233 The influence of different cryoprotectants on mitochondrial function in vitrified bovine oocytes

There are concerns about the impact of high concentrations of cryoprotectants such as dimethyl sulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG) during oocyte vitrification. It has been established that DMSO can cause spindle disorganization and PG and EG can cause in vitro chromosomal damage, and all three can alter Ca²⁺ levels. Oocyte mitochondria are vital for the regulation of calcium, and appropriate calcium regulation is vital for mitochondrial function. It has been hypothesised that cryoprotectants can harm the mitochondria within the oocyte, causing poor calcium regulation, which subsequently leads to decreased embryo viability. Therefore, in this study oocyte mitochondria function was assessed after cryopreservation to determine the effect of different cryoprotectants. Bovine oocytes were purchased from a commercial supplier (DeSoto Biosciences) and matured for 20 h during shipment; mature oocytes were selected for vitrification. Oocytes were partially denuded for vitrification, and cumulus cells were removed completely before staining. Vitrification and warming media were prepared in HEPES-buffered M199 with 20% FBS with either DMSO + EG or PG + EG. Groups of 5 oocytes were incubated in equilibration medium consisting of 7.5% of each cryoprotectant for 2.5 minutes, washed in vitrification medium consisting of 15% of each cryoprotectant and 1 M sucrose, and loaded onto a Cryolock device before being plunged into liquid nitrogen. For warming, a Cryolock with oocytes was plunged into warming medium with 1 M sucrose and incubated for 1 minute. Oocytes were then transferred to dilution medium with 0.5 M sucrose for 3 minutes and incubated in IVM medium (IVF Biosciences) for 2 h for post-warming recovery. To measure mitochondria function, overall reactive oxygen species (ROS) were detected with 2′-7′-dichlorodihydrofluorescein diacetate and mitochondrial membrane potential with JC-1. Imaging was performed on a Nikon fluorescent microscope and intensities were recorded and analysed using ImageJ. One hundred forty-seven oocytes were used for the ROS staining and 106 oocytes were used for the JC-1 treatment. The same oocytes were not used for both ROS and JC-1 staining. The resulting data were not normally distributed, so a Mann–Whitney test was used (GraphPad Prism) with a significance at P ≤ 0.05. Oocytes vitrified with DMSO and EG had a median ROS of 331.3 (n = 71) and JC-1 results had a median ratio of fluorescence of 2.850 (n = 52), compared with those vitrified with PG and EG, which had a median ROS of 367.5 (n = 76) and JC-1 median ratio of fluorescence of 2.391 (n = 54). Oocytes vitrified with DMSO and EG resulted in decreased ROS levels and increased mitochondrial membrane potential compared with those vitrified with PG and EG. Our findings will be useful in future studies to minimize damage during oocyte cryopreservation. Subsequent studies will focus on subsequent development of cryopreserved oocytes after fertilization.