Super Open Pulled Straw Vitrification of Porcine Blastocysts: Effect of Centrifugation and Cytoskeletal Stabilization.

Abstract

The objective of the present study was to evaluate the effect of centrifugation and cytoskeletal stabilization with cytochalasin-B before vitrification on the survival and cytoskeleton integrity of vitrified porcine blastocysts. Unhatched blastocysts were collected by laparotomy from weaned crossbred sows (n=10) on day 6 of the estrous cycle. A subset of blastocysts (n=22) was not vitrified and cultured to evaluate the in vitro development of fresh embryos. The remaining blastocysts were randomly allocated to different experimental groups: a) blastocysts vitrified without pre-treatments (n=25); b) blastocysts centrifuged at 13.000 x g for 12 min (n=28); c) blastocysts equilibrated in 7.5 µg/ml cytochalasin-B for 20 min (n=30) and d) blastocysts equilibrated in cytochalasin-B for 20 min and then centrifuged at 13.000 × g for 12 min. Pretreated and non-pretreated blastocysts were vitrified (n=28). The basic medium for vitrification and warming (TCM-NBCS) was TCM199 Hepes supplemented with 20% (v/v) of newborn calf serum (NBCS) and 0.5 mM of L-Glutamine. Groups of 4 to 6 embryos were equilibrated in TCM-NBCS with 7.5% (v/v) dimethyl-sulfoxide (DMSO) and 7.5% (v/v) ethylene glycol (EG) for 3 min and in TCM-NBCS with 17% (v/v) DMSO, 17% (v/v) EG and 0.4 M sucrose for 1 min. During the last step, embryos were loaded into the narrow end of a superfine open pulled straw (SOPS) by capillary action and plunged into the liquid nitrogen. Straws were warmed using a one step dilution procedure. The narrow of the straw was immersed vertically in a well containing 800 µL TCM-NBCS with 0.13 M sucrose for 5 min. Fresh and vitrified-warmed blastocysts were cultured for 24 h in TCM199 supplemented with 10% fetal calf serum at 39°C, in 5% CO2 in humidified air. After the culture, embryos were assessed by stereomicroscopy and classified as viable or degenerated. Blastocysts that reformed their blastocoelic cavities after warming, displaying a normal zona pellucida and excellent appearance, were considered viable. The in vitro survival rate was defined as the ratio of viable embryos divided by the total number of embryos cultured. The hatching rate was determined as the ratio of the number of embryos hatched to the total number embryos cultured. Some vitrified and fresh embryos were labeled after culture, with Alexa Fluor 488 phalloidin and wheat germ agglutinin-Alexa Fluor 594 conjugate, to assess cytoskeleton quality. Data were analyzed by Chi-square test and expressed as mean values ± SEM. The embryo survival (81.8 ± 1.2%) and the hatching rate (36.4 ± 8.6) for fresh embryos were not significantly different to those obtained with vitrified embryos (ranging from 75.0±8.3% to 80.0±8.2% and from 20.0 ± 8.2% to 39.3 ± 9.4%, respectively). No differences were found for in vitro suvirval, hatching rates and cytoskeleton quality among vitrification groups Embryos classified as degenerated by stereomicroscopy displayed wide cytoskeletal disruption. Vitrified embryos classified as viable by stereomicroscopy displayed similar cytoskeleton quality than fresh blastocysts, showing only few cells with microfilament disruption. In conclusion, the pretreatment with cytochalasin-B and/or the centrifugation prior vitrification did not improve the blastocysts developmental competence and the cytoskeletal quality after SOPS-vitrification. Supported by CICYT-FEDER (AGL2004-07546), CARM (2I05SU0012), MEC (BES-2005-8894) and Seneca Foundation (04543/GERM/07).