Resumption of ATP levels and mitochondrial functionality in vitrified/warmed ovine oocytes.

Alterations in mitochondrial functionality and ATP production have been documented in cryopreserved oocytes. However the capability of vitrified-warmed oocytes to recover these damages associated to cryopreservation has not been established yet. The aim of this study was to evaluate the resumption of mitochondrial activity in vitrified ovine oocytes during the post-warming culture. Metaphase II (MII) oocytes were vitrified according to standard procedure used in our laboratory. After warming, oocytes were cultured in 5% CO2 in air at 39°C. At fixed time-points (0, 2, 4 and 6 hours) the oocytes were processed for: 1) determination of intracellular ATP levels by capillary electrophoresis; 2) assessment of active mitochondrial distribution using MitoTracker Red CM-H2XRos probe and analyzed through confocal microscopy. For each time-points, n=20 vitrified-warmed oocytes plus n=20 fresh MII oocytes (as a control) were used for the analyses above described. Differences in mitochondrial activity and ATP concentration between control and vitrified oocytes were assessed by ANOVA, while differences in mitochondrial distribution patterns were analyzed by Chi square test. ATP levels were higher (P˂0.01) in fresh than in vitrified-warmed oocytes. Moreover differences have been found in vitrified oocytes during post-warming culture. Indeed, from 0 to 2h of culture we observed a decline of ATP levels without significant differences, while at 4 and 6h post- warming the ATP concentration increased significantly but did not reach the higher value seen in fresh oocytes (P<0.01). Also mitochondrial activity showed higher value in fresh oocytes compared to vitrified ones (P<0.01). In particular during the culture post-warming, the value was significantly lower at 0 and 6h compared to fresh oocytes and to 4h of culture, while at 2h the value was intermediate between the time point 0 and 4h (P<0.01). Referring to mitochondrial distribution patterns, we classified it in three groups: 1) Pattern A: homogeneous FINE; 2) Pattern B: homogeneous GRANULAR; 3) Pattern C: heterogeneous CLUSTERED. In fresh group a lowerpercentages of oocytes have shown the pattern A compared to vitrified oocytes (P<0.05). For pattern B no significantly differences were evidenced among groups. For pattern C, fresh and warmed oocytes at 6h have shown the higher percentages compared to warmed oocytes at 0, 2, and 4h of culture (P<0.01). No differences were evidenced between fresh and warmed oocytes at 6 h. In conclusion mitochondrial function and ATP level increased significantly in vitrified/warmed ovine oocyte within 6h of in vitro culture. Time of mitochondrial activity resumption after vitrification could be used to standardize the vitrification protocols and to improve the developmental competence in vitrified/warmed oocyte. This study was supported by MIGLIOVINGENSAR project.