Effects of cryoprotectant toxicity and temperature sensitivity on the embryos of black tiger shrimp ( Penaeus monodon)

Abstract

The response of black tiger shrimp ( Penaeus monodon) embryos in terms of hatching success to cryoprotectants and temperature sensitivity was investigated for basic knowledge to enhance cryopreservation success of P. monodon embryos. Three stages of embryonic development (early-, mid- and late-stage embryos corresponding to 2, 6 and 10 h post-spawning) were chosen and exposed to various cryoprotectants, namely methanol, ethanol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), acetamide, formamide, glycerol and sucrose, at four concentrations (5, 10, 15 and 20%) for an equilibration period of 10 or 20 min. Toxicity tolerance of P. monodon embryos varied with developmental stage, cryoprotectant concentration and equilibration period. Late-stage embryos were more resistant to prolonged contact with cryoprotectants, as compared to the other two embryonic stages. Early-stage embryos were very sensitive to cryoprotectant concentrations above 5%. Mid- and late-stage embryos tolerated cryoprotectants to 10%, although a higher hatching percentage was observed in late- than mid-stage embryos. Hatching percentage declined significantly ( P < 0.05) with an increase in cryoprotectant concentration and equilibration time. DMSO, acetamide, glycerol and sucrose were relatively less toxic, compared to other cryoprotectants. The effect of temperature on the three stages of embryonic development was investigated by exposing embryos to pre-chilled seawater at 6, 11, 16, 21 and 25 °C for periods of 30, 60, 90, 120 and 150 min. Late-stage embryos were also the most tolerance stage to chilling. Hatching did not occur at 6 °C but above this, success improved with water temperature. Based on the sensitivity to cryoprotectant exposure and temperature, late-stage embryos of P. monodon are an appropriate stage for further cryopreservation.