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 30 min at 22 掳C and then were loaded into 0.5 ml 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 掳C/min, 0.4 掳C/min, 1 掳C/min, 2 掳C/min, 4 掳C/min and 7 掳C/min from 20 掳C to seeding temperature (鈭?.5 掳C for 2 M methanol). Manual seeding and hold for 5 min, freezing from seeding temperature to 鈭?0 掳C at 2 掳C/min, from 鈭?0 掳C to 鈭?0 掳C at 10 掳C/min and hold for 10 min, samples were then plunged in liquid nitrogen at 鈭?96 掳C and held for at least 10 min. Samples were thawed using a water bath at 28 掳C. Removal
of cryoprotectant was conducted in four steps. The results from the present study indicated that the tissue fragments can be incubated in 2 M methanol + 20% FBS in L-15 medium for 30 min at room temperature and then frozen to 鈭?96 掳C at post-seeding cooling rate 4 掳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 卤 2.3% for stage I and 68.2 卤 1.9% for stage II using TB staining and 48.2 卤 2.9% for stage I and 54.2 卤 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 Bedfordshire
Conflict of interest: None declared.