114 CRYOTOP VITRIFICATION FOR IN VITRO-PRODUCED BUFFALO (BUBALUS BUBALIS) EMBRYOS

Abstract

The aim of this study was to compare the efficiency of different combinations of cryoprotectants for vitrification of IVP buffalo (Bubalus bubalis) embryos by the cryotop method (Kuwayama et al. 2005 RBM Online 11, 300–308). In group A, we evaluated the vitrification and warming solutions previously used to vitrify buffalo embryos in French straws (Gasparrini et al. 2001 Theriogenology 55, 307). Embryos were equilibrated in 1.4 M glycerol for 5 min and in 1.4 M glycerol and 3.6 M ethylene glycol (EG) for an additional 5 min. After being transferred into 3.4 M glycerol and 4.6 M EG for 25 s, individual embryos were picked up in an extremely small volume (<0.1 µL) of vitrification solution and placed on the top of a very fine polypropylene strip (0.4 mm wide × 20 mm long × 0.1 mm thick) attached to a hard plastic handle, kindly provided by M. Kuwayama. Each embryo was placed onto the thin strip of the Cryotop and immediately submerged into liquid nitrogen. For warming, the strip of the Cryotop was immersed directly into a 0.5 M sucrose solution; embryos were retrieved and transferred into 0.25 M sucrose for 5 min before culture in SOF medium. In group B, we examined the vitrification and warming solutions previously used for OPS vitrification of buffalo embryos (De Rosa et al. 2006 Reprod. Fertil. Dev. 18, 153). Embryos were equilibrated in 7.5% EG + 7.5% dimethyl sulfoxide (DMSO) for 3 min before transfer into 16.5% EG + 16.5% DMSO + 0.5 M sucrose. After 25 s, they were placed on the cryotop, as previously described, and submerged into liquid nitrogen. For warming, embryos were recovered into a 0.25 M sucrose solution for 1 min, transferred into 0.15 M sucrose for 5 min, and cultured in SOF. IVP buffalo embryos of excellent quality that, by Day 7 of culture (Day 0 = in vitro fertilization), had reached the blastocyst stage (n = 44 and 53 for groups A and B, respectively), over 6 replicates, were vitrified. Embryo survival rate was determined as the percentage of vitrified-warmed embryos undergoing further development during a 24-h in vitro culture period. Differences between methods were analyzed by chi-square test. A significantly higher embryo survival rate was recorded in Group B compared to Group A (67.9 vs. 43.2% respectively; P < 0.05). In conclusion, it was demonstrated that cryotop vitrification, with the combination of cryoprotectants used in group B, is a valid tool to cryopreserve IVP buffalo blastocysts.