Developmental rates of in vivo and in vitro produced bovine embryos cryopreserved in ethylene glycol based solutions by slow freezing or solid surface vitrification

The aim of this study was to compare in vitro survival rates of in vivo and in vitro-produced bovine embryos by slow freezing or solid surface vitrification. In vivo-produced blastocysts (n = 210) and in vitroproduced blastocysts (n = 445) were randomly allocated in two cryopreservation groups. Group 1 embryos were exposed to 1.5 M ethylene glycol (EG) for 5 min, loaded in 0.5 ml straws, frozen at -6.5oC and seeded. After 10 min of equilibration, straws were cooled at -0.6oC/min until 35oC, and then plunged into liquid nitrogen (-196oC). Group 2 embryos were exposed to a 15% EG + 0.25 M trehalose solution for 1 min and then a 30% EG + 1 M trehalose solution for 30 sec to be vitrified using the Cryologic Vitrification Method (CVM). After at least one week of storage, embryos in the slow freezing group were thawed in a water bath at 30°C for 12 sec and then placed in holding medium for 5 min and transferred into SOF culture media. Vitrified embryos were placed directly into a 0.25 M sucrose solution for 5 min then cultured in SOF medium. Re-expansion and hatching rates were evaluated at 24 and 72 h, respectively. In vivo-produced embryos had higher (P < 0.01) re-expansion (179/210, 81% vs. 244/445, 54%) and hatching rates (159/210, 72% vs. 177/445, 39%) than in vitro-produced embryos, regardless of the cryopreservation method. However, re-expansion and hatching rates were higher (P < 0.01) for in vitro-produced vitrified embryos (155/223, 69% and 132/223, 59%) than in vitro-produced embryos cryopreserved by slow freezing (89/222, 40% and 45/222, 20%). Although similar re-expansion rates were obtained with in vivoproduced embryos cryopreserved by the two systems, hatching rates tended to be lower (P = 0.09) with in vivo-produced embryos that were vitrified as compared to slow freezing. In conclusion, solid surface vitrification improved the cryosurvival rates of in vitroproduced embryos compared to the conventional slow freezing procedure.