Effect of different treatments on the chorion permeability to DMSO of turbot embryos (Scophthalmus maximus)

Abstract

Abstract While cryopreservation techniques have been largely established for mammalian embryos, attempts to freeze fish embryos are still unsuccessful because of basic and technical problems. Insufficient dehydration and cryoprotectant penetration of fish embryos due to their relative large size and the presence of membranes and compartments with different water and cryoprotectant permeabilities are the main problems. In our approach we have assayed the efficiency and toxicity of different methods for chorion permeabilization in turbot. We have checked two different chorion treatments: enzymatic digestion and chemical treatment. Embryos at different developmental stages were exposed to several concentrations of pronase E, type XIV of Streptomyces griseus , (0.1, 0.01 and 0.001 mg/ml in Ringer solution) or to chlorine solutions (Ca(OCl) 2 0.01% and 0.001% and Na(OCl) 2 from 0.01% to 0.0001%) and the embryo viability was recorded. Exposure to pronase for longer than 10 min decreased the survival rate to 32.4% with 0.1 mg/ml, 54.1% with 0.01 mg/ml and 64.7% with 0.001 mg/ml for 35 min of exposure. Sodium hypochlorite concentrations over 0.005% reduced the survival rate to approximately 60%. Treatments with calcium hypochlorite were toxic at all the tested concentrations. The permeabilization of the chorion with pronase and sodium hypochlorite was assessed by changes in embryo volume and by the evaluation of DMSO entry through this envelope by high performance liquid chromatography (HPLC). The concentration of DMSO in permeabilized and nonpermeabilized embryos was determined in three embryo developmental stages—E, F and G. Results showed that DMSO concentration inside the chorion was stage dependent, obtaining for E stage 11.8 mM, for F stage 46.8 mM and for G stage 113.2 mM DMSO when embryos were exposed to a 2 M DMSO solution. The chorion of turbot embryos was slightly permeable to DMSO and the tested permeabilization methods did not significantly increase permeability to this cryoprotectant.