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KMID : 1094020130300060442
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Journal of Veterinary Clinics
2013 Volume.30 No. 6 p.442 ~ p.448
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Dog Sperm Cryopreservation Using Glucose in Glycerol-free TRIS: Glucose Concentration, Exposure Time
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Yu Il-Jeoung
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Abstract
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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 4oC. Ejaculates of six dog sperm were cooled in glycerol-free TRIS through 4oC for 100 min, cooled at 4oC 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 37oC 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 4oC 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 0.3M 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 4oC. 줄이기
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KEYWORD
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Dog, sperm, cryopreservation, Glycerol-free TRIS, glucose
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