Improved solution for vitrification ofProchilodus lineatusembryos 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 -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 ll 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.

Morphological evaluation of Prochilodus lineatus embryos after vitrification–thawing in high‐osmolarity cryoprotectant solution

“…This chemical has the ability to make hydrogen bonds with water molecules, thereby interfering with the crystallization process, which facilitates glass formation (Kirichek, Soper, Dzyuba, & Holt, ). It is important that considering the above discussion, PROP also presents low toxicity to P. lineatus embryos, permitting both long embryonic exposure (Costa, Souza, Senhorini, Ribeiro, et al, ) and its association with dimethyl sulphoxide (Me 2 SO) and sucrose (SUC) (Kirichek et al, ; Valdez et al, ; Zhang & Rawson, ).…”

“…Both techniques reduce, by means of linear or complex curves, the temperature of the samples, normally reaching a similar stock temperature of −196ºC (that of liquid nitrogen) (Robles, Cabrita, Acker, & Herráez, 2009). However, vitrification differs in the use of a high concentration of cryoprotectants and a marked curve for the reduction in temperature (Costa, Souza, Senhorini, Ribeiro, et al, 2018;Dinnyés, Urbányi, Baranyai, & Magyary, 1998;Hagedorn, Peterson, Mazur, & Kleinhans, 2004;Rubinsky & Pegg, 1988;Zhang & Rawson, 1995).…”

Fatty acid influence onProchilodus lineatus(Characiformes, Prochilodontidae) embryo cryopreservation parameters.

“…Solutions of 1,2‐propanediol at concentrations of 5 and 6 M were used. In previous studies of this research group, it is considered the most appropriate solution for embryonic vitrification processes, because heat exchange between the system (PROP 6 M/embryos/liquid nitrogen) was faster than other cryoprotectants and combinations; had low toxicity, promoted 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 (Costa, Souza, Senhorini, Veríssimo‐Silveira, & Ninhaus‐Silveira, ; Costa, Souza, Senhorini, Ribeiro, ). The embryos were allocated into 24‐well culture plates (6 × 4), and a single embryo was placed into each well.…”

Cooling of curimba (Prochilodus lineatus) embryos using different concentrations of dimethyl sulphoxide and methanol

“…Cryoprotectant substances are considered to have a key role in the cryopreservation process, but it is necessary to determine both the type and ideal concentration necessary to prevent possible embryo damage by exposure to low temperatures (Costa et al, 2018). The fundamental characteristics for an effective permeating cryoprotective agent are low molecular weight, high capacity to cross the cell membrane, and low toxicity.…”