89 OPEN PULLED STRAW 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 at different developmental stages by the open pulled straw (OPS) method. In method 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 before being placed into 1.4 M glycerol + 3.6 M ethylene glycol (EG) for 5 min. Then, embryos were transferred into 3.4 M glycerol + 4.6 M EG for 25 s and loaded into the OPSs. For warming, OPSs were briefly immersed in a 0.5 M sucrose solution; the embryos were exposed to 0.25 M sucrose for 5 min before transfer to SOF medium for culture. In Method B, we examined the vitrification and warming solutions previously used for OPS vitrification of cattle embryos (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Buffalo embryos were equilibrated in 7.5% EG + 7.5% dimethyl sulfoxide (DMSO) for 3 min before transfer into 16.5% EG + 16.5% DMSO and 0.5 M sucrose. After 25 s, they were loaded into the OPSs. For warming, embryos were recovered in a 0.25 M sucrose solution and transferred into a 0.15 M sucrose solution for 5 min before being placed in SOF medium. A total of 293 IVP buffalo embryos (eight replicates) were vitrified at Day 7 of culture (Day 0 = in vitro fertilization). Embryos were vitrified at the following developmental stages: early blastocyst (eBL, n = 26 and 34 with methods A and B, respectively), blastocyst (Bl, n = 31 and 35 for Methods A and B, respectively), expanded blastocyst (XBl, n = 29 and 38 for Methods A and B, respectively), and hatched blastocyst (HBl, n = 46 and 54 for Methods A and B, respectively). 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 ANOVA following arcsine transformation of data. The overall embryo survival rate recorded at 24 h was not significantly different between Methods A and B (70% vs. 62%, respectively). Specifically, no differences were observed in embryos vitrified at the eBL (70% vs. 73%, A and B, respectively), Bl (69% vs. 70%, A and B, respectively), and HBl (46% vs. 36%, A and B, respectively) stages. In contrast, a significantly higher survival rate was recorded for XBl-stage embryos vitrified-warmed by Method A as compared to Method B (90% vs. 53%, respectively; P < 0.01). In Method A, survival rate of XBl was significantly higher than that of HBl (P < 0.05), but it was not different from that of eBl and Bl. Within Method B, the survival efficiency was similar for eBL, BL, and XBl, whereas survival rate of HBl was significantly lower (P < 0.05). In conclusion, although overall embryo survival in vitro was similar between methods, the combination of cryoprotectants used in Method A seemed more suitable for vitrification of IVP buffalo embryos at the XBl stage.