358 MORPHOLOGICAL CHANGES IN VITRIFIED WATER BUFFALO CUMULUS–OOCYTE COMPLEXES AND THEIR IN VITRO NUCLEAR MATURATION

Abstract

Morphological changes and in vitro nuclear maturation of buffalo cumulus–oocyte complexes (COCs) was evaluated subsequent to their cryopreservation by vitrification in solutions containing 4 M, 6 M, 8 M, and 10 M concentrations of glycerol (G) or ethylene glycol (EG) or their combination. COCs collected from buffalo ovaries by aspiration (n = 1342) were equilibrated in 50% of the vitrification solution and then placed in the vitrification solution (Dulbecco's phosphate-buffered saline+0.5M sucrose + 0.5% BSA + cryoprotectant). COCs were transferred to empty semen straws, kept over LN vapor for 2–3 min, and then plunged into LN. After 7–10 days of storage, COCs were warmed and evaluated for morphological damage. Morphologically normal COCs were cultured in vitro (9 replicates each with 5–10 oocytes in 50–100-µL culture drops) in TCM-199 medium supplemented with 5µgmL-1 FSH, 5µgmL-1 LH, and 1 ngmL-1 estradiol with 25mM HEPES, 0.25mM pyruvate, and antibiotics. The COCs were incubated for 24 h at 38±1°C and 5% CO₂ in humidified air in a CO₂ incubator and evaluated for nuclear maturation at the end of 24 h of culture. Freshly collected COCs were also matured in vitro and kept as controls (n=142). The proportions of COCs retrieved in morphologically normal form were compared by chi-square test; the arcsin transformed data of the proportions of oocytes matured was compared by Duncan's new multiple range test. The proportions of oocytes recovered in a morphologically normal form were highest in the 6M EG group (95.23%), followed by 8M EG (94.0%) and 6M G (90.6%) groups. At 10M concentration, a significantly (P <0.05) lower percentage of oocytes was morphologically normal. The morphological abnormalities recorded were change in shape, rupture of zona pellucida, and leakage of oocyte contents. A significantly higher (65.62%; P <0.05) proportion of fresh oocytes reached metaphase-II compared to oocytes vitrified in all concentrations of G and EG. The proportion of oocytes reaching metaphase-II increased with increasing concentrations of both G and EG, but at 10M concentration the proportion of oocytes reaching metaphase-II decreased. The proportions of COCs reaching metaphase-II in 4M, 6 M, 8M, and 10M glycerol were 6.9%, 21.2%, 25.7%, and 5.5%, respectively. The respective proportions of COCs reaching metaphase-II in 4M, 6 M, 8M, and 10M ethylene glycol were 21.9%, 34.3%, 40.8%, and 7.5%. No significant benefit of in vitro maturation of oocytes was seen for oocytes vitrified in a combination of both G and EG. It was concluded that although vitrification brings about some damage to the oocytes, yet it appears to be a good tool for oocyte cryopreservation, and 8M concentration of either G or EG appears to be optimum for vitrification of buffalo oocytes.