Effect of Cryoprotectants on Activity of Selected Enzymes in Fish Embryos

Abstract

Cryoprotectants, which are essential for minimizing cryoinjury during freezing, can be toxic to biological systems. Monohydric alcohols, dimethyl sulfoxide (Me 2SO), and ethylene glycol (EG), are known to denature enzymes at room temperature. In this study, rosy barb ( Puntius conchonius) and zebra fish ( Brachydanio rerio) embryos at cleavage, epiboly, and closure of blastopore stages were exposed to Me 2SO and EG at 1.0, 2.0, 3.0, and 4.0 M for 0.25. 0.5, 1.0, 2.0, and 3.0 at room temperature. The cryoprotectants were then removed and the activities of two glycolytic enzymes, lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH), were determined. Cryoprotectant concentration and equilibration period had a significant ( P < 0.05) effect on the total activity of the two enzymes. In both species the decline in enzymatic activity was more pronounced for G-6-PDH, and EG was more toxic than Me 2SO. Zebra fish embryos were more resistant to cryoprotectant enzymatic denaturation than rosy barb embryos. For both species the total LDH activity measured after 3.0 h equilibration in 4.0 M Me 2SO and EG declifted sharply. In zebra fish cleavage, epiboly, and closure of blastopore embryos, the total LDH activity (or control value) when Me 2SO and EG were used was reduced by 70.0 and 86.1, 79.2 and 83.0, and 57 and 75% for the three embryonic stages, respectively. Under similar conditions G-6-PDH activity was reduced by 100 and 100, 88.3 and 100, and 69.0 and 100%, respectively, for the same embryonic stages. Similarly for rosy barb embryos, the total LDH activity was reduced by 89.2 and 98.8, 85.0 and 87.1, and 80.6 and 95.4% when equilibrated in Me 2SO and EG. In embryos at the closure of blastopore stage. G-6-PDH activity was reduced by 71.2% when equilibrated in 4 M Me 2SO. The reduction in the total activity of these enzymes was probably due to the damage to the perivitelline membrane and blastoderm caused by the osmotic stress and partial denaturation of the leached enzymes within the perivitelline space.