Optimization of Ovine-Oocyte Vitrification Utilizing Calcium Depletion by Adding EGTA to Freezing Solution

Vitrification affects intracellular calcium, fertilization ability, and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether reducing calcium of vitrification solution could improve the fertilization and developmental competence of ovine oocytes.

COCs were collected from the ovine ovary. MII oocytes were divided into 5 groups, one non-vitrified (control) and four vitrified groups 24 hours after COC culture. Vitrified groups were designed according to the presence or absence of EGTA (a calcium chelator) and/or calcium in base media, including mPB1+ (modified PBS with Ca2+), mPB1- (modified PBS without Ca2+), mPB1+/EGTA (mPB1+ containing EGTA), mPB1-/EGTA (mPB1- containing EGTA). Fertilization rate and in vitro development were evaluated after embryo thawing. Also, blastocyst quality was assessed using differential staining. Data analysis was carried out using one-way analysis variance.

There was no significant difference in the viability rate between vitrified groups. Fertilization and the developmental rate decreased in the presence of calcium (p<0.05) but in the calcium-free medium with the EGTA supplementation group, the developmental rate obviously increased. On the other hand, blastocyst cell count in the control group was similar to vitrified groups.

Using a calcium-free cryoprotectant by adding EGTA can improve the quality of vitrified-thawed ovine MII oocyte and also a higher developmental rate in obtained embryos.