Study on Characteristics of Intracellular Ice Formation Behavior for Haliotis Diversicolor Eggs and Porcine Oocytes by a Thermal Electric Cooled Cryomicroscope

This study aimed to develop a cryomicroscope system based on thermoelectric cooling (TEC) and to use the TEC cryomicroscope system to investigate intracellular ice formation (IIF) behaviors of small abalone eggs and germinal vesicle (GV) stage porcine oocytes. The TEC cryomicroscope system can achieve accurate control of cooling rate up to 70degreeC/min and heating rate up to 112degreeC/min. The isothermal control can be achieved between 70degreeC and -55degreeC with root-mean-squared-error less than 0.4degreeC. According to the IIF experiments of small abalone eggs, IIF were well suppressed at the cooling rates of 1.5, 3, 7, 12degreeC/min in the presence of 2.0, 2.5, 3.0 and 4.0 M dimethyl sulfoxide (DMSO), respectively. Thus, 2.0 M DMSO was selected to be the suspension solution for considering the solution toxicity effect (STE)，and the eggs were cooled at 1.5 degreeC/min to perform osmotic observation. The results showed 48.8% of small abalone eggs was osmotically active after thawing and higher freezing rate reduced the percentage of osmotically active eggs. By separate experiment of fertility test with 1.5degreeC/min cooling rate in the presence of 2.0 M DMSO as cryopreservation protocol, a normalized hatching rate of 23.7% was obtained. As to IIF experiments of germinal vesicle (GV) stage porcine oocytes , five constant cooling rates of 24, 12, 6, 3 and 1.5degreeC/min were tested in experiments in freezing GV porcine oocytes from 20degreeC to -50degreeC in an NCSU-23 medium plus 2.0 M DMSO. 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 1.5degreeC/min, respectively. 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. The survival was defined as porcine oocytes developed from the GV stage to the MII stage by using Hoechst 33258 staining. 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 6degreeC/min, respectively. The experimental results indicate that slow freezing is a feasible approach for cryopreservation of small abalone eggs and 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 small abalone eggs and GV porcine COCs by using a TEC cryomicroscope system.