Effect of bovine oocyte vitrification with EGTA and post-warming recovery with resveratrol on meiotic spindle, mitochondrial function, reactive oxygen species, and developmental competence.

The present study aimed to determine the effects of the addition of EGTA to vitrification solutions and a post-warming recovery period supplemented with 1 µM resveratrol on meiotic spindle integrity, mitochondrial activity, ATP content, reactive oxygen species (ROS) levels, and developmental potential of partially denuded, vitrified-warmed bovine oocytes. Results of microtubule distribution and chromosomal arrangement indicated that resveratrol supplementation, irrespective to EGTA addition, reduced the incidence of abnormal meiotic spindles to similar levels of the control group. Mitochondrial membrane potential was similar in all groups, but ATP content was negatively affected by the vitrification-warming procedure and failed to recover after 4 h of post-warming culture. Resveratrol caused the reduction of ROS to lower levels of the control group, and showed the lowest ROS levels when combined with EGTA treatment. Oocytes in all vitrification groups presented lower developmental potential when compared to fresh oocytes. However, oocytes that underwent vitrification supplemented with EGTA and post-warming culture along with resveratrol showed higher developmental competence compared with vitrified-warmed oocytes not supplemented with resveratrol. The results of our study indicate that submitting vitrified-warmed, partially denuded bovine oocytes to a post-warming recovery period supplemented with 1 µM resveratrol improves vitrification outcomes. However, the benefits of EGTA on vitrification and warming of bovine oocytes need to be further investigated.

Recovery of spindle morphology and mitochondrial function through extended culture after vitrification-warming of bovine oocytes.

The present study aimed to determine the effects of vitrification on the meiotic spindle and mitochondrial function of bovine oocytes submitted to different times of post-warming culture. Partially denuded cumulus-oocyte complexes were vitrified at different maturation times (18-, 20-, and 24-h) using a two-step cryoprotectant addition protocol and submitted to 6-, 4-, or 0-h of post-warming extended culture in maturation medium. Microtubule configuration and chromosomal arrangement were analyzed after 0- and 6-h of extended culture, whereas mitochondrial membrane potential and ATP content were measured at 0-, 4-, and 6-h of post-warming recovery. Results of meiotic spindle integrity revealed that vitrified-warmed oocytes that underwent 6-h of culture had similar incidence of normal microtubule configuration and chromosomal arrangement as compared to fresh oocytes, but higher than oocytes in the vitrification control group (no culture). Mitochondrial membrane potential was not different in all the vitrification groups, but the oocytes that were cultured for 4-h after warming had similar levels compared to fresh oocytes. ATP concentration in all vitrification groups was lower than the control group. However, oocytes cultured for 6-h had the lowest rate of ATP depleted oocytes among the vitrification groups. The results of this study indicate that extended culture after warming promotes the recovery of the meiotic spindle and, to some extent, mitochondrial function of vitrified-warmed metaphase II bovine oocytes.