Effect of different
treatments on the chorion permeability to DMSO of turbot embryos (Scophthalmus
maximus)
E.
Cabrita, O. Chereguini, M. Luna, P. de Paz, M.P. Herráez-2003
Aquaculture, 221(1-4): 593-604
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.
(Department of Cell Biology, Faculty of Biology,
University of Leon, 24071, Leon, Spain, e-mail of M.P. Herráez: dbcmho@unileon.es)