Superfine open pulled straws vitrification of porcine blastocysts does not require pretreatment with cytochalasin B and/or centrifugation

Abstract

The present study investigated the in vitro development of and cytoskeletal disruption suffered by in vivoderived porcine blastocysts subjected to superfine open pulled straws (SOPS) vitrification. Blastocystswere either untreated prior to SOPS vitrification or were subjected to one of the following three pretreatment protocols: (1) centrifugation (12 min, 13 000g); (2) 25 min equilibration with 7.5μgmL-1 cytochalasin B; or (3) equilibration with cytochalasin B followed by centrifugation. After 24 h culture, fresh (n=32) and vitrified-warmed (n=188) blastocysts were evaluated by stereomicroscopy, with survival and hatching rates recorded. Some blastocysts were stained with 4′,6′-diamidino- 2-phenylindole and processed for cytoskeletal evaluation. Three cytoskeletal patterns were identified: Grade I, intact cytoskeleton; Grade II, gross maintenance of integrity, but with some clumps of actin within the cytoplasm; and Grade III, a highly disrupted cytoskeleton. There were no differences in the survival, hatching and cell death rats, total cell number or cytoskeletal integrity between the different vitrification groups. Cell death was greater for vitrified blastocysts than for fresh blastocysts (3.6±0.4% v. 0.4±0.7%, respectively; P <0.05) and the percentage of blastocysts with a Grade I cytoskeletal pattern was lower for vitrified compared with fresh blastocysts (60.8% v. 92%, respectively; P <0.05). The vitrified-warmed blastocysts that hatched during culture exhibited a Grade I cytoskeletal pattern. In conclusion, successful SOPS vitrification of porcine blastocysts does not require pretreatment with cytochalasin B and/or centrifugation. © 2010 CSIRO.