Bovine cyropreserved epididymal spermatozoa viability after incubation by different periods of time in IVF medium

The recovery of epididymal spermatozoa, their cryopreservation and posterior use on IVP of bovine embryos, has become an important tool to store genetic material from animals that have died unexpectedly or that have acquired reproductive inability. The present study aimed to evaluate the viability of spermatozoa from epididymides after their incubation on IVF medium supplemented or not with heparin. Spermatozoa were recovered from epididymis of bulls (n=3) and were cryopreserved. In each replicate one straw from each animal was thawed to form a pool, which was centrifuged in percoll 45% at 700g for 10 minutes. After centrifugation, one sperm sample was taken (0h) to assess total and progressive motility (CASA), morphology (phase contrast), capacitation (chlortetracycline-CTC), membrane integrity (6-carboxy-fluorescein diacetate and propidium iodide-FDA-IP) and acrosome integrity (isothiocyanate-conjugated peanut agglutinin and PI, iodide de propídeo-PNA-FITC-IP). The remaining sample was incubated in IVF medium with or without heparin (hep=10μg/ml) for 3, 6 e 9 h (3h-hep, 3h+hep, 6h-hep, 6h+hep, 9h-hep and 9h+hep, respectively). Three replicates were performed and data were analyzed byANOVA and Tukey test (P<0.05). No differences were detected among groups regarding morphology (44.7% to 61.7%), capacitated cells (15.3% to 26.4%) and intact acrosome (21.5% to 33.6%). The percentage of cells with intact membrane was similar between the groups 0h (43.8±6.6%), 3h-hep (33.4±4.8%), 3h+hep (31.5±8.0%) and 6h-hep (31.5±8.05%). However, group 6h+hep (26.5±4.4%) showed a lower percentage compared to group 0h, but was similar to groups 9h-hep (22.5±5.3%) and 9h+hep (26.5±7.9%). Compared to 0h group (75.3±14.0%), a decreased in total motility was observed at 6 h of culture (6h-hep=35.7±8.4%, 6h+hep=45.0±4.0%), which was similar to that observed at 9 h with (30.0±4.9%) or without heparin (34.7±11.2%). A significant decrease in progressive motility was only observed at 9h of culture (0h=39.0±10.8%, 9h-hep=23.3±9.2% e 9h+hep=22.7±10.1%), and no effect of heparin was also detected for this parameter. It can be concluded that spermatozoa from epididymides, when cultured in IVF medium, maintained their viability characteristics unchanged in the first hours of culture, regardless the presence of heparin. In addition, up to 9 h of culture only motility and membrane integrity values were altered.