Cryopreservation of semen in the dog: use of ultra-freezers of −152 °C as a viable alternative to liquid nitrogen

This experimental work was carried out to validate the use of a −152 °C ultra-low temperature freezer to freeze and store canine semen. The semen of three dogs was pooled and processed to obtain a final dilution with a concentration of 100×106 spermatozoa/mL, glycerol at 5% and Equex at 0.5%. Then, four freezing protocols were tested to evaluate the cryosurvival of sperm at 1, 7, 30, 60 and 120 days after freezing: (I) semen was frozen and stored in liquid nitrogen; (II) semen was frozen in liquid nitrogen and stored in the ultra-low freezer at −152 °C; (III) semen was frozen in the vapour of liquid nitrogen and stored in the ultra-low freezer at −152 °C; (IV) semen was frozen and stored in the ultra-low freezer at −152 °C. Data were statistically analyzed by repeated measures analysis of variance to determine the effect of the freezing protocol and time on the sperm characteristics assessed. The percentages of sperm motility and of dead/live spermatozoa were similar throughout the experimental period, with no significant differences (P<0.05) to be observed between four different freezing techniques tested. At 120 days after freezing, the percentage of abnormal cells and the percentage of sperm cells with abnormal acrosome were not significantly different between the freezing techniques. Although the number of dogs used was slightly low, in vitro results of this preliminary study showed that the use of ultra-freezers at −152 °C to freeze and store canine semen could be a viable alternative to liquid nitrogen.