Maintaining canine sperm function and osmolyte content with multistep freezing protocol and different cryoprotective agents

Cryopreservation procedures cause osmotic stress to spermatozoa following cryoinjury and reduce their content of osmolytes. Conventional method for cryoprotectant loading and dilution on canine semen freezing which could be categorized in single step protocol, makes decreasing in sperm performance such as motility, morphology and viability. Therefore, the objective of the present study was to determine if a multistep protocol using glycerol or ethylene glycol can be used to overcome the osmotic sensitivity of canine spermatozoa, and to identify osmolytes that were involved in regulation of osmotic stress. A multistep protocol, comprising serial loading and dilution of cryoprotective agents by dividing the total volume of extender into 4 steps (14%, 19%, 27%, and 40%) every 30 s, was compared to a single step method. Frozen-thawed spermatozoa in the multistep group showed superior quality (P < 0.05) compared with the single step process in progressive motility (23.3 ± 1.3% vs. 12.5 ± 1.6%), intact membranes (66.5 ± 2.8% vs. 49.5 ± 2.6%) and bent tail (29.2 ± 3.2% vs. 46.2 ± 1.9%). Multistep also succeeded in minimizing loss of the osmolytes carnitine (20.6 ± 2.0 nmol/U G6PDH vs. 10.8 ± 2.1 nmol/U G6PDH) and glutamate (18.4 ± 1.6 nmol/U G6PDH vs. 14.4 ± 0.8 nmol/U G6PDH) compared to the single step group. Moreover, glycerol with multistep was more advantageous for maintaining sperm quality than ethylene glycol. In conclusion, the multistep protocol with glycerol can be used to improve the morphology, motility and osmolytes content of frozen-thawed canine spermatozoa.