Determination of appropriate cryopreservation protocols for epididymal cat spermatozoa.

Abstract

Effects of Equex and glycerol additions and sample dilution step on frozen-thawed epididymal cat spermatozoa were investigated. The epididymal sperm pellets were resuspended in extenders using one- (groups III and IV) or two- (groups I, II, V and VI) step dilution. For one-step dilution, the pellets were resuspended in plain egg yolk-Tris medium (EYT)+5% glycerol with (IV)/without (III) 0.5% Equex and cooled (4(°) C, 1h). For two-step dilution, the pellets were resuspended in EYT (I and V) and in EYT+3% glycerol (II and VI), cooled and further diluted with EYT+10% glycerol with (I)/without (V) 1% Equex and with EYT+7% glycerol with (II)/without (VI) 1% Equex. Immediately after freeze-thawing, no differences (p>0.05) were found in the motility, viability and membrane integrity (HOST) among the groups except the lowest HOST in IV (p=0.005 to p=0.04). The acrosome integrity (FITC) in group I was comparable to that in group II (p>0.05) and was higher than the rest (p<0.001 to p=0.02). At 2h after thawing, the motility, viability and HOST were comparable among the groups (p>0.05) except the lower percentages of viability in III (p=0.008 to p=0.3) and of HOST in IV (p=0.005 to p=0.2). Two-step dilutions with Equex (I, II) were more beneficial for the FITC at 2h than without Equex (V) (p=0.005 and p=0.02) and than one-step dilutions (III, IV) (p<0.001 to p=0.02). In conclusion, epididymal cat sperm quality after freeze-thawing could be improved when Equex was added and two-step dilution was performed during freezing. The extenders prepared for the first step of dilution could be with (3%) or without (0%) glycerol.