Toxic Effects of Cryoprotectants on oyster gametes and embryos: A preliminary step towards establishing cryopreservation protocols

Abstract

Aquaculture development is dependent on continuous seed production, regardless of the spawning season. Cryopreservation can therefore, be a valuable tool for achieving seed availability. The benefits of cryopreservation have been demonstrated for mammals, fish and some achievements have been reported for invertebrates; however, cryopreservation has not yet contributed significantly to oyster rearing. The literature on this topic demonstrates that an optimal cryopreservation method developed for one species is not always applicable to another. One of the reasons is that there are differences among species in the toxicological responses of gametes and embryos to cryoprotectants. The aim of this study was to determine the toxic effects of cryoprotectants dimethylsulfoxide (Me2SO), glycerol (G), ethylene glycerol (EG), propylene glycerol (PG) and methanol (MET) on Crassostrea rhizophorae gametes and embryos). Gametes (oocytes and spermatozoa) and embryos (trochophores) were exposed to a range of concentrations of each cryoprotectant for 10, 20 and 30 minutes. Based on the EC50-24h values there were no significant differences (p > 0.05) among the exposure times in toxic effects to either gametes or embryos. The trochophores were relatively resistant to cryoprotectant exposure, while oyster gametes became increasingly susceptible to the cryoprotectants as concentration levels were increased. Critical values (EC50-24h) of cryoprotectants were markedly different for gametes and embryos. For gametes, both G and MET were more toxic (EC50-24h respectively of 3.46 and 4.52% for oocytes, and 2.07 and 11.21% for spermatozoa) than EG, PG and Me2SO. However, PG (EC50-24h = 23.56%) and EG (EC50-24h = 45.18%) were more toxic for trochophores than Me2SO (EC50-24h = 54.25) or MET(EC50-24h = 55.63%). These results show the importance of previous toxicological studies for cryoprotectant selection as a preliminary step towards establishing cryopreservation protocols.