spermatozoa go to the culture medium). In order to obtain the fraction rich in spermatozoa, the top layer is collected. It is still widely used and useful in normozoospermia. It allows to obtain fractions with more than 90% of PR spermatozoa. Density gradients. In this technique, a tube is filled with layers of liquids of different densities and semen is placed on the top layer. Then, The tube goes through a centrifugation to filter cell debris and non motile cells. After the centrifugation, healthy sperm are on the very bottom layer of the liquid in the tube, while debris and non-motile spermatozoa are in upper layers. This procedure takes approximately 60 minutes and it is specially indicated in oligozoospermia, asthenozoospermia and abundant debris samples. At the end, all the cells will arrive to the bottom, but those with more motility will arrive sooner. This procedure is often called just the "Percoll method", since Percoll was frequently used as the density medium, but other density mediums are now used. Filtration. It consist in a filter that does not allow every sperm to pass. It is less used nowadays and only spermatozoa with better motility will pass through the filter. However, nowadays there are new techniques which surpass these ones. For instance, we have PICSI, MACS or microfluidic chips. Measurement Numerous methods have been developed to assess the degree to which sperm cells are undergoing capacitation in vitro. Computer-aided sperm analysis (CASA) was developed in the 1980s for measuring sperm kinematics. CASA uses phase-contrast microscopy combined with sperm tracking software to analyze sperm motility parameters. Certain parameters such as curvilinear velocity (VCL), straightline velocity (VSL), average path velocity (VAP), and the amplitude of lateral head displacement (ALH) have been shown to be positively correlated with the acquisition of fertilization competency and are thus used to identify hyperactive sperm cell motility. While motility measurements are critical for identifying the presence of hyperactive motility, additional methods have been developed to identify the occurrence of the acrosome reaction. A simple method uses Coomassie brilliant blue G250 to stain cells, providing visual evidence of intact or reacted acrosomes. More advanced techniques employ fluorescent or electron microscopy methods. Fluorescein-conjugated Peanut agglutinin (FITC-PNA) or Pisum sativum agglutinin (FITC-PSA) can be used to fluorescently tag the acrosome of sperm cells, which can be then used to assess the status of the acrosome using a fluorescent microscope. Discovery The discovery