| dc.description.abstract | Cryopreservation has increased the possibilities in animal genetic breeding and reproduction technologies. In recent years, vitrification has emerged as a promising substitute for conventional cryopreservation techniques, especially for oocytes and embryos. The rapid cooling/warming rates associated with vitrification prevent the formation of intracellular ice crystals and improve the survivability of oocytes. However, this method relies on the exposure of oocytes to high concentrations of cryoprotective agents (CPAs) that can cause irreversible damage to the oocyte structure. Lipid droplets are a vital part of energy metabolism in oocytes and are highly sensitive to CPA toxicity and low temperatures. This study aimed to identify the optimal concentrations of CPAs that cause the least visible change to lipid droplets in bovine oocytes before vitrification. Additionally, the survival rate of bovine oocytes that are exposed to selected CPAs after the vitrification process was examined. In order to determine the optimal CPAs, a CPA stress test was performed on bovine oocytes (n = 985) using ethylene glycol (EG), propylene glycol (PG), glycerol (GLY), methanol (MeOH), and dimethyl sulfoxide (DMSO) as CPAs. Two concentrations of each individual CPA were used in the stress test in addition to different combinations of these CPAs. From each treatment group, five oocytes were stained with BODIPY 493/503 to visualize lipid droplets and approximately 20 oocytes were fertilized to evaluate the development. Four treatments of PG 20%, GLY 20%, DMSO 7.5% + EG 7.5%, and EG 7.5% + PG 7.5% were selected as optimal CPAs due to their high embryo development and low impact on lipid droplets. Vitrification was performed (n = 153) using the droplet method with the selected CPAs and 0.5% sucrose. Upon thawing, approximately eight oocytes were stained with BODIPY 493/503 to visualize lipid droplets and the remaining oocytes were fertilized to evaluate development post vitrification. All oocytes appeared degenerated after fertilization. Confocal imaging of lipid droplets showed significant damage to the oocytes incubated with PG, GLY, and DMSO + EG. Another vitrification process was conducted (n = 35) with EG + PG which yielded the same cleavage result of 0.0%. However, out of 35 vitrified/warmed oocytes, 29 appeared degenerated and six appeared not cleaved. The conclusion of this study was that the irreversible damage to cellular structures caused by CPAs and low temperature lead to a decreased survival rate. The addition of more equilibrium stages and increased exposure to vitrification media in future experiments may result in oocyte survival post-thaw. | |
