78 Radio electric asymmetric conveyer treatment during prolonged cold storage of ovaries enhances developmental competence of oocytes in the domestic cat model

Abstract

Hypothermic storage (4°C) of ovaries for long-distance transport holds great potential to expand access to fertility preservation in animals and humans (Duncan et al. 2016 Reproduction 152, 201-210). However, storage for prolonged periods (over 24 h) leads to structural/functional changes in the ovarian tissue with a critical reduction of follicular viability and oocyte quality (Piras et al. 2018 Reprod. Biol. Endocrinol. 10, 16-76; Isachenko et al. 2009 Fertil. Steril. 91, 1556-1559). The radio electric asymmetric conveyer (REAC) is a novel technology platform for neuro and bio-modulation that optimize the ion fluxes and the mechanisms driving cellular asymmetry and polarization in biological structures (Maioli et al. 2016 Sci. Rep. 6, 28682). The REAC has already proved to be effective in promoting cell differentiation and reprogramming and to counteract the biological mechanisms linked to aging and the degenerative process (Berlinguer et al. 2017 Reprod. Biol. Endocrinol. 15, 11; Maioli et al. 2014 Age 36, 9-20). In the present study, the domestic cat was used as an experimental model to evaluate the effect of REAC treatment during ovary storage at 4°C for 48 h on in vitro developmental ability of oocytes retrieved from antral follicles. Ovaries harvested from healthy domestic queens during ovariectomy were randomly assigned to the REAC-treated (R: n = 13) and untreated (C: n = 13) groups. In detail, ovaries were maintained in 4 mL of phosphate-buffered saline at 4°C for 48 h. The REAC device was set at 2.4 GHz, and its conveyer electrodes were immersed into the phosphate-buffered saline. After 48 h, ovaries were sliced to release cumulus-oocyte complexes, which were selected according to their morphological characteristics (Johnston et al. 1991 Biol. Reprod. 45, 898-906) for IVM (R: n = 130; C: n = 133). Matured oocytes were fertilised (IVF) with frozen-thawed epididymal spermatozoa and presumptive zygote were in vitro cultured (IVC) for 7 days. On Day 2 and Day 7 of IVC, respectively, the number of embryos cleaved and developing to the blastocyst stage was determined. The IVM, IVF, and IVC were performed according to the procedure of Piras et al. (2018 Reprod. Biol. End. 16, 76). Data were analysed by chi-square test with STATA\IC 11.0. Maturation rate of oocytes did not differ between groups (R: n = 59/130, 45.4%; C: n = 66/133, 49.6%). Cleavage rate was higher (P < 0.05) in the R group (n = 34/59, 57.6%) compared with the C group (n = 25/66, 37.9%). The percentages of blastocyst formation relative to the number of cleaved embryos (R: n = 12/34, 35.3%; C: n = 3/25 12.0%) and to the total number of MII oocytes (R: n = 12/59, 20.4%; C: n = 3/66, 4.5%) increased (P < 0.05) after REAC treatment compared with the untreated counterpart. In conclusion, REAC treatment during cold storage of cat ovaries for 48 h positively affected the quality of oocytes as assessed by in vitro embryo production outcome. The REAC technology could provide a useful approach for the optimization of ovarian tissue transport conditions for fertility preservation especially for endangered species and patients with fertility-threatening conditions.