Effect of vitrification on meiotic maturation, mitochondrial distribution and glutathione synthesis in immature silver fox cumulus oocyte complexes

The present study was designed to investigate the effects of vitrifying oocytes obtained from silver foxes on nuclear maturation, mitochondrial distribution and glutathione (GSH) synthesis after in vitro culture for 72 h. Immature oocytes were randomly divided into three groups: (1) fresh GV (germinal vesicle) oocytes (Control group), (2) exposure to the equilibration and vitrification solution but without being plunged into liquid nitrogen (exposed group), and (3) vitrification by the cryoloop method (vitrified-warmed group). The number of survival oocytes was not decreased by either being exposed to the cryoprotectant or being vitrified-warmed compared with the control group (P > 0.05). After IVM, the percentage of resumption of meiosis for vitrified-warmed oocytes (41.9%) was significantly lower than in the control (81.2%) and exposed (79.1%) groups (P < 0.05). However, the proportion of oocytes reaching the metaphase II (MII) stage was similar among the different groups (11.4%, 9.3% and 5.2%, respectively, P > 0.05). The translocation of active mitochondria during fox oocyte maturation was revealed using MitoTracker Red staining and confocal laser microscopy. For fresh oocytes at the GV stage, active mitochondria were distributed around the entire cortex with small granulations and various-sized cavities (no MitoTracker signals). After IVM, the mitochondria formed large granulations and clumps throughout the cytoplasm. Vitrification significantly decreased the proportion of MII oocytes with normal mitochondrial distribution compared with the control and exposed groups (35.4%, 71.9% and 59.2%, respectively, P < 0.05). Similarly, the GSH content was significantly lower in vitrified-warmed oocytes compared with the control and exposed oocytes after IVM (3.4, 5.7 and 4.7 pM/oocyte, respectively, P < 0.05). However, no significant difference was observed between the cryoprotectant exposed and control groups with regard to the normal mitochondrial distribution or GSH content (P > 0.05). These results indicate that vitrification of fox immature oocytes using a cryoloop allows them to resume meiosis and develop to the MII stage. The damage to mitochondria and the GSH synthesis deficiency may be associated with the reduced developmental competence of cryopreserved oocytes.