Cryopreservation of ovine in vitro produced embryos using centrifugation and cytocalasin D

Abstract

The cryosurvival of ovine in vitro produced embryos are still low, thus preventing its routine transfer. Attempts have been made to override this problem by decreasing embryos lipid content or protecting their structure with cytoskeletal stabilizers. In this study we used embryo mechanical delipidation through centrifugation in the presence or absence of cytocalasin D testing its effect on embryo quality and cryosurvival. Mature ovine oocytes (n=1146) were inseminated using fresh semen of a Merino ram. After assessing cleavage, embryo development proceeded until the blastocyst stage. Prior to vitrification, embryos were randomly distributed to the following groups: control (n= 20), without treatment; centrifugation (n=18), blastocysts were centrifuged at 15000g; cytochalasin D (n=20), embryos were treated with 5 µg mL-1 cytocalasin D; centrifugation+cytocalasin D (n=17), embryos were treated with both centrifugation and cytocalasin D. Embryos integrity and re-expansion were assessed post-warming and after 3 hours of culture. Post-warming integrity rate was lowest (p=0.04) in embryos of centrifugation group. No differences were identified among groups for re-expansion rates. A possible role of cytocalasin D in protecting mechanical damage of centrifuged embryos during cryopreservation was identified. However this stabilizer alone did not improve the quality of warmed embryos when compared to control.