Osmotic tolerance and freezability of isolated caprine early-staged follicles

Isolated caprine early-staged follicles were submitted to osmotic tolerance tests in the presence of sucrose, ethylene glycol (EG), or NaCl solutions and were exposed to and cryopreserved (by slow or rapid cooling) in MEM alone or MEM supplemented with sucrose, EG (1.0 or 4.0 M), or both. When follicles were exposed to 1.5 M NaCl, only 2% of the follicles were viable, whereas 87% of the follicles were viable after exposure to 4.0 M EG. Regarding exposure time, the highest percentage of viable follicles was obtained when follicles were exposed for 10 min to 1.0 M EG + 0.5 M sucrose; exposure for 60 s to 4.0 M EG + 0.5 M sucrose also maintained high percentage viability in follicles. Slow cooling in the presence of 1.0 M EG + 0.5 M sucrose (75%) or rapid cooling in the presence of 4.0 M EG + 0.5 M sucrose (71%) resulted in a significantly higher proportion of viable follicles than all other treatments (P < 0.05). A 24-h culture of frozen-thawed follicles was used to assess survival; only slow-frozen follicles showed viability rates similar to control follicles (64% vs. 69% respectively; P > 0.05). Interestingly, the percentage of viable rapid-cooled follicles (59%) was similar to that obtained after in vitro culture of conventional slow-cooled follicles but was significantly lower than that in controls. Thus, in addition to determining improved procedures for the exposure of follicles to EG and sucrose before and after freezing of caprine early-staged follicles, we report the development of rapid- and slow-cooling protocols.