Cryopreservation of Bovine Oocyte using Vitrification Solution and Cryotop Techniques

Abstract

Cryopreservation is used to preserve biological samples over an extended period at ultra-low temperatures. This process evolved into vitrification, a more advanced and superior technology in which fluids or water molecules form a glass-like structure without forming ice crystals. Unlike fresh cells, cryopreservation is reported to reduce oocyte viability and developmental competency. This study employed two vitrification techniques, vitrification solution (VS) and Cryotop, to investigate the meiotic resumption in bovine. Oocytes were extracted from cow ovaries collected from slaughterhouses in Banting and Shah Alam, Selangor, Malaysia. The oocytes were grouped (A, B, and B’) based on cumulus morphology and matured in vitro in a culture dish (humidified 5% carbon dioxide incubator at 38.5°C) for 20 to 24 hr. Oocytes were vitrified after maturation using straws or aids of Cryotop sheets, then submerged in liquid nitrogen and stored for five days before defrosting for cryoprotectant elimination. By using Giemsa staining, the maturation state of fresh and vitrified bovine oocytes was evaluated through five parameters: zygotene, pachytene, diakinesis, metaphase I, and metaphase II. The maturation rate demonstrated only slight differences in the three groups of oocytes treated with VS (A: 44.79%; B: 30.97%; B’: 20.70%) and Cryotop (A: 39.42%; B: 37.27%; B’: 28.97%), which were significantly lower than fresh oocytes (A: 55.83%; B: 44.82%; B’: 56.17%). Both VS and Cryotop methods were viable options for cryopreserving oocytes, but the Cryotop technique was more effective in increasing the meiotic competence of poor-quality oocytes.