samples for artificial insemination.

Dog sperm swimming parameters analysed by computer-assisted semen analysis of motility reveal major breed differences.

Dogs have undergone an intensive artificial selection process ever since the beginning of their relationship with humans. As a consequence, a wide variety of well-defined breeds exist today. Due to the enormous variation in dog phenotypes and the unlikely chance of gene exchange between them, the question arises as to whether they should still be regarded as a single species or, perhaps, they be considered as different taxa that possess different reproductive traits. The aim of this study was therefore to characterize some male reproductive traits, focusing on kinematic characteristics of dog spermatozoa from several breeds. Thirty-seven dogs from the following breeds were used: Staffordshire Bull Terrier, Labrador Retriever, Spanish Mastiff, Valencian Rat Hunting Dog, British Bulldog and Chihuahua. Semen samples were obtained via manual stimulation and diluted to a final sperm concentration of 50 million/ml, and they were subsequently analysed by the computer assisted semen analysis (CASA-Mot) ISAS® v1 system. Eight kinematic parameters were evaluated automatically. All parameters showed significant different values among breeds and among individuals within each breed. The fastest sperm cells were those of Staffordshire Bull Terriers and the slowest were recorded in Chihuahuas. The intra-male coefficient of variation (CV) was higher than the inter-male CV for all breeds with the Staffordshire Bull Terrier showing the lowest values. When taking into consideration the cells by animal and breed, discriminant analyses showed a high capability to predict the breed. Cluster analyses showed a hierarchical classification very close to that obtained after phylogenetic studies with genome markers. In conclusion, future workers on dog spermatozoa should bear in mind major differences between breeds and realize that results cannot be extrapolated from one to another. Because sperm characteristics are associated with breed diversity, dogs may represent a good model to examine changes in reproductive parameters associated with selection processes.

Combined effect of type and capture area of counting chamber and diluent on Holstein bull sperm kinematics.

The evaluation of sperm motion is crucial for processing of seminal doses for artificial insemination. Here, the combined effect of the type and capture area of three counting chambers, together with the type of diluent employed, on sperm motility was analysed. Ejaculates from thirteen Holstein bulls were used for sperm kinematic analysis with the ISAS® v1 CASA-Mot system, using two capillary-loaded counting chambers (Leja® and Cell-Vu® ) and one drop displacement chamber (Makler® ). Nine fixed positions were analysed per chamber type, considering central and lateral and three longitudinal fields. Independent of the diluent used, differences were found between the three chambers. Independent of the extender, no differences in x-axis were observed with Cell-Vu® , while using Leja® , some parameters showed lower values in the centre than in lateral areas. In both counting chambers, the lowest values were observed in the distal area. Results obtained with the two diluents were highly different with a very low correlation between them. In conclusion, the capture area inside the chambers leads to significant changes in sperm kinematic parameters and different dilution media introduce considerable differences in the motility patterns. It is necessary to optimise sampling methods and specific set-ups to be used with CASA-Mot technology.

Effect of frame rate capture frequency on sperm kinematic parameters and subpopulation structure definition in boars, analysed with a CASA-Mot system.

Motility is the most widely used indicator of sperm quality. Computer-Assisted Semen Analysis (CASA) allows the objective evaluation of sperm motility parameters. CASA technology is a common tool to predict semen doses in farm animal reproduction. The kinds of video cameras used until now for image acquisition have presented limited frame rates (FR), which have a negative influence on the quality of the obtained data. The aim of the present work was to define the optimal frame rate for a correct evaluation of boar sperm motility and its subpopulation structure. Eighteen ejaculates from nine mature boars of the Pietrain breed were used. Using the ISAS® v1 CASA-Mot system, with a video camera working up to 200 Hz, six FRs (25, 50, 75, 100, 150 and 200 fps) were compared. ISAS® D4C20 counting chambers, warmed to 37°C, were used. FR affected all the kinematic parameters, with curvilinear velocity (VCL) and BCF the most sensitive ones. All the parameters showed differences among animals. Non-linear correlation showed the asymptotic level for VCL at 212 fps, being the highest FR for all the parameters. For future studies based just on progressive motility, almost 100 fps FR for 0.5 s must be used, while when kinematics must be considered, almost 212 fps for one-second should be analysed. Three principal components were obtained (velocity, progressivity and oscillation), being similar at 50 and 200 fps. Cells were grouped in four subpopulations but with different kinematic and cellular distribution at both FRs.

Effect of counting chamber depth on the accuracy of lensless microscopy for the assessment of boar sperm motility.

Sperm motility is one of the most significant parameters in the prediction of male fertility. Until now, both motility analysis using an optical microscope and computer-aided sperm analysis (CASA-Mot) entailed the use of counting chambers with a depth to 20µm. Chamber depth significantly affects the intrinsic sperm movement, leading to an artificial motility pattern. For the first time, laser microscopy offers the possibility of avoiding this interference with sperm movement. The aims of the present study were to determine the different motility patterns observed in chambers with depths of 10, 20 and 100µm using a new holographic approach and to compare the results obtained in the 20-µm chamber with those of the laser and optical CASA-Mot systems. The ISAS®3D-Track results showed that values for curvilinear velocity (VCL), straight line velocity, wobble and beat cross frequency were higher for the 100-µm chambers than for the 10- and 20-µm chambers. Only VCL showed a positive correlation between chambers. In addition, Bayesian analysis confirmed that the kinematic parameters observed with the 100-µm chamber were significantly different to those obtained using chambers with depths of 10 and 20µm. When an optical analyser CASA-Mot system was used, all kinematic parameters, except VCL, were higher with ISAS®3D-Track, but were not relevant after Bayesian analysis. Finally, almost three different three-dimensional motility patterns were recognised. In conclusion, the use of the ISAS®3D-Track allows for the analysis of the natural three-dimensional pattern of sperm movement.

CASA-Mot technology: how results are affected by the frame rate and counting chamber.

For over 30 years, CASA-Mot technology has been used for kinematic analysis of sperm motility in different mammalian species, but insufficient attention has been paid to the technical limitations of commercial computer-aided sperm analysis (CASA) systems. Counting chamber type and frame rate are two of the most important aspects to be taken into account. Counting chambers can be disposable or reusable, with different depths. In human semen analysis, reusable chambers with a depth of 10µm are the most frequently used, whereas for most farm animal species it is more common to use disposable chambers with a depth of 20µm . The frame rate was previously limited by the hardware, although changes in the number of images collected could lead to significant variations in some kinematic parameters, mainly in curvilinear velocity (VCL). A frame rate of 60 frames s-1 is widely considered to be the minimum necessary for satisfactory results. However, the frame rate is species specific and must be defined in each experimental condition. In conclusion, we show that the optimal combination of frame rate and counting chamber type and depth should be defined for each species and experimental condition in order to obtain reliable results.

Comparison of sperm motility subpopulation structure among wild anadromous and farmed male Atlantic salmon (Salmo salar) parr using a CASA system.

Atlantic salmon (Salmo salar) is an endangered freshwater species that needs help to recover its wild stocks. However, the priority in aquaculture is to obtain successful fertilisation and genetic variability to secure the revival of the species. The aims of the present work were to study sperm subpopulation structure and motility patterns in wild anadromous males and farmed male Atlantic salmon parr. Salmon sperm samples were collected from wild anadromous salmon (WS) and two generations of farmed parr males. Sperm samples were collected from sexually mature males and sperm motility was analysed at different times after activation (5 and 35s). Differences among the three groups were analysed using statistical techniques based on Cluster analysis the Bayesian method. Atlantic salmon were found to have three sperm subpopulations, and the spermatozoa in ejaculates of mature farmed parr males had a higher velocity and larger size than those of WS males. This could be an adaptation to high sperm competition because salmonid species are naturally adapted to this process. Motility analysis enables us to identify sperm subpopulations, and it may be useful to correlate these sperm subpopulations with fertilisation ability to test whether faster-swimming spermatozoa have a higher probability of success.

Anti-leishmanial evaluation of C2-aryl quinolines: mechanistic insight on bioenergetics and sterol biosynthetic pathway of Leishmania braziliensis.

A series of diverse simple C2-aryl quinolines was synthesized de novo via a straightforward synthesis based on the acid-catalyzed multicomponent imino Diels-Alder reactions. Seven selected quinolines were evaluated at different stages of Leishmania braziliensis parasite. Among them, the 6-ethyl-2-phenylquinoline 5f was able to inhibit the growth of promastigotes of this parasite without affecting the mammalian cells viability and decreasing the number of intracellular L. braziliensis amastigotes on BMDM macrophages. The mechanism of action studied for the selected compound consisted in: (1) alteration of parasite bioenergetics, by disrupting mitochondrial electrochemical potential and alkalinization of acidocalcisomes, and (2) inhibition of ergosterol biosynthetic pathway in promastigote forms. These results validate the efficiency of quinoline molecules as leishmanicide compounds.