33 Effect of extended culture after vitrification-warming of bovine oocytes on mitochondrial function

This study aimed to determine the effect of extended culture (EC) on the recovery of mitochondrial function after vitrification-warming of bovine oocytes. Abattoir-derived, IVM bovine oocytes were randomly divided into four treatments: Control group (fresh oocytes), vitrification + 6-h culture (EC6), vitrification + 4-h culture (EC4), and vitrification without culture (Vit). The experiment was replicated four times. Oocytes in the control group were completely denuded after 24 h of maturation and prepared for analysis. Prior to vitrification, oocytes were partially denuded of cumulus cells by brief exposure to hyaluronidase (1 mg mL⁻¹) diluted in HEPES-Tyrode’s albumin lactate pyruvate (TALP) (HM). Oocytes in the EC6, EC4, and Vit groups were vitrified at 18, 20, and 24 h of maturation, respectively. A cohort of five oocytes was placed in equilibration solution (ES; 7.5% ethylene glycol, 7.5% dimethyl sulfoxide (DMSO)) for 9 min followed by brief exposure to vitrification solution (VS; 15% ethylene glycol, 15% DMSO, 0.5 M sucrose). While in VS, oocytes were loaded onto a Cryolock® (Biotech Inc.) and plunged into liquid nitrogen in less than 1 min. Warming was carried out by placing a Cryolock in dilution solution 1 (DS1; 0.5 M sucrose) for 3 min, followed by another 3 min in dilution solution 2 (DS2; 0.25 M sucrose). Finally, oocytes were rinsed and placed in HM until all the oocytes from the same treatment were warmed. After warming, oocytes in EC6 and EC4 were submitted to culture in IVM medium (BO-IVM, IVF-Bioscience) for 6 and 4 h, respectively. Oocytes in the vitrification groups were completely denuded and prepared for analysis after EC. Vitrification and warming were carried out at 38.5°C, and phosphate-buffered saline (PBS) + 20% fetal bovine serum (FBS) was used as base medium. The parameters measured included mitochondrial membrane potential and ATP content. Mitochondrial membrane potential was determined through fluorescence microscopy using the JC-1 dye (CS0390, Sigma Aldrich) counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and values were reported as the ratio of red to green fluorescence. Only oocytes that presented an extruded polar body were considered matured and used for analysis. The ATP content was determined using an ATP bioluminescence assay kit (FLASC, Sigma Aldrich). Concentration of ATP (pmol) per oocyte was generated from a 10-point standard curve. Only oocytes with more than 1 pmol of ATP were deemed viable and used for analysis. Data were analysed by ANOVA with post hoc Tukey using SAS 9.4 (SAS Institute Inc.). Membrane potential results revealed no differences between the vitrification treatments (P > 0.05). The control group (0.943 ± 0.298) was not different from the EC4 (0.836 ± 0.176) group, but it was significantly different than the EC6 (0.785 ± 0.092) and Vit (0.817 ± 0.098) groups. For ATP concentration, the control group differed from the vitrification groups (P < 0.0001); however, no differences were seen among the vitrification groups (P > 0.05). Interestingly, there was an increasing tendency of oocytes deemed as nonviable as the culture hours decreased. In conclusion, ATP concentration of oocytes among the vitrification groups was not different; however, oocytes that underwent 4 h of culture after warming were similar to the control group in regards to mitochondrial membrane potential.