Cryotop vitrification for in vitro produced buffalo (Bubalus bubalis) embryos.

The aim of this study was to compare the efficiency of different combinations of cryoprotectants for vitrification of IVP buffalo 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 further 5 min. After being transferred into 3.4 M glycerol and 4.6 M EG for 25 sec, 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 Kuwayama M. The embryo was placed onto the thin strip of the Cryotop and immediately submerged into liquid nitrogen (LN2). 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 (1,2): 153). Embryos were equilibrated in 7.5 % EG and 7.5 % dimethyl sulfoxide (DMSO) for 3 min before transfer into 16.5 % EG and 16.5 % DMSO and 0.5 M sucrose. After 25 sec, they were placed on the cryotop, as previously described, and submerged into LN2. 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 tight morula (TM; n= 8 and 13 for groups A and B respectively) and the blastocyst stage (Bl; 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 (P<0.05) higher embryo survival rate was recorded, regardless of the stage of development, in group B compared to group A (59.1 % vs 38.5 respectively). The overall efficiency in both groups was lowered by the poor survival of the TM (12.5 and 23.1 % respectively for groups A and B). Blastocyst survival to vitrification was improved in group B vs Group A (67.9 vs 43.2 % respectively; P<0.05). Although it appears that the stage of development affects the freezability of the embryos, the numbers are limited and we cannot rule out that the lower efficiency of the TM is simply due to the worse quality of the embryos, indicated by their delayed development. 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.