Effect of different permeable and non-permeable cryoprotectants on the hatching rate of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) embryos

The purpose of the present study was to investigate the toxicity of cryoprotectants on the hatching rate of rainbow trout (Oncorhynchus mykiss) embryos. Epiboly and first eye pigmentation stage embryos were immersed in six permeable cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), propylene glycol (PG), ethylene glycol (EG), and acetamide (Ac), in concentrations of 1–5 M for 5 or 10 min and two non-permeable cryoprotectants, sucrose (Suc) (10%, 15%, 20%) and polyvinyl pyrrolidone (PVP) (5%, 10%, 15%) for 5 min. The embryos were then washed and incubated until hatching occurred. The toxicity of the cryoprotectant was assessed by the hatching rate. The results illustrated that permeable cryoprotectant toxicity for rainbow trout embryos increased in the order of PG < DMSO < MeOH < Gly < EG < Ac. The hatching rate of the embryos treated with permeable cryoprotectants decreased (P < 0.05) with increased concentration and duration of exposure. There were no significant decreases in hatching rate of embryos treated with sucrose and PVP with the increase of concentration; sucrose had higher hatching rates than PVP. Rainbow trout embryos at first eye pigmentation stage exhibited greater tolerance to cryoprotectants than embryos at epiboly stage.     

Preliminary investigation to determine the toxicity of various cryoprotectants on striped gourami (Trichogaster fasciata) embryos

As a study of cryoprotectant toxicity is an essential prerequisite for the development of a cryopreservation protocol, this study focuses on determining the toxicity of four permeable cryoprotectants: dimethyl sulfoxide (DMSO), propylene glycol (PG), methanol (MeOH), and acetamide (Ac). In cryoprotectant toxicity experiments, striped gourami (Trichogaster fasciata) embryos at three different developmental stages (multi cell, 100% epiboly, and proliferation of somites) were exposed to cryoprotectant solutions with concentrations from 1 to 4 M for a period of 5 and 15 min. Following these treatments, the embryos were incubated until the evaluation of hatching rate. Embryos were tolerant to low concentrations of all cryoprotectants tested in the range of 1 to 2 M for all developmental stages. Early stage embryos were more vulnerable to high concentration (3 and 4 M) than late stage embryos. Results also showed that as concentration and duration of exposure increased, the hatching rate significantly decrease (P < 0.05). On a molar-equivalent basis, DMSO appeared to be less toxic to PG, MeOH, and Ac in general. Exposure to cryoprotectants revealed a stage-dependent sensitivity. Toxicity increased in the order of MeOH < DMSO < PG < Ac in multi cell stage and DMSO < MeOH < PG < Ac in 100% epiboly and proliferation of somites stages. The proliferation of somites stage embryos was less sensitive to exposure to cryoprotectants than multi cell and 100% epiboly stages. These findings could be important for designing cryopreservation protocols for this demanding ornamental species.