Comparison of Cryopreservation of Bovine Ovarian Tissue: Slow and Rapid Cryopreservation

Cryopreservation of ovarian cortical is a good way to preserve a large number of oocytes when they are expected to disappear due to chemotherapy, radiotherapy, or other predictive causes of premature ovarian failure. Cryopreservation of ovarian tissues has generally been performed using slow-freezing methods with a programmed freezer. Recently, a rapid, simple and economical cryopreservation method, rapid freezing, has been applied to ovarian tissues. These methods are preformed in several species. The aim of our investigations was to compare rapid freezing and slow freezing on bovine ovary cortex by immunohistochemistry examination of apoptosis and necrosis of ovarian cells. This cross-sectional study was performed to compare the two laboratory methods. The cow ovary cortex was cut around 0.5mm thick and 1x5 mm square. 20 patches were cut. Ovarian tissues were placed in 10ml of cryoprotectant and allowed to be incubated at 4°C for 30 minutes on a roller mixer. After equilibration tissues were transferred to the cryovials, half of ovarian patches were frozen by slow program and half of them were frozen by rapid freezing. In slow method vials were placed in programmable freezer (Planer Biomed, Sunbury, and Middlesex, UK) and temperature slow down gradually to - 140°C. After finishing the program the samples unloaded and plunged in to nitrogen. For rapid freezing vial directly plunged in to liquid nitrogen. The vials remove from the liquid nitrogen and thawed in the beaker of water at the room temperature. Thin sections were transferred to a small petri dish contain Caxboxy flurenscein diacetate, succinmidylester (CFDA) and incubated in 37°C for 20 minutes. The thin slides were examined under fluroscopic light. The proportion of necroses was significantly increased in rapid-frozen ovaries 36% compared to 12% in slow frozen (P< 0.05). Conventional slow freezing is the method of choice for the cryopreservation of ovarian fragments, resulting in a much better preservation of all types of follicles than that by the rapid freezing.