In vitro capacitation of fresh, chilled and frozen–thawed dog spermatozoa assessed by the chlortetracycline assay and changes in motility patterns

The effect of preservation on capacitation status of dog spermatozoa was investigated. Split ejaculates from six dogs were assessed as fresh, chilled for 24 h and rewarmed, and frozen–thawed samples. Capacitation-like status was assessed using the chlortetracycline (CTC)-assay and the measurement of sperm motility patterns using a computer-assisted sperm analyzer. Evaluations were performed on washed spermatozoa immediately after dilution in a Tris–fructose–citrate buffer (TFC) or in canine capacitation medium (CCM), and at 2-h intervals during 8 h of incubation in 5% CO2 in air, at 37°C. Preservation decreased significantly the proportion of uncapacitated spermatozoa. In TFC, at hour 0, chilled–rewarmed and frozen–thawed samples had a significantly lower proportion of uncapacitated, viable spermatozoa than the fresh samples (P<0.05) according to the CTC-assay. The time course of capacitation was accelerated in the preserved samples, compared to the fresh ones. During incubation in CCM, the mean time from hour 0 to when, according to the CTC-assay, the highest proportion of capacitated spermatozoa was present in the samples (time-to-peak), was 4 h for fresh and 2 h for chilled–rewarmed and frozen–thawed samples (P<0.1). The highest values for curvilinear line velocity (VCL) and lateral head displacement (LHD), thought to be descriptive of sperm hyperactivation, were also observed 4 and 2 h after incubation began, in the fresh and the preserved samples, respectively. The difference in time-to-peak for VCL and LHD between fresh, chilled–rewarmed and frozen–thawed semen samples was statistically significant (P<0.02). It can be concluded that based on the CTC-assay and the analysis of motility patterns, capacitation-like changes in dog semen seem to be both initiated and accelerated by the preservation procedures.