341 IN VITRO MATURATION OF GOAT FOLLICULAR OOCYTES SUBSEQUENT TO THEIR CRYOPRESERVATION BY VITRIFICATION

Abstract

The in vitro maturation, fertilization, and morphological changes of goat cumulus–oocyte complexes (COCs) cryopreserved by vitrification 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. Oocytes were cryopreserved by vitrification by holding them over LN vapor for 2-3 min and then plunging them into LN. After 7–10 days of storage, the COCs were rewarmed and used for in vitro maturation or fertilization in 2 experiments to record morphological damage due to vitrification, nuclear maturation 28 h after culture (9 replicates), and fertilization 24 h after insemination (10 replicates). Oocytes were matured in vitro in 50–100 �L microdrops of TCM-199 supplemented with 5 �g mL-1 FSH, 5 �g mL-1 LH, and 1 ng mL-1 estradiol, 25 mM HEPES, 0.25 mM pyruvate, and antibiotics for 28 h at 38 � 1�C and 5% CO2 in a CO2 incubator. Matured oocytes were fertilized using frozen–thawed buck semen prepared as per previous methods (Palomo et al. 1999 Theriogenology 51, 927). Freshly collected COCs were matured and fertilized simultaneously and kept as controls. The arcsin transformed data of the proportions of oocytes matured or fertilized was compared by Duncan's new multiple range test. 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 = 856) and 2 (n = 1204), nuclear maturation and fertilization of oocytes was significantly higher (P < 0.05) for fresh oocytes (M-II: 65.62%; fert: 40.8%) than for vitrified oocytes. Nuclear maturation and fertilization of vitrified oocytes increased with increasing concentration of both G [M-II: 4 M (22.7%), 6 M (34.2%), 8 M (41.9%); fert: 4 M (15.2%), 6 M (25.7%), 8 M (31.5%)] and EG [M-II: 4 M (8.0%), 6 M (21.7%), 8 M (25.0%); fert: 4 M (5.5%), 6 M (14.7%), 8 M (17.2%)] 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 vitrification 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 vitrification brings about morphological damage to oocytes. The optimum vitrification cryoprotectant is up to 8 M concentration of G and EG, with EG being better.