Studies on cryoprotectant toxicity to early stage zebrafish (Danio rerio) ovarian follicles

Cryoprotectants are substances characterised by their ability to reduce cryoinjury of biological materials during the course of freezing. Unfortunately cryoprotectants can be toxic for cells. The aim of this study is to investigate the toxicity of cryoprotectants to early stage ovarian follicles of zebrafish (Danion rerio) before designing protocols for their cryopreservation. Commonly used cryoprotectants methanol, dimethyl sulfoxide (DMSO), propylene glycol (PG) and ethylene glycol (EG) were studied. Experiments were conducted with stage I and II zebrafish ovarian follicles, which were incubated in 50% L-15 medium containing different concentrations of cryoprotectants (0.25-5M) for 30 min at room temperature. Stage III zebrafish ovarian follicles were also used as comparisons. Two different tests were used to assess ovarian follicle viability: trypan blue (TB) and Fluorescein diacetate (FDA) + propidium iodide (PI) staining. Both TB and FDA+PI tests indicated that cryoprotectant toxicity to ovarian follicles increased in the order of methanol, DMSO, PG and EG. FDA+PI test was shown to be more sensitive than TB staining. No Observed Effect Concentrations (NOECs) for stage I and II follicles were 2M, 1M, 0.5M, and 0.25M for methanol, dimethyl sulfoxide (DMSO), propylene glycol (PG) and ethylene glycol (EG) respectively when assessed with FDA+PI. Stage III ovarian follicles appeared to be more sensitive than stage I and II ovarian follicles.