In the field of assisted reproductive techniques (ART), computer-assisted sperm analysis (CASA) systems have proved their utility and potential for assessing sperm quality, improving the prediction of the fertility potential of a seminal dose. Although most laboratories and scientific centers use commercial systems, in the recent years certain free and open-source alternatives have emerged that can reduce the costs that research groups have to face. However, these open-source alternatives cannot analyze sperm kinetic responses to different stimuli, such as chemotaxis, thermotaxis or rheotaxis. In addition, the programs released to date have not usually been designed to encourage the scalability and the continuity of software development. We have developed an open-source CASA software, called OpenCASA, which allows users to study three classical sperm quality parameters: motility, morphometry and membrane integrity (viability) and offers the possibility of analyzing the guided movement response of spermatozoa to different stimuli (useful for chemotaxis, thermotaxis or rheotaxis studies) or different motile cells such as bacteria, using a single software. This software has been released in a Version Control System at Github. This platform will allow researchers not only to download the software but also to be involved in and contribute to further developments. Additionally, a Google group has been created to allow the research community to interact and discuss OpenCASA. For validation of the OpenCASA software, we analysed different simulated sperm populations (for chemotaxis module) and evaluated 36 ejaculates obtained from 12 fertile rams using other sperm analysis systems (for motility, membrane integrity and morphology modules). The results were compared with those obtained by Open-CASA using the Pearson’s correlation and Bland-Altman tests, obtaining a high level of correlation in all parameters and a good agreement between the different used methods and the OpenCASA. With this work, we propose an open-source project oriented to the development of a new software application for sperm quality analysis. This proposed software will use a minimally centralized infrastructure to allow the continued development of its modules by the research community.

No abstract

Reactive oxygen species (ROS) play an essential role in mammalian sperm capacitation. NADPH oxidase 5 (NOX5) has been described as the main source of ROS production in some mammalian spermatozoa, such as human and equine. On the other hand, melatonin can decrease cellular ROS levels and regulates NOX activity in somatic cells. Therefore, the objectives of this work were (1) to identify NOX5 in ram spermatozoa and analyze its possible changes during in vitro capacitation and (2) to investigate the effect of melatonin on NOX5 expression and localization and on superoxide levels in capacitated ram spermatozoa. Protein bands associated with NOX5 were detected by Western blot analysis. Likewise, indirect immunofluorescence (IIF) revealed six different immunotypes for NOX5, which varied throughout in vitro capacitation. Superoxide (O2⋅–), evaluated by DHE/Yo-Pro-1, rose after in vitro capacitation and in the presence of the calcium ionophore A23187 but decreased in the presence of the NOX inhibitor GKT136901. GKT also reduced the percentage of capacitated and acrosome-reacted spermatozoa that had increased during incubation in capacitating conditions. The presence of melatonin at micromolar concentrations avoided the increment in O2⋅– and the changes in NOX5 immunotypes provoked by capacitation. In conclusion, NOX5 is present in ram spermatozoa and the changes in its distribution, associated with sperm capacitation, can be prevented by melatonin. To this extent, it could imply that melatonin exerts its antioxidant role, at least in part, by modulating NOX5 activity during ram sperm capacitation.

Testicular ultrasound is a non-invasive technique that could be very useful for predicting ram seminal quality. Recent software developments allow macroscopic and microscopic evaluation of testicular parenchyma. Thus, the aim of this study was to evaluate the testicular echotexture using ultrasound-video analysis and investigate its possible correlation with semen quality. Nine rams were evaluated for one year using a portable ultrasound scanner and the echotexture was analyzed with ECOTEXT® software. The number of black (Ec1), white (Ec2), and grey pixels (Ec3), tubular density (TD), lumen area (LA), and lumen diameter (LD) were analyzed. Semen was collected by an artificial vagina the same day and the sperm concentration, morphology, motility, viability, phosphatidylserine (PS) translocation, reactive-oxygen-species (ROS) levels, DNA damage and capacitation state were evaluated. Ec2 and Ec3 correlated positively with “bad quality” sperm parameters (the percentage of spermatozoa with high ROS levels, with PS translocation and proximal cytoplasmic droplets), and negatively with motility. In contrast, TD and LA showed a positive correlation with “good quality” parameters (motility or normal morphology) and a negative correlation with spermatozoa with high ROS levels, with DNA fragmentation, and proximal or distal cytoplasmic droplets. Thus, echotexture analysis by ultrasound-video analysis could be a valuable tool for assessing ram fertility.

No abstract