Climate change has increased the incidence of coral bleaching events, resulting in the loss of ecosystem function and biodiversity on reefs around the world. As reef degradation accelerates, the need for innovative restoration tools has become acute. Despite past successes with ultra-low temperature storage of coral sperm to conserve genetic diversity, cryopreservation of larvae has remained elusive due to their large volume, membrane complexity, and sensitivity to chilling injury. Here we show for the first time that coral larvae can survive cryopreservation and resume swimming after warming. Vitrification in a 3.5 M cryoprotectant solution (10% v/v propylene glycol, 5% v/v dimethyl sulfoxide, and 1 M trehalose in phosphate buffered saline) followed by warming at a rate of approximately 4,500,000 °C/min with an infrared laser resulted in up to 43% survival of Fungia scutaria larvae on day 2 post-fertilization. Surviving larvae swam and continued to develop for at least 12 hours after laser-warming. This technology will enable biobanking of coral larvae to secure biodiversity, and, if managed in a high-throughput manner where millions of larvae in a species are frozen at one time, could become an invaluable research and conservation tool to help restore and diversify wild reef habitats.

Our goal was to develop a standardized approach for sperm vitrification of marine fishes that can be applied generally in aquatic species. The objectives were to: 1) estimate acute toxicity of cryoprotectants over a range of concentrations; 2) evaluate the properties of vitrification solutions (VS); 3) evaluate different thawing solutions, and 4) evaluate sperm quality after thawing by examination of motility and membrane integrity. Sperm were collected from red snapper (Lutjanus campechanus), spotted seatrout (Cynoscion nebulosus), and red drum (Sciaenops ocellatus). A total of 29 combinations of cryoprotectants were evaluated for toxicity and glass formation. Samples were loaded onto 10-µL polystyrene loops and plunged into liquid nitrogen. There was a significant difference (P < 0.05) in post-thaw motility among VS and among species when using the same VS. The sperm in VS of 15% DMSO + 15% ethylene glycol + 10% glycerol + 1% X-1000™ + 1% Z-1000™ had an average post-thaw motility of 58% and membrane integrity of 19% for spotted seatrout, 38% and 9% for red snapper, and 30% and 19% for red drum. Adaptations by marine fish to high osmotic pressures could explain the survival in the high cryoprotectant concentrations. Vitrification offers an alternative to conventional cryopreservation.

protocols to bank precious germplasm from endangered species, [1,4] manage biodiversity, provide year-round access to embryos to grow and harvest important aquaculture species, [5,6] and support biomedical research [7,8] and gene banking. [9] One of the best model systems for studying teleost embryo cryopreservation is the zebrafish (Danio rerio) embryo. Since becoming an increasingly important model system for biomedical research, [10,11] researchers have enhanced this model into tens of thousands of mutant, transgenic, and wild-type zebrafish lines. [12-14] Since preserving all these valuable genotypes through live fish colonies is expensive, risky, and beyond the capacity of even the largest stock centers, cryopreservation is an important technique for insuring against the loss of genetically important lines due to catastrophic events, such as the spread of an infectious disease or reproductive failures within a breeding facility. [10,11] Further, it can reduce significant long-term cost and space needs by banking valuable zebrafish lines that are not routinely used. [10,11] In last decade many other aquatic species such as medaka, [15] Xiphophorus, [16] and Xenopus [17] have also become increasingly valuable biomedical models and require robust embryo cryopreservation protocols for long term maintenance of transgenic and mutant lines. [10] Cryopreservation relies on two key techniques. First, a certain amount of intracellular water must be replaced with viscous antifreeze molecules, or cryoprotective agents (CPA). Second, the cooling and rewarming of specimens to and from cryogenic temperatures must occur at rates faster than the rates of ice crystallization. These "critical cooling" and "critical warming" rates and the CPA type and concentration used for vitrification are inversely related, i.e., the higher the CPA concentration is, the lower the critical cooling and warming rates. In addition, critical warming rates exceed critical cooling rates by at least an order of magnitude in both cryoprotectant media and biological systems. [18,19] The main challenges with applying the these well-known principles of cryopreservation to zebrafish embryos are i) the relatively large size of the embryo (1000 times bigger in volume than the mammalian gametes that are routinely cryopreserved), resulting in a low surface-to-volume ratio that impedes water and CPA efflux/influx; ii) the presence of multiple compartments, such as the blastoderm and yolk, with differing This study shows for the first time the ability to rewarm cryopreserved zebrafish embryos that grow into adult fish capable of breeding normally. The protocol employs a single injection of cryoprotective agents (CPAs) and gold nanorods (GNRs) into the yolk and immersion in a precooling bath to dehydrate the perivitelline space. Then embryos are encapsulated within CPA and GNR droplets, plunged into liquid nitrogen, cryogenically stabilized, and rewarmed by a laser pulse. Postlaser nanowarming, embryos (n = 282) exhibit intact structure by 1 h (40%), continued ...