Development of cryopreservation protocols for early stage zebrafish (Danio rerio) ovarian follicles using controlled slow cooling

Cryopreservation of germplasm of aquatic species offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful fish sperm cryopreservation has been achieved with many species, there has been no report of successful cryopreservation of fish embryos and late stage oocytes which are large, chilling sensitive and have low membrane permeability. In the present study, cryopreservation of early stage zebrafish ovarian follicles was studied for the first time using controlled slow freezing. The effect of cryoprotectant, freezing medium, cooling rate, method for cryoprotectant removal, post-thaw incubation time and ovarian follicle developmental stage were investigated. Stages I and II ovarian follicles were frozen in 4 M methanol and 3 M DMSO in either L-15 medium or KCl buffer. Ovarian follicle viability was assessed using trypan blue, FDA + PI staining and ADP/ATP assay. The results showed that KCl buffer was more beneficial than L-15 medium, methanol was more effective than DMSO, optimum cooling rates were 2-4 °C/min, stepwise removal of cryoprotectant improved ovarian follicle viability significantly and stage I ovarian follicles were more sensitive to freezing. The results also showed that FDA + PI staining and ADP/ATP assay were more sensitive than TB staining. The highest follicle viabilities after post-thaw incubation for 2 h obtained with FDA + PI staining were 50.7 ± 4.0% although ADP/ATP ratios of the cryopreserved follicles were significantly increased indicating increased cell damage. Studies are currently being carried out on in vitro maturation of these cryopreserved ovarian follicles.