The purpose of this study was to improve the quality of frozen-thawed canine spermatozoa through the optimization of glycerol concentration (GC) and freezing rate in the semen freezing protocol. Ejaculates from nine dogs were diluted with an extender containing 0%, 1.5%, 3%, 6%, or 9% glycerol. The suspensions were loaded into 0.25 ml straws, frozen in nitrogen vapor in a closed box, and immersed in liquid nitrogen (LN