Dog sperm cryopreservation using cryovials and different dilution steps

• Published in: Cryo-Letters Vol. 45; no. 1; p. 16
• Main Authors: Ibrahim, S, Hamad Talha, N A, Cho, J, Jeon, Y, Yu, I J
• Format: Journal Article
• Language: English
• Published: England 01.01.2024
• Subjects: Acrosome; Animals; Cryopreservation - methods; Cryopreservation - veterinary; Cryoprotective Agents - pharmacology; Dogs; Freezing; Male; Semen; Semen Preservation - methods; Semen Preservation - veterinary; Sperm Motility; Spermatozoa
• ISSN: 0143-2044
• DOI: 10.54680/fr24110110312

The conventional sperm freezing method for dog sperm is with straws and includes two-step dilution and a long equilibration time. To develop a more efficient freezing method using cryovials. Three freezing protocols using cryovials (0.5 mL) were conducted with dog spermatozoa at 1 x 10 sperm/mL: Group 1 spermatozoa were cooled in cryovials and extender 1 (E1) and extender 2 (E1 +1 M glycerol) at 4 degree C for 50 min and then frozen over LN for 20 min; Group 2 sperm was cooled and frozen in cryovials with a mixture of E1 and E2 (1:1) in a deep freezer (-80 degree C) for 30 min; Group 3 sperm in cryovials and E1 were cooled at 4 degree C for 20 min, cooled for an additional 20 min after addition of E2 (E1:E2, 1:1), and then frozen using LN vapour for 20 min. The control (Group 4) consisted of spermatozoa in straws being frozen using the conventional freezing method using two-step dilution. All groups were plunged and stored in LN after freezing and their functional performance and gene expression determined. Progressive motility and acrosome integrity were highest (P < 0.05) in Groups 2, 3 and 4 (only acrosome integrity). Viability in Group 3 was significantly better that in the other Groups, and the reactive oxygen species (ROS) level and phosphatidylserine (PS) translocation index were significantly lower in Group 2 than the other Groups. The expression of sperm mitochondria-associated cysteine-rich protein (SMCP) and anti-apoptotic B-cell lymphoma 2 (BCL2) genes was highest (P < 0.05) in Group 2 and the expression of pro-apoptotic Bcl2-associated X protein (BAX) was lowest (P < 0.05) in Group 4. The sperm frozen using cryovials, one step dilution and the deep freezer (Group 2) proved to be a simple and suitable cryopreservation method for dog sperm. https://doi.org/10.54680/fr24110110312.