Improved solution for vitrification of Prochilodus lineatus embryos based on the reduction in risk factors: Toxicity, osmotic responses and ice-nucleation

Abstract

The objective of our work was to describe a low toxicity cryoprotectant solution that allowed vitreous solid formation. Embryos of Prochilodus lineatus were submitted to sensitivity evaluations of six internal cryoprotectants (dimethyl sulphoxide - Me2SO, dimethyl acetamide - DMA, dimethyl formamide - DMF, methanol - MET, glycerol - GLY and 1,2-propanediol - PROP) at concentrations of 1-6 M; and two external cryoprotectants (sucrose - SUC and glucose - GLU) at concentrations of 0.1-1 M for 20 min. The capacity of the cryoprotectant solutions to exchange heat with the medium and to form glassy solids was evaluated by immersing 10 mu l of cryoprotectant in liquid nitrogen. The PROP had a high survival rate at all concentrations evaluated, and was the only substance that allowed a vitreous solid formation. Thus, it is concluded that the PROP-6 M was the most adequate solution for embryonic vitrification processes, because heat exchange between the system (PROP 6 M/embryos/liquid nitrogen) was faster than for other cryoprotectants and combinations thereof; has low toxicity, promote high rates of dehydration in short periods, and reach the vitreous state, being a good candidate to be used in the tests of embryonic vitrification.