Specific activation requirements of in vitro-matured sheep oocytes following vitrification-warming

Oocyte vitrification and assisted oocyte activation have increasingly important roles in assisted reproductive technology. Yet, an important area of concern with matured oocyte cryobiology is that elements of oocytes intimately involved in metaphase-II arrest may be modified by cryopreservation. By comparing different cellular characteristics of unvitrified, vitrified-warmed, and unvitrified-activated oocytes, the present study investigated how vitrification-warming process may affect developmental competence of in vitro-matured sheep oocytes following parthenogenetic activation. Structural, ultrastructural, and molecular analyses indicated that the characteristics of vitrified-warmed oocytes vastly differed from fresh oocytes, instead resembling unvitrified-activated oocytes. For unvitrified oocytes, the highest blastocyst yield (41.8 ± 0.6%) was achieved using the maximum ionomycin concentration (5 µM), and importantly, the duration of ionomycin treatment was not of utmost importance at this concentration. In contrast, the maximum blastocyst yield of vitrified-warmed oocytes (28.4 ± 1.4%) was achieved with a minimal duration of ionomycin treatment (1 min), and further extending the duration dramatically reduced developmental potential of vitrified-warmed oocytes. These results suggested that vitrified-warmed oocytes may need an activation protocol different from unvitrified oocytes. In this respect, unvitrified oocytes were more sensitive to the concentration rather than the duration of ionomycin treatment when compared with vitrified oocytes, which were sensitive to the treatment duration. These results may provide a platform to improve the potential applications of vitrified oocytes in medicine and agriculture.