Alternative procedures for the cryopreservation of brown bear ejaculates depending on the flexibility of the >in cooling> period (5°C)

The adaptability of cryopreservation protocols for brown bear spermatozoa collected under field conditions and frozen in a nearby laboratory (transported for a few hours) or shipped to a reference laboratory for sex sorting (transported for a few days) was evaluated. Forty-nine electroejaculates from 15 mature brown bears were extended to 100×106sperm/mL in a TES-Tris-Fructose based extender and cryopreserved (-20°C/min to -100°C and stored at -196°C). After thawing, the quality of the seminal samples was assessed for total (TM), progressive (PM) motility and kinetic parameters - by CASA -, and viability (VIAB), viable and non-apoptotic status (YOPRO-), high membrane mitochondrial potential (MIT) and intact acrosomes (iACR) - by flow cytometry - In Experiment 1, we assessed different storage times (0, 0.5, 1 - control -, 4-5, 7-8 and 11-12h) at 5°C from final dilution to freezing. After thawing, non-equilibrated samples (0h) showed lower values of iACR, TM and PM. No significant differences were found for the different periods of equilibration tested. In Experiment 2, we evaluated three long-term storage times (24, 48 and 72h) at 5°C before freezing using storage for 1h as control. The post-thawing quality of brown bear spermatozoa declined markedly after 48-72h of pre-freezing. In conclusion, our findings suggest the possibility of extending the pre-freezing cooling period up to 24h post-collection without freezing. This knowledge should enable the adaptation of the freezing protocols for when a special handling conditions are required such as the shipment of seminal samples to technological centers for the pre-freezing application of enhancer spermatic biotechnologies.This work was supported in part by MICINN (CGL2010-19213/BOS) and CANTUR S.A.Peer Reviewe