Avaliando os efeitos das proteínas anticongelantes na viabilidade de embriões bovinos produzidos in vitro vitrificados

Abstract

Embryo cryopreservation is an important tool that enables the logistics of storage and transfer of blastocysts of high genetic merit. There are currently two main distinct cryopreservation methodologies, slow freezing and vitrification, developed in order to avoid cryoinjuries. Vitrification is a rapid freezing technique that transforms the liquid into a glassy state, reducing the negative effects of ice crystal formation. However, the vitrification technique can still cause damage related to the formation of ice crystals. Some proteins have been identified as inhibitors of intracellular ice formation, called antifreeze proteins (AFP). These proteins have action in modifying the structure of the ice crystal, decreasing the freezing point and inhibiting the recrystallization activity. Based on the above, this study aimed to evaluate the cryoprotective effects of AFP extracted from the grass Lolium perenne (LpAFP) and AFP extracted from the larva of the Tenebrio molitor beetle (TmAFP) on the vitrification of bovine embryos. For this, in vitro produced bovine blastocysts were vitrified using the cryotop method in both experiments. In the first experiment, during the vitrification process, the blastocysts were randomly separated into two experimental groups, the control group (GC) containing 0 ng/mL LpAFP and the treatment group (GT) supplemented with 500 ng/mL LpAFP. Vitrification was carried out by transferring the blastocysts to the equilibrium solution: 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 2 min, then to the vitrification solution: 15% EG, 15 % dimethyl sulfoxide (DMSO) and then deposited on the cryotop rod and submerged in liquid nitrogen. Heating was carried out in three steps with decreasing concentrations of sucrose (1.0, 0.5 and 0.5 M sucrose). After heating, the blastocysts were cultured for 24 hours and analyzed for survival rate (expansion and/or hatching), stained with Hoechst to evaluate the total number of cells and analyzed under transmission electron microscopy for ultrastructural evaluation. In the second experiment, in vitro produced blastocysts were randomly separated into three experimental groups and vitrified with stabilization and vitrification medium supplemented with different concentrations of TmAFP: 0 ng/mL; 500 ng/mL and 1000 ng/mL. The vitrification and heating process took place in the same way as in the first experiment. After 24 hours of post-warming cultivation, the survival rate (expansion and/or hatching) was analyzed and ultrastructural analysis of the expanded embryos was performed. In experiment 1, the results showed that there was no significant difference in the reexpansion rate 24 hours after heating, however there was variation (P < 0.05) in the hatching rate in the GT. The total number of cells 24 hours after heating was significantly higher in the GT when compared to the GC (GT 114.87 ± 7.24 vs. GC 91.81 ± 4.94). The ultrastructural analysis showed a decrease in cytoplasmic lesions, mainly in mitochondria and rough endoplasmic reticulum in GT. In experiment two, the group supplemented with 500 ng/mL of TmAFP (500TmAFP) had a higher survival rate when compared to the other two groups, control (0 ng/mL of TmAFP) and 1000TmAFP (1000 ng/mL of TmAFP), also a higher rate of blastocoel expansion was observed in the 500TmAFP group. Ultrastructural lesions were observed in all vitrified embryos, however embryos from the 500TmAFP and 1000TmAFP groups showed less cytoplasmic lesions when compared to the control group. In conclusion, experiment 1 demonstrated that the addition of 500 ng/mL of LpAFP during the vitrification of in vitro produced bovine embryos proved to be favorable in improving the survival and development of blastocysts after heating. In addition, experiment 2 showed that TmAFP supplementation can mitigate cellular changes, which involve organelles and cellular components essential for proper functioning and viability after heating of vitrified in vitro produced bovine embryos.