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  • Title: Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols.
    Author: Demirci B, Lornage J, Salle B, Frappart L, Franck M, Guerin JF.
    Journal: Fertil Steril; 2001 Apr; 75(4):754-62. PubMed ID: 11287031.
    Abstract:
    OBJECTIVE: To test the toxicity of cryoprotectant in sheep ovarian tissue and to determine optimal conditions for freezing hemiovary cortex. DESIGN: Small follicles (<60 microm 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, Hôpital Edouard Herriot, Lyon, France. ANIMAL(S): Lambs 5 to 6 months of age. INTERVENTION(S): Two-millimeter 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 cryoprotectant 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 8.4% (2 M 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 degrees C/min) without seeding and the standard very slow cooling protocol (0.3 degrees C/min) were similar. CONCLUSION(S): Optimal survival of primordial follicles in the sheep was obtained by using a slow cooling protocol with semiautomatic seeding at 2 M of DMSO.
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