State of actin cytoskeleton and development of slow-frozen and vitrified rabbit pronuclear zygotes

This study was focused on the effect of cryopreservation on the state of actin cytoskeleton and development of rabbit pronuclear zygotes. Zygotes were collected from superovulated females and immediately used for 1) slow-freezing in a solution containing 1.5 M 1,2-propanediol and 0.2 M sucrose, or 2) vitrification in a solution containing 42.0% (v/v) of ethylene glycol, 18.0% (w/v) of dextran and 0.3 M sucrose as cryoprotectants. After thawing or warming, respectively, zygotes were evaluated for 1) actin distribution, 2) in vitro or 3) in vivo development to blastocyst. Comparing actin filaments distribution, a significantly higher number of vitrified zygotes with actin distributed in cell border was observed (55 ± 7.7 vs. 74 ± 6.1% for slow-frozen vs. vitrified, respectively). After 24 and 72 h of in vitro development, significant differences in the cleavage and morula rate among the groups were observed (9 ± 2.4 and 3 ± 1.3 vs. 44 ± 3.0 and 28 ± 2.7% for slow-frozen vs. vitrified, respectively). None of the slow-frozen zygotes reached the blastocyst stage, in contrast to the vitrified counterparts (11 ± 1.9%). Under in vivo culture conditions, a significant difference in blastocyst rate was observed between vitrified and fresh embryos (6 ± 1.5 vs. 35 ± 4.4% respectively). Our results showed that alterations in actin cytoskeleton and deteriorated development are more evident in slow-frozen than vitrified pronuclear zygotes. Vitrification method seems to be a more effective option for rabbit zygotes cryopreservation, although pronuclear zygotes manipulation per se resulted in a notable decrease in embryo development.