Assessing Spermatozoa Motility with CASA

Spermatozoa motility was assessed by the computer-assisted sperm assay (CASA) method according to World Health Organization guidelines48 . After 24 h of treatment, boar spermatozoa were incubated at 37.5 °C for 30 min then samples were placed in a pre-warmed counting chamber (MICROPTIC S.L., Barcelona, Spain). After euthanized, spermatozoa was collected from cauda epididymis of mice and suspended in DMEM/F12 medium with 10% FBS and incubated at 37.5 °C for 30 min then samples were placed in a pre-warmed counting chamber49 (link). The Micropic Sperm class analyzer (CASA system) was used in this investigation. It was equipped with a 20-fold objective, a camera adaptor (Eclipse E200, Nicon, Japan), and a camera (acA780-75gc, Basler, Germany), and it was operated by an SCA sperm class analyzer (MICROPTIC S.L.). The classification of sperm motility was as follows: grade A linear velocity >22 μm s⁻¹; grade B <22 μm s⁻¹ and curvilinear velocity >5 μm s⁻¹; grade C curvilinear velocity <5 μm s⁻¹; and grade D immotile spermatozoa48 .