Optimizing thawing conditions are required to maximize recovery of semen capacities after cryopreservation. This study aimed to assess the effect of thawing procedures on motility recovery and maintenance of semen capacities over time. A fractional factorial design approach was used to reduce the number of repetitions and simultaneously analyze the different interactions. Thirty canine frozen semen samples were thawed under different thawing conditions. Motility and velocity parameters were recorded using a computer-assisted semen analyzer up to 6 hours after thawing. Ten quadratic models were found to be significant, with the most significant effects observed on the velocity parameters. Second, this study allowed us to evaluate the effect of the proAKAP4 marker on frozen/thawed semen, with regard to motility recovery. ProAKAP4 is the precursor of A-kinase anchor protein 4. It has been studied in several species as a marker to assess sperm quality. In our study, the expression of proAKAP4 was determined using flow cytometry. No correlation was found between post-thaw motility parameters and proAKAP4 levels. However, the effects of thawing temperature, incubation time, and straw size were significant and similar to those observed for velocity parameters.
Keywords: canine; cryopreservation; flow cytometry; motility; proAKAP4; spermatozoa.