• Journal of Veterinary Clinics
• /
• v.30 no.6
• /
• pp.442-448
• /
• 2013

The aim of the present study was to develop glycerol-free TRIS extender using glucose for dog sperm cryopreservation. We determined the appropriate concentration of glucose in glycerol-free TRIS and the exposure time in glycerol-free TRIS containing 0.3 M glucose at $4^{\circ}C$. Ejaculates of six dog sperm were cooled in glycerol-free TRIS through $4^{\circ}C$ for 100 min, cooled at $4^{\circ}C$ in TRIS with different glucose concentrations 0 M, 0.04 M, 0.1 M, 0.2 M and 0.3 M, respectively for 30 min followed by cryopreservation. After thawing at $37^{\circ}C$ for 25 sec, membrane and acrosome integrities of dog sperm were evaluated. In addition, the effect of exposure time (10, 30, 50 and 70 min) of sperm to glycerol-free TRIS containing 0.3 M glucose at $4^{\circ}C$ on progressive motility, viability, and DNA integrity following sperm cryopreservation was studied. Membrane integrity and acrosome integrity were assessed by 6-carboxyfluoresceindiacetate (6-CFDA)/propidium iodide (PI) fluorescent staining and Pisum sativum agglutinin conjugated to fluorescein isothiocyanate, respectively. DNA integrity was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling, using flow cytometry. Sperm frozen in glycerol-free TRIS supplemented with 0.2 M or 0.3 M glucose have an intact plasma membrane (CFDA+/PI-) after cryopreservation than sperm frozen in the extenders with lower glucose concentrations (p<0.05). Acrosome integrity was significantly higher in the 0.3 M group than less than 0.1 M groups (p<0.05). The sperm DNA fragmentation index did not differ according to exposure time, although progressive motility was significantly higher in the 50 min exposure group than the other groups (p<0.05). These results indicate that cryopreservation of dog sperm is feasible and yields more motile sperm following freezing and thawing in glycerol-free TRIS containing 0.3 M glucose with the exposure time for 50 min at $4^{\circ}C$.

• Journal of Veterinary Clinics
• /
• v.11 no.2
• /
• pp.545-552
• /
• 1994

This experiment was carried out to investigate the renditions to maintain good post-thaw motility and viability of canine spermatozoa when the semen was frozen using methanol. The semen from two male dogs which had been proven to be fertile in the previous one year was treated with different compositions of semen diluent and was frozen at different freezing temperatures, When canine semen was frozen at-2$0^{\circ}C$, -6$0^{\circ}C$, or -8$0^{\circ}C$, the spermatozoa frozen and stored at -2$0^{\circ}C$ showed very low post-thaw motility and viability from day 2 to 7 and showed no viability since day 15 after freezing. The spermatozoa frozen and stored at -6$0^{\circ}C$ or -8$0^{\circ}C$ showed higher post-thaw motility and viability on day 2, 1, 15 and 30 after freezing than that frozen and stored at-2$0^{\circ}C$(p<0.01), with no difference between two groups. Among different composition groups of the semen diluents of control(tris + egg yolk + glycerol), egg yolk-free, 히ycerol-free, and tris-free, Prior to freezing, the egg yolk-free diluent showed significantly love. motility and viability than the other diluents(p<0.05). On each thawing day (from day 2 to 15 after freezing), control group showed considerably higher motility and viability than the other groups(p<0.01). The canine spermatozoa frozen and stored at -6$0^{\circ}C$ and -8$0^{\circ}C$ showed gradual decrease of motility from day 2 to 30 after freezing and the spermatozoa of these two groups thawed on day 30 showed considerably love. motility than those thawed on day 2 after freezing, respectively(p<0.01). These results indicate that the freezing temperature of either -6$0^{\circ}C$ or -8$0^{\circ}C$ can be applicable to the freezing method using methanol and also all of the components of the semen diluent including cryoprotectant, buffer and cold-shock buffer are very important to maintain motility and viability of canine spermatozoa in the freezing and thawing procedure.