Sperm motility analysis

The sperm motility parameters of the ejaculates were measured using computer-assisted sperm analysis (CASA) as described previously [22]. Video recordings of motile sperm were captured using a microscope with a 4 x negative phase objective and a Sony CCD black-and-white video camera (SPTM108CE). MicroCell 50-μm-deep, two-chamber slides (Conception Technologies, San Diego, USA) were mounted on a heated stage (37°C). Semen samples were diluted in BIOXcell extender (IMV Technologies, L'aigle, France) at a ratio of 1:40 prior to motility measurements. Sperm motility parameters were analysed using a Hobson Sperm Tracker (Hobson Vision Ltd., Baslow, UK) as described previously [23]. Each sample was analysed in duplicate. For further analysis, the percentage of total motile sperm was assessed, in addition to the movement trajectory parameters: VSL, VCL, ALH, VAP, and LIN.