Appraisal and standardization of curvilinear velocity (VCL) cut‐off values forCASAanalysis of Japanese quail (Coturnix japonica) sperm

Abstract

One of the basic steps in objective analysis of sperm motility is the subdivision of a motile sperm population into slow, medium and rapid categories based on their velocity. However, for CASA analysis of quail sperm, the velocity values for categorization of slow, medium and rapid sperm have not yet been standardized. To identify the cut-off values of "velocity curvilinear" (VCL) for quail sperm categorization, we captured and analysed 22,300 tracks of quail sperm using SCA -CASA. The median and mean VCL values were 85 and 97 μm/s. To define the VCL cut-off values, we used two methods. In the first, we identified the upper (rapid sperm) and lower (slow sperm) cut-off values using: (i) median VCL ± 25% or ± 50% or ± 75% of median VCL value; (ii) first and third quartile values of VCL data (i.e. 25% cut-off setting); and (iii) 33% and 66% of VCL data. Among these settings, sperm categories and their corresponding motility characteristics recorded using the "25%" setting (i.e. slow ≤36 ≤ medium ≤154 ≤ rapid) were found the most realistic and coherent with male ranking by fertility. In the second method, we calculated heteroscedasticity in the total VCL data using PCA and the two-step clustering method. With this approach, the mean of the high and low clusters was 165 and 51 μm/s, respectively. Together, the mean from two methods suggested that, for SCA -CASA categorization of quail sperm, sperm should be classed as "rapid" at VCL ≥160 μm/s and "slow" at VCL ≤45 μm/s.

Morphometric analysis of sperm used for IVP by three different separation methods with spatial light interference microscopy

“…All the other motility parameters were statistically different between the groups, with only the VCL between the electro-channel and Swim-Up showing no difference. The VCL in a previous paper was identified as an important parameter to evaluate the motility (Farooq et al 2017). That paper and our results both showed a higher VCL value for the Percoll group compared the other two groups.…”

Age‐related declines in ejaculate quality and sperm kinematics vary among strains of Japanese Quail (Coturnix japonica)

“…To measure sperm concentration, 1.5 μl neat semen was diluted with 598.5 μl formalin buffer (2% formalin in PBS buffer) in a 1 ml vial, mixed well and transferred to a polystyrene cuvette. Absorbance at 560 nm in a spectrophotometer (Metertech UV/VIS SP8001) was compared with a standard curve established in our laboratory for measuring sperm concentration in the emu and in Japanese quail (FarooqMalecki, Mahmood, & Martin, ; Sood, Malecki, Tawang, & Martin, ).…”

“…Semen samples were diluted in a medium comprising motility buffer (150 mM NaCl and 20 mM N‐tris‐[hydroxymethyl]‐methyl‐2‐aminoethane‐sulphonic acid, 35 mM glucose and 0.1% bovine serum albumin adjusted to pH 8.0 at 25°C) and 10% proctodeal gland foam extract (Farooq et al, ). The foam extract had been prepared as described by Farooq et al, ().…”

“…The CASA equipment has been described previously (Farooq et al, ). In brief, we used the motility module of the Sperm Class Analyzer (SCA ® ) version 5.2 (Microptic S.L., Barcelona, Spain).…”

“…Kinematic measures of sperm motility are widely considered important for assessing ejaculates and fertility in a wide variety of species (Amann, ; Bakst & Cecil, ; Farrell, Foote, Simkin, Clegg, & Wall, ; Holt, Holt, Moore, Reed, & Curnock, ; Klimowicz, Nizanski, Batkowski, & Savic, ; Long et al, ; Moore & Akhondi, ). The CASA technology has been used to evaluate potential fertility in a variety of poultry species, including the broiler chicken and turkey, as well as quail (Farooq, Malecki, Mahmood, & Martin, , ; King, Holsberger, & Donoghue, ; Long et al, ). Moreover, changes in sperm motility parameters with advancing age have been documented in humans (Johnson, Dunleavya, Gemmell, & Nakagawa, ; Sloter et al, ).…”