Simple SummaryNew cryopreservation methods for dog semen are constantly under investigation because the fertility results after insemination with frozen–thawed semen are still not satisfying. Most researchers do not investigate the fertilization ability of spermatozoa after the modification of cryopreservation protocols. In this study, we investigated whether spermatozoa membrane fluidity modification by use of cholesterol-loaded cyclodextrins (CLC) can improve kinematic parameters and also this method’s effects on capacitation status and fertilization ability. The lowest concentration of CLC increased motility parameters and percentage of live spermatozoa without cholesterol efflux compared to the control. There was no change in capacitation status but the binding ability of the sperm to the oocyte was decreased. Overall, these data suggest that improvement of kinematic parameters does not necessarily improve the zona pellucida binding ability results of canine spermatozoa.AbstractEven though the search for methods improving cryopreservation of canine spermatozoa led to an improvement of post-thaw quality, fertilizing results after insemination with frozen–thawed semen are still not satisfying. In this study, we focused on modification of spermatozoa membrane fluidity and investigated whether kinematic parameters as assessed by computer-assisted semen analyzer (CASA) can be improved. The primary aim of our study was to investigate whether the use of cholesterol-loaded cyclodextrins (CLC; 0.5 mg, 1 mg, 2 mg) and 2-Hydroxypropyl-ß-cyclodextrin (HBCD; 1 mg) positively influence capacitation status as examined by tyrosinphosphorylation, cholesterol efflux and zona binding assay (ZBA) of spermatozoa. The use of 0.5 mg of CLC increased the percentage of motile, progressive and rapid spermatozoa compared to the control. Addition of HBCD decreased motility and progressive motility of spermatozoa and the population with rapid movement in comparison to the control. The percentage of live spermatozoa without efflux of cholesterol compared to the control was increased when extender with 0.5 mg of CLC was used. There was no change in capacitation status. The zona binding ability of spermatozoa was significantly lower in the group with 0.5 mg of CLC than in the control. In conclusion, these results suggest that improvement of kinematic parameters does not necessarily coincide with better zona pellucida binding ability of spermatozoa.