Preliminary studies on the cryopreservation of gilthead seabream (Sparus aurata) embryos

Vitrification could provide a promising tool for the cryopreservation of fish embryos. In order to achieve successful cryopreservation, several parameters should be taken into account in the design of a vitrification protocol. In the present study some relevant factors were investigated (permeable and non-permeable cryoprotectant toxicity, toxicity of vitrificant solutions, adequate container for embryo loading and temperature of thawing) using two gilthead seabream embryonic development stages (tail bud and tail-bud-free). Permeabilized embryos were incubated in dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG) and 1,2-propanediol (PROH) in concentrations ranging from 0.5 to 6 M for 10 and 30 min and in 5%, 10% and 15% polyvinyl pyrrolidone (PVP), 10%, 15% and 20% sucrose or 0.1%, 1% and 2% X1000® for 2 min. After treatment, embryos were washed and incubated in seawater until hatched. The toxicity of permeable cryoprotectants increased with concentration and exposure time. EG was best tolerated by the embryos. Exposure to non-permeable cryoprotectants did not affect the hatching rate except at F stage. Six vitrificant solutions (DMSO—V1, V2 and V3 and EG—V1, V2 and V3) were tested using a stepwise incorporation protocol. The DMSO-based solutions contained 5 M DMSO + 2 M MeOH + 1 M EG plus 5% PVP, 10% sucrose or 2% X1000® and the EG-based solutions contained 5 M EG + 2 M MeOH + 1 M DMSO plus 5% PVP or 10% sucrose. Before loading the embryos into 0.5 ml straws or 1 ml macrotubes, toxicity tests were effected with these solutions. Our results demonstrated that DMSO-based solutions were better tolerated by seabream embryos than EG-based solutions. After thawing (water bath, 0 or 25 °C), embryos were evaluated by stereoscopic microscopy and the percentage of embryos with intact morphology was registered. The highest percentage of embryos with intact morphology (28%) was observed in samples frozen in macrotubes and thawed at 25 °C. Several malformations associated with ice crystal formation inside the embryos were detected. None of these embryos achieved hatching. Our results suggest that the absence of a proper incorporation of cryoprotectants prior to vitrification is the main problem that must be overcome. This procedure should be optimized in order to avoid ice crystal formation inside embryo compartments.