Viability of small preantral ovarian follicles from domestic cats after cryoprotectant exposure and cryopreservation

Abstract

About 1500 preantral follicles can be recovered from a single cat ovary by mechanical dissection. This is a potentially rich source of genetic material if ova could be preserved and grown in vitro, especially from rare or endangered species that die abruptly or are ovariectomized for medical reasons. The aims of this study were to examine cryoprotectant toxicity and then the potential of successfully cryopreserving preantral cat follicles. In the initial toxicity trial, isolated cat follicles (40-90 μm) were exposed to dimethylsulfoxide, glycerol, 1,2-propandiol or ethylene glycol at 0°C for 15 min. Follicle viability was assessed by supravital staining using a combination of Trypan blue and Hoechst 33258 at 0 h, and after 18 h and 1 week of culture. Percentages of follicles with intact oocytes and granulosa cells were similar (P > 0.05) among control (no cryoprotectant), dimethylsulfoxide, 1,2-propandiol and ethylene glycol treatments at all time points, but were reduced (P < 0.05) after glycerol exposure. On the basis of this finding, dimethylsulfoxide and 1,2-propandiol were used to cryopreserve intact follicles, and post-thaw viability was assessed by supravital staining and 5-bromo-2'-deoxyuridine uptake into oocytes and granulosa cells during culture. Of control (noncryopreserved) follicles, 31.4% ± 2.9%, 18.8% ± 1.9% and 16.2% ± 1.6% were intact after 0 h, 18 h and 1 week of culture, respectively. Uptake of 5-bromo-2'-deoxyuridine occurred in approximately 20% of follicles at all time points. On the basis of the presence of both a healthy oocyte and granulosa cells, cryopreservation in dimethylsulfoxide or 1,2-propandiol allowed approximately 19% of follicles to survive. Approximately 10% demonstrated clear evidence of cell activity that was sustainable for 1 week. In conclusion, the cat ovary contains a population of preantral follicles that are not adversely affected by short-term exposure to most conventional cryoprotectants. Furthermore, there is a subpopulation of these follicles capable of surviving cryopreservation, remaining structurally intact and physiologically active after thawing.