125 IN VITRO-PRODUCED BOVINE EMBRYOS VITRIFIED IN GLASS CAPILLARIES USING TCM-199 OR DULBECCO'S PHOSPHATE-BUFFERED SALINE AS VITRIFICATION-WARMING MEDIA

Abstract

In previous studies (unpublished data) we observed that the replacement of open pulled straws (OPS) with glass capillaries (GC) did not affect the embryo survival rate after vitrification–warming. The aim of this work was to evaluate the post-Cryopreservation survival rate of in vitro-produced bovine blastocysts (B) and expanded blastocysts (eB) using Dulbecco's phosphate-buffered saline (PBS) or TCM-199 (TCM; Sigma-Aldrich, St Louis, MO, USA) as holding medium during vitrification and warming in glass capillaries. Cumulus–oocyte complexes were in vitro-matured and fertilized as previously described (Mucci et al. 2006 Theriogenology 65, 1551–1562), and cultured in 4 well plates in groups of 50 in 400-µL drops in serum-free CR1aa under low oxygen condition. Grade B1, B2, and eB were selected at Day 7 post-insemination and allocated to 3 groups: vitrification in TCM, vitrification in PBS, and control (without vitrification). Vitrification and warming were performed according to Vajta et al. (1998, Mol. Reprod. Dev. 51, 53–58), replacing OPS with GC (Tecnon Argentina S.A., Buenos Aires, Argentina); 75 mM length, 1.4 mM internal diameter, 1.6 mM external diameter). Briefly, B and eB were incubated in 1.78 M ethylene glycol (EG) and 1.3 M dimethyl sulfoxide (DMSO) in TCM or PBS supplemented with 20% estrous cow serum (ECS) for 3 min. Embryos were then transferred for 25 s to TCM or PBS supplemented with 3.56 M EG, 2.6 M DMSO, 0.5 M sucrose, and 20% ECS (vitrification solution: VS). Loading of embryos (2 per capillary) was performed by touching a 1-µL drop of VS with the capillary. After this, each capillary was immediately submerged into and stored in liquid nitrogen. Warming was performed by placing the capillary tip directly into TCM or PBS supplemented with 0.25 M sucrose for 5 min. Embryos were then transferred to TCM or PBS containing 0.15 M sucrose and 20% ECS for 1 min. After warming, embryos were cultured for 72 h in CR1aa + 5% ECS to evaluate embryo survival (hatching rate). Data was analyzed using the CATMOD procedure (SAS Institute, Inc., Cary, NC, USA). No interaction was found between holding media and embryo stage. Vitrified-warmed embryos had a significantly lower hatching rate compared with the control group (P < 0.05), whereas no differences were found between TCM and PBS. Expanded blastocysts had a higher hatching rate than blastocysts (P < 0.05). In conclusion, TCM can be replaced with PBS for its use in vitrification procedures. This protocol modification allows a simplified use of this technique in field conditions.