83 IMPROVED SURVIVAL TO ONE-STEP REHYDRATION OF VITRIFIED–WARMED VERSUS FROZEN–THAWED IN VITRO-PRODUCED BOVINE BLASTOCYSTS

Abstract

Vitrification allows cryopreservation of embryos while avoiding the detrimental effects derived from the intracellular ice formation during slow freezing. However, while slow freezing allows direct transfer of embryos, vitrification usually requires 1 or 2 rehydration steps after warming. The aim of this work was to analyze survival rates and quality of vitrified or slow frozen in vitro-produced (IVP) embryos, after warming/thawing by one-step procedures. Bovine blastocysts were produced in vitro, and on Day 7 and 8 excellent- and good-quality expanded blastocysts were selected for slow freezing (n = 175) or vitrification (n = 176) in 4 replicates. Slow freezing was performed in phosphate buffered saline containing ethylene glycol (1.5 M) and sucrose (0.1 M). Embryos were placed in a Biocool chamber (Biocool II, FTS^® Systems Inc.) at –7°C for 5 min and seeded. After 5 min, embryos were cooled at –0.3°C min^–1 until –32°C and plunged in LN₂. Embryos were thawed in a water bath at 37°C for 30 s. Vitrification was performed in fibreplugs as previously described (Trigal et al. 2012 Theriogenology 10.1016/j.theriogenology.2012.06.018). For warming, embryos were incubated for 5 min in TCM199–HEPES, 20% FCS, and 0.25 M sucrose. Thawed and warmed embryos were washed and subsequently cultured in mSOFaaci + 6 g L^–1 BSA + 10% FCS for 48 h. Re-expansion (RE) (at 2, 24, and 48 h in culture) and hatching rates (HR; at 24 and 48 h in culture) were recorded. Total cells were counted in blastocysts that hatched at 24 and 48 h after fixation and bisbenzimide staining. Data were analyzed by ANOVA and are presented as least squares means ± standard error. No differences were found within RE at 2 and 24 h, and HR at 24 h (2-h RE: 94.1 ± 4.9 v. 95.4 ± 4.9; 24-h RE: 92.6 ± 4.9 v. 94.5 ± 4.9; HR: 21.0 ± 7.0 v. 20.6 ± 7.0, for slow frozen and vitrified embryos, respectively; P > 0.05). However, at 48 h, vitrified embryos hatched at higher rates than did slow-frozen embryos (53.6 ± 10.2 v. 32.5 ± 10.2; P < 0.05). Vitrified embryos (143.5 ± 12.7) had higher (P < 0.05) cell numbers than did slow-frozen embryos (106.1 ± 9.6) after hatching. Our results show that vitrification of IVP embryos in fibreplugs followed by a one-step warming is a promising candidate procedure to replace slow freezing for direct transfer on field. These results must be completed with embryo transfers to analyze pregnancy rates.

RTA2011-00090 (FEDER-INIA) is acknowledged. Muñoz, Trigal, and Correia are sponsored by RYC08-03454, Cajastur, and FPU2009-5265, respectively.