Melatonin promotes the in vitro maturation of cat vitrified oocytes

The combination of immature oocyte cryopreservation and in vitro maturation (IVM) allows exploiting the genetic material of valuable individuals, domestic or wild, after spaying or death, but results still need improvements. Preserved oocytes mature to lower rates than fresh ones and oxidative stress and apoptosis hinder survival and development of cryopreserved oocytes. Melatonin (MLTN) was suggested to exert antioxidant and antiapoptotic effects on in vitro matured oocytes of different mammalian species, but it remains to be investigated on cat oocytes. Thus, the aim of this study was to test if MLTN could be beneficial for the IVM of cat oocytes vitrified by Cryotop. Fresh oocytes were firstly used to determine the best concentration of MLTN. Ovaries were obtained at spaying and cumulus oocytes complexes (COCs) were matured as fresh for 24 h in a controlled atmosphere (38.5°C and 5% CO2 in air) in Medium 199 with 3 mg/mL bovine serum albumin, 10 ng/mL epidermal growth factor (EGF), 0.6 mM cysteine and 0.5 IU/mL follicle-stimulating hormone (FSH) + 0.5 IU/mL luteinizing hormone (LH), supplemented with four concentrations (10-7 M, 10-9 M, 10-11 M, 0 M as control) of MLTN. The best concentration was added during oocyte vitrification-warming and/or IVM of vitrified oocytes. Chromatin configurations were assessed by bis-benzimide (Hoechst 33342) staining after IVM. Data were analyzed by Fisher’s exact test (significance at p<0.05). Fresh control COCs reached full maturation (telophase I-metaphase II, TI-MII) at 51.48% (18/35 COCs), 10-11 M MLTN lowered it to 38.71% (12/31), and the addition of melatonin 10-7 and 10-9 M brought it to 58.06% (18/31; p=0.63 vs fresh COCs). The lowest concentration with beneficial effects (10-9 M) was used for vitrified oocytes. While TI-MII rate of control vitrified COCs (no MLTN) was 12.12% (4/33) and the addition of MLTN only during vitrification-warming or IVM slightly increased the rate to 22.58% (7/31; p=0.33), its addition both during cryopreservation and IVM significantly improved it, giving 48.39% (15/31) mature COCs (p<0.01). In summary, MLTN 10-9 M improved the IVM of cat vitrified oocytes. The use of MLTN during cat oocyte vitrification and culture could contribute to improve the developmental competence of biobanked oocytes, bringing it closer to fresh gametes and allowing the achievement of better outcomes from the genetic material of valuable subjects. Supported by UNIMI “PSR Linea 2” 2020 and 2021.