Effects of Rapid Cooling Prior to Freezing on the Quality of Canine Cryopreserved Spermatozoa

The aim of this study was to evaluate the effects of rapid cooling prior to freezing on frozen-thawed canine sperm quality.In experiment 1, centrifuged ejaculates from 6 dogs were pooled, split into 4 aliquots and cryopreserved by the Uppsalaprocedure using different cooling rates (control, cooling speed 18 C/90 min and average cooling rate 0.2 C/min; rapid,cooling speed 18 C/8 min and average cooling rate 2.25 C/min) in combination with 2 glycerol addition protocols (fractionatedor unfractionated). In experiment 2, centrifuged ejaculates from 4 dogs were processed individually using the same coolingrates described in experiment 1 in combination with an unfractionated glycerol addition protocol. Each of the experiments wasreplicated 5 times. Sperm quality was evaluated after 30 and 150 min of post-thawing incubation at 38 C. Total motility (TM),progressive motility (PM) and quality of movement parameters were assessed using a computerized system, and sperm viability(spermatozoa with intact plasma and acrosome membranes) was assessed using flow cytometry (H-42/PI/FITC-PNA). Values for TM,PM, viable spermatozoa and the quality of movement parameters after thawing were not significantly affected by the coolingrate. The interaction between the cooling rate and the added glycerol protocol was not significant. There were significantdifferences among the males (P<0.01) in the sperm quality parameters evaluated after thawing. The interaction between themales and the cooling rate was not significant. In conclusion, canine spermatozoa can be cryopreserved using the Uppsalamethod at an average cooling rate of 2.25 C/min prior to freezing together with addition of fractionated or unfractionatedglycerol.