Effect of slide type to evaluate motility parameters of frozen-thawed equine sperm using computer aided sperm analysis

In equine industry, motility assessment is the primary parameter currently used to evaluate sperm quality in an ejaculate. Motility is determined either by visual subjective assessment or objectively via computer aided sperm analysis (CASA) system. Various types of slides and coverslips are used for sperm motility assessment. Two main methods are a fixed coverslip slide (e.g. Leja ® slide) or a drop coverslip slide (e.g. Cell-Vu ® slide or glass slide). Both these slides can have a fixed chamber depth of 20 microns. Previous studies in our laboratory demonstrated a negative effect on total and progressive motility with fixed coverslip slides compared to drop coverslip slides for analysis of fresh semen using CASA. Objective was to determine if there is a difference in CASA derived motility parameters of frozen-thawed stallion sperm based on slide/chamber type. We evaluated the postthaw motility of 30 ejaculates of frozen semen. Samples were thawed at 37C for 30 seconds and placed into a prewarmed 5 ml tube. Concentration was determined using a NucleoCounter and samples were diluted to 30 x 106 /ml in 2 ml standard skim milk semen extender. Samples were incubated for 10 minutes and evaluated using the Ceros II CASA system. Then, 5 µl sample was placed in each chamber on either fixed coverslip slide or drop coverslip slide. To avoid any possible influence of an increased incubation time, the first slide type evaluated alternated with every sample. A minimum of 5 fields were selected for each sample and analysis continued until a minimum of 400 motile sperm were examined. Each field was limited to have a maximum number of 150 objects per screen in order to minimize the impact of collisions. Motility parameters evaluated were total motility (TM), progressive motility (PM), path velocity (VAP), curvilinear velocity (VCL), straight line velocity (VSL), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), linearity (LIN), and straightness (STR). A sperm was considered progressively motile if it had a STR of > 75% and a VAP > 50 µm/sec. A Student’s paired t-test was used to evaluate the difference in motility values between 2 slides and p < 0.05 indicated significance. There was a difference (p < 0.0001) for TM and PM between 2 slide types. Both motility values were higher in drop coverslip slides. Average TM for drop coverslip slides was 52% versus fixed coverslip slides was 35%. Average PM for drop coverslip slides was 38% versus fixed coverslip slides was 24%. There was no difference between cell types for VAP, VSL, VCL, STR, LIN, ALH, and BCF. These data agree with previous results in our laboratory obtained with fresh semen. Although drop coverslip slides and fixed coverslip slides utilized 20 micron chamber for motility analysis, these results indicate that slide type has substantial impact on sperm motility and comparison of motility measures between samples using various chamber types is not valid.

Keywords: Equine sperm, motility, CASA, fixed coverslip, drop coverslip

This manuscript was originally published in the journal Clinical Theriogenology Vol 12(3) Sept 2020.  Clinical Theriogenology is the official journal of the Society for Theriogenology (SFT) and the American College of Theriogenologists (ACT).  This content has been reproduced on the IVIS website with the explicit permission of the SFT/ACT.