Ivos 12

The sperm motility indices were analyzed using a CASA system (IVOS12.1, Hamilton-Thorne Biosciences, Beverly, MA, USA) as described previously [15 (link)]. Before the analysis of CASA-based sperm indices, the frozen-thawed semen was diluted in tris buffer that gives 40X10⁶ sperm/ml. One μl of diluted semen was loaded in a Leja slide (8 chambers with 20μm depth) and 5 optical fields were analyzed by software. The motility and kinetic parameters such as total motility (TM, %), straight linear velocity (VSL, μm/s), average path velocity (VAP, m/s), curvilinear velocity (VCL, μm/s), average lateral head displacement (ALH, μm/s), beat cross frequency (BCF, Hz), straightness (STR,%) and linearity (LIN,%) were recorded. The CASA software settings, including frame rate, frames acquired, VAP cut-off, and STR cut-off, were identical for all experiments.

The spermatozoa incubated with pesticide metabolites (OPPMs) were subject to computer-assisted sperm analysis (CASA) for estimating the velocity and motion parameters of the buffalo spermatozoa. Computer-assisted sperm analyzer (IVOS12.1, Hamilton-Thorne Biosciences, Beverly, MA, USA) was used to evaluate the kinetic characteristics. The motility and movement parameters like the curvilinear velocity (VCL, µm/s), linear velocity (VSL, µm/s), average path velocity (VAP, µm/s), the mean amplitude of lateral head displacement (ALH, µm), the percentage of linearity i.e. the ratio between VSL and VCL (LIN, %), the straightness coefficient which is the ratio between VSL and VAP (STR, %) and the frequency with which the actual sperm trajectory crossed the average path trajectory (BCF, Hz) were recorded in duplicates for all the experimental groups. The CASA software settings were as follows: temperature = 38 °C, frame rate = 60 Hz, frames acquired = 30, minimum contrast = 35, minimum cell size = 5 pixels, cell size = 9 pixels, cell intensity = 110 pixels, progressive cells (VAP cut-off = 50 m/s, STR cut-off = 70%), slow cells (VAP cut-off = 30/s and VSL cut-off = 15/s). The spermatozoa (N = 500) were observed in a minimum of five optical fields around the central reticulum of the chamber for sperm motility analysis.