Effect of different cryoprotectant concentrations for ultrarapid freezing of immature goat follicular oocytes on their subsequent maturation and fertilization in vitro

The in vitro maturation, fertilization, and morphological changes of goat cumulus–oocyte complexes (COCs) cryopreserved by ultrarapid freezing using DPBS + 0.5% sucrose + 0.4% BSA and 4 M, 6 M, 8 M, or 10 M concentrations of glycerol (G) or ethylene glycol (EG) were studied. COCs were cryopreserved by ultrarapid freezing by holding them over LN2 vapor for 2-3 min and then plunging them into LN2. After 7–10 d of storage, the COCs were rewarmed and used for in vitro maturation or fertilization in 2 experiments to record morphological damage due to ultrarapid freezing, nuclear maturation 28 h after culture, and fertilization 24 h after insemination. Freshly collected COCs were matured and fertilized simultaneously and kept as controls. The proportions of COCs that were recovered in morphologically normal form were highest for oocytes vitrified in 6 M concentrations of both G (90.8%) and EG (95.0%). The proportions of oocytes in morphologically normal form increased with increasing concentrations of both G and EG up to 8 M but at 10 M, the proportion of normal oocytes decreased significantly (70.7% for G and 72.0% for EG; P < 0.05). At the end of experiments 1 (n = 566) and 2 (n = 1019), nuclear maturation and fertilization of oocytes was significantly higher (P < 0.05) for fresh oocytes (M-II: 65.62%; Fert: 40.8%) than for oocytes frozen by ultrarapid freezing (M-II:8.0%,21.1%, and 25.0% for 4MG, 6MG and 8MG and, 22.6%, 34.3% and 41.9% for 4MEG, 6MEG and 8MEG respectively; Fert: 5.4%, 14.6% and 17.2% for 4MG, 6MG and 8MG and, 15.2%, 25.7% and 31.5% for 4MEG, 6MEG and 8MEG respectively). Nuclear maturation and fertilization of vitrified oocytes increased with increasing concentration of both G and EG up to 8 M concentration, but at 10 M concentration, the proportion of oocytes matured (6.4% for G, 8.4% for EG) or fertilized (4.2% for G, 5.6% for EG) decreased significantly (P < 0.05). EG was found to be a better cryoprotectant for ultrarapid freezing of goat oocytes as evident by significantly higher proportions (P < 0.05) of oocytes maturing and fertilizing in EG compared to G at all concentrations tested. It was concluded that ultrarapid freezing causes morphological damage to oocytes. The optimum ultrarapid freezing cryoprotectant is up to 8 M concentration of G and EG, with EG exceeding G.