Towards cryopreservation of Greenshell mussel (Perna canaliculus) oocytes.

Cryopreservation is a powerful tool for selective breeding in aquaculture as it enables genetic material from selected stock to be stored and crossed at will. The aim of this study was to develop a method for cryopreserving oocytes of the Greenshelltrade mark mussel (Perna canaliculus), New Zealand's main aquaculture species. The ability of oocytes to be fertilized post-thawing was used as the criterion for success in initial experiments and then subsequently, the ability of frozen oocytes to develop further to D-stage larvae was assessed. Ethylene glycol, propylene glycol, dimethyl sulphoxide and glycerol were evaluated at a range of concentrations with and without the addition of 0.2M trehalose using post-thaw fertilization as the endpoint. Ethylene glycol was most effective, particularly when used in combination with trehalose. A more detailed investigation revealed that ethylene glycol at 9% or 10% in the presence of 0.2-0.4M trehalose afforded the best protection. In experiments varying sperm to egg ratio and egg density in post-thaw fertilization procedures, D-larval yield averaged less than 1%. Following these results, a detailed experiment was conducted to determine the damaging steps in the cryopreservation process. Fertilization losses occurred at each step whereas D-larval yield approximately halved following CPA addition and was almost zero following cooling to -10 degrees C. Cryomicroscopy studies and fertilization results suggest that the inability of oocytes to develop to D-larvae stage after cooling to -10 degrees C and beyond are most likely related to some form of chilling injury rather than extracellular ice triggering intracellular ice formation. Further research is needed to determine the causes of this injury and to reduce CPA toxicity and/or osmotic effects.

Cholesterol addition and removal in pacific oyster oocytes does not improve cryopreservation success.

The optimal cholesterol content in cells could provide the benefit of lowering or eliminating the lipid phase transition temperature, while maintaining membrane fluidity and strength; thus, making cells less sensitive to chilling injury and more amenable to cryopreservation. Such effects were shown in some gametes and embryos of certain mammalian species, however, some other cell types, benefited from cholesterol removal. The experiments developed in this study aimed to determine the effect of incubating Pacific oyster (Crassostrea gigas) oocytes in cholesterol-addition or removal solutions prior to cryopreservation on their post-thaw fertilization ability. The results showed a positive association of cholesterol with the oocytes when assessed by fluorescent microscopy. However, this uptake was not reflected by an increase in cholesterol as determined by colorimetric analysis or in the post-thaw fertilization rate of treated oocytes. It is presumed either that oyster oocytes already contain a substantial amount of cholesterol or other lipids in their plasma membranes and do not benefit from any additional cholesterol or there is no lipid phase transition temperature in oyster oocytes.

Determination of the membrane permeability characteristics of Pacific oyster, Crassostrea gigas, oocytes and development of optimized methods to add and remove ethylene glycol.

In order for cryopreservation to become a practical tool for aquaculture, optimized protocols must be developed for each species and cell type. Knowledge of a cell's osmotic tolerance and membrane permeability characteristics can assist in optimized protocol development. In this study, these characteristics were determined for Pacific oyster oocytes and modified methods for loading and unloading ethylene glycol (EG) were tested. Oocytes were found to behave as ideal osmometers and their osmotically inactive fraction (V(b)) was calculated to be 0.48. Oocytes exposed to NaCl solutions of 0.6 to 2.3 Osm fertilized at rates equivalent to oocytes left in seawater. This corresponds to volume changes of +27.3 and -38.1+/-1.2%. The permeability of the oocytes to water (L(p)) was determined to be 3.8+/-0.4 x 10(-2), 5.7+/-0.8 x 10(-2), and 13.2+/-1.3 x 10(-2) microm min(-1)atm(-1), when measured at temperatures of 5, 10 and 20 degrees C. The respective EG permeability values (P(s)) were 9.5+/-0.1 x 10(-5), 14.6+/-1.2 x 10(-5), and 41.7+/-2.4 x 10(-5) cm min(-1). The activation energies for L(p) and P(s) were determined to be 14.5 and 17.5 kcal mol(-1), respectively. Different models for EG loading and unloading from oocytes were developed and tested. Post-thaw fertilization did not differ significantly between a published step addition method and single step addition at 20 degrees C. This represents a considerable reduction in handling. The results of this study demonstrate that the cryobiological characteristics of a given cell type should be taken into account when developing cryopreservation methods.

Survival of Pacific oyster, Crassostrea gigas, oocytes in relation to intracellular ice formation.

The effect of IIF in Pacific oyster oocytes was studied using cryo and transmission electron microscopy (TEM). The viability of oocytes at each step of a published cryopreservation protocol was assessed in an initial experiment. Two major viability losses were identified; one when oocytes were cooled to -35 degrees C and the other when oocytes were plunged in liquid nitrogen. Although the cryomicroscope showed no evidence of IIF in oocytes cooled with this protocol, TEM revealed that these oocytes contained ice crystals and were at two developmental stages when frozen, prophase and metaphase I. To reduce IIF, the effect of seven cooling programmes involving cooling to -35 or -60 degrees C at 0.1 or 0.3 degrees C min(-1) and holding for 0 or 30 min at -35 or -60 degrees C was evaluated on post-thaw fertilization rate of oocytes. Regardless of the cooling rate or holding time, the fertilization rate of oocytes cooled to -60 degrees C was significantly lower than that of oocytes cooled to -35 degrees C. The overall results indicated that observations of IIF obtained from cryomicroscopy are limited to detection of larger amounts of ice within the cells. Although the amount of cellular ice may have been reduced by one of the programmes, fertilization was reduced significantly; suggesting that there is no correlation between the presence of intracellular ice and post-thaw fertilization rate. Therefore, oyster oocytes may be more susceptible to the effect of high solute concentrations and cell shrinkage than intracellular ice under the studied conditions.