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Purpose Temporary and reversible downregulation of metabolism may improve the survival of tissues exposed to nonphysiological conditions during transport, in vitro culture, and cryopreservation. The objectives of the study were to (1) optimize the concentration and duration of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP-a mitochondrial uncoupling agent) exposures for biopsies of domestic cat ovarian tissue and (2) examine the effects of FCCP preexposures on follicle integrity after tissue culture and/or cryopreservation. Methods Biopsies of cat ovarian tissue were first treated with various concentrations of FCCP (0, 10, 40, or 200 nM) for 10 or 120 min to determine the most suitable pre-exposure conditions. Based on these results, tissues were pre-exposed to 200 nM FCCP for 120 min for the subsequent studies on culture and cryopreservation. In all experiments and for each treatment group, tissue activity and integrity were measured by mitochondrial membrane potential (relative optical density of rhodamine 123 fluorescence), follicular viability (calcein assay), follicular morphology (histology), granulosa cell proliferation (Ki-67 immunostaining), and follicular density.Results Ovarian tissues incubated with 200 nM FCCP for 120 min led to the lowest mitochondrial activity (1.17 ± 0.09; P < 0.05) compared to control group (0 nM; 1.30 ± 0.12) while maintaining a constant percentage of viable follicles (75.3 ± 7.8 %) similar to the control group (71.8 ± 11.7 %; P > 0.05). After 2 days of in vitro culture, percentage of viable follicles (78.8 ± 8.9 %) in similar pre-exposure conditions was higher (P < 0.05) than in the absence of FCCP (61.2 ± 12.0 %) with percentages of morphologically normal follicles (57.6 ± 17.3 %) not different from the fresh tissue (70.2 ± 7.1 %; P > 0.05). Interestingly, percentages of cellular proliferation and follicular density were unaltered by the FCCP exposures. Based on the indicators mentioned above, the FCCP-treated tissue fragments did not have a better follicle integrity after freezing and thawing. Conclusions Pre-exposure to 200 nM FCCP during 120 min protects and enhances the follicle integrity in cat ovarian tissue during short-term in vitro culture. However, FCCP does not appear to exert a beneficial or detrimental effect during ovarian tissue cryopreservation.

Transplantation of cryopreserved ovarian tissue is a fertility preservation technique that results in live births in patients who have undergone pelvic chemo- or radiotherapy. It can be used for conservation purposes in endangered animals by transplanting the cryopreserved ovarian tissue of animals with highly valuable genetics into the immunodeficient animal to grow the follicles. This study aimed to examine the preantral follicular viability and K i-67 proliferation index of the preantral follicles within fresh and cryopreserved cat ovarian tissue transplanted into nude mice as a model for endangered felids. Adult female nude mice (n = 9) were chosen to be the hosts of ovarian tissues from 2-year-old domestic cat. Ovarian cortical tissues were cut into 66 small pieces (1 × 1 × 0.2 mm3). Half of the fragments were cryopreserved using slow-freezing method with 1.5 M dimethyl sulfoxide (DMSO) and 0.1 M sucrose and thawed using thawing medium with 0.75 M DMSO and 0.5 M sucrose. Three pieces of fresh and cryopreserved tissues were transplanted into the subcutaneous pocket of the left and right side of the back of the nude mouse, respectively. Follicular viability and proliferation index were investigated after graft retrieval on Day 15 post-transplantation. Evaluation of preantral follicle viability was based on the integrity of the basement membrane, pyknotic bodies, and oocyte integrity. Proliferation index was determined by percentage of preantral follicle that had K i-67 immunopositive granulosa cells. Preantral follicle viability data was analysed using Kruskal-Wallis test and proliferation index data were analysed using general linear model test of least squares means. The percentages of viable follicles of all stages were decreased at Day 15 (Table 1). The percentage of proliferating preantral follicles in the fresh ovarian fragments was higher after transplantation (11 ± 2% and 46 ± 24% before and after transplantation, respectively; P < 0.05). However, the percentages of proliferating follicles were not different between before and after cryopreserved tissue transplantation (35 ± 8% and 45 ± 33%, respectively). In conclusion, our findings showed the possibility of domestic cat ovarian tissue transplantation into the nude mouse. although cryopreserved ovarian tissue did not tolerate the short-term transplantation as the fresh tissue. This approach needs further investigation to optimize the transplantation technique in terms of ischaemic reperfusion and neovascularization. Table 1.Percentage of viable follicles per area of 0.0625 mm2 within fresh and cryopreserved cat ovarian fragments at Day 0 and Day 15 of the study

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