123 Development of cryopreservation protocol for zebrafish ovarian fragments using controlled slow cooling

Cryopreservation of gametes is an important method of conservation of germplasm and has wide range of applications in aquaculture and fisheries management. Cryopreservation is the most efficient method for long term storage of genetic materials. Though fish sperm cryopreservation has been a success, developing successful protocols for eggs and embryo cryopreservation still remain elusive. Several studies have been undertaken on cryopreservation of isolated zebrafish ovarian follicles at different stages yet the protocols used lead to compromised viability. Cryopreservation of follicles within ovarian tissue has attracted considerable attention in recent years. It has become a valid alternative to cryopreservation of human oocytes. The present study was undertaken to determine the effect of cryopreservation on zebrafish ovarian tissue fragments containing the stage I and stage II follicles. The effect of cryopreservation medium, the effect of cryoprotectants, cooling rate and ovarian follicle viability assessment after controlled slow cooling were investigated. Ovarian follicle membrane integrity was assessed by trypan blue (TB) and fluorescein acetate-propidium iodide (FDA-PI) staining. ATP levels were measured to assess the metabolic status following cryopreservation. In these experiments ovarian tissue fragments were exposed to cryoprotectant solutions for 30min at 22°C and then were loaded into 0.5ml plastic straws before placing in a programmable cooler (Planer KRYO 550). Ovarian tissue fragments incubated in cryoprotectant-free L-15 medium or KCl buffer were used as controls. The following cooling protocol was used: cooling at 0.3°C/min, 0.4°C/min, 1°C/min, 2°C/min, 4°C/min and 7°C/min from 20°C to seeding temperature (−7.5°C for 2M methanol). Manual seeding and hold for 5min, freezing from seeding temperature to −40°C at 2°C/min, from −40°C to −80°C at 10°C/min and hold for 10min, samples were then plunged in liquid nitrogen at −196°C and held for at least 10min. Samples were thawed using a water bath at 28°C. Removal of cryoprotectant was conducted in four steps. The results from the present study indicated that the tissue fragments can be incubated in 2M methanol+20% FBS in L-15 medium for 30min at room temperature and then frozen to −196°C at post-seeding cooling rate 4°C/min and the removal of the cryoprotectant in four steps. After cryopreservation the survival rate of the follicles was assessed using TB staining, FDA+PI staining and ATP assay. The survival rate after the cryopreservation procedure were 55.4±2.3% for stage I and 68.2±1.9% for stage II using TB staining and 48.2±2.9% for stage I and 54.2±2.6% for stage II follicles using FDA+PI staining. The results obtained from ATP assay showed compromised survival of the ovarian follicles. The present study also indicated that the use of non-permeating cryoprotectants is not beneficial for the zebrafish ovarian tissue cryopreservation since it decreased the survival rate of the follicles when compared to the use of permeating cryoprotectants. The results obtained after the in-vitro culture of the cryopreserved fragments showed that there was no growth of follicles obtained after in-vitro culture treatment and the survival rate of the cryopreserved follicles were significantly lower to those of the non-cryopreserved follicles. The results obtained in this study provide useful information for future cryopreservation protocol development.Source of funding: Funding support for the programme of research was provided by the strategic research funds of the iBEST, University of BedfordshireConflict of interest: None [email protected]