Cryopreservation of Fish Spermatogonial Cells: The Future of Natural History Collections.

As global biodiversity declines, the value of biological collections increases. Cryopreserved diploid spermatogonial cells meet two goals: to yield high-quality molecular sequence data; and to regenerate new individuals, hence potentially countering species extinction. Cryopreserved spermatogonial cells that allow for such mitigative measures are not currently in natural history museum collections because there are no standard protocols to collect them. Vertebrate specimens, especially fishes, are traditionally formalin-fixed and alcohol-preserved which makes them ideal for morphological studies and as museum vouchers, but inadequate for molecular sequence data. Molecular studies of fishes routinely use tissues preserved in ethanol; yet tissues preserved in this way may yield degraded sequences over time. As an alternative to tissue fixation methods, we assessed and compared previously published cryopreservation methods by gating and counting fish testicular cells with flow cytometry to identify presumptive spermatogonia A-type cells. Here we describe a protocol to cryopreserve tissues that yields a high percentage of viable spermatogonial cells from the testes of Asterropteryx semipunctata, a marine goby. Material cryopreserved using this protocol represents the first frozen and post-thaw viable spermatogonial cells of fishes archived in a natural history museum to provide better quality material for re-derivation of species and DNA preservation and analysis.

Producing Coral Offspring with Cryopreserved Sperm: A Tool for Coral Reef Restoration.

Cryopreservation is an important conservation tool, which may help reef-building coral survive. However, scaling-up from small, laboratory-sized experiments to higher-throughput restoration is a major challenge. To be an effective restoration tool, the cryopreservation methods and husbandry to produce new offspring must be defined. This study examined small and larger-scale in vitro reproduction and settlement for Acropora tenuis and Acropora millepora and found that: 1) cryopreservation of coral sperm reduced sperm motility and fertilization success in half, thus fresh sperm, capable of becoming highly motile, is key; 2) the sperm-to-egg ratio and the concentration of the cryoprotectant treatments affected fertilization success in small- and larger-scale reproduction trials using cryopreserved sperm (p < 0.05); 3) cryopreservation did not affect settlement success, as larvae produced with fresh or cryopreserved sperm had the same settlement success (p > 0.05); and 4) the residence time of the sperm within the bank was not important as the fertilization success of sperm frozen for less than 1 month was similar to that frozen up to 2 years (p > 0.05). These results described the first settlement for coral larvae produced from cryopreserved sperm and established important ground-work principles for the use of cryopreserved coral sperm for future reef restoration efforts.

Seasonal Preservation Success of the Marine Dinoflagellate Coral Symbiont, Symbiodinium sp.

Coral reefs are some of the most diverse and productive ecosystems on the planet, but are threatened by global and local stressors, mandating the need for incorporating ex situ conservation practices. One approach that is highly protective is the development of genome resource banks that preserve the species and its genetic diversity. A critical component of the reef are the endosymbiotic algae, Symbiodinium sp., living within most coral that transfer energy-rich sugars to their hosts. Although Symbiodinium are maintained alive in culture collections around the world, the cryopreservation of these algae to prevent loss and genetic drift is not well-defined. This study examined the quantum yield physiology and freezing protocols that resulted in survival of Symbiodinium at 24 h post-thawing. Only the ultra-rapid procedure called vitrification resulted in success whereas conventional slow freezing protocols did not. We determined that success also depended on using a thin film of agar with embedded Symbiodinium on Cryotops, a process that yielded a post-thaw viability of >50% in extracted and vitrified Symbiodinium from Fungia scutaria, Pocillopora damicornis and Porites compressa. Additionally, there also was a seasonal influence on vitrification success as the best post-thaw survival of F. scutaria occurred in winter and spring compared to summer and fall (P < 0.05). These findings lay the foundation for developing a viable genome resource bank for the world's Symbiodinium that, in turn, will not only protect this critical element of coral functionality but serve as a resource for understanding the complexities of symbiosis, support selective breeding experiments to develop more thermally resilient strains of coral, and provide a 'gold-standard' genomics collection, allowing for full genomic sequencing of unique Symbiodinium strains.

Cryobiology of coral fragments.

Around the world, coral reefs are dying due to human influences, and saving habitat alone may not stop this destruction. This investigation focused on the biological processes that will provide the first steps in understanding the cryobiology of whole coral fragments. Coral fragments are a partnership of coral tissue and endosymbiotic algae, Symbiodinium sp., commonly called zooxanthellae. These data reflected their separate sensitivities to chilling and a cryoprotectant (dimethyl sulfoxide) for the coral Pocillopora damicornis, as measured by tissue loss and Pulse Amplitude Modulated fluorometry 3weeks post-treatment. Five cryoprotectant treatments maintained the viability of the coral tissue and zooxanthellae at control values (1M dimethyl sulfoxide at 1.0, 1.5 and 2.0h exposures, and 1.5M dimethyl sulfoxide at 1.0 and 1.5h exposures, P>0.05, ANOVA), whereas 2M concentrations did not (P<0.05, ANOVA). A seasonal response to chilling was observed in the coral tissue, but not in the zooxanthellae. During the winter when the fragments were chilled, the coral tissue remained relatively intact (∼25% loss) post-treatment, but the zooxanthellae numbers in the tissue declined after 5min of chilling (P<0.05, ANOVA). However, in the late spring, coral tissue (∼75% loss) and zooxanthellae numbers declined in response to chilling alone (P<0.05, ANOVA). When a cryoprotectant (1M dimethyl sulfoxide) was used in concert with chilling it protected the coral against tissue loss after 45min of cryoprotectant exposure (P>0.05, ANOVA), but it did not protect against the loss of zooxanthellae (P<0.05, ANOVA). The zooxanthellae are the most sensitive element in the coral fragment complex and future cryopreservation protocols must be guided by their greater sensitivity.

Oxidative stress in zebrafish (Danio rerio) sperm.

Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely cryopreserve a genetic line.

Zebrafish reproduction: revisiting in vitro fertilization to increase sperm cryopreservation success.

Although conventional cryopreservation is a proven method for long-term, safe storage of genetic material, protocols used by the zebrafish community are not standardized and yield inconsistent results, thereby putting the security of many genotypes in individual laboratories and stock centers at risk. An important challenge for a successful zebrafish sperm cryopreservation program is the large variability in the post-thaw in vitro fertilization success (0 to 80%). But how much of this variability was due to the reproductive traits of the in vitro fertilization process, and not due to the cryopreservation process? These experiments only assessed the in vitro process with fresh sperm, but yielded the basic metrics needed for successful in vitro fertilization using cryopreserved sperm, as well. We analyzed the reproductive traits for zebrafish males with a strict body condition range. It did not correlate with sperm volume, or motility (P>0.05), but it did correlate with sperm concentration. Younger males produced more concentrated sperm (P<0.05). To minimize the wastage of sperm during the in vitro fertilization process, 10(6) cells/ml was the minimum sperm concentration needed to achieve an in vitro fertilization success of ≥ 70%. During the in vitro process, pooling sperm did not reduce fertilization success (P>0.05), but pooling eggs reduced it by approximately 30 to 50% (P<0.05). This reduction in fertilization success was due not to the pooling of the females' eggs, but to the type of tools used to handle the eggs. Recommendations to enhance the in vitro process for zebrafish include: 1) using males of a body condition closer to 1.5 for maximal sperm concentration; 2) minimizing sperm wastage by using a working sperm concentration of 10(6) motile cells/ml for in vitro fertilization; and 3) never using metal or sharp-edged tools to handle eggs prior to fertilization.

Analysis of internal osmolality in developing coral larvae, Fungia scutaria.

Coral species throughout the world are facing severe local and global environmental pressures. Because of the pressing conservation need, we are studying the reproduction, physiology, and cryobiology of coral larvae with the future goal of cryopreserving and maintaining these organisms in a genome resource bank. Effective cryopreservation involves several steps, including the loading and unloading of cells with cryoprotectant and the avoidance of osmotic shock. In this study, during the time course of coral larvae development of the mushroom coral Fungia scutaria, we examined several physiologic factors, including internal osmolality, percent osmotically active water, formation of mucus cells, and intracellular organic osmolytes. The osmotically inactive components of the cell, V(b), declined 33% during development from the oocyte to day 5. In contrast, measurements of the internal osmolality of coral larvae indicated that the internal osmolality was increasing from day 1 to day 5, probably as a result of the development of mucus cells that bind ions. Because of this, we conclude that coral larvae are osmoconformers with an internal osmolality of about 1,000 mOsm. Glycine betaine, comprising more than 90% of the organic osmolytes, was found to be the major organic osmolyte in the larvae. Glycerol was found in only small quantities in larvae that had been infected with zooxanthellae, suggesting that this solute did not play a significant role in the osmotic balance of this larval coral. We were interested in changes in cellular characteristics and osmolytes that might suggest solutes to test as cryoprotectants in order to assist in the successful cryopreservation of the larvae. More importantly, these data begin to reveal the basic physiological events that underlie the move from autonomous living to symbiosis.

Betaines and dimethylsulfoniopropionate as major osmolytes in cnidaria with endosymbiotic dinoflagellates.

Most marine invertebrates and algae are osmoconformers whose cells accumulate organic osmolytes that provide half or more of cellular osmotic pressure. These solutes are primarily free amino acids and glycine betaine in most invertebrates and small carbohydrates and dimethylsulfoniopropionate (DMSP) in many algae. Corals with endosymbiotic dinoflagellates (Symbiodinium spp.) have been reported to obtain from the symbionts potential organic osmolytes such as glycerol, amino acids, and DMSP. However, corals and their endosymbionts have not been fully analyzed for osmolytes. We quantified small carbohydrates, free amino acids, methylamines, and DMSP in tissues of the corals Fungia scutaria, Pocillopora damicornis, Pocillopora meandrina, Montipora capitata, Porites compressa, and Porites lobata (all with symbionts) plus Tubastrea aurea (asymbiotic) from Kaneohe Bay, Oahu (Hawaii). Glycine betaine, at 33-69 mmol/kg wet mass, was found to constitute 90% or more of the measured organic solutes in all except the Porites species. Those were dominated by proline betaine and dimethyltaurine. DMSP was found at 0.5-3 mmol/kg in all species with endosymbionts. Freshly isolated Symbiodinium from Fungia, P. damicornis, and P. compressa were also analyzed. DMSP and glycine betaine dominated in the first two; Porites endosymbionts had DMSP, proline betaine, and dimethyltaurine. In all specimens, glycerol and glucose were detected by high-performance liquid chromatography only at 0-1 mmol/kg wet mass. An enzymatic assay for glycerol plus glycerol 3-phosphate and dihydroxyacetone phosphate yielded 1-10 mmol/kg. Cassiopeia andromeda (upside-down jelly; Scyphozoan) and Aiptasia puchella (solitary anemone; Anthozoan) were also analyzed; both have endosymbiotic dinoflagellates. In both, glycine betaine, taurine, and DMSP were the dominant osmolytes. In summary, methylated osmolytes dominate in many Cnidaria; in those with algal symbionts, host and symbiont have similar methylated amino acids, as do congeners. However, little glycerol was present as an osmolyte and was probably metabolized before it could accumulate.