Enabling the cryopreservation of embryos from aquatic species using ice recrystallization inhibitors

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In most human and animal systems, sperm cryopreservation is well defined and has successful outcome protocols. However, embryo cryopreservation does not exist except for humans and few agriculturally important species. Little cryopreservation success has been observed with marine embryos and larvae and only within the past year has this been changing for some aquatic organisms. The development of embryo cryopreservation technologies revolutionized breeding of domestic animals as well as human reproductive technologies over the past three decades. We will examine two aquatic embryo or larval systems, including coral and fish. Coral reefs are threatened around the world and their conservation would benefit enormously from increased understanding and application of their cryobiology. We believe that our recent work with novel cryopreservation technologies could help transform these cryopreservation processes in these two systems that are currently stalled with low cryopreservation success. The most significant cause of decreased viability and impaired function during cryopreservation is physical and chemical damage resulting from ice crystal formation and growth. In the context of embryo cryopreservation, a low surface area / volume ratio and a low membrane permeability to water and cryoprotectant makes it difficult to accumulate high enough concentrations of cryoprotectant into the embryo to avoid intracellular / interstitial ice formation. Consequently, new approaches are urgently required. Recently, large chemical libraries of small molecule ice recrystallization inhibitors (iRis) have been developed and used in the cryopreservation of mammalian cells. Ice recrystallization is the process that occurs during freezing and thawing where by large ice crystals grow larger at the expense of smaller ones and is a major cause of cellular damage that reduces postthaw viability and potency. We hypothesize that controlling ice formation using novel small molecule ice recrystallization inhibitors will reduce cryoinjury and enhance postthaw viability and cell function of embryos from aquatic species. Our multidisciplinary team has set four complementary aims to improve our understanding of the effectiveness of IRI as cryoprotectants and their beneficial impact on embryos from aquatic species. Embryos from established fresh and saltwater model systems [ie. zebrafish (Danio rerio) and coral (Fungia scutaria) will be evaluated. We will evaluate the cryoprotective effectiveness of using ice recrystallization inhibitors to control ice as a novel approach to the practical cryopreservation of marine and freshwater aquatic species. Secondarily we will develop knowledge on cryobiological properties of embryos which will help inform further optimization of embryo cryopreservation protocols, identify potential mechanism of cryoinjury and aid in the translation of this for use in human health research and conservation efforts.