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In recent decades, mortality caused by ostreid herpesvirus (OsHV-1) and a variant (OsHV-1 μvar) has severely impacted the production of Pacific oyster, Crassostrea gigas, chronologically in France, New Zealand (NZ) and Australia (Jenkins et al., 2013; Keeling et al., 2014; Segarra et al., 2010). This study was conceived based on observations made in several commercial hatcheries and research institutes in these countries where vertical transmission of OsHV-1 μvar has been absent or extremely rare. We have collated data from the production of Pacific oysters across these countries to assess the risk associated with breeding oysters from broodstock grown in OsHV-1 μvar-infected waters. The aim is to provide a reference point for regulatory authorities to use when developing or updating biosecurity management policy associated with the hatchery production of Pacific oysters.

The chelating agent ethylenediaminetetraacetic acid (EDTA) is used throughout the world to improve the yield of early stage D-larvae during bivalve hatchery production. Adding EDTA (12 μM) to seawater significantly increases the survival of Greenshell™ mussel (Perna canaliculus) larvae during their first 48 h of development. However, whether there are benefits of continuing to use EDTA beyond this first stage of larval development remain unknown and were tested in this study. After rearing for 48 h in the presence of EDTA, P. canaliculus larvae were experimentally raised to 22-day post-fertilisation in seawater with and without 12 μM EDTA. The survival, shell length growth, algal ingestion rate, swimming activity and potential toxic metal accumulation by the larvae were compared over this period. There were minimal benefits from continuing addition of EDTA. However, significant changes in metal concentrations within the larvae were observed. Zinc, cadmium and mercury were detected at significantly lower concentrations in 22-day-old larvae reared with EDTA versus those without EDTA. Collectively, the results indicate that the use of EDTA is critical only during the first 48 h of larval development, during which time larval shell formation is initiated and appears highly vulnerable to interference by heavy metal ions.

Farmed adult Greenshell™ mussels were exposed to a range of conditions immediately after harvest to evaluate the effects on emersion survival and the implications for live transport. Two experiments were performed, coinciding with the mussel harvest season during summer and winter 2017. In the summer experiment, a wide range of conditions were tested to identify alternatives to be evaluated in more detail during the winter experiment. The postharvest experimental conditions selected included transient re-immersion at different temperatures, cold and heat-shock, tidal simulation, and packing with ice. These treatments aimed to either reduce postharvest stress and/or give the mussels a potentially beneficial physiological and behavioural advantage to help them cope with emersion. The mussels' responses in terms of gaping and survival differed between summer and winter, and among and within treatments. Sub-ambient temperature seawater exposure after harvest tended to improve the survival time of the mussels in both seasons, the effect being dependent on both temperature and duration of the exposure. Some of the differences observed could be explained by the conditions (in the wild and in the laboratory) to which the mussels were exposed before emersion and the different capability that each mussel has to cope with stress. However, the exact mechanisms driving these changes are not fully understood and require further investigation. This study, nevertheless, indicates that emersion survival (i.e. live mussel shelf-life) could be extended by re-immersion after harvest combined with a sub-lethal cold-shock followed by immersed recovery before transportation. If the commercial shelf-life of live product can be significantly extended, the implications for food quality and safety will also need to be evaluated. A similar approach could be applied in other shellfish such as other species of mussels as well as oysters or clams to improve shelf-life, considering their physiological capabilities and behaviour and how those are affected by environmental conditions in different seasons or regions.

Cryopreservation of larvae of Greenshell™ mussel Perna canaliculus, the most cultivated species in New Zealand, can provide flexibility for selective breeding programmes and enhance its global production. In this study, we set out to develop a reliable protocol for freezing D-stage larvae of Greenshell™ mussels that ensured long-term survival for successful rearing of thawed larvae in the hatchery. The effects of different combinations of cryoprotecting agents (CPA), varying CPA equilibration times, larval concentrations per straw as well as different larval development stages (48 h vs 72 h old) were evaluated by assessing the behavioural response (swimming activity, algal consumption), shell size and survival of larvae, up to 4 days post-thawing. The protocol yielding the best larval performances was a combination of the following CPA (final concentrations): 14% ethylene-glycol (EG) + 0.6 M trehalose (TRE) + 1% polyvinyl-pyrrolidone (PVP), prepared with Milli-Q water. Stocking densities ranging from 50,000 to 150,000 larvae per straw (0.25 mL) and a 20 min equilibration time gave the best results, while no significant differences in fitness were found between larvae cryopreserved at 48 h nor 72 h-old. Using the improved cryopreservation protocol, over 50% of previously cryopreserved D-larvae were able to survive after 4 days of rearing, compared with 65% in the unfrozen control. More importantly, about one third of thawed larvae were able to swim and feed, and to potentially develop further. These findings contribute to enhance the selective breeding programmes for this species. Xunta de Galicia Ministry for Business, Innovation and Employment (New Zealand) Shellfish Aquaculture Ref. CAWX1801

International audience; Pacific oyster Crassostrea gigas is one major species for aquaculture, and the development of breeding programs and the need for preservation of wild stock genetic resources prompted the need for larvae cryopreservation. The objective of the present study was to choose the most reliable protocol from several existing publications, to test its biological and practical limitations, and to adapt it to hatchery conditions. The selected protocol was characterized by a very slow freezing rate without seeding, and by the use of ethylene glycol and sucrose as cryoprotectant. The best survivals after thawing and rearing up to 48 h post fertilization (hpf) were obtained with larvae that were frozen at late trochophore (20 hpf) and early-D (24 hpf) stages. Increasing the larvae concentration in the straws and using high throughput straw filling and freezing devices did not alter the cryopreservation outcome. The whole procedure was applied to cryopreservation in a commercial hatchery (Satmar, France), and the thawed larvae yielded 9.4 ± 4.5% survivals at 12 days post fertilization. The overall success was dampened by some variability in the larvae survival that is likely due to the physiological status of the larvae. In all, the proposed procedure is robust and reliable and can be used for cryobanking of oyster genetic resources.

The sea urchin, Echinometra lucunter, is widely used in embryo-larval tests for ecotoxicological studies in Brazil and other countries. For each test, sea urchins are collected from the wild and this can cause impact on wild populations and it is limited by the weather and season which in turn limits the ability to carry out the tests. Cryopreservation is a method of live biological material storage at low temperature and can be used for long periods with little decline in viability, reducing the number of animals taken from the wild and enabling testing to be carried out on demand, irrespective of spawning season or location. In this study, 15 combinations of cryoprotective agents (CPAs) were evaluated on spermatozoa, subjected to a rapid cooling curve followed by immersion in liquid nitrogen. Twenty-four CPA combinations were evaluated on eggs subjected to a more gradual cooling curve in nitrogen vapor down to −35 °C and then plunging in liquid nitrogen. Fertilization tests using cryopreserved spermatozoa gave high pluteus larvae yields (≈80%) when concentrations of 10.5% or 13.65% ME2SO or 13.65% ME2SO+15.75% sucrose were used. The higher concentrations of ME2SO plus sucrose were more effective at maintaining the fertilization capacity of spermatozoa post-thawing. Egg cryopreservation was not successful with 0% fertilization observed post-thawing. The results suggest that it is feasible to implement spermatozoa cryopreservation as technological innovation to create a sperm bank for E. lucunter, which can be used in ecotoxicological tests, bringing benefits for researches and contributing to the conservation of the species.

A comprehensive study, incorporating histology, light microscopy, scanning electron microscopy, immunochemistry and confocal microscopy, was performed to investigate embryogenesis and larval development of the New Zealand Greenshell™ mussel, Perna canaliculus. Detailed observations with this multi-technique approach revealed a gastrula stage at 18 hours post-fertilization, with the appearance of a blastopore, apical sense organ and enclosing vegetal pole. Early D-stage larvae showed limited alimentary organogenesis and clear initiation of a developing nervous system. Shell morphology of D-larvae was characterized by a flat, hinged, pitted–punctate prodissoconch I shell, followed closely by commarginal growth lines within the prodissoconch II shell. Early umbo larvae had a protruding functioning velum, and well-developed posterior adductor and velar retractor muscles. Significant progression in neuronal development occurred just before the umbo stage with noticeable paired cerebral, pedal and visce...

Cryopreservation of genetic material from farmed aquatic species is a valuable technique to advance selective breeding programs for stock improvement. In this study, effects of cryopreservation on development of trochophore and D-stage larvae of Greenshell™ mussel (Perna canaliculus) were evaluated through histology, light microscopy, scanning electron microscopy, and confocal microscopy. Larvae of both life stages were motile immediately post-thawing, but survival declined rapidly from 4 days post-fertilisation (dpf). At 18 dpf, ~23% of non-cryopreserved control larvae had progressed to the pediveliger stage, while

The flat oyster, Ostrea chilensis, native to New Zealand (NZ) and Chile is considered an important ecological, cultural and fisheries resource. Currently, commercial landings of this species in NZ are restricted due to low population numbers caused by ongoing mortalities resulting from the presence of the haplosporidian parasite, Bonamia exitiosa. More recently, the arrival of B. ostreae in NZ led to major mortalities in farmed stocks. To understand how diseases caused by Bonamia spp. affect this oyster species, a more complete understanding of its biology, physiology and immune system is needed. The present study characterized, for the first time, hemocytes of adult O. chilensis, from the Foveaux Strait, NZ, using flow cytometry (FCM) and histology. Based on the internal complexity of the hemocytes, two main circulating hemocyte populations were identified: granulocytes and hyalinocytes (accounting for ~30% and ~70% of the total circulating hemocyte population, respectively). These were further divided into two sub-populations of each cell type using FCM. A third sub-population of granulocytes was identified using histology. Using FCM, functional and metabolic characteristics were investigated for the two main hemocyte types. Granulocytes showed higher phagocytic capabilities, lysosomal content, neutral lipid content and reactive oxygen species production compared to hyalinocytes, indicating their important role in cellular immune defence in this species. Methods of hemocyte sampling and storage were also investigated and flow cytometric protocols were detailed and verified to allow effective future investigations into the health status of this important species.

Sea urchins are excellent model organisms useful for several lines of biotechnological research. Sea urchins are typically collected from the sea and kept in research facilities all over the world for such purposes. Cryopreservation can be a very powerful tool to enhance the use of sea urchins as a model species for research. The development of cryopreservation protocols for different sea urchin gametes, embryos, and larvae allows year round access to high quality material outside the natural reproductive season. It also reduces the uncertainty and variability that may be caused by changing oceanic, meteorological, and environmental conditions. Access to cryopreserved gametes and embryos will allow using these model organisms in laboratories all around the world, regardless of their facilities or their proximity to a natural population of sea urchins. Cryopreservation is a very useful tool for aquaculture production, fisheries conservation, and wild stock enhancement allowing spat supply all year round without the need of conditioning broodstock for out of season reproduction-which is expensive, time consuming, and often unfruitful. It will also provide flexibility for selective breeding programs by allowing crossings of individuals with different reproductive seasons. Although cryopreservation protocols have been successfully developed for many valuable fisheries species such as sturgeons, salmonid fishes, and other marine invertebrates (e.g., oysters), only a small amount of research has been carried out regarding sea urchin cryopreservation. In this chapter, we outline protocols for the cryopreservation of sea urchin cells.

The production of valuable metabolites from microalgae represents a potentially sustainable source of a range of products that can be difficult to synthesise directly. Microalgae respond to the dynamic and often subtly shifting growth environment in a complex way. The optimal conditions for growth can be quite different to those needed for optimal product generation, depending on the nature of the biosynthesis of that product. This is especially so for secondary metabolites. A combination of a multi-vessel photobioreactor array, where certain growth conditions can be monitored and controlled precisely together with an experimental design matrix has been used to determine the optimal combination of temperature, irradiance and pH for a group of xanthophylls including fucoxanthin in the algae Tisochrysis lutea in continuous culture. Continuous culture as a mode is more suited for industrial production than batch mode in which the media constituents and algal population change dramatically over time. ...

The purpose of this study was to determine the effects of adding ethylenediaminetetraacetic acid (EDTA) into the fertilization (50 μM) and embryo incubation (25 μM) seawater of Perna canaliculus. With EDTA, 78.6% of oocytes developed to D-larvae in comparison to 1.2% without EDTA (P Statement of relevance EDTA is routinely used in many shellfish hatcheries to reduce possible metal toxicity. However, the underlying biological mechanisms associated with EDTA use are poorly understood. We investigate the biological impacts of EDTA on P. canaliculus embryos and show for the first time that without EDTA embryos show oxidative stress and ultrastructure abnormalities. This study adds significantly to our understanding of the beneficial role(s) of EDTA in aquaculture.

Sea urchins are excellent model organisms useful for several lines of biotechnological research. Sea urchins are typically collected from the sea and kept in research facilities all over the world for such purposes. Cryopreservation can be a very powerful tool to enhance the use of sea urchins as a model species for research. The development of cryopreservation protocols for different sea urchin gametes, embryos, and larvae allows year round access to high quality material outside the natural reproductive season. It also reduces the uncertainty and variability that may be caused by changing oceanic, meteorological, and environmental conditions. Access to cryopreserved gametes and embryos will allow using these model organisms in laboratories all around the world, regardless of their facilities or their proximity to a natural population of sea urchins. Cryopreservation is a very useful tool for aquaculture production, fisheries conservation, and wild stock enhancement allowing spat supply all year round without the need of conditioning broodstock for out of season reproduction-which is expensive, time consuming, and often unfruitful. It will also provide flexibility for selective breeding programs by allowing crossings of individuals with different reproductive seasons. Although cryopreservation protocols have been successfully developed for many valuable fisheries species such as sturgeons, salmonid fishes, and other marine invertebrates (e.g., oysters), only a small amount of research has been carried out regarding sea urchin cryopreservation. In this chapter, we outline protocols for the cryopreservation of sea urchin cells.

The therapeutic benefits of Greenshell™ mussel (GSM; Perna canaliculus) preparations have been studied using in vitro test systems, animal models, and human clinical trials focusing mainly on anti-inflammatory and anti-arthritic effects. Activity is thought to be linked to key active ingredients that include omega-3 polyunsaturated fatty acids, a variety of carotenoids and other bioactive compounds. In this paper, we review the studies that have been undertaken in dogs, cats, and horses, and outline new research directions in shellfish breeding and high-value nutrition research programmes targeted at enhancing the efficacy of mussel and algal extracts. The addition of GSM to animal diets has alleviated feline degenerative joint disease and arthritis symptoms, and chronic orthopaedic pain in dogs. In horses, GSM extracts decreased the severity of lameness and joint pain and provided improved joint flexion in limbs with lameness attributed to osteoarthritis. Future research in this area should focus on elucidating the key active ingredients in order to link concentrations of these active ingredients with their pharmacokinetics and therapeutic effects. This would enable consistent and improved efficacy from GSM-based products for the purpose of improved animal health.

Oxidative damage to proteins and lipids, the enzymatic and nonenzymatic antioxidants' response, and the fertilization and development capability of Perna canaliculus oocytes were investigated at critical treatment steps in a previously published controlled-rate cryopreservation protocol. The cryoprotectant (CPA) from this protocol comprises 10% ethylene glycol (v:v) and 0.2 M trehalose (wt/vol) final concentration. Critical treatment steps included (1) seawater control, (2) CPA addition, (3) CPA addition followed by cooling to −6 °C, (4) CPA addition and cooling to −10 °C, and (5) CPA addition and cooling to −35 °C and immersion in liquid nitrogen (LN). The percentage of fertilized oocytes was 53.8 ± 13.3% in the seawater control but was reduced to 26.0 ± 15.6% after −35 °C + LN treatment, whereas development to D-larvae was 21.0 ± 6.4% in the seawater control reduced to 4.8 ± 2.9% after cooling to −6 °C, and was zero at all the subsequent cooling steps. All oxidative damage biomarkers, protein carbonyls (PCs) and lipid hydroperoxides (LPs), and antioxidants, superoxide dismutase (SOD), catalase, glutathione peroxidase, percent reduced glutathione (%GSH), and total glutathione (defined as glutathione; reduced [GSH] plus glutathione disulphide; derived from two molecules of GSH [GSSG]) were measured over all treatments on unfertilized oocytes over a post-treatment recovery period of 0 to 240 minutes in seawater. An ANOVA showed that both treatment and post-treatment periods had significant effects on the concentrations of all biomarkers (P

Cryopreservation experiments were conducted on D-stage larvae of the Pacific oyster (Crassostrea gigas) to investigate the effects of two cryoprotectant solutions and three cooling rates on larval development from 1 to 22 days post-fertilisation. Cryoprotectant solutions were made up to final concentrations (after 1:1 dilution with larvae) of 10% ethylene glycol, 1% polyvinylpyrrolidone and either 0.2 or 0.4 M trehalose. Three cooling rates (0.5, 1 and 2 °C min−1 between −10 and −35 °C post-holding) were tested in an orthogonal design with the two cryoprotectants. Results indicate that control larvae out-performed all cryopreservation treatments for survival, feeding consumption and shell length parameters. However, larvae exposed to 0.4 M trehalose did considerably better than those exposed to 0.2 M trehalose, regardless of cooling rate conditions. Scanning electron and light microscopy observations were used to assess larval morphology and organogenesis, indicating that treatments with surviving larvae ...

Female broodstock of Pacific oyster (Crassostrea gigas) were conditioned using different diets to determine the effect of diet on oocyte cryopreservation success. Oocyte biochemical composition, fatty acid profile, and fertilization success before and after cryopreservation were determined. Broodstock were conditioned in 2 stages: a cold preconditioning stage during which animals were either starved or fed live microalgae, and a warm conditioning stage during which animals were again either starved or fed live microalgae, AlgaMac-2000, or a mixture of corn and rice flour. Overall, oysters that were fed microalgae during cold preconditioning produced oocytes with significantly more lipid than oysters that were starved. Similarly, those that were fed during warm conditioning produced oocytes with more lipid than starved oysters, regardless of diet. There was no effect of conditioning regime on the level of polyunsaturated fatty acids (PUFAs) in the polar lipid fraction. However, oysters that were starved or fed AlgaMac-2000 during warm conditioning had higher PUFA levels than oysters fed microalgae or flour. Broodstock conditioning regime had no effect on the fertilization success of oocytes prior to cryopreservation, but those that were fed during cold preconditioning produced oocytes that had significantly higher fertilization success after thawing.

The Greenshell™ mussel (Perna canaliculus) is the main shellfish species farmed in New Zealand. The aim of this study was to evaluate the effects of cryoprotectant concentration, loading and unloading strategy as well as freezing and thawing method in order to develop a protocol for cryopreservation of trochophore larvae (16–20 h old). Toxicity tests showed that levels of 10–15% ethylene glycol (EG) were not toxic to larvae and could be loaded and unloaded in a single step. Through cryopreservation experiments, we designed a cryopreservation protocol that enabled 40–60% of trochophores to develop to D-larvae when normalized to controls. The protocol involved: holding at 0 °C for 5 min, then cooling at 1 °C min−1 to −10 °C, holding for a further 5 min, then cooling at 0.5 °C min−1 to −35 °C followed by a 5 min hold and then plunging into liquid nitrogen. A final larval rearing experiment of 18 days was conducted to assess the ability of these frozen larvae to develop further. Results showed that only 2.8% of the frozen trochophores were able to develop to competent pediveligers.

The effect of cryopreservation on aspects of sperm function was investigated for sperm of the Greenshell™ mussel ( Perna canaliculus ). Fertility, DNA integrity, viability and motility were measured before and after cryopreservation. Cryopreservation had a significant effect on fertility with many cryopreserved samples failing to achieve 50% oocyte fertilisation at the highest sperm concentration measured. Sperm DNA integrity, measured using the Sperm Chromatin Structure Assay, showed a small but significant increase in DNA damage as a result of cryopreservation. Sperm viability, measured using propidium iodide/SYBR-14 dual staining, decreased significantly following cryopreservation (69.9 ± 3.5% for fresh sperm versus 25.3 ± 2.9% for cryopreserved sperm). Aspects of motility were similarly affected. The number of sperm tracked, curvilinear velocity (VCL), average path velocity (VAP), variation of straight line velocity (PFT), mean angular displacement (MAD), beat cross frequency (BCF), amplitude of lateral head displacement (ALH) and dance mean (DMN) all decreased following cryopreservation. Correlation analyses between the various aspects of sperm function were carried out. However, no single aspect of sperm function was significantly correlated with sperm fertility following cryopreservation. In addition, the technique for fertility measurement is relatively quicker and less dependent on high tech equipment than the other parameters studied. Fertility therefore remains the most important and relevant end point to measure. Further optimisation of the cryopreservation protocol for Greenshell™ mussel sperm is needed to improve post-thaw fertility.

A series of eight experiments was conducted to determine the most suitable cryoprotectants (CPAs), dilution rates and methods of CPA addition as well as freezing method for the cryopreservation of Greenshell™ mussel ( Perna canaliculus ) sperm. The experiments utilized a fertilization assay to determine the treatments that gave the highest post-thaw fertility. The assays revealed that dimethyl sulphoxide (DMSO) gave the best results of the seven CPAs tested. A concentration of DMSO of approximately 12% [volume: volume] in Milli-Q water provided the best results. A dilution rate of 1:1 (sperm: CPA solution) was superior to 1:3 indicating that more concentrated sperm had higher post-thaw fertility. The time that sperm were exposed to the CPA solution prior to freezing had no effect over the range tested which gives some flexibility in the amount of time sperm can be exposed to CPA prior to freezing. The addition of the CPA in either one step or in 4 equal volume steps over two min had no significant effect on fertility and the two different freezing methods (programmable freezer or rack floating above liquid nitrogen) produced similar results. The post-thaw fertility of mussel sperm using the final protocol developed (12% DMSO plus 0.2 M trehalose, sperm diluted 1:1, loaded into 0.5 mL straws and cooled on a rack 3 cm above liquid nitrogen followed by immersion in liquid nitrogen) was significantly reduced in comparison to fresh sperm. On average, approximately 363 x more sperm were required to achieve an equivalent level of fertilization as that of fresh sperm. The method is being used in mussel selective breeding, however, there is considerable between-animal variability. Further refinement to the method may help to improve post-thaw fertility.

A culture collection of freshwater planktonic and benthic cyanobacteria collected from sites across New Zealand has been established at the Cawthron Institute, Nelson, New Zealand. Limited resources led to uncertainty regarding the long‐term maintenance of this collection. The present study demonstrates cryopreservation to be a viable method for long‐term storage of cyanobacteria. Seventeen of 20 strains evaluated were successfully cryopreserved using the permeating cryopreservation agent dimethyl sulfoxide (Me2SO), at a final concentration of 15% (v/v). Cyanotoxin analysis was undertaken on selected strains known to produce microcystins, nodularin, anatoxin‐a, and saxitoxins. All strains retained their ability to produce these toxins following cryopreservation.

In order for cryopreservation to become a practical tool for aquaculture, optimized protocols must be developed for each species and cell type. Knowledge of a cell's osmotic tolerance and membrane permeability characteristics can assist in optimized protocol development. In this study, these characteristics were determined for Pacific oyster oocytes and modified methods for loading and unloading ethylene glycol (EG) were tested. Oocytes were found to behave as ideal osmometers and their osmotically inactive fraction (V(b)) was calculated to be 0.48. Oocytes exposed to NaCl solutions of 0.6 to 2.3 Osm fertilized at rates equivalent to oocytes left in seawater. This corresponds to volume changes of +27.3 and -38.1+/-1.2%. The permeability of the oocytes to water (L(p)) was determined to be 3.8+/-0.4 x 10(-2), 5.7+/-0.8 x 10(-2), and 13.2+/-1.3 x 10(-2) microm min(-1)atm(-1), when measured at temperatures of 5, 10 and 20 degrees C. The respective EG permeability values (P(s)) were 9.5+/-0.1 x 10(-5), 14.6+/-1.2 x 10(-5), and 41.7+/-2.4 x 10(-5) cm min(-1). The activation energies for L(p) and P(s) were determined to be 14.5 and 17.5 kcal mol(-1), respectively. Different models for EG loading and unloading from oocytes were developed and tested. Post-thaw fertilization did not differ significantly between a published step addition method and single step addition at 20 degrees C. This represents a considerable reduction in handling. The results of this study demonstrate that the cryobiological characteristics of a given cell type should be taken into account when developing cryopreservation methods.

This review is focused on the applications of genome cryobanking of aquatic species including freshwater and marine fish, as well as invertebrates. It also reviews the latest advances in cryobanking of model species, widely used by the scientific community worldwide, because of their applications in several fields. The state of the art of cryopreservation of different cellular types (sperm, oocytes, embryos, somatic cells and primordial germ cells or early spermatogonia) is discussed focusing on the advantages and disadvantages of each procedure according to different applications. A special review on the need of standardization of protocols has also been carried out. In summary, this comprehensive review provides information on the practical details of applications of genome cryobanking in a range of aquatic species worldwide, including the cryobanks established in Europe, USA, Brazil, Australia and New Zealand, the species and type of cells that constitute these banks and the utilization of the samples preserved. Statement of relevance This review compiles the last advances on germplasm cryobanking of freshwater and marine fish species and invertebrates, with high value for commercial aquaculture or conservation. It is reviewed the most promising cryopreservation protocols for different cell types, embryos and larvae that could be applied in programs for genetic improvement, broodstock management or conservation of stocks to guarantee culture production., SI

Microalgae and seaweeds have a long history and increasingly important applications as both food ingredients and animal feed. The vast majority of algal species have yet to be evaluated for these applications. However, due to their extensive diversity, it is likely that they will lead to the discovery of many new algal products and processes in the future. This chapter covers algae as food, feed, nutraceuticals, functional food and food ingredients as well as production systems for food from algae.

Cryopreservation of P. canaliculus oocytes has not yet been achieved.The present study is to investigate whether the incorporation of: DMSO (0.09%), α-tocopherol (0.1 mM) plus taurine (1 mM) and ethylenediaminetetraacetic acid (EDTA; 0.1 mM), is beneficial during cryopreservation.These three additives were incorporated to both the cryoprotectant (CPA) and recovery media, and evaluated in terms of development and oxidative stress at three key stages of cryopreservation: 1) cryoprotectant addition [10% v/v ethylene glycol plus 0.2M trehalose; final concentration], 2) cooling to -6 degrees C, and 3) cooling to -35 degrees C and liquid nitrogen immersion.Over all treatments (including controls) progressive cryopreservation steps resulted in a decrease in fertilization and development to D-larvae, an increase in macromolecular oxidative damage markers (protein carbonyls, lipid hydroperoxides and oxidized DNA), and a decrease in enzymatic (superoxide dismutase, catalase, glutathione S-transferase and glutathione reductase) and non-enzymatic antioxidants.Whilst results varied, the major effects of the additives were the improved percentage fertilization and a decrease in macromolecular damage.

Investigation into fish oocyte membrane permeability is essential for developing successful protocols for their cryopreservation. The aim of the present work was to study the permeability of the zebrafish (Danio rerio) oocyte membrane to water and cryoprotectants before cryopreservation protocol design. The study was conducted on stage III and stage V zebrafish oocytes. Volumetric changes of stage III oocytes in different concentrations of sucrose were measured after 20 min exposure at 22 degrees C and the osmotically inactive volume of the oocytes (Vb) was determined using the Boyle-van't Hoff relationship. Volumetric changes of oocytes during exposure to different cryoprotectant solutions were also measured. Oocytes were exposed to 2 M dimethyl sulphoxide (DMSO), propylene glycol (PG), and methanol for 40 min at 22 degrees C. Stage III oocytes were also exposed to 2 M DMSO at 0 degrees C. Oocyte images were captured on an Olympus BX51 cryomicroscope using Linkham software for image recording. Scion Image was used for image analysis and diameter measurement. The experimental data were fitted to a two-parameter model using Berkeley Madonna 8.0.1 software. Hydraulic conductivity (L(p)) and solute (cryoprotectant) permeability (Ps) were estimated using the model. The osmotically inactive volume of stage III zebrafish oocytes was found to be 69.5%. The mean values+/-SE of Lp were found to be 0.169+/-0.02 and 0.196+/-0.01 microm/min/atm in the presence of DMSO and PG, respectively, at 22 degrees C, assuming an internal isosmotic value for the oocyte of 272 mOsm. The Ps values were 0.000948+/-0.00015 and 0.000933+/-0.00005 cm/min for DMSO and PG, respectively. It was also shown that the membrane permeability of stage III oocytes decreased significantly with temperature. No significant changes in cell volume during methanol treatment were observed. Fish oocyte membrane permeability parameters are reported here for the first time. The Lp and Ps values obtained for stage III zebrafish oocytes are generally lower than those obtained from successfully cryopreserved mammalian oocytes and higher than those obtained with fish embryos and sea urchin eggs. It was not possible to estimate membrane permeability parameters for stage V oocytes using the methods employed in this study because stage V oocytes experienced the separation of outer oolemma membrane from inner vitelline during exposure to cryoprotectants.

This paper describes a simple method for cryopreserving sperm of the Pacific oyster ( Crassostrea gigas Thunberg) in quantities suitable for commercial spat production. Experiments to refine the cryoprotectant mixtures demonstrated the key role of trehalose. Trehalose alone (at 0.45 M final concentration) was an effective cryoprotectant. The addition of 2.5–15% dimethyl sulphoxide (DMSO) in combination with 0.45 M trehalose gave only modest improvement in fertility over trehalose alone ( p =0.056). There was no significant difference in fertility among DMSO concentrations ( p =0.611). Seawater (SW) without cryoprotectant gave very poor results, but yielded some fertilization at very high sperm concentrations (7±1% at 10 7 sperm mL −1 , 21±2% at 3.2×10 7 sperm mL −1 , mean±S.E., n =3). The fertility of unfrozen sperm was 30- to 100-fold higher than that of sperm cryopreserved with DMSO and/or trehalose. For sperm cryopreserved in 4.5-mL cryovials, two simplified freezing methods gave fertilization rates equivalent to sperm cryopreserved by controlled rate freezing ( p =0.386). These methods involved securing the cryovials to aluminium canes and then either placing them into a bath of methanol chilled with dry ice, or holding them on a floating rack 3 cm above liquid nitrogen. A third technique of plunging the cryovials directly into liquid nitrogen gave reduced and variable fertility relative to the methanol/dry ice bath method ( p =0.032). The commercial applicability of the protocols was demonstrated on a batch of 30 million eggs. Fertilization with cryopreserved sperm yielded 81% fertilization, and larval rearing by normal commercial practises yielded 3.7 million settled spat, which was comparable to the 2.5 million spat from a parallel batch fertilized with unfrozen sperm.

Summary A method was developed to evaluate the mitochondrial function and membrane integrity of marine invertebrate sperm using flow cytometry in combination with rhodamine 123 (R123) and propidium iodide (PI) staining. The method was evaluated with sperm of sea urchins (Evechinus chloroticus), mussels (Perna canaliculus) and abalone (Haliotis iris). Using R123 and PI simultaneously, it was possible to distinguish live sperm with functioning mitochondria and intact membranes from dying or dead sperm. The mitochondrial inhibitor rotenone was used to confirm that R123 was only accumulated by sperm with functioning mitochondria. The stain combination was further validated using known ratios of fresh to killed sperm. Among individual males evaluated, the percentage of sperm identified as live varied considerably, irrespective of the species studied. Among samples from the same male, however, the percentage of sperm identified as live was similar. The method developed has application for assessing the quality ...

Development of effective cryopreservation protocols relies on knowledge of the fundamental cryobiological characteristics for a particular cell type. These characteristics include osmotic behaviour, membrane permeability characteristics, and osmotic tolerance limits. Here, we report on measures of these characteristics for unfertilized and fertilised eggs of the sea urchin (Evechinus chloroticus). In NaCl solutions of varying osmolalities, sea urchin eggs behaved as ideal linear osmometers. The osmotically inactive volume (vb) was similar for unfertilized and fertilised eggs, 0.367+/-0.008 (mean+/-SE) and 0.303+/-0.007, respectively. Estimates of water solubility (Lp) and solute permeability (Ps) and their respective activation energies (Ea) for unfertilized and fertilised eggs were determined following exposure to cryoprotectant (CPA) solutions at different temperatures. Irrespective of treatment, fertilised eggs had higher values of Lp and Ps. The presence of a CPA decreased Lp. Among CPAs, solute permeability was highest for propylene glycol followed by dimethyl sulphoxide and then ethylene glycol. Measures of osmotic tolerance limits of the eggs revealed unfertilized eggs were able to tolerate volumetric changes of -20% and +30% of their equilibrium volume; fertilised eggs were able to tolerate changes +/-30%. Using membrane permeability data and osmotic tolerance limits, we established effective methods for loading and unloading CPAs from the eggs. The results of this study establish cryobiological characteristics for E. chloroticus eggs of use for developing an effective cryopreservation protocol. The approach we outline can be readily adapted for determining cryobiological characteristics of other species and cell types, as an aid to successful cryopreservation.

Cryopreservation is a valuable technique for aquaculture as it enables a library or bank of genetically valuable animals to be maintained in a cost-effective manner. Here, we describe a method to cryopreserve the sperm of the Greenshell™ mussel (Perna canaliculus) and how to use the sperm post-thawing to maximize larval production from thawed sperm in selective breeding.

Cryopreservation is a valuable technique for aquaculture as it enables a library or bank of genetically valuable animals to be maintained in a cost-effective manner. Here, we describe a method to cryopreserve the sperm of the Greenshell™ mussel (Perna canaliculus) and how to use the sperm post-thawing to maximize larval production from thawed sperm in selective breeding.

Oysters and mussels are among the most farmed species in aquaculture industry around the world. The aim of this study was to test the toxicity of cryoprotective agents to trochophore larvae from two different species of bivalves and develop an improved cryopreservation protocol to ensure greater efficiency in the development of cryopreserved trochophores (14 h old oyster larvae and 20 h old mussel larvae) to normal D-larvae for future developments of hatchery spat production. The cryopreservation protocol producing the best results for oyster trochophores (60.0 ± 6.7% normal D-larvae) was obtained by holding at 0 °C for 5 min then cooling at 1 °C min(-1) to -10 °C and holding for 5 min before cooling at 0.5 °C to -35 °C, holding 5 min and then plunging into liquid nitrogen (LN), using 10% ethylene glycol. For mussel experiments, no significant differences were found when cooling at 0.5 °C min(-1) or at 1 °C min(-1) for CPA combinations with 10% ethylene glycol and at 0.5 °C min(-1). Using these combinations, around half of trochophores were able to develop to normal D-larvae post-thawing (48.9 ± 7.6% normal D-larvae)., Sí

Cryopreservation is a powerful tool for selective breeding in aquaculture as it enables genetic material from selected stock to be stored and crossed at will. The aim of this study was to develop a method for cryopreserving oocytes of the Greenshelltrade mark mussel (Perna canaliculus), New Zealand's main aquaculture species. The ability of oocytes to be fertilized post-thawing was used as the criterion for success in initial experiments and then subsequently, the ability of frozen oocytes to develop further to D-stage larvae was assessed. Ethylene glycol, propylene glycol, dimethyl sulphoxide and glycerol were evaluated at a range of concentrations with and without the addition of 0.2M trehalose using post-thaw fertilization as the endpoint. Ethylene glycol was most effective, particularly when used in combination with trehalose. A more detailed investigation revealed that ethylene glycol at 9% or 10% in the presence of 0.2-0.4M trehalose afforded the best protection. In experiments varying sperm to egg ratio and egg density in post-thaw fertilization procedures, D-larval yield averaged less than 1%. Following these results, a detailed experiment was conducted to determine the damaging steps in the cryopreservation process. Fertilization losses occurred at each step whereas D-larval yield approximately halved following CPA addition and was almost zero following cooling to -10 degrees C. Cryomicroscopy studies and fertilization results suggest that the inability of oocytes to develop to D-larvae stage after cooling to -10 degrees C and beyond are most likely related to some form of chilling injury rather than extracellular ice triggering intracellular ice formation. Further research is needed to determine the causes of this injury and to reduce CPA toxicity and/or osmotic effects.

The effect of IIF in Pacific oyster oocytes was studied using cryo and transmission electron microscopy (TEM). The viability of oocytes at each step of a published cryopreservation protocol was assessed in an initial experiment. Two major viability losses were identified; one when oocytes were cooled to -35 degrees C and the other when oocytes were plunged in liquid nitrogen. Although the cryomicroscope showed no evidence of IIF in oocytes cooled with this protocol, TEM revealed that these oocytes contained ice crystals and were at two developmental stages when frozen, prophase and metaphase I. To reduce IIF, the effect of seven cooling programmes involving cooling to -35 or -60 degrees C at 0.1 or 0.3 degrees C min(-1) and holding for 0 or 30 min at -35 or -60 degrees C was evaluated on post-thaw fertilization rate of oocytes. Regardless of the cooling rate or holding time, the fertilization rate of oocytes cooled to -60 degrees C was significantly lower than that of oocytes cooled to -35 degrees C. The overall results indicated that observations of IIF obtained from cryomicroscopy are limited to detection of larger amounts of ice within the cells. Although the amount of cellular ice may have been reduced by one of the programmes, fertilization was reduced significantly; suggesting that there is no correlation between the presence of intracellular ice and post-thaw fertilization rate. Therefore, oyster oocytes may be more susceptible to the effect of high solute concentrations and cell shrinkage than intracellular ice under the studied conditions.

The ability to routinely cryopreserve micro-algal species reduces costs associated with maintaining large culture collections and reduces the risks of losing particular strains or species through contamination and genetic drift. Cryopreservation is also a useful adjunct in aquaculture hatcheries for strains of micro-algae where the nutritional status may change as a result of continuous sub-culture. In this study, cryopreservation of isolates from seven micro-algal classes was investigated. Successful candidates included the marine dinoflagellates Amphidinium carterae, Amphidinium trulla, and Gymnodinium simplex, and the haptophytes Chrysochromulina simplex, Prymnesium parvum, Prymnesium parvum f. patelliferum, Isochrysis galbana, and Pavlova lutheri. Also successfully cryopreserved were the planktonic diatoms Chaetoceros calcitrans, Chaetoceros muelleri, Chaetoceros sp., and the benthic Nitzschia ovalis, the chlorophyte Chlamydomonas coccoides, the rhodophyte Porphyridium purpureum, the prasinophytes Tetraselmis chuii, and Tetraselmis suecica, and the cyanophytes Raphidiopsis sp., and Aphanizomenon flos-aquae. All species were successfully cryopreserved using 15% Me2SO.

Effects of cryopreservation on development of trochophore (18 h post-fertilisation) and D-stage larvae (48 h post-fertilisation) of the New Zealand greenshell™ mussel, Perna canaliculus were evaluated over a 21 day rearing period. Examination of development was undertaken through histology, scanning electron microscopy (SEM), confocal microscopy, light microscopy, and visual observations. Larvae were cryopreserved in 10% ethylene glycol and 0.4 M trehalose which had been made up at twice the final concentration using Milli-Q water. Larvae diluted 1:1 with cryoprotectant solution were loaded into 0.5 mL straws and frozen in a controlled rate freezer that was programmed to hold at 0 °C for 5 min then cool at 1 °C min−1 to −10 °C, hold for 5 min then cool at 0.5 °C min−1 to −35 °C before being plunged into liquid nitrogen. Straws were held for 1–2 h in liquid nitrogen then thawed in a 28 °C water bath. Thawed larvae were then reared under standard hatchery conditions and compared with larvae which were not cryopreserved (controls). Results showed that there were significant differences in survivability, shell length, and feeding consumption between controls and cryopreserved treatments, but no comparable differences among the two larval stages cryopreserved after the 21-day development period. At 18 days post-fertilisation, ∼23% of control larvae had progressed to competent pediveliger, while

The effect of external medium composition on chorion and plasma membrane permeability of zebrafish (Danio rerio) embryos was investigated in this study. Initially, survival of embryos spawned into varying strengths (10-40%) of Hank's solution (HBSS) was assessed. Development and hatching rates for embryos spawned into 30% and 40% HBSS were significantly lower than those obtained with embryos spawned into system water. The effect of embryo survival in 30% HBSS with different calcium levels was then investigated. Embryo survival in calcium free 30% HBSS or 30% HBSS with 10x the standard calcium concentration was similar to survival in standard 30% HBSS. Membrane water permeability was determined by measuring the floatation time of embryos in test solutions made up with heavy water (D2O) instead of deionized water. Intact embryos at early developmental stages were less permeable than later stages irrespective of the external medium that they were spawned into. In system water, the floatation time of embryos at one-cell and two-cell stages were 1323+/-83 and 1189+/-55 s, respectively, compared to 432+/-6 and 353+/-10 s at the high and 50% epiboly stages. Change of external medium composition had no effect on membrane permeability of intact embryos at early developmental stages. However, at later stages embryos spawned into 30% HBSS were less permeable than embryos spawned into system water, irrespective of calcium concentration. The flotation time of embryos at the high stage increased from 432+/-6s in system water to 468+/-10s in 30% HBSS. The study on dechorionated embryos showed that change of external medium composition had no effect on plasma membrane permeability.

Larvae of the sea urchin, Evechinus chloroticus, at varying stages of development, were assessed for their potential to survive cryopreservation. Ethylene glycol (EG) and dimethyl sulphoxide (Me2SO), at concentrations of 1-2 M, were evaluated as cryoprotectants (CPAs) in freezing regimes initially based on methods established for freezing larvae of other sea urchin species. Subsequent work varied cooling rate, holding temperature, holding time, and plunge temperature. Ethylene glycol was less toxic to larvae than Me2SO. However, no larvae survived freezing and thawing in EG. Larvae frozen in Me2SO at the gastrula stage and 4-armed pluteus stage regained motility post-thawing. The most successful freezing regime cooled straws containing larvae in 1.5 M Me2SO from 0 to -35 degrees C at 2.5 degrees C min(-1), held at -35 degrees C for 5 min, then plunged straws into liquid nitrogen. Motility was high 2-4 h post-thawing using this regime but decreased markedly within 24 h. Some 4-armed pluteus larvae that survived beyond this time developed through to metamorphosis and settled. Different Me2SO concentrations and supplementary trehalose did not improve long-term survival. Large variation in post-thaw survival was observed among batches of larvae produced from different females.

Geoducks are large salt water clams. They are currently farmed in North America. In New Zealand, there is a small wild fishery for the native geoduck, Panopea zelandica. More recently, there is increasing interest in farming this high value species in New Zealand. Already a small batch has been reared and is ready to be experimentally on-grown on existing mussel farms. Cryopreservation can be a powerful tool for hatcheries and selective breeding programmes for aquaculture. In hatcheries, cryopreservation allows year-round spat supply without the need to condition broodstock for out of season production. This is particularly useful with new species where optimized conditioning regimes have not been established. Spawning techniques may also need development and cryopreservation can enable hatcheries to capitalize on single sex spawnings where gametes would otherwise be wasted. In selective breeding, cryopreservation enables breeders to make crosses on demand; providing them with complete flexibility and enabling faster genetic gains. Towards this goal, we investigated cryopreservation and fertility of gametes from the New Zealand geoduck, Panopea zelandica. Geoducks were induced to spawn during their natural spawning season using thermal shock and serotonin injection. Sperm was collected as concentrated as possible by aspiration and stored at 4 °C before being used in experiments. Oocytes were maintained at either room temperature or 4 °C. For both sperm and oocytes, three cryoprotectants, ethylene glycol, dimethyl sulphoxide and propylene glycol were made up in Milli-Q water and evaluated at final concentrations of 10% and 15% with or without 0.2 M trehalose. For sperm, a simple freezing method utilizing a rack attached to a polystyrene frame which floated the rack 3 cm above the liquid nitrogen surface was evaluated as a method of freezing. Sperm were diluted 1:1 with cryoprotectant solutions and loaded into 0.5 mL straws and frozen on the rack for 10 min before plunging into liquid nitrogen. For oocytes, two cooling regimes were evaluated. Oocytes were diluted 1:1, loaded into 0.25 mL straws and placed into controlled rate freezers that were programmed to hold at 0 °C for 5 min then cool at 1 °C min−1 to −10 °C, hold for 5 min then cool at either 0.3 or 0.5 °C min−1 to −35 °C. Upon reaching −35 °C, straws were plunged in liquid nitrogen. Straws containing sperm were thawed at room temperature. Straws containing oocytes were thawed at 28 °C until the ice had melted. Overall, unfrozen sperm obtained from serotonin injection had slightly higher fertility with fresh oocytes than sperm obtained from thermal shock (70% vs 50% at sperm concentration of 105 mL−1) The addition of bovine serum albumin to the fertilization assay improved sperm fertility by 30%. Sperm frozen in either 10% dimethyl sulphoxide or 10% or 15% propylene glycol gave the highest post-thaw fertilization rates (>70% at sperm concentration of 106 mL−1). Oocytes were lysed upon thawing prior to dilution for fertilization. Further work will investigate: optimization and development of cryopreservation methods for both gametes and larvae; the longevity of geoduck gametes and a larval assay to assess subsequent development.

Cryopreservation can be a powerful tool in shellfish aquaculture for selective breeding programmes and hatchery production. Most species that are commercially farmed are broadcast spawners, releasing sperm and eggs into the environment where they fertilize and develop. However, some species are brooders (e.g. flat oysters (Ostrea chilensis) ), which retain and rear embryos until they reach later developmental stages. Sperm cryopreservation has been successfully achieved for several farmed species including the Pacific oyster ( Crassostrea gigas ), greenshell™ mussel ( Perna canaliculus ) and abalone ( Haliotis iris ). In general, dimethyl sulphoxide and trehalose are the preferred cryoprotectants (CPAs) and simple methods to control cooling rate (e.g. floating rack on liquid nitrogen; methanol/dry ice bath) have worked well for our main species of interest (Pacific oyster, greenshell™ mussel and abalone). Interestingly, sperm perform better in CPAs that are prepared in Milli-Q water as opposed to seawater, indicating that a lower salt concentration during freezing is beneficial. Recent studies with flat oysters and geoducks ( Panopea zelandica ) have also resulted in successful sperm cryopreservation. Shellfish oocyte and larvae cryopreservation has proven more challenging, however, successful cryopreservation of Pacific oyster oocytes has been achieved. Pacific oyster oocytes are able to be fertilized and develop to normal spat post-thawing. However, for other species (e.g. greenshell™ mussel) and for larvae, cryo-injuries are not always immediately apparent post-thawing but manifest during development and are often lethal. greenshell™ mussel oocytes are irretrievably damaged when they reach -6 to -8°C and attempts to overcome this sub-zero chilling injury by vitrification have been unsuccessful. Towards understanding these injuries and their effects, we investigated the effect of cryopreservation on the meiotic spindle of greenshell™ mussel oocytes. Results show that the meiotic spindle undergoes major changes during both CPA addition and cooling. However, the spindle is able to re-form normally in most instances given sufficient time post thawing. For a proportion of oocytes, cryopreservation appears to trigger oocyte activation. The effect of cryopreservation on oxidative stress in greenshell™ mussel oocytes has also been investigated. Although, there are changes in oxidative stress parameters as a result of cryopreservation and strong correlations between fertilization, development and oxidative stress parameters (lipid hydroperoxides, superoxide dismutase, protein carbonyls, total glutathione and glutathione state), preliminary attempts to mitigate oxidative stress using antioxidants have so far failed to enable normal development following cooling, suggesting that oxidative stress may be a secondary rather than primary stress event. Research on shellfish larval cryopreservation utilizing SEM and confocal microscopy has also been carried out for greenshell™ mussel. Later larval stages (D-larvae) appear more resilient to cryopreservation than trochophore larvae, but abnormalities are still apparent (e.g.shell abnormalities and delayed organogenesis). Future studies are aimed toward elucidating mechanisms of injury using new tools such as proteomics and then targeting susceptible cellular processes for protection during cryopreservation. Source of funding: This research was funded by the New Zealand Ministry of Business Innovation and Employment (CAWX0802). Conflict of interest: None declared. serean.adams@cawthron.org.nz