39 Role of antifreeze protein type I in feline ovarian tissue cryopreservation

Ovarian tissue cryopreservation is emerging as a crucial technique for fertility preservation by leveraging the extensive follicular reserve. This study assessed the effects of two concentrations of antifreeze protein type I (AFP I) on feline follicles following ovarian cryopreservation, focusing on their metabolism and viability after 7 days of in vitro culture. Ovaries were collected from queens (n = 8) after routine ovariectomy, transported to the laboratory at 4°C in 0.9% saline, sectioned into 5 × 3 × 3 mm fragments, and kept in TCM-199 medium with 20% fetal bovine serum (base medium, BM) for 15 min. Then, fragments from each ovary were randomly assigned to (1) fresh control (FC); (2) vitrification control without any AFP (VC); (3) vitrification with 10 µg mL⁻¹ (V10); or (4) 50 µg mL⁻¹ (V50) of AFP in vitrification solution. Fragments were equilibrated in a petri dish containing BM with 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 10 min at 4°C. Then, they were transferred to the vitrification solution for 10 min more before being moved into cryogenic tubes and stored in liquid N₂. Warming was performed in a four-step protocol at 37°C, with decreasing concentrations of sucrose, all diluted in BM, for 5 min each: 1 M, 0.5 M, 0.25 M, and 0 M (only BM). Then, fragments were stained with fluorescence dyes for glutathione (GSH), reactive oxygen species levels (ROS), and mitochondrial activity (values presented as relative fluorescence arbitrary unit/µm²). Fragments were cultured in agarose gel with TCM-HEPES supplemented with 1% BSA; 2 mM l-glutamine, 1% insulin, transferrin, and selenium; 1% antibiotic/antimycotic; 1% sodium pyruvate; 100 ng mL⁻¹ of FSH; and 100 ng mL⁻¹ of EGF. Culture was 7 days at 38.5°C and 5% CO₂, with half of the medium replaced every 48 h. Follicle viability assay was performed by incubation on Neutral Red solution at 50 μg mL⁻¹ in PBS for 30 min, followed by three washes in PBS for 10 min. Follicles were considered viable when cells surrounding the oocyte as well as the oocyte itself were both stained red. Data were submitted to the Shapiro-Wilk normality test and Levene homoscedasticity test and compared by ANOVA followed by Tukey post-hoc test. A P < 0.05 was considered significant, and data are presented as mean ± standard error of the mean (SEM). Reduced mitochondrial activity was noted in V50 (0.69 ± 0.02) compared with FC (1.00 ± 0.07), but not for VC (0.88 ± 0.05) and V10 (0.93 ± 0.04), which were similar to each other. An increase in ROS levels (P < 0.05) was observed in V0 (1.07 ± 0.01) and V10 (1.09 ± 0.1) compared with FC (1.00 ± 0.02) but not V50 (1.01 ± 0.01). No significant differences were observed in GSH levels among groups. A total of 3226 follicles were evaluated (70.1 ± 4.8 per fragment). The percentage of viable preantral follicles in VC (50.3 ± 2.8%) and V50 (51.9 ± 1.1%) groups were lower compared with the FC group (66.2 ± 2.8%), but no significant difference was observed in the V10 group (53.7 ± 1.7%) compared with the FC or vitrified groups. In conclusion, the addition of 10 µg mL⁻¹ of AFP I could maintain mitochondrial activity and follicle viability after cryopreservation and in vitro culture of feline ovarian fragments.

Support for this study was provided by FAPERJ and CNPq.