Developmental rates of in vivo and in vitro produced bovine embryos cryopreserved in ethylene glycol based solutions by slow freezing or solid surface vitrification
Rodriguez Villamil, PLozano, DOviedo, J MOngaratto, F LBó, G A
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 v itrification. In vivo -produced blastocysts (n = 210) and in vitro - produced 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 wate r 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% a nd 45/222, 20%). Although similar re-expansion rates were obtained with in vivo - produced 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 vitro - produced embryos compared to the conventional slow freezing procedure.(AU)
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