Is the protective effect of egg yolk against osmotic and cryogenic damage on dog spermatozoa dose-dependent?

Egg yolk (EY) is conventionally used to reduce sperm cryodamage, however, there has not be evaluation of whether there is a dose-dependent effect with inclusion of EY in semen extender. To enhance the knowledge about the protective effect of EY during cryopreservation of dog semen, a specific study was designed to evaluate the dose-dependent protection of the EY against osmotic and cryogenic damage of dog sperm. In the first experiment, sperm stored in an extender that contained graded EY concentrations (0 %, 5 %, 10 %, and 20 %) were diluted with hypo- or hyper-osmotic solutions (final osmolality of 75, 150, 300, 500, 1000 mOsm/kg). Results from sperm kinetic, membrane integrity (MI), mitochondrial activity, and normal morphology evaluations indicated osmotic stress has especially marked effects on the kinetic capacity of spermatozoa, however, there were no direct effects on mitochondrial activity. In both hypo- and hyper-osmotic conditions, EY had a protective effect regardless of concentration. In the second experiment, semen samples were diluted in extenders at increasing EY concentrations (0 %, 5 %, 10 %, and 20 %) and cryopreserved. Effects on sperm kinetics, membrane and acrosome integrity and mitochondrial membrane potential indicated there was improved sperm viability after thawing when the EY concentration was 5 % and 10 %, and lesser viability when it was 20 %. These results indicate, for the first time, that EY reduces osmotic and cryogenic damage when used at 5 % or 10 % concentrations, and that these concentrations can be used to protect dog spermatozoa more effectively than the conventionally used concentration (20 %).