Sperm motility and sperm concentration were objectively assayed as previously described (Santiago Moreno et al., 2012) using a computer aided sperm analyses (CASA) system coupled to a phase contrast contrast microscope (Nikon Eclipse model 50i; negative contrast) and employing Sperm Class Analyzer (SCA R ) v.4.0 software (Microptic S.L., Barcelona, Spain). Sperm samples were diluted 1:41 (v:v) in Lake and Ravie medium and loaded onto a warmed (38oC) 20 μm LejaR 8-chamber slide (Leja Products B.V., Nieuw-Vennep, The Netherlands). The percentage of immotile sperm (i.e., not moving at all), the percentage of sperm showing non-progressive motility (sperm moving their tails but not moving forward, or swimming slowly but in a curve) and the percentage showing progressive motility (sperm swimming quickly forward in a straight line) were recorded. Sperm movement characteristics - straight-line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP) - were also analyzed by CASA. According to their general velocity, spermatozoa were classified as slow (<10 μm/sec), medium (10–50 μm/sec) or rapid (>50 μm/sec). The amplitude of lateral head displacement (ALH) and beat-cross frequency (BCF) were also analyzed. A minimum of three fields and 500 sperm tracks were examined at 10x for each sample chamber (image acquisition rate 25 frames/s).
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