Possibility of cryopreservation of medaka eggs using liquid meniscus

Abstract

The cryopreservation of fish eggs is an important subject in the field of fishery and preservation of biological species. Thus far, there has been no success in the preservation of fish eggs because of the large size of the eggs and the thick external shell. This paper discusses the effectiveness of using the liquid meniscus formed around the egg for protecting its morphology. Freezing and thawing experiments of medaka eggs were performed under different freezing conditions, and the hatching rate of the egg was examined. Before freezing, the eggs were dehydrated at room temperature in order to reduce the effect of volume expansion caused by freezing. It was confirmed that 100% of the eggs dehydrated by 15% or less were successfully hatched. In the freezing process, a medaka egg was placed on a hydrophobic cooling plate and a thin liquid meniscus was formed around the hydrophilic egg surface. An aqueous solution of trehalose was used as the liquid meniscus as well as a cryoprotectant to prevent damage caused by freezing. Cryopreservation of the egg was not successfully performed for all processes, including intracellular freezing; however, 80% of the eggs were alive even after freezing of the external meniscus. Therefore, it is confirmed that the liquid meniscus is effective for the cryopreservation of the external shell. The liquid meniscus can reduce the physical stress due to extracellular ice growth. Moreover, since the liquid meniscus system has a low heat capacity, the thermal process is easy to control compared to the conventional method. We concluded that the present method can be used for the cryopreservation of fish eggs.