26 Effect of different cryoprotectant concentrations on vitrification of in vitro-matured bovine oocytes in paper containers

Abstract

A great challenge for successful oocyte vitrification is the development of a low-cytotoxic cryoprotectant solution in a safe device allowing ultra-rapid cooling. This study compared different concentrations of cryoprotectants for bovine IVM-oocyte vitrification in a safe paper container device on oocyte survival and cleavage rates. Abattoir ovaries were obtained and cumulus–oocyte complexes (COCs) were recovered by aspirating follicles of 3 to 6 mm in diameter. A total of 470 COCs with homogeneous cytoplasm oocytes, surrounded by several layers of cumulus cells were selected, in 5 replicates. Groups of ∼50 COCs were matured in 500 µL of semi-defined IVM medium for 22 h at 38.8°C in a humidified atmosphere with 5% CO₂. After IVM, COCs were allocated to 1 of 3 groups of 20 to 30 COCs, differing only in final concentration of cryoprotectants. A nonvitrified control group (CG) was also tested, totalling 4 groups. Before vitrification, each group was transferred to 500 µL of TCM-199 HEPES with 20% fetal bovine serum (FBS) (Base medium, BM) for 5 min at 34°C, and COCs were partially denuded by gentle pipetting. Vitrification followed a 3-step protocol at room temperature and groups of 4 to 5 COCs were transferred to BM solution drops containing (1) 5% ethylene glycol (EG) + 5% dimethyl sulfoxide (DMSO) for 30 s; (2) 10% EG + 10% DMSO + 0.25 M sucrose for 30 s; and (3) vitrification solution (VS), according to each group: high (HG), 20% EG + 20% DMSO + 0.5 M sucrose; medium (MG), 15% EG + 15% DMSO + 0.5 M sucrose; or low (LG), 10% EG + 10% DMSO + 0.5 M sucrose for 30 s. Afterwards, COCs were loaded in <1 µL of solution and placed in a homemade paper container device, and immediately plunged in liquid nitrogen. Warming was performed placing the paper container in 3 mL of 1 M sucrose in BM for 2 min. After warming, a 3-step protocol was conducted and COCs were transferred to (1) 500 µL of 0.5 M sucrose in BM for 2 min; (2) 500 µL of 0.25 M sucrose for 2 min; (3) 500 µL of BM for 2 min. Then, COCs from each group were transferred to 250 µL of semi-defined IVF medium. Motile sperm were recovered by Percoll washing from one bull and added to IVF medium (Day 0) at final concentration of 10⁶ sperm mL⁻¹ for 18 h. At Day 1, all presumptive zygotes were cultured in 25 µL of SOF medium with 5% FBS under mineral oil at 38.8°C with 5% CO₂ and 5% O₂. Normal data were subjected to ANOVA and post hoc Tukey test. Cleavage rate was recorded at Day 2 after IVF. Oocyte survival rate was similar (P > 0.05) among vitrified groups (HG, 80%; MG, 86%; LG,87%). Cleavage rate differed (P < 0.05) in all vitrified groups compared with control (CG, 82%; HG, 10%; MG, 16%; LG, 16%). Although no difference (P > 0.05) was observed among vitrified groups, MG and LG showed a slightly increased oocyte survival and cleavage rates compared with HG. In conclusion, the use of either medium or low concentrations of cryoprotectants may be a less toxic alternative for vitrification of IVM bovine oocytes on paper device.

This research was funded by CAPES/COFECUB (#88881.142966/2017-01).