Oxidative damage to proteins and lipids, the enzymatic and nonenzymatic antioxidants' response, and the fertilization and development capability of Perna canaliculus oocytes were investigated at critical treatment steps in a previously published controlled-rate cryopreservation protocol. The cryoprotectant (CPA) from this protocol comprises 10% ethylene glycol (v:v) and 0.2 M trehalose (wt/vol) final concentration. Critical treatment steps included (1) seawater control, (2) CPA addition, (3) CPA addition followed by cooling to -6 °C, (4) CPA addition and cooling to -10 °C, and (5) CPA addition and cooling to -35 °C and immersion in liquid nitrogen (LN). The percentage of fertilized oocytes was 53.8 ± 13.3% in the seawater control but was reduced to 26.0 ± 15.6% after -35 °C + LN treatment, whereas development to D-larvae was 21.0 ± 6.4% in the seawater control reduced to 4.8 ± 2.9% after cooling to -6 °C, and was zero at all the subsequent cooling steps. All oxidative damage biomarkers, protein carbonyls (PCs) and lipid hydroperoxides (LPs), and antioxidants, superoxide dismutase (SOD), catalase, glutathione peroxidase, percent reduced glutathione (%GSH), and total glutathione (defined as glutathione; reduced [GSH] plus glutathione disulphide; derived from two molecules of GSH [GSSG]) were measured over all treatments on unfertilized oocytes over a post-treatment recovery period of 0 to 240 minutes in seawater. An ANOVA showed that both treatment and post-treatment periods had significant effects on the concentrations of all biomarkers (P < 0.05). Protein carbonyls and LPs increased very little after CPA addition and cooling treatments, when compared with the seawater control, but large increases up to sixfold occurred between 0 and 240 minutes for the -35 °C + LN treatment. Concentrations of SOD, catalase, total glutathione, and %GSH at 0 minutes decreased by -31.2%, -26.9%, -21.9%, and -25.0%, respectively, between the seawater control and the -35 °C + LN treatment. In contrast, glutathione peroxidase concentrations at 0 minutes increased by 34.3% between the seawater control and the -35 °C + LN treatment. Although most biochemical biomarkers showed an increasing trend over time (0-240 minutes), total glutathione decreased in concentration over time in all treatments, with the greatest decrease after the -35 °C + LN treatment (-41.2%). Significant correlations between biomarkers and D-larvae yield were negative for LPs and positive for SOD, total glutathione, and %GSH (P < 0.05). This is the first report of an investigation using these oxidative stress biomarkers and antioxidant responses on mussel oocytes, and the results have proved useful in characterizing cellular injury during the cryopreservation process.
Keywords: Chilling sensitivity; Embryo development; Ethylene glycol; Reactive oxygen species; Shellfish.
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