Cryopreservation of rabbit spermatozoa using acetamide in combination with trehalose and methyl cellulose

Glycerol is not an effective cryoprotectant for rabbit spermatozoa; therefore, rabbit spermatozoa were used as a model for developing cryopreservation procedures for other cell types which also freeze poorly when glycerol is used as the cryoprotectant. Experiments were conducted to 1) compare several published protocols for cryopreserving rabbit spermatozoa; 2) determine if removal of seminal granules, required for flow cytometry analysis, affects the motility of rabbit spermatozoa; and 3) determine if using a combination of cell permeating cryoprotectants (acetamide) with cell nonpermeating cryoprotectants (trehalose and methyl cellulose; MC), can increase the recovery of viable rabbit spermatozoa after cryopreservation. Media containing acetamide as a cryoprotectant were found to be most effective for rabbit spermatozoa. The cryoprotectants ethylene glycol, dimethylsulfoxide and glycerol were not effective for cryopreserving rabbit spermatozoa. Second, rabbit spermatozoa could be centrifuged through a Percoll gradient composed of equal volumes of Prcoll and a HEPES-buffered sperm medium. This centrifugation removed all seminal granules without affecting the percentage of motile spermatozoa after initial sperm dilution (85 vs 74%) or after cryopreservation (35 vs 30%), when sperm were either centrifuged or not centrifuged, respectively. The substitution of trehalose in the cryopreservation medium for raffinose did not improve recovery of motile cells following cryopreservation (P > 0.05). However, addition of MC resulted in higher percentages of motile sperm after cryopreservation (43 vs 31%; P < 0.05). In addition, sperm viability and acrosomal integrity were simultaneously evaluated using flow cytometry. The addition of both trehalose and MC to media containing acetamide resulted in higher percentages of live acrosome-intact cells than acetamide alone (53 vs 37%; P < 0.05). These results indicate that a combination of permeating and nonpermeating cryoprotectants (acetamide, trehalose and MC) were more effective in preserving rabbit spermatozoa than acetamide alone and that analyzing multiple sperm characteristics, by flow cytometry, can assess sperm damage not detected by analyzing sperm motion characteristics.