Artificial Shrinkage Improves Embryo Survival and Qualities of Cryopreserved Bovine Blastocysts.

Abstract

Cryopreservation of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production by using embryo transfer technique. However, the low efficiency of frozen-thawed embryos survival and further development is a crucial problem. Thus, we examined the effect of artificial shrinkage before vitrification and slow freezing of bovine blastocysts on the survival rate and apoptosis of frozen-thawed embryos. Bovine blastocysts were vitrified and slow-freezed after artificial shrinkage, which was performed by puncturing the blastocoele with a pulled pasteur pipet. The shrunken (Group A) and not shrunken (Group B) vitrified embryos were exposed to cryoprotectant 15% ethylene glycol-15% DMSO and 0.5 M sucrose for 1min, and the shrunken (Group C) and not shrunken (Group D) slow-freezed embryos were exposed to cryoprotectant 1.5 M ethylene glycol and 0.1 M sucrose for 5 min. Blastocysts (Group A and B) were placed in a small volume of vitrification solution and plunged into liquid nitrogen on a cryotop. Also blastocysts (Group C and D) were loaded 0.25 ml plastic straws and transferred into a controlled rate freezer and plunging into liquid nitrogen for storage. Then, after thawing, blastocysts derived from Group A and B were diluted in 1.0 M, 0.5 M, 0.25 M, and 0 M sucrose for 1, 3, 5, and 5 min, and blastocysts derived from Group C and D were washed three times each in Hepes buffered TCM-199 for 5min, respectively. After thawing, differences in survival and hatching rates of blastocysts derived from shrunken and not shrunken embryos were found in both vitrification (Group A vs B: 95.3±4.3 vs 81.1±7.1% for survival rate, 72.8±10.3 vs 58.9±8.3% for hatching rate P<0.05) and slow freezing (Group C vs D: 89.6±7.6 vs 53.1±12.4% for survival rate, 41.6±9.7 vs 21.4±12.9% for hatching rate, P<0.05) groups. Furthermore, we also found that the total cell numbers of blastocysts in artificial shrinkage groups were increased (Vitrification: 130.5±2.6 vs 113.7±3.5, Slow freezing: 116.7±2.4 vs 101.5±4.3, P<0.05) and the numbers of blastomeres undergoing apoptosis in artificial shrinkage groups were decreased (Vitrification: 8.5±1.5 vs 14.5±1.4, P<0.05; Slow freezing: 13.4±1.0 vs 14.3±2.6). Consistent with the results, qPCR data showed that the expression of anti-apoptotic Bcl-xl gene was greatly increased by shrunken groups, whereas the expression of pro-apoptotic Bax was decreased. Our results showed that survival and qualities in the both vitrified and slow-freezed bovine blastocysts could be improved by reducing the fluid content after artificial shrinkage. Therefore, we suggest that artificial shrinkage with pulled pasteur pipet is an effective pretreatment technique for the both vitrification and slow freezing methods of bovine blastocysts. (poster)