The cryoprotective effect of Ficoll on the rabbit spermatozoa quality

The aim of this study was to evaluate the effect of the addition of Ficoll 70 into the cryopreservation medium containing sucrose and dimethyl sulfoxide (DMSO) on rabbit spermatozoa characteristics following freezing/thawing. This large molecular weight polymer elevates the viscosity of medium and, therefore, could better protect spermatozoa during the freezing process. Only ejaculates of good initial motility (>80%) were used in the experiments. Heterospermic pools were diluted in a freezing medium composed of commercial diluent, 16% dimethyl sulphoxide (DMSO) and 2% sucrose (control) or in the same medium enriched with 4% Ficoll 70 (Ficoll) and frozen in liquid nitrogen vapours for 10 min before being plunged in liquid nitrogen. The quality of fresh and frozen/thawed spermatozoa samples was evaluated in vitro using the Computer Assisted Semen Analysis (CASA) system, fluorescent probes (peanut agglutinin (PNA)-Alexa Fluor®; annexin V-FLOUS) and by electron microscopy. Better cryoprotective effect was observed when Ficoll 70 was added, compared with the semen cryopreserved with sucrose and DMSO only. The higher values (P < 0.05) of motile and progressively moving spermatozoa immediately after thawing and at 30 min following incubation at 37°C were obtained in the Ficoll group. Moreover, the higher number (P < 0.05) of acrosome intact sperm was found in the Ficoll compared with the control group. Furthermore, no significant differences in kindling rates and number of pups born between frozen/thawed and fresh semen group were found. In conclusion, our study showed that the addition of Ficoll 70 might improve several characteristics of rabbit spermatozoa measured in vitro following freezing/thawing.