Cryopreservation of canine spermatozoa at -80°C

The freezing and storage in liquid nitrogen (LN₂) is the technique used most for the preservation of canine and other domestic species semen, for long periods of time. This study was designed to validate the use of deep freezer at -80^oC to freeze and store canine semen in straws. Three protocols were tested (n = 10): Control: sperm frozen in N₂L and stored; Exp 1: sperm frozen and stored -80°C, and Exp.2: semen frozen at -80 ° C and stored in N₂L. Post-thawing was assessed by flow cytometry and the viability of plasma membrane integrity (SYBR-14/PI), mitochondrial membrane potential (ΨΔm, JC-1), acrosome membrane integrity (PSA / FITC-PI), translocation of phosphatidylserine (Annexin -V-FITC/PI) and DNA fragmentation (TUNEL). No significant differences (P <0.05) between the Control, Exp.2 and Exp.1groups regarding: intact plasma membrane integrity (42.2 ± 5.3, 35.57 ± 10.3 and 40.76 ± 12.1, respectively), ΨΔm normal (54.7 ± 20.6, 44.4 ± 15.8 and 43.4 ± 15.3, respectively), intact acrosomal membrane integrity (42.9 ± 11, 1, 53.2 ± 15.8 and 48.7 ± 20.1, respectively) and fragmented DNA (0.87 ± 0.41, 0.75 ± 0.16, 0.79 ± 0.27, respectively). However, the average motility of the post-thawing of Exp.1 and Exp.2 groups (37.0 ± 4.4% 36.8 ± 4.1% respectively) showed significant differences (P <0.05) than the Control group (55.9 ± 8.7%). The results obtained in this study showed that the use of deep freezer at -80°C for freezing and storing canine sperm has potential use in veterinary medicine as an alternative to the use of N₂L.