An improved method for vitrification of in vitro matured ovine oocytes; beneficial effects of Ethylene Glycol Tetraacetic acid, an intracellular calcium chelator.

Vitrification affects 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 addition of Ethylene Glycol Tetraacetic acid (EGTA) to vitrification solution could improve quality and developmental competence of in vitro matured ovine oocytes. Vitrified groups were designed according to the presence or absence of EGTA 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). In vitro development, numerical chromosome abnormalities, hardening of zona pellucida, mitochondrial distribution and function of viable oocytes were evaluated and compared between groups. Quality of blastocysts was assessed by differential and TUNEL staining. Also, mRNA expression levels of six candidate genes (KIF11, KIF2C, CENP-E, KIF20A, KIF4A and KIF2A), were quantitatively evaluated by RT-PCR. Our results showed that calcium-free vitrification and EGTA supplementation can significantly increase the percentage of normal haploid oocytes and maintain normal distribution and function of mitochondria in vitrified ovine oocytes, consequently improving developmental rate after in vitro fertilization. qRT-PCR analysis showed no significant difference in mRNA expression levels of kinesin genes between vitrified and fresh oocytes. Also, the presence of calcium in vitrification solution significantly increased zona hardening. In conclusion, we have shown for the first time that supplementation of vitrification solution with EGTA, as a calcium chelator, improved the ability of vitrified ovine oocytes to preserve mitochondrial distribution and function, as well as normal chromosome segregation.

Developmental competence of in vitro matured ovine oocytes vitrified in solutions with different concentrations of trehalose.

This study aimed to determine the optimum concentration of trehalose in solutions used for vitrification of in vitro matured (IVM) ovine oocytes. IVM oocytes were randomly divided into four experimental (vitrified) and one control (fresh) groups. Experimental groups were treated with different concentrations (0.0, 0.25, 0.5 and 1.0 M) of trehalose. After warming, some viable oocytes were exposed to 0.25% pronase to test zona pellucida hardening, whereas the others were fertilized and cultured in vitro for 8 days to evaluate their developmental competence. Blastocysts quality was assessed by differential staining and TUNEL test. Survival and developmental rates of oocytes vitrified in the presence of 0.5 M trehalose were significantly higher than those of the other vitrified groups. Furthermore, there was a significant difference between fresh and vitrified groups in total blastocyst rate. Analysis of blastocysts quality also revealed a significant difference between the group treated with 0.5 M trehalose and other groups in terms of apoptotic index. Furthermore,zona pellucida digestion time period was longer in trehalose-free (0.0 M) group compared to other groups. In conclusion, we found that IVM ovine oocytes vitrified in solutions containing 0.5 M trehalose are fertilization-competent and are able to produce good-quality blastocysts with an apoptotic index comparable to that of the fresh oocytes. Therefore, 0.5 M may be considered the optimum concentration of trehalose to be used in solutions prepared for vitrification of oocytes.