Studies on cryopreservation of zebrafish (Danio rerio) oocytes using controlled slow cooling and vitrification

A thesis submitted to the University of Bedfordshire in partial fulfillment of the requirements for the degree of Doctor of PhilosophyCryopreservation of gametes provides a promising method to preserve fish genetic materials, which offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful cryopreservation of spermatozoa of about 200 fish species has been achieved, systematic studies on cryopreservation of fish oocytes have only recently been undertaken. The objective of the present studies was to use zebrafish as a model system to develop a cryopreservation protocol for fish oocytes and to develop reliable viability assessment methods for monitoring zebrafish oocyte viability both before and after cryopreservation. A simple and rapid enzymatic method for zebrafish oocytes isolation was developed and the investigations on cryopreservation of zebrafish oocytes using improved controlled slow cooling and vitrification were carried out. Oocyte viability following cryopreservation was investigated by ATP assay, oocyte viability molecular signature (OVMS) and cryomicroscopic observation in addition to staining methods. The optimum conditions for oocyte enzymatic separation were identified as 0.4mg/ml collagenase or 1.6mg/ml hyaluronidase treatment for 10min at 22ºC and this method can be used for oocytes at all stages. The use of sodium free medium (KCl buffer), fast warming and 4-step removal of cryoprotectants in an improved controlled slow cooling protocol significantly enhanced oocyte viability (67.5 ± 1.7%) when compared with a previous study (16.3 ± 2.3%) in this laboratory. Mixtures of cryoprotectants (methanol, Me2SO and propylene glycol), stepwise addition and removal of cryoprotectants in combination of a new vitrification system (CVA65 vitrification system) were used in vitrification studies. Oocyte survivals after vitrification assessed by trypan blue staining were relatively high (76.5 ± 6.3%) shortly after warming in KCl buffer. Furthermore, the result of ATP assay showed that ATP levels in oocytes decreased significantly after cryopreservation indicating the bioenergetic systems of oocytes were damaged. Cryomicroscopic observations demonstrated that Intracellular ice formation (IIF) is the main factor causing injuries during cryopreservation of zebrafish oocytes. The results provided by the present study will assist successful protocol design for cryopreservation of fish oocytes in the future