Ivos 2

Specimens were collected for sperm kinetics testing using a disposable 4-cell Leja 20-mm CE chamber (Leja Products, Nieuw-Vennep, Netherlands) combined with (CASA; Hamilton Thorne, IVOS II, USA). Computer-assisted sperm analysis is a computer system with a high-resolution camera connected to a phase-contrast microscope. Analysis for one field of view takes only 1 s. The sperm kinetic parameters measured in this study were the percentage of motility, the percentage of progressive sperm, and the average velocity of sperm in their movement path (average path velocity, average pathway velocity [VAP], μm/s).

The base media used for this study was kept as simple as possible (phosphate buffered saline (PBS) supplemented with 0.3% bovine serum albumin (BSA)) to avoid effects of nutrients in the media obscuring effects of seminal plasma supplementation. Samples were diluted to a standard concentration of 20 × 10⁶ spermatozoa mL⁻¹ prior to measurement of sperm kinematic traits using computer assisted sperm analysis (CASA; Hamilton-Thorne IVOS II, Beverly, MA, USA). Sub-samples (5.5 µL) of diluted ejaculates were placed on pre-warmed (37 °C) CASA slides (Cell Vu; Millenium Sciences, Mulgrave, Vic., Australia), and 8 replicate videos (minimum of 200 cells) captured using factory ram settings (Animal Breeder software version 1.8). Parameters measured were: total motility, average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), linearity (LIN), straightness (STR), wobble (WOB), amplitude of lateral head displacement (ALH), and beat-cross frequency (BCF). Three replicate subsamples of each ejaculate at each timepoint were measured, and an average value calculated to minimise measurement error.

Semen from DKO pigs and WT control pigs were collected and returned to the laboratory in a 17°C incubator for testing their quality. The detection system was Hamilton-Thorne Research IVOS II computer-assisted sperm analyzer to measure the concentration, motility, and velocity distribution of the sperm.

Sperm were isolated by shredding cauda epididymis in 2 mL HTF media (2002, InVitroCare). After incubating at 37 °C for 20 min, sperm were diluted 3-fold with HTF media. CASA was done using IVOS II (Hamilton Thorne, USA). At least 600 sperm from each sample were examined. Motion parameters of motility (%), progressive motility (%), average path velocity (VAP), straight-line velocity (VSL) and curvilinear velocity (VCL) were collected.

Male mice were sacrificed by cervical dislocation, and cauda epididymides were carefully dissected and squeezed on a clean slide to harvest spermatozoa, which were incubated in M2 medium (SIGMA, M7167) for sperm parameters testing by using the IVOS II (Hamilton Thorne Inc., Beverly, MA) computer-assisted sperm analysis system^{64 (link)}. The concentration and motility of sperm could be automatically assessed by this system.

The sperm count and motility was quantified by Computer Assisted Sperm Analysis detection with the IVOS II™ system (Hamilton Thorne, United States). Briefly, sperm from Tmprss12^−/− and WT mice were extracted from the cauda epididymis and incubated in human tubal fluid (HTF) culture medium (Easy Check, M1130) at 37°C. The sperm suspension was added to a counting chamber for analysis.