Effect of post-thaw incubation on semen characteristics of ram spermatozoa cryopreserved under controlled and uncontrolled rate of cooling

Ano: 2009 | Volume: 6 | Número: 4

Autores: D. Kumar, A. Joshi, S.M.K. Naqvi

Palavras-chave: acrosome, frozen ram semen, spermatozoa, sheep

Resumo Inglês:

Exposure of frozen-thawed spermatozoa to a thermal resistance test reveals damages, which are not apparent immediately after thawing but are useful to assess the fertilizing ability of ram spermatozoa. Our earlier study has shown that cryopreservation of ram spermatozoa under controlled rate of cooling and freezing significantly improves the post-thaw motility and acrosomal integrity, compared to uncontrolled rate of cooling prior to controlled rate of freezing. The purpose of this study was to assess the effect of post-thaw in vitro incubation on motion characteristics and acrosomal integrity of ram spermatozoa cryopreserved under controlled (Group 1) and uncontrolled rate of cooling (Group 2) followed by programmable freezing. Semen samples of good initial motility obtained from adult Malpura rams were pooled, diluted to 1 x 109 spermatozoa per ml with Egg yolk-Test-glycerol extender and packaged in 0.25 ml straws. Straws representing Group 1 were cooled in a programmable cell freezer from 25 to 5°C at the rate of 0.15°C per minute followed by a holding time of 2 h for equilibration, while straws of Group 2 were allowed to cool slowly up to 5°C and equilibrate for 2 h in the cold cabinet. After equilibration, straws of Group 2 were also loaded in the cell freezer for freezing straws of both the treatment groups simultaneously from 5 to –125°C at the rate of 25°C per minute. Thawing of straws was done at 50°C for 10 seconds and thawed spermatozoa were subjected to a thermal resistance test at 37°C for 4 h. Samples were assessed immediately after thawing and at hourly interval for sperm motion characteristics by computer-aided semen analysis technique. Post-thaw incubated spermatozoa were also evaluated at 0, 1, 2, 3, and 4 h for acrosomal integrity after staining the dried semen smears with Giemsa stain. The % motility, % rapid moving spermatozoa, % linearity and % sperm with normal acrosome were significantly (P < 0.05) higher in Group 1 compared to Group 2. The effect of incubation time was also significant (P < 0.05) on % motility, fraction of rapid motile spermatozoa, % linearity, curvilinear velocity, average path velocity, straight line velocity, area of sperm head, lateral head displacement and % spermatozoa with normal acrosome. The % motility, % rapid motile spermatozoa, sperm velocity, lateral head displacement and % spermatozoa with normal acrosome progressively declined during 4 h of incubation but the decline in all the traits was less in Group 1 compared to Group 2. The results showed that controlled rate of cooling conferred better cryopreserving ability to ram spermatozoa for post-thaw thermoresistance test compared to uncontrolled rate of cooling prior to programmable freezing.