Determinants Survival Aspects of Vitrified In Vitro Produced Bovine Embryos.

Abstract

Since the birth of the first calf from an in vitro produced (IVP) embryo, there have been great advances in IVP systems. Currently, IVP is successfully applied commercially to bovine embryos. However, a major obstacle for dissemination of this technology is the great sensitivity of IVP embryos to cryopreservation. Characteristics such as lipid accumulation, very electron-dense cytoplasm, loose blastomeres, buoyant density, slower growth, excessive metabolism of energy substrates, and high thermal sensibility are indicators that the IVP embryos are more sensitive to cryopreservation. The aim of the present study was to evaluate the relationship between some embryo development parameters such as apoptosis rate, lipid content, quality degree score, and cell number, with blastocyst survival after vitrification. Slaughterhouse's ovaries were used to obtain oocytes which were matured and fertilized in vitro (Day 0). The presumptive zygotes were cultured on SOFaa as described by Sudano et al. Embryos recovery from superstimulated cows were also used. Embryo quality degree score and cell number was obtained after seven days of culture. Apoptosis in blastocysts was accessed through TUNEL reaction. A sample of blastocysts was stained with Sudan Black B for lipid quantification. Blastocysts were vitrified on Day 7 using the "two steps" technique with the 0.25mL straw. After warming, embryos were placed into culture for 12 h under standard conditions. The survival after vitrification was defined as a reduced apoptosis rate in vitrified blastocysts. For the statistical analysis, Pearson's correlation test were calculated between embryo development parameters (apoptosis, lipid content, quality degree score and cell number) and blastocyst survival after vitrification using PROC CORR (SAS, Cary, NC). Apoptosis in fresh blastocysts was strongly correlated (r= 0.83, n= 195; P<0.0001) with the survival after vitrification, while the parameters of lipid content (r= 0.53, n= 195; P<0.0001), quality degree score (r=0.62, n=195; P<0.0001) and cell number (r= 0.53, n= 195; P<0.0001) were moderately correlated with embryo survival after vitrification. In conclusion, apoptosis in fresh blastocyst was a more determinant embryo development parameter than lipid content, quality degree score and cell number was to predict the survival after vitrification. Acknowledgment: FAPESP 09/54513-3 (poster)