Cryopreservation of Germinal Vesicle Stage Porcine Oocytes Based on Intracellular Ice Formation Assessment

This study aimed at evaluating the feasibility of slow freezing for cryopreservation of germinal vesicle (GV) stage porcine oocytes. In this study, intracellular ice formation (IIF) characteristics of GV porcine oocytes were investigated by using a thermoelectric cooling (TEC) cryomicroscope system. This cryomicroscope system used a thermoelectric cooling (TEC) chip in its cold stage as a heat sink and employed a PID control algorithm to achieve accurate temperature control. The temperature was controlled to a range between 70°C and 55°C with an accuracy of ±0.5°C. Five constant cooling rates of 24, 12, 6, 3 and 1.5°C/min were tested in experiments in freezing GV porcine oocytes from 20°C to -50°C in an NCSU-23 medium plus 2.0 M DMSO. The IIF temperature of each individual oocyte was recorded and cumulative IIF probabilities were calculated for each cooling rate. The total cumulative probabilities of IIF temperature distribution were 100%, 100%, 50.0%, 54.3% and 58.6% at cooling rates of 24, 12, 6, 3 and 1.5°C/min, respectively. A Weibull distribution model was found to adequately describe the distribution of IIF temperatures of GV porcine oocytes for the cooling rates tested (R² = 0.858±0.09). The IIF experimental results indicate that cooling rates of 6, 3 and 1.5°C/min could be considered as possible cryopreservation protocols. Further experiments were performed to examine the feasibility of using these protocols to cryopreserve GV porcine oocytes. After 44 h of in-vitro maturation in NCSU-23, the survival of thawed oocytes was checked. Porcine oocytes developed from the GV stage to the MII stage by using Hoechst 33258 staining, followed by Lacmoid staining as a secondary check. Normalized survival rates of 37.7±4.6%, 45.0±4.4% and 45.4±5.9% were obtained for GV oocytes frozen at 1.5, 3 and 6°C/min, respectively. The experimental results indicate that slow freezing is a feasible approach for cryopreservation of GV porcine oocytes when cooling rate is properly selected. This study also demonstrated an efficient approach for investigating optimal cooling rates by assessing the IIF characteristics of GV porcine COCs.