Comparison of different semen extenders and cryoprotectant agents to enhance cryopreservation of rabbit spermatozoa

Abstract

The purpose of this research was to find a suitable protocol to enhance frozen rabbit sperm preservation analysing the role that seminal plasma (SP) plays and the effect of different cryoprotectant agents on sperm quality 0 and 2 h after thawing. Sperm samples were pooled and divided in eight fractions. Four of them were diluted with BotuCrio<sup>®</sup> (extender A), INRA 96<sup>®</sup> plus 6% glycerol (extender B), 6% N, N-dimethylformamide (extender C) and 6% N-methyl-2-pyrrolidone (extender D), respectively. The other four fractions were centrifuged and the supernatant was discarded in order to eliminate SP. Each sample was then resuspended with extender A, B, C and D. Samples were cooled progressively, loaded into 0.5 ml freezing straws and frozen with liquid nitrogen vapour. Thawing was performed by placing the straws into a bain-marie at 37°C for 21 s. Straws were dried and sperm samples placed into Eppendorf tubes to be analyzed by ISAS software, vitality test, HOS test and acrosome integrity test. The best motility and velocity parameters were obtained by extender A (P < 0.050) even when the motility parameter was compared with previous studies using other diluents. Additionally, sperm quality decreased over incubation time (P < 0.050) and no differences were found in samples processed with or without SP. This research revealed that BotuCrio<sup>®</sup> could be used for rabbit sperm cryopreservation and moreover the improvement of the cryopreservation process of rabbit sperm due to the demonstration that SP removing is not required.