The aim of this study was to evaluate the effect of freezing/thawing procedure on two different feline species semen, Leopardus tigrinus (n = 5, 15 ejaculates) and L. pardalis (n = 5, 17 ejaculates) by using two different freezing extenders, TRIS-EQUEX (tris + 20% egg yolk + 7% glycerol) and MP-50 (egg yolk + 3% glycerol + 2% dimetilformamide). Anesthetic protocols for electroejaculation were constituted of: 1)-Ketamine (15mg/Kg) + Midazolam (0,5 mg/ Kg), 2) Ketamine (15 mg/ Kg) + Midazolam (0,5 mg/Kg) + Butorphanol, 3) Tiletamine/Zolazepam (10 mg/Kg), via intramuscular. Isoflurane was utilized to anesthesia maintenance. Semen samples were obtained by electroejaculation and processed by the following protocol: centrifugation (300g/10min), extending with cryodiluent, packed into 0,25mL straws (5x10⁶ sptz) and storage at 5°C for one hour (< 0,5°C/ min); put over nitrogen vapor for 20 minutes and then immersed in nitrogen liquid. Thawing was achieved at 46°C for 15 seconds. Ultrastructure of spermatozoa was analyzed to detect injuries caused by cryopreservation. Data were analyzed by Wilcoxon´s non-parametric test for comparing freezing extenders efficiency.

Cryopreserved and thawed semen of L. tigrinus exhibited a marked decrease on sperm motility (mean 55%) and progressive motility (mean 34,7%). In L. pardalis, semen evaluation showed a slighter decrease on sperm motility (mean 21%) and progressive motility (mean 19%). Both semen species presented elevation on the percentage of major defects (mean 52% and 30,5%, respectively) due to the increase of acrosomal injuries. Sperm contamination by urine was a remarkable factor on L. tigrinus (53% of the ejaculates) and L. pardalis (59% of the ejaculates) that occasioned a high incidence of bent and coiled tails (23,4%--tigrinus, and 13,3%--ocelots) in addition to cause loss of spermatic resistance to face cryopreservation/thawing stress. Evaluation of spermatic cell ultrastructure revealed that, after cryopreservation, semen of two species showed severe acrosomal damages, which might be a limiting factor for the cryopreserved semen fertility.

In conclusion, the results appointed that there were not any evident differences between the two cryodiluents for both species.