64 CRYOPRESERVATION OF CAT OVARIAN TISSUE BY VITRIFICATION

Abstract

The cryopreservation of ovarian tissue is linked to a wide range of possible applications, from oocyte harvesting to allo- and xenotransplantation. These procedures have significant potential for the preservation of valuable genetic material and endangered-species conservation. The objectives of the present study were to (1) compare viability of preantral follicles obtained from fresh v. vitrified feline ovarian cortex, (2) evaluate the effect of apoptotic inhibitors (ROCK inhibitor v. glutathione) on viability of follicles from vitrified samples, and (3) determine the optimal inhibitor concentration for follicle viability. In Experiment 1, 5 × 5 × 1 mm cortical tissue samples were obtained from excised cat ovaries and assigned to either the fresh control or vitrification group. Fresh samples were processed through a 230-micron-pore dissection strainer to collect preantral follicles. Follicles were then stained in Trypan blue to determine membrane integrity and survival rates. Vitrification samples were first equilibrated in 7.5% dimethyl sulfoxide and 7.5% ethylene glycol at ~22°C and then in vitrification solution consisting of 20% dimethyl sulfoxide, 20% ethylene glycol, and 0.5 M sucrose. They were then vitrified on a thin, perforated, metal strip (Cryotissue, Kitazato Biopharma, Fujinomiya, Japan). Samples were later warmed in 1.0 M sucrose at 38°C. Follicles were then collected and assessed for survival. In Experiment 2, follicles were collected from samples vitrified/warmed in cryo-media supplemented with either 3 × 104 nM ROCK inhibitor or 6 nM glutathione. Follicles from samples vitrified/warmed without inhibitor treatment were used as controls. In Experiment 3, tissue samples were vitrified/warmed in cryo-media supplemented with 0, 2, 6, or 10 nM glutathione before follicle viability was determined. Data were evaluated by chi square analysis. In Experiment 1, 637 and 340 follicles were collected from fresh and vitrified samples, respectively. Overall, survival was higher in freshly collected follicles when compared to those from the vitrified group (67 v. 18%, respectively; P < 0.05). Evaluation of apoptotic inhibitors was determined through collection of 314, 354, and 506 follicles from inhibitor-free, ROCK inhibitor, and glutathione-treated media, respectively. Follicles from samples vitrified in inhibitor-free media and in ROCK inhibitor survived at a lower rate than those from glutathione-treated samples (10 and 13% v. 18%, respectively; P < 0.05). In Experiment 3, a total of 539, 641, 625, and 632 follicles were collected from samples treated in 0, 2, 6, and 10 nM glutathione, respectively. There were no statistical differences in follicle survival among the 0, 2, and 6 nM groups. However, follicles treated in 10 nM glutathione survived at a higher rate than those vitrified/warmed in the absence of glutathione (20 v. 14%; P < 0.05). In summary, viability of preantral follicles from ovarian cortical tissue was significantly reduced by vitrification. Despite this, tolerance of such follicles to cryopreservation was improved by vitrifying and warming in cryo-media containing 10 nM glutathione. Partially funded by the LSU/ACRES Collaborative Project.