Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols

Objective: To test the toxicity of cryoprotectant in sheep ovarian tissue and to determine optimal conditions for freezing hemiovary cortex. Design: Small follicles (<60 <mu>m in diameter) were isolated enzymatically for viability testing. Dead and live follicles were identified by using trypan blue staining, and follicle morphology was examined histologically. Setting: Centre hospitalo-universitaire de Biologie de la Reproduction, Hopital Edouard Herriot, Lyon, France. Animal(s): Lambs 5 to 6 months of age. Intervention(s): Two-millimetre slices of hemiovarian cortex were prepared for cryoprotectant toxicity tests and freezing procedures. Main Outcome Measure(s): Follicular mortality and histologic structure. Result(s): For freezing procedures, the concentration of cryoprotectant was increased to 2 M on the basis of results of cyoprotectant toxicity tests in fresh tissues. Follicular mortality rates were 4.6% with of 2 M dimethyl sulfoxide (DMSO) and 3.8% with 2 M of propylene glycol (PROH). After freezing with semiautomatic seeding, follicular mortality rates were 8.38 (2 hi of DMSO) and 12.4% (2 M of PROH). Tissue morphology was well preserved with 1.5 M of DMSO or PROH. With 1.5 M DMSO, results of the slow cooling protocol (2 degreesC/min) without seeding and the standard very slow cooling protocol (0.3 degreesC/min) were similar. Conclusion(s): Optimal survival of primordial follicles in the sheep was obtained by using a slow tooling protocol with semiautomatic seeding at 2 hi of DMSO, (Fertil Steril (R) 2001;75:753-62. (C) 2001 by American Society for Reproductive Medicine.).