49 SUCCESSFUL CRYOPRESERVATION USING LOW ETHYLENE GLYCOL CONCENTRATION FOR IN VITRO-PRODUCED BOVINE EMBRYOS

Abstract

In vitro-produced (IVP) bovine embryos tend to have a lower survival rate after cryopreservation than in vivo embryos do. Therefore, the freezing medium (FM) and concentration of cryoprotectant are very important factors. This study was to investigate the effect of 1.2 M ethylene glycol (EG) with 0.1 M sucrose (SUC) on survival of IVP embryos after freezing. The COC were matured in 25 mM HEPES-buffered TCM199 (TCM199) supplemented with 5% calf serum (CS) and 0.02 AU mL⁻¹ FSH. Oocytes (20 to 25) were cultured in 100-μL droplets of maturation medium for 20 h. After 6 h of gamete co-culture (5 × 10⁶ sperm/mL), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% CS for 9 days (fertilization = Day 0). Only the expanded blastocysts from Days 7 to 9 were used in this experiment and separated into 3 treatment groups. The first and second groups were frozen in Dulbecco’s phosphate-buffered saline (D-PBS) supplemented with 20% CS, 0.1 M SUC, and 1.2 or 1.5 M EG (groups 1.2 or 1.5 M EG), respectively. The third group was D-PBS supplemented with 20% fetal calf serum (FCS), 0.25 M SUC, and 1.4 M glycerol (group GLY). In each group, embryos were equilibrated with their FM for 10 min and loaded into 0.25-mL straws individually. These straws were placed into the cooling chamber of a programmable freezer precooled to −7°C. After 2 min, the straws were seeded and then held for a further 13 min at −7°C. Then, the straws were cooled to −30°C at −0.3°C/min before being plunged into liquid nitrogen. The cryopreserved embryos were thawed by allowing the straws to stand in air for 7 s and then warming them in a 30°C water bath for 20 s. The thawed embryos were washed twice using 38°C D-PBS supplemented with 20% FCS. Subsequently, they were immersed in the same medium, held at 38°C for 10 min, and then each embryo was cultured in 20-μL droplets of TCM199 supplemented with 20% FCS and 0.1 mM β-mercaptoethanol for 72 h. The rates of embryos developing to the re-expanded and hatching blastocyst stages were determined 72 h after thawing. All data were analysed by the chi-squared test with Yates’ correction. The re-expanded and hatching rates of frozen-thawed embryos after 72 h in culture were not significantly different between 1.2 M EG (n = 39: 71.8% and 69.2%), 1.5 M EG (n = 38: 76.3% and 63.2%), and 1.4 M GLY (n = 37: 75.7% and 64.9%) groups (P > 0.05). Survival and hatching rates according to embryo quality were also not significantly different between 1.2 M EG (good n = 18: 88.9% and 88.9%; fair n = 21: 57.1% and 52.4%), 1.5 M EG (good n = 19: 89.5% and 84.2%; fair n = 19: 63.2% and 42.1%), and 1.4 M GLY (good n = 18: 77.8% and 66.7%; fair n = 19: 73.7% and 63.2%) (P > 0.05). In conclusion, cryoprotectant type and concentration did not affect embryo survival or development after cryopreservation in this study. Therefore, the ethylene glycol concentration used for the cryopreservation of IVP embryos can be reduced.