A Semi-Automated Workflow for the Cryopreservation of Coral Sperm to Support Biobanking and Aquaculture.

Coral reefs are facing a crisis as the frequency of bleaching events caused by ocean warming increases, resulting in the death of corals on reefs around the world. The subsequent loss of genetic diversity and biodiversity can diminish the ability of coral to adapt to the changing climate, so efforts to preserve existing diversity are essential to maximize the resources available for reef restoration now and in the future. The most effective approach to secure genetics long-term is cryopreservation and biobanking, which permits the frozen storage of living samples at cryogenic temperatures in liquid nitrogen indefinitely. Cryopreservation of coral sperm has been possible since 2012, but the seasonal nature of coral reproduction means that biobanking activities are restricted to just a few nights per year when spawning occurs. Improving the efficiency of coral sperm processing and cryopreservation workflows is therefore essential to maximizing these limited biobanking opportunities. To this end, we set out to optimize cryopreservation processing pathways for coral sperm by building on existing technologies and creating a semi-automated approach to streamline the assessment, handling, and cryopreservation of coral sperm. The process, which combines computer-assisted sperm analysis, barcoded cryovials, and a series of linked auto-datasheets for simultaneous editing by multiple users, improves the efficiency of both sample processing and metadata management in the field. Through integration with cross-cutting research programs such as the Reef Restoration and Adaptation Program in Australia, cryopreservation can play a crucial role in large-scale reef restoration programs by facilitating the genetic management of aquaculture populations, supporting research to enhance thermal tolerance, and preventing the extinction of coral species. The described procedures will be utilized for coral cryopreservation and biobanking practitioners on reefs worldwide and will provide a model for the transition of cryopreservation technologies from research laboratories to large-scale applications.

Cryopreservation of the collector urchin embryo, Tripneustes gratilla.

The collector urchin, Tripneustes gratilla, is an ecologically important member of the grazing community of Hawai'i's coral reefs. Beyond its ability to maintain balance between native seaweeds and corals, T. gratilla has also been used as a food source and a biocontrol agent against alien invasive algae species. Due to overexploitation, habitat degradation, and other stressors, their populations face local extirpation. However, artificial reproductive techniques, such as cryopreservation, could provide more consistent seedstock throughout the year to supplement aquaculture efforts. Although the sperm and larvae of temperate urchins have been successfully cryopreserved, tropical urchins living on coral reefs have not. Here, we investigated the urchin embryos' tolerance to various cryoprotectants and cooling rates to develop a cryopreservation protocol for T. gratilla. We found that using 1 M Me2SO with a cooling rate of 9.7 °C/min on gastrula stage embryos produced the best results with survival rates of up to 85.5% and up to 50.8% maturation to the 4-arm echinopluteus stage, assessed three days after thawing. Continued research could see cryopreservation added to the repertoire of artificial reproductive techniques for T. gratilla, thereby assisting in the preservation of this ecologically important urchin, all while augmenting aquaculture efforts that contribute to coral reef restoration.

Conduction-Dominated Cryomesh for Organism Vitrification.

Vitrification-based cryopreservation is a promising approach to achieving long-term storage of biological systems for maintaining biodiversity, healthcare, and sustainable food production. Using the "cryomesh" system achieves rapid cooling and rewarming of biomaterials, but further improvement in cooling rates is needed to increase biosystem viability and the ability to cryopreserve new biosystems. Improved cooling rates and viability are possible by enabling conductive cooling through cryomesh. Conduction-dominated cryomesh improves cooling rates from twofold to tenfold (i.e., 0.24 to 1.2 × 105  °C min-1 ) in a variety of biosystems. Higher thermal conductivity, smaller mesh wire diameter and pore size, and minimizing the nitrogen vapor barrier (e.g., vertical plunging in liquid nitrogen) are key parameters to achieving improved vitrification. Conduction-dominated cryomesh successfully vitrifies coral larvae, Drosophila embryos, and zebrafish embryos with improved outcomes. Not only a theoretical foundation for improved vitrification in µm to mm biosystems but also the capability to scale up for biorepositories and/or agricultural, aquaculture, or scientific use are demonstrated.

Cryopreservation Cooling Rate Impacts Post-Thaw Sperm Motility and Survival in Litoria booroolongensis.

The cryopreservation and storage of gametes (biobanking) can provide a long-term, low-cost option for the preservation of population genetic diversity and is particularly impactful when applied to manage selective breeding within conservation breeding programs (CBPs). This study aimed to develop a sperm cryopreservation protocol for the critically endangered Booroolong frog (Litoria booroolongensis) to capture founder genetics within the recently established (est. 2019) CBP for this species. Hormone-induced sperm release was achieved using established protocols, and spermic urine samples were collected over a 6-h period. Pooled spermic urine samples (n = 3 males) were divided equally between two cryoprotectant (CPA) treatments and diluted by 1:5 (sperm:CPA) with either 15% (v/v) dimethyl sulfoxide + 1% (w/v) sucrose in simplified amphibian Ringer's (SAR; CPAA) or 10% (v/v) dimethylformamide + 10% (w/v) trehalose dihydrate in SAR (CPAB). The samples were cryopreserved in 0.25 mL straws using either a programmable freezer (FrA) or an adapted dry shipper method (FrB). The thawed samples were activated via dilution in water and assessed for viability and motility using both manual assessment and computer-assisted sperm analysis (CASA; 0 h, 0.5 h post-thaw). Upon activation, the survival and recovery of motility (total motility, forward progression and velocity) of cryopreserved sperm suspensions were higher for sperm preserved using FrB than FrA, regardless of CPA composition. This work supports our long-term goal to pioneer the integration of biobanked cryopreserved sperm with population genetic management to maximize restoration program outcomes for Australian amphibian species.

Cryopreservation and revival of Hawaiian stony corals using isochoric vitrification.

Corals are under siege by both local and global threats, creating a worldwide reef crisis. Cryopreservation is an important intervention measure and a vital component of the modern coral conservation toolkit, but preservation techniques are currently limited to sensitive reproductive materials that can only be obtained a few nights per year during spawning. Here, we report the successful cryopreservation and revival of cm-scale coral fragments via mL-scale isochoric vitrification. We demonstrate coral viability at 24 h post-thaw using a calibrated oxygen-uptake respirometry technique, and further show that the method can be applied in a passive, electronics-free configuration. Finally, we detail a complete prototype coral cryopreservation pipeline, which provides a platform for essential next steps in modulating post-thaw stress and initiating long-term growth. These findings pave the way towards an approach that can be rapidly deployed around the world to secure the biological genetic diversity of our vanishing coral reefs.

Tank fouling community enhances coral microfragment growth.

Anthropogenic stressors threaten reefs worldwide and natural in situ coral reproduction may be inadequate to meet this challenge. Land-based culture can provide increased coral growth, especially with microfragments. We tested whether culture methods using different algal fouling communities could improve the growth and health metrics of microfragments of the Hawaiian coral, Porites compressa. Culture method fouling communities were: (1) similar to a reef environment (Mini Reef); (2) clean tanks managed to promote crustose coralline algae (Clean Start); and (3) tanks curated beforehand with poorly-competing algae (Green Film) assessed in winter and summer months. The Green Film method during the winter produced the fastest microfragment mean growth at 28 days until the first row of new polyps developed, and also the highest tank and plate metric health scores. Time efficient, standardized methods for land-based culture designed to maximize growth and production of coral fragments will contribute considerably to the success of large-scale restoration efforts.

Pharmacologic inhibition of NLRP3 reduces the levels of α-synuclein and protects dopaminergic neurons in a model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is characterized by a progressive degeneration of dopaminergic neurons, which leads to irreversible loss of peripheral motor functions. Death of dopaminergic neurons induces an inflammatory response in microglial cells, which further exacerbates neuronal loss. Reducing inflammation is expected to ameliorate neuronal loss and arrest motor dysfunctions. Because of the contribution of the NLRP3 inflammasome to the inflammatory response in PD, we targeted NLRP3 using the specific inhibitor OLT1177®. METHODS: We evaluated the effectiveness of OLT1177® in reducing the inflammatory response in an MPTP neurotoxic model of PD. Using a combination of in vitro and in vivo studies, we analyzed the effects of NLRP3 inhibition on pro-inflammatory markers in the brain, α-synuclein aggregation, and dopaminergic neuron survival. We also determined the effects of OLT1177® on locomotor deficits associated with MPTP and brain penetrance. RESULTS: Treatment with OLT1177® prevented the loss of motor function, reduced the levels of α-synuclein, modulated pro-inflammatory markers in the nigrostriatal areas of the brain, and protected dopaminergic neurons from degeneration in the MPTP model of PD. We also demonstrated that OLT1177® crosses the blood-brain barrier and reaches therapeutic concentrations in the brain. CONCLUSIONS: These data suggest that targeting the NLRP3 inflammasome by OLT1177® may be a safe and novel therapeutic approach to arrest neuroinflammation and protect against neurological deficits of Parkinson's disease in humans.

Solar radiation, temperature and the reproductive biology of the coral Lobactis scutaria in a changing climate.

Coral reefs worldwide are at risk due to climate change. Coral bleaching is becoming increasingly common and corals that survive bleaching events can suffer from temporary reproductive failure for several years. While water temperature is a key driver in causing coral bleaching, other environmental factors are involved, such as solar radiation. We investigated the individual and combined effects of temperature, photosynthetically active radiation (PAR), and ultraviolet radiation (UVR) on the spawning patterns and reproductive physiology of the Hawaiian mushroom coral Lobactis scutaria, using long-term experiments in aquaria. We examined effects on spawning timing, fertilisation success, and gamete physiology. Both warmer temperatures and filtering UVR altered the timing of spawning. Warmer temperatures caused a drop in fertilisation success. Warmer temperatures and higher PAR both negatively affected sperm and egg physiology. These results are concerning for the mushroom coral L. scutaria and similar reproductive data are urgently needed to predict future reproductive trends in other species. Nonetheless, thermal stress from global climate change will need to be adequately addressed to ensure the survival of reef-building corals in their natural environment throughout the next century and beyond. Until then, reproduction is likely to be increasingly impaired in a growing number of coral species.

Contrasting reproductive strategies of two Hawaiian Montipora corals.

Sessile invertebrates often engage in synchronized spawning events to increase likelihood of fertilization. Although coral reefs are well studied, the reproductive behavior of most species and the relative influence of various environmental cues that drive reproduction are not well understood. We conducted a comparative examination of the reproduction of the well-studied Hawaiian coral Montipora capitata and the relatively unknown reproduction of its congener, Montipora flabellata. Both are simultaneous hermaphroditic broadcast spawners that release egg-sperm bundles with external fertilization. Montipora capitata had a distinct reproductive pattern that resulted in coordinated gamete maturation and the synchronized release of thousands of egg-sperm bundles across two spawning pulses tightly coupled to consecutive new moon phases in June and July. Montipora flabellata exhibited a four month reproductive season with spawning that was four-fold less synchronous than M. capitata; its spawning was aperiodic with little linkage to moon phase, a broadly distributed release of only dozens or hundreds of bundles over multiple nights, and a spawning period that ranged from late June through September. The reproductive strategy of M. flabellata might prove detrimental under climate change if increased frequency and severity of bleaching events leave it sparsely populated and local stressors continue to degrade its habitat.

Cryopreservation and Flow Cytometric Analysis of Ovarian Tissue in Murray River Rainbowfish, Melanotaenia fluviatilis.

Freshwater fish populations are declining with many small, Australian fish species at risk of extinction within the next twenty-years. Cryopreservation of reproductive cells and tissues makes it possible to reproduce individuals from a species even after they are extinct in the wild. We describe the successful cryopreservation of ovarian tissue in the Murray River Rainbowfish, Melanotaenia fluviatilis (Order: Atheriniformes). Histology showed that oogonia are 13.70 µm ± 1.75 µm in size, stain positive for germ-line marker Vasa, and represent approximately 2.29 ± 0.81% of cells in the ovary. Flow cytometry was used to analyse ovarian cell suspensions, requiring an optimised tissue digestion protocol. We found that 0.25% trypsin with 1.13 mM EDTA produced cell suspensions with the highest viability (76.28 ± 4.64%) and the highest number of cells recovered per gram of tissue (1.2 × 108 ± 4.4 × 107 cells/g). Subsequent sorting of ovarian cell suspensions by flow cytometry increased oogonial cells in suspension from 2.53 ± 1.31% in an unsorted sample to 5.85 ± 4.01% in a sorted sample (p = 0.0346). Cryopreservation of ovarian tissue showed DMSO-treated samples had higher cell viability post-thaw (63.5 ± 18.2%) which was comparable to fresh samples (82.5 ± 7.1%; p = 0.36). Tissue cryopreserved in 2.0 M DMSO had the highest cell viability overall (76.07 ± 3.89%). This protocol could be applied to bio-banking programs for other species in the Melanotaeniidae, and perhaps species in other families and orders of Australian fish.

Reproductive plasticity of Hawaiian Montipora corals following thermal stress.

Ocean warming, fueled by climate change, is the primary cause of coral bleaching events which are predicted to increase in frequency. Bleaching is generally damaging to coral reproduction, can be exacerbated by concomitant stressors like ultraviolet radiation (UVR), and can have lasting impacts to successful reproduction and potential adaptation. We compared morphological and physiological reproductive metrics (e.g., sperm motility, mitochondrial membrane integrity, egg volume, gametes per bundle, and fertilization and settlement success) of two Hawaiian Montipora corals after consecutive bleaching events in 2014 and 2015. Between the species, sperm motility and mitochondrial membrane potential had the most disparate results. Percent sperm motility in M. capitata, which declined to ~ 40% during bleaching from a normal range of 70-90%, was still less than 50% motile in 2017 and 2018 and had not fully recovered in 2019 (63% motile). By contrast, percent sperm motility in Montipora spp. was 86% and 74% in 2018 and 2019, respectively. This reduction in motility was correlated with damage to mitochondria in M. capitata but not Montipora spp. A major difference between these species is the physiological foundation of their UVR protection, and we hypothesize that UVR protective mechanisms inherent in Montipora spp. mitigate this reproductive damage.

Freezing on the beach: A robust coral sperm cryopreservation design.

Cryopreservation of coral sperm requires reliable, travel-ready, inexpensive hardware. To this end, we developed and tested a robust, second-generation, conduction-based cryovial cooling rack assembled from 3D-printed and commercially available parts. Cooling rates from -10 to -80 °C were found to be repeatable at -22.9 ± 1.9 (rate ± SD) °C/min for 1-mL samples and -35.4 ± 3.3 °C/min for 0.5-mL samples. This represents an improvement on the variability of cooling rates in an older design, which was found to be -31.8 ± 7.1 °C/min for 1-mL samples. Design files and a manual were produced to encourage widespread use and the development of derivative designs.

Assessing coral sperm motility.

The declining reproductive viability of corals threatens their ability to adapt to changing ocean conditions. It is vital that we monitor this viability quantitatively and comparatively. Computer-assisted sperm analysis (CASA) systems offer in-depth analysis used regularly for domestic and wildlife species, but not yet for coral. This study proposes quality control procedures and CASA settings that are effective for coral sperm analysis. To resolve disparities between CASA measurements and evaluations by eye, two negative effects on motility had to be resolved, slide adhesion (procedural) and sperm dilution (biological). We showed that the addition of bovine serum albumin, or caffeine, or both to fresh sperm reduced adhesion in the CASA cassettes, improved motility and motile sperm concentration (P < 0.0001), yet these additions did not affect measurements of total sperm concentration. Diluting coral sperm reduced sperm motility (P = 0.039), especially from heat-stressed corals. We found CASA concentration counts comparable to haemocytometer and flow cytometer measures (P = 0.54). We also found that motile sperm per egg is a useful predictor of fertilisation success, using cryopreserved sperm. Standard measurements of coral reproductive characteristics inform our understanding of the impacts of climate change on reef populations; this study provides a benchmark to begin this comparative work.

Cryopreservation of testicular tissue from Murray River Rainbowfish, Melanotaenia fluviatilis.

Globally, fish populations are in decline from overfishing, habitat destruction and poor water quality. Recent mass fish deaths in Australia's Murray-Darling Basin highlight the need for improved conservation methods for endangered fish species. Cryopreservation of testicular tissue allows storage of early sperm precursor cells for use in generating new individuals via surrogacy. We describe successful isolation and cryopreservation of spermatogonia in an Australian rainbowfish. Testis histology showed rainbowfish spermatogonia are large (> 10 µm) and stain positive for Vasa, an early germ line-specific protein. Using size-based flow cytometry, testis cell suspensions were sorted through "A" (> 9 µm) and "B" gates (2-5 µm); the A gate produced significantly more Vasa-positive cells (45.0% ± 15.2%) than the "B" gate (0.0% ± 0.0%) and an unsorted control (22.9% ± 9.5%, p < 0.0001). The most successful cryoprotectant for "large cell" (> 9 µm) viability (72.6% ± 10.5%) comprised 1.3 M DMSO, 0.1 M trehalose and 1.5% BSA; cell viability was similar to fresh controls (78.8% ± 10.5%) and significantly better than other cryoprotectants (p < 0.0006). We have developed a protocol to cryopreserve rainbowfish testicular tissue and recover an enriched population of viable spermatogonia. This is the first step in developing a biobank of reproductive tissues for this family, and other Australian fish species, in the Australian Frozen Zoo.

Cryopreservation and Laser Nanowarming of Zebrafish Embryos Followed by Hatching and Spawning.

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 development after 3 h (22%), movement after 24 h (11%), hatching after 48 h (9%), and swimming after Day 5 (3%). Finally, from fish that survives till Day 5, two larvae are grown to adulthood and spawned, yielding survival comparable to an unfrozen control. Future efforts will focus on improving the survival to adulthood and developing methods to cryopreserve large numbers of embryos for research, aquaculture, and biodiversity preservation.

New directions in assisted breeding techniques for fish conservation.

Fish populations continue to decline globally, signalling the need for new initiatives to conserve endangered species. Over the past two decades, with advances in our understanding of fish germ line biology, new exsitu management strategies for fish genetics and reproduction have focused on the use of germ line cells. The development of germ cell transplantation techniques for the purposes of propagating fish species, most commonly farmed species such as salmonids, has been gaining interest among conservation scientists as a means of regenerating endangered species. Previously, exsitu conservation methods in fish have been restricted to the cryopreservation of gametes or maintaining captive breeding colonies, both of which face significant challenges that have restricted their widespread implementation. However, advances in germ cell transplantation techniques have made its application in endangered species tangible. Using this approach, it is possible to preserve the genetics of fish species at any stage in their reproductive cycle regardless of sexual maturity or the limitations of brief annual spawning periods. Combining cryopreservation and germ cell transplantation will greatly expand our ability to preserve functional genetic samples from threatened species, to secure fish biodiversity and to produce new individuals to enhance or restore native populations.

First instance of settlement by cryopreserved coral larvae in symbiotic association with dinoflagellates.

Coral reefs are suffering on a global scale due to human impacts, thereby necessitating cryopreservation efforts. The objective of this study was to develop a suitable vitrification and laser warming protocol for larvae of the scleractinian coral Seriatopora caliendrum, which inherit their dinoflagellate algal symbionts vertically. Toxicity experiments were conducted with the cryoprotectants (CPAs) ethylene glycol (EG), propylene glycol (PG), dimethyl sulfoxide (DMSO), glycerol (GLY), and methanol (METH; listed in order from least to most toxic), and larvae were subjected to vitrification and laser warming using 2 M EG + 1 M PG and 2 M EG + 1 M DMSO. Vitrification and laser warming (300 V, 10 ms pulse width, 2 mm beam diameter) using a vitrification solution of 2 M EG + 1 M PG, 40% w/v Ficoll, and 10% v/v gold nanobars (GNB) at a final concentration of 1.2 × 1018 GNB/mL and a characteristic wavelength of 535 nm resulted in larvae with vitality and settlement percentages of 55 and 9%, respectively. This represents the first successful instance of cryopreservation of coral larvae that proceeded to settle upon warming, and suggests that the vitrification and ultra-fast laser warming approach may be applicable to other threatened marine species.

Cryopreservation as a Tool for Reef Restoration: 2019.

Throughout the world coral reefs are being degraded at unprecedented rates. Locally, reefs are damaged by pollution, nutrient overload and sedimentation from out-dated land-use, fishing and mining practices. Globally, increased greenhouse gases are warming and acidifying oceans, making corals more susceptible to stress, bleaching and newly emerging diseases. The coupling of climate change impacts and local anthropogenic stressors has caused a widespread and well-recognized reef crisis. While the establishment and enforcement of marine protected areas and preventing the acceleration of climate change are essential to management of these stressors, the inexorable impacts of climate change will continue to cause declines in genetic diversity and population viability. Gamete cryopreservation has already acted as an effective insurance policy to maintain the genetic diversity of many wildlife species, and has now begun to be explored and applied to coral conservation. Cryopreservation can act to preserve reef biodiversity and genetic diversity. To date, we have had a great deal of success with cryopreserving sperm from ~30 coral species of coral species. Moreover, we are creating the basic science to freeze and thaw coral larvae that can soon be used to help secure and restore reefs. Building on these successes, we have established genetic banks using frozen samples and use those samples to help mitigate threats to the Great Barrier Reef and other areas.

Challenges Related to the Implementation of an EMS-Administered, Large Vessel Occlusion Stroke Score.

INTRODUCTION: There is considerable interest in triaging victims of large vessel occlusion (LVO) strokes to comprehensive stroke centers. Timely access to interventional therapy has been linked to improved stroke outcomes. Accurate triage depends upon the use of a validated screening tool in addition to several emergency medical system (EMS)-specific factors. This study examines the integration of a modified Rapid Arterial oCcclusion Evaluation (mRACE) score into an existing stroke treatment protocol. METHODS: We performed a retrospective review of EMS and hospital charts of patients transported to a single comprehensive stroke center. Adult patients with an EMS provider impression of "stroke/TIA," "CVA," or "neurological problem" were included for analysis. EMS protocols mandated the use of the Cincinnati Prehospital Stroke Score (CPSS). The novel protocol authorized the use of the mRACE score to identify candidates for triage directly to the comprehensive stroke center. We calculated specificity and sensitivity for various stroke screens (CPSS and a mRACE exam) for the detection of LVO stroke. The score's metrics were evaluated as a surrogate marker for a successful EMS triage protocol. RESULTS: We included 312 prehospital charts in the final analysis. The CPSS score exhibited reliable sensitivity at 85%. Specificity of CPSS for an LVO was calculated at 73%. For an mRACE score of five or greater, the sensitivity was 25%. Specificity for mRACE was calculated at 75%. The positive predictive value of the mRACE score for an LVO was estimated at 12.50%. CONCLUSION: In this retrospective study of patients triaged to a single comprehensive stroke center, the addition of an LVO-specific screening tool failed to improve accuracy. Reliable triage of LVO strokes in the prehospital setting is a challenging task. In addition to statistical performance of a particular stroke score, a successful EMS protocol should consider system-based factors such as provider education and training. Study limitations can inform future iterations of LVO triage protocols.