To develop selection programs for the great scallop, artificial reproduction of the species needs to be improved by gamete cryobanking. Here, a set of four experiments was designed in order to define the basic elements of a cryopreservation protocol for scallop sperm, including extender composition (experiment 1), cryoprotectant selection (2), cooling rate (3), and assessment of the effects of sperm cryopreservation on sperm motility and fertilization capacity (4). Sperm was collected after serotonin injection (100 μl of a 10-mM solution) and frozen in 500-μl straws. For the first three experiments, the percentage of motile fresh sperm (80 ± 4%, mean ± SEM) was significantly higher than that observed for thawed sperm (11 ± 2%). During the first experiment, no significant difference of the percentage of motile thawed sperm was observed among the three saline extenders tested: seawater, calcium-free Hanks’ balanced salt solution (Ca-free HBSS) and DCSB4 solution. However, a complementary experiment (2) showed that a significantly higher percentage of motile thawed sperm was recorded using DCSB4 than with Ca-free HBSS as an extender. During the third experiment, sperm motility was higher when using polyethylene glycol (10% PEG) as a cryoprotectant, than when using dimethyl sulphoxide, DMSO (10 and 20%), methanol (10%) and ethylene glycol (10% EG). A higher D-larval rate was obtained during the fourth experiment, using fresh sperm than thawed sperm, and using a 500:1 sperm-to-egg ratio compared with a 50:1 ratio. There is some evidence of inter-individual variations in sperm tolerance to cryopreservation. In conclusion, the highest survival of great scallop sperm after thawing was recorded using the following conditions: DCSB4 extender (1:3 vol/vol sperm to extender dilution), PEG cryoprotectant (10%) and straws maintained at 5.5 cm above liquid nitrogen.

The toxic dinoflagellate Alexandrium catenella recurrently blooms on thecoasts of France and produces Paralytic Shellfish Toxins (PSTs) that accumulate inbivalves. These toxins can affect various physiological functions including reproduction.The present study aims to validate measurements of sperm viability, DNA content andmitochondrial membrane potential in Pacific oyster Crassostrea gigasusing flow cytometry coupled with fluorescent markers, and to use thesemeasurements to assess the cellular parameters of sperm from Pacific oysters exposed toA. catenella. These parameters may influence fertilization,embryogenesis and larval development in free-spawning shellfish. Sperm viability and DNAcontent estimation were assessed using SYBR-14, which only penetrates cells with intactmembranes. Cell mortality was measured with propidium iodide (PI), which penetrates cellswith membrane damage. Mitochondrial membrane potential, used as an estimate ofmitochondrial function, was measured using JC-1 dye, which selectively enters intomitochondria and reversibly changes colour from green to orange as the membrane potentialincreases. To assess the effect of toxic algae on oyster sperm, broodstock (ripe oysters)were fed toxic (A. catenella) or non toxic (Heterocapsatriquetra) dinoflagellates at 250 cell ml-1 for 9 days. After thisexposure period, mature oysters were stripped and cellular responses of sperm analysed.Average DNA staining, as measured by SYBR-14, appeared lower and more variable in gametesfrom A. catenella-exposed oysters than in those from control oysters fedH. triquetra. Additionally, mitochondrial membrane potential of spermfrom A. catenella-exposed oysters was significantly higher (1.5 fold)than that of sperm produced by oysters fed H. triquetra. Both theincrease of mitochondrial membrane potential and the modification of DNA structure can beexpected to impact spermatozoa ability to fertilize oocytes and could thus impact relatedreproductive processes.

Sperm characteristics of scallops have not been well described in the scientificliterature. The effects of sperm release technique (thermal shock versus serotonininjection), of sperm collection technique (testis sampling versus serotonin injection), ofsperm sampling location along the genital tract, of in vitro sperm maturation, and of timepost activation on scallop sperm characteristics were assessed in the present work.Whatever sperm release technique used, no significant differences were observed regardingthe percentage of motile spermatozoa and the velocity of the average path (VAP). Comparedto testicular sperm, a higher percentage of motile spermatozoa, VAP and intracellularadenosine triphosphate (ATP) content were observed for sperm shed after serotonininjection. From the distal part of testes up to the gonopore, an increase of thepercentage of motile spermatozoa and VAP was assessed, suggesting a sperm ‘maturationprocess’ along the genital ducts. A higher increase in the percentage of motile sperm wasrecorded during a 5 min incubation of testicular sperm in seawater containing 2 mMserotonin and seawater containing 10 mM caffein compared to seawater (control). Inaddition, a higher VAP was assessed, incubating testicular sperm in caffein, compared tocontrol or serotonin. Then, the percentage of motile spermatozoa, VAP and intracellularATP content exhibited a progressive reduction during the 10 h swimming period. Mean valuesof the percentage of motile spermatozoa, VAP, sperm track linearity (LIN) andintracellular ATP content recorded at the beginning of the movement period for spermsamples collected after intragonadal serotonin injection, were 82 ± 7%,162 ± 15μm s-1, 0.33 ± 0.12 and 212 ± 133 nmol × 10-9 spermatozoa (n = 9 males), respectively. The presentstudy confirms the existence of a sperm “maturation process” along scallop genital ducts.In addition, the cessation of scallop sperm movement can be explained by the exhaustion ofATP content at the end of the movement phase.

Cryopreservation is a valuable tool for genetic improvement programs. Several bivalvemollusc species have already been the subject of such programs and the Tahitian blackpearl oyster industry is now planning the development of selective breeding for desirabletraits in Pinctada margaritifera. The ability to cryopreserve spermatozoawould, therefore, offer significant benefits to the cultured black pearl industry.Spermatozoa were cryopreserved with cryoprotectant agent (CPA) 0.7 M trehalose in 0.8 MMe2SO and a two-step freezing process was used: straws were first maintainedin nitrogen vapour for 10 minutes, then directly plunged into liquid nitrogen and storedfor one week before use. The viability of thawed sperm was 23% lower than that of freshsperm. When using thawed sperm, therefore, a higher sperm/egg ratio of 100 000:1 wasrequired to reach 80% oocyte fertilization, compared with 100:1 for fresh sperm.Nevertheless, this first demonstration of cryopreserved sperm fertility in black pearloyster confirms the hatchery applicability of the cryopreservation technique defined here.Monitoring for larval viability during the first 23 days of life revealed no significantdifferences between the progeny produced with cryopreserved sperm and that produced usingfresh sperm.

A reliable anaesthesia and sampling protocol for Pacific oysters will enable experiments to be conducted without sacrificing animals and will facilitate successive sampling of individuals for gametogenesis studies. As no such techniques were available for Crassostrea gigas, the present study aimed to define suitable anaesthetic conditions for use with this species. Three groups of ten oysters (mean weight ± SD, 32.1 ± 9.0 g) were anaesthetised in 5 L containers. Among different chemicals: benzocaine, eugenol and three different types of magnesium chloride (a laboratory one – Flucka® – and two designed for agriculture – DEUSA International® and Dead Sea Works®) and concentrations tested, one laboratory (concentration: 72 g L−1) and one agricultural (Dead Sea Works® 50 g L−1) type of magnesium chloride were the most effective, respectively inducing anaesthesia in 73 ± 3% and 80 ± 3$% after three hours. Lower oyster weight and a two day period of starving prior to treatment significantly increased the number of anaesthetised animals. Using this protocol, losses of 0 to 10% of oysters were observed one week after anaesthesia. Increasing anaesthesia duration from 3 to 16 h resulted in a significant increase in the number of anaesthetised oysters (from 50 ± 10 to 97 ± 7%) but no increase in mortality (7 ± 11%). On the other hand, reducing water temperature from 19.5 °C to 15.3 °C, resulted in a significant decrease in anaesthesia efficacy. A reliable anaesthesia protocol was developed: 100% of Pacific oysters are anaesthetised using 50 g L−1MgCl2 Dead Sea Work®for a 16 h duration. This long duration facilitates tissue sampling and does not increase the working time needed. This protocol was validated by monthly anaesthesia and gonad sampling during a three month period with the loss of only a single oyster. It can thus be used for routine applications.

Because of natural complexity, field studies are often inconclusive with regards to the ultimate cause of a given change observed in wild animal populations. As a consequence, there is scope to develop an Experimental Ecology Approach (EEA). In this paper, we favour the use of experimental ecology studies to evaluate the effects of environmental factors on fish biology. We identify the advantages of EEA: disentangling the effects of several factors and identifying their respective roles, generating artificial scenarios and increasing our capability to collect and analyse data. This study emphasises the constraints of this approach: fish biological performances may be altered by rearing conditions, by domestication and by the prevention of some behaviours. It also considers the extrapolation of experimental results with regard to the sea including the realism of experimental design and the constraints of scaling or extending the results to larger scales. We also propose some experimental ecosystems which are well adapted to the requirements of EEA and a careful selection of fish species which fit this approach very well. Atlantic Cod (Gadus morhua) is identified as a good candidate for experimental studies and we present examples of the contribution of EEA to the description and quantification of the effects of environmental factors in this species. We conclude by highlighting the respective contribution of field studies (describing a situation in the wild and suggesting some possible causes) and EEA (allowing an active investigation of phenomena and identifying their final causes).

At present, European marine fish farming is based on sea bream and sea bass. A trend for diversification is sustained by the diversity of environmental conditions, by the availability of new production techniques such as recirculating systems, by an increase in rearing yields, by new market trends and by the possibilities to reduce risks of disease outbreak. Already reared fish species were chosen considering a limited number of criteria such as a high selling price and the availability of juveniles or breeders in the wild. This paper proposes a new selection method of fish species as candidates for aquaculture development on the French Atlantic, the Channel and the North Sea coasts. Using a three-phase procedure, candidates were selected among 20000 fish species. Final classification was carried out using a panel of 22 criteria, taking into account inquiries conducted in France with aquaculturists but also with the main actors of distribution and transformation channels and with consumers. Cod (Gadus morhua) was selected by the present work as the first candidate for aquaculture development on the western coasts of France. This work also highlights the high interest of Gadoids for aquaculture development in this area.

The effect of different parameters on short-term storage capacity of turbot ova was assessed over a 45-h period after ova collection for fertilization rates and over a 9-h period after ova collection for hatching rates. Increasing the volume of ova sampling from 0.5 to 2.5 mL, as well as adding an antibiotic-antimicotic solution or oxygen did not significantly change the storage capacity of ova. Regarding the hatching rates, a higher storage ability was recorded at 8 and 13 °C, compared to 3 °C. The mean composition of the ovarian fluid was determined (n = 57 spawns). Use of a diluent mimicking the ovarian fluid significantly decreased the storage ability as assessed by the fertilization rates but did not modify the hatching rates. Diluting ova in an artificial ovarian fluid deprived of calcium significantly decreased the fertilization and hatching rates during the storage period. Furthermore, addition or not of soybean trypsin inhibitor (Sigma T 9003) to the artificial ovarian fluid deprived of calcium did not significantly change the results. Storage capacity of control batches of ova was low: at 13 °C, without any diluent and when ova were fertilized 3 h after stripping, the hatching rate was lowered to 62.4 ± 29.4 % (mean ± SD) of the initial value.

A sperm cryopreservation protocol adapted from turbot, was tested on sea bass using either 250-μL straws or 1.5-mL cryovials. A dilution to 1/3 in Mounib's extender and a cooling rate of −65 °C·min−1 allowed frozen sperm to recover an initial motility similar to that of fresh sperm at thawing; however, significant differences in motility (P < 0.001, n = 10 fish semen) were observed at further post-activation times, the motility decrease being faster in thawed sperm. At the experimental scale, triplicate inseminations of 2-mL aliquots (approximately 2 000 eggs) showed a significant fertility decay of thawed sperm compared to that of fresh sperm (P < 0.01, n = 12 fish semen) when a discriminating 35·103 spermatozoa to egg ratio was applied. When 70·103 and 200·103 spermatozoa per egg were provided in the same experimental conditions, no significant difference appeared between the fertilisation rates of fresh and thawed sperm. In order to validate the procedure for production or cryobank purpose, a scaled-up protocol was established. Two and 50 mL batches of eggs (approximately 2·103 and 50·103 eggs, respectively) were inseminated in triplicate using either fresh or thawed individual sperms of 5 males with 200·103 spermatozoa per egg. The mean fertility decreased by 23.5 % due to cryopreservation. This decline was explained by the loss of fertility of only one sperm, and only in large-volume conditions, probably due to the delay of use after thawing.

The survival of turbot eggs and the rearing capacities of larvae stemmed from artificial fertilization practices using frozen-thawed spermatozoa were evaluated. Furthermore, the viability of sperm samples stored during a 9 month period in liquid nitrogen was assessed. No significant difference in the fertilization rate, hatching rate, survival and wet weight of 10-day old larvae were observed using fresh or frozen-thawed spermatozoa. The motility recorded at 10 s and 60 s post-activation and the fertilization capacity of frozen-thawed spermatozoa were not significantly decreased during a 9 month storage period in liquid nitrogen. These results confirm the high quality of the turbot spermatozoa stemmed from the cryopreservation process, allowing their use for routine aquaculture practices.

Short-term storage over several days as well as cryopreservation of turbot (Scophthalmus maximus) sperm were studied. Two extenders, Ringer 200 and artificial seminal liquid (ASL2), are suggested for semen collection in order to avoid the deleterious effect of urine contamination, and for the purpose of short-term storage between 0 and 15 °C. Oxygen atmosphere is not suitable for turbot sperm storage. Turbot spermatozoa undergo cryopreservation with a high rate of success especially in a sucrose solution with 10% dimethyl sulfoxide (DMSO) and 10% egg yolk.

This paper presents some features of turbot sperm and compares them with data reported in the literature on other freshwater and marine fish. In captivity, turbot spermiation period lasts 6 months. When compared to other fish species, turbot males are characterized by low values of maximum gonadosomatic index (0.6-0.8%) and are poor sperm producers in terms of volume (0.2-2.2 ml), concentration (0.7-11.0 × 109 spermatozoa/ml) and total sperm number released per stripping (0.2-12.0 × 109 spematozoa). Compared to most freshwater species, the duration of cell movement is long (1-17 min). The spermatozoon of turbot can be considered to be of a primitive type. A very high protein content (8.8 mg/ml) is found in the seminal fluid of turbot. Increasing collection frequency of turbot males, from monthly to fortnightly and weekly stripping, results in the release of successive samples presenting decreasing semen volume and spermatozoa concentration. Similar spermatozoa concentration and total volume of semen produced per turbot, during a 2 month experimental period, are released from males submitted to an annual light and temperature cycle, compared to those receiving a 6 month contracted light and temperature schedule.