Computer-Assisted Sperm Motility Analysis

Spermatozoa motility was assessed using a computer-assisted sperm assay (CASA) method according to World Health Organization guidelines (46 (link)). After euthanasia, spermatozoa were collected from the cauda epididymis of mice and suspended in Dulbecco’s modified Eagle medium (DMEM)-F-12 medium with 10% FBS and incubated at 37.5°C for 30 min; samples were then placed in a prewarmed counting chamber. The Microptic sperm class analyzer (CASA system) was used in this investigation. It was equipped with a 20-fold objective, a camera adaptor (Eclipse E200; Nikon, 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 linear velocity, <22 μm s⁻¹; curvilinear velocity, >5 μm s⁻¹; grade C curvilinear velocity, <5 μm s⁻¹; and grade D, immotile spermatozoa. The spermatozoa motility data represented only grade A and grade B since only these two grades are considered to be functional.