Sperm dilution
The semen extender containing 300 mM glucose, 10% dimethyl
sulfoxide (DMSO) in 100 ml distilled water was prepared according
to Gwo et al. [13] as the basic extender. Collected semen from
males that showing >80% motility was pooled into equal aliquots
according to the required semen volume and sperm density
needed to eliminate effects of individual variability of the donors.
The basic semen extender was diluted separately using either
0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 mg/120 million sperm CLC at 37 C as
experimental groups. Semen extenders containing CLC were vortexed
and sonicated until completely dissolved to obtain a final
working CLC solution. Control was made without cholesterolloaded
cyclodextrin (CLC). Adjusted and pooled semen according
to same sperm concentrations from ten males was diluted at ratio
of 1:3 (v/v) with the CLC experiment groups at 37 C. The diluted
samples were incubate