Classification of differentiating oocytes during ovarian cycle in the giant freshwater prawn, Macrobrachium rosenbergii de man

Based on the light microscopic observations of cells' sizes, chromatin patterns, amount of lipid droplets and yolk granules, the female germ cells could be classified into four different phases, which include 1) oogonia (Oog), 2) primary oocytes (pOc), 3) secondary oocytes (sOc), and 4) mature oocyte (mOc). Oog are small oval-shaped cells with irregular-shaped nuclei sizing 4–6 μm in diameter. They rest on the connective tissue germinal cord at the tip of each ovarian pouch (lobule). Oogonia increase their number through mitotic division, and the daughter cells move into ovarian pouch where they undergo first meiotic division to become primary oocytes, which have various steps of 1st meiotic prophase accumulating at the innermost zone of the ovarian pouch. The primary oocytes are small oval-shaped cells (8.5–10 μm in diameter) with large nuclei containing chromatin in various states of condensation that finally transform into chromatids. Their nuclei are surrounded by thin rim of faint blue-stained cytoplasm. The secondary oocytes derived from 2nd meiosis and comprise five steps: Oc1 and Oc2, classified as previtellogenic oocytes, Oc3 and Oc4, classified as vitellogenic oocytes, and mature oocyte (mOc) The zones of ovarian pouch are defined based on the accumulation of various steps of developing oocytes, namely, oogenic, previtellogenic, vitellogenic and mature zones, respectively. The ovarian cycle is divided into five stages based on the number and types of oocytes present in each stage. Stage 0 and I are spawn and spent stages. Stage II and III are proliferative and premature stages, while stage IV is mature stage. During ovarian stage I, each ovarian pouch contains primarily oogonia, primary oocytes, Oc1 and a few Oc2. In stage II, the pouch contains mainly Oc2 and Oc3, while in stage III the predominant cells are Oc4. Mature oocytes appear synchronously, in stage IV. The ovulating mature oocytes pass through the thin disrupted wall of ovarian pouch into subcapsular space, that leads into the oviduct situated on the ventro-lateral side of the ovarian lobe. At spawning, the ovarian pouches break down and only connective sheaths and hemolymph sinuses remain. The germinal cords and islets of oogonia remain in the central area of stage 0 ovary. The ovarian capsule, including the muscular layer, becomes attenuated as the ovary progresses from stage 0 to IV. The hemolymph vessels become highly convoluted in the central area of the ovary, and they branch radially into smaller hemolymph sinuses around each oogenic pouch.     

Serotonin induces ovarian maturation in giant freshwater prawn broodstock, Macrobrachium rosenbergii de Man

This study investigated the effects of serotonin (5-hydroxytryptamine or 5HT) on ovarian development in Macrobrachium rosenbergii de Man. Adult female prawns at the ovarian stage I (spent) were injected with 5HT at 1, 5, 10, 20 and 50 μg g^− 1 body weight (BW) intramuscularly on days 0, 5 and 10, and sacrificed on day 15. The doses as related to the effect could be categorized into three levels: low (1 and 5 μg g^− 1 BW of 5HT), medium (10 and 20 μg g^− 1 BW of 5HT) and high (50 μg g^− 1 BW of 5HT). The low-dose, especially at 1 μg g^− 1 BW, caused prawns to exhibit a significant increase in ovarian index (ovarian weight/body weight × 100) (5.79 ± 0.09%) as compared to the control (1.49%). The ovaries of most of these prawns could develop to stage IV (mature) and contained synchronously mature oocytes while most of the control ovaries remained at stage I and II (proliferative), and contained only oogonia to previtellogenic (Oc1, Oc2) and early vitellogenic oocytes (Oc3). The medium- and high-dose treated prawns exhibited ovaries that could reach stages III and IV and contained various types of oocytes of different maturity. Pretreatment with 5HT receptor antagonist, cyproheptadine (CYP), at 10 μg g^− 1 BW before 5HT injection significantly suppressed the effect of 5HT. Intramuscular injection of the 5HT-primed thoracic ganglion culture medium into CYP-pretreated prawns resulted in the increase of ovarian index about 5–6 times more than in the control, and in the groups injected with 5HT-primed media from muscle strip, eyestalk and brain. The ovaries of most prawn could develop up to stage IV and contained synchronously developed vitellogenic (Oc4) and mature oocytes (Oc5). These findings suggest that 5HT indirectly induces ovarian development and oocytes maturation in M. rosenbergii, probably via a putative ovarian stimulating factor released from the thoracic ganglia.     

Temporal progression in migratory status and sexual maturation in European silver eels during downstream migration

The onset of downstream migration of European eels is accompanied by a cessation of feeding and the start of sexual maturation which stresses the link between metabolism and sexual maturation, also suggesting an important role for exercise. Exercise has been tested with eels in swim tunnels and was found to stimulate the onset of sexual maturation. In this study, we have investigated the interplay between migration and maturation in the field during the downstream migration of female silver eels. Temporal changes in migratory status and sexual maturation among silver eels of the upstream Rhine River system over 3 months of the migration season (August, September and October) were determined in biometrical parameters, plasma 17β-estradiol and calcium levels, oocyte histology and gonadal fat levels. Furthermore, the ecological relevant parameters age as determined by otolithometry and health aspects indicated by haematocrit, haemoglobin and swim-bladder parasite load were measured. Silver eels were estimated to be 14 years old. A strong temporal progression in migratory stage was shown over the months of downstream migration. Catches probably represented a mix of reproductive migrants and feeding migrants of which the ratio increased over time. Furthermore, this study confirmed our hypothesis linking the migratory stage to early maturation as indicated by enlargement of the eyes, oocyte growth and fat deposition in the oocytes, exactly the same changes as found induced by exercise but not ruling out environmental influences. Migrants show extensive fat uptake by the oocytes, probably stimulated by the swimming exercise. In addition, at least 83% of the silver eels in this spawning run may have suffered from negative effects of swim-bladder parasites on their swimming performance.     

Swimming physiology of European silver eels (<Emphasis Type="Italic">Anguilla anguilla</Emphasis> L.): energetic costs and effects on sexual maturation and reproduction

The European eel migrates 5,000–6,000 km to the Sargasso Sea to reproduce. Because they venture into the ocean in a pre-pubertal state and reproduce after swimming for months, a strong interaction between swimming and sexual maturation is expected. Many swimming trials have been performed in 22 swim tunnels to elucidate their performance and the impact on maturation. European eels are able to swim long distances at a cost of 10–12 mg fat/km which is 4–6 times more efficient than salmonids. The total energy costs of reproduction correspond to 67% of the fat stores. During long distance swimming, the body composition stays the same showing that energy consumption calculations cannot be based on fat alone but need to be compensated for protein oxidation. The optimal swimming speed is 0.61–0.67 m s⁻¹, which is ~60% higher than the generally assumed cruise speed of 0.4 m s⁻¹ and implies that female eels may reach the Sargasso Sea within 3.5 months instead of the assumed 6 months. Swimming trials showed lipid deposition and oocyte growth, which are the first steps of sexual maturation. To investigate effects of oceanic migration on maturation, we simulated group-wise migration in a large swim-gutter with seawater. These trials showed suppressed gonadotropin expression and vitellogenesis in females, while in contrast continued sexual maturation was observed in silver males. The induction of lipid deposition in the oocytes and the inhibition of vitellogenesis by swimming in females suggest a natural sequence of events quite different from artificial maturation protocols.     

Prey extracts evoke swimming behavior in juvenile Atlantic halibut (Hippoglossus hippoglossus)

This study tested the efficacy of prey extracts to induce food search behavior in juvenile (15–25 cm total length, 0 year group) Atlantic halibut (Hippoglossus hippoglossus). In square culture tanks, halibut responded to shrimp and squid extract by tightly turning towards the source of the stimulus and by swimming in circles for 2–5 min following stimulus delivery. Cod extract, or a synthetic mixture consisting of glycine, proline, betaine, arginine, and alanine, failed to evoke this behavior.     

Modification of essential fatty acid composition in broodstock of cultured European eel Anguilla anguilla L

Farmed eels had lower levels of arachidonic acid (20:4 n‐6) (ARA) and higher ratios of eicosapentaenoic acid (20:5 n‐3) (EPA):ARA compared to wild European eels collected from the Baltic Sea and southern Norwegian coast. Eels fed a formulated feed (JD) with a distribution of essential fatty acids (EFA) resembling wild European eel were sampled after 0, 5, 10, 14 and 44 weeks of feeding to examine changes in fatty acid composition (FAC) in ovaries, visceral fat and muscle. The results showed a slow but steady incorporation of EFA. Lipids are incorporated in the oocytes early in oogenesis, and the leading cohort of oocytes is rich in lipid droplets before the onset of vitellogenesis. This indicates that feeding with optimized broodstock feeds should start early to allow the incorporation of EFA in the first cohort of oocytes. At least 14 weeks of feeding is required to change lipid EFA in broodstock eel to resemble EFA in the diet or in wild fish. After 44 weeks of feeding, ARA was significantly higher in the neutral lipids of ovaries (1.9%) compared to visceral fat (1.2%) or muscle (1.0%). EPA:ARA ratios decreased two‐ to threefold in all tissues examined during that time. ARA and docosahexaenoic acid (22:6 n‐3) (DHA) had accumulated in ovarian polar lipids.     

Pharmacokinetics and the active metabolite of enrofloxacin in Chinese mitten-handed crab (Eriocheir sinensis)

The pharmacokinetics and active metabolite of enrofloxacin were estimated after single intramuscular administration (10.0 or 20.0 mg/kg body weight) to the Chinese mitten-handed crab (Eriocheir sinensis) in fresh water at 25.0 ± 1.0 °C. Levels of enrofloxacin and its metabolite ciprofloxacin in the main tissues (hemolymph, hepatopancreas, muscle, ovary and spermary) were simultaneously detected by HPLC. Enrofloxacin concentration–time profiles for the hemolymph in both tests were described by a two-compartment open model with first-order absorption. Distribution half-time (T_1/2), elimination half-time (T_1/2β), body clearance (CL/F), mean residence time (MRT_0–∞), area under the concentration–time curve from 0 to ∞ h (AUC_0–∞) and apparent volume of distribution (Vd/F), which derived from the pharmacokinetic model, were 0.427 h, 21.3 h, 0.133 l/h/kg, 60.0 h, 96.9 μg/ml/h and 4.08 l/kg, respectively, at a dose of 10.0 mg/kg body weight, and 0.216 h, 12.3 h, 0.189 l/h/kg, 85.8 h, 187 μg/ml/h and 3.35 l/kg, respectively, at a dose of 20.0 mg/kg body weight. Similarities were found between the hemolymph concentration–time curves of the two tests; for example, instant absorption process followed by the distribution phrase, and a second absorption peak at 6 h post-treatment. After intramuscular administration of 10.0 mg/kg body weight, absorption of enrofloxacin was observed in the main edible tissues (hepatopancreas, muscle, ovary and spermary), and the drug residue was the highest in the hepatopancreas, where the ‘drug sink’ phenomenon occurred. Comparative pharmacokinetics showed fast absorption, broad distribution and fast elimination of enrofloxacin in E. sinensis after intramuscular dosing. Regarding ciprofloxacin, the main active metabolite of enrofloxacin, though relatively low levels were detected in all the main tissues of the crab, its kinetics in the hemolymph in the two tests were not described by a one- or two-compartment open model.     

Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica

Repeated injections of salmon pituitary extract (20 mg per fish per week) induced vitellogenesis in feminized, cultivated Japanese eels (Anguilla japonica). Oocytes were attained at the migratory nucleus stage after 11 or 12 injections. Addition of 17,20-dihydroxy-4-pregnen-3-one (DHP) into the incubation medium induced germinal vesicle breakdown (GVBD) in the oocytes at the migratory nucleus stage. An injection of DHP (2 µg g^-1 BW), given 24h after an injection of salmon pituitary extract (20 mg fish^-1), succeeded in inducing maturation and ovulation in females which contained occytes at the migratory nucleus stage. Most fish ovulated 15–18h following the DHP injection. Eggs that were ovulated within 15h after the DHP injection showed high fertility and hatchability, but eggs ovulated 18 or 21h after the DHP injection, showed considerably lower fertility and hatchability. A delay between ovulation and stripping of the eggs rapidly decreased both the fertility and hatchability within 6–9h after ovulation, indicating that artificial fertilization must be carried out immediately after ovulation. Repeated injections of human chorionic gonadotropin (hCG) at a concentration of 1 IU g^-1 BW week^-1 induced spermatogenesis, spermiation, and the acquisition of potential for sperm motility in cultivated males. Most males spermiated after the fifth or sixth injection of hCG, and the milt weight gradually increased and remained constant (1–2 g) from the 11th to 31th injection. Sperm motility peaked 24h after each weekly injection, and decreased from the 3^rd day after the injection. Potassium ions are an essential constituent for the maintenance of motility in the eel spermatozoa. Artificial seminal plasma containing 15.2 mM KCl is applicable as a milt diluent. Using these techniques developed for female and male eels, we have succeeded in obtaining many fertilized eggs from cultivated eels.     

Controlled reproduction in the wild European eel (Anguilla anguilla): two populations compared

This study aimed to describe the response variability of female silver eels in terms of gonad development and eggs production to a standardized gonadotropic treatment (Carp pituitary extracts—CPE), and to relate this variability to population characteristics. For this purpose, sexual maturation, ovulation, and fertilization were induced in two eel populations coming from different locations in Adriatic Sea (Comacchio—CM and Marano-Grado—MG lagoons), and after that, their reproductive capacity was valuated. External (Silver index—SI, Eye index—EI, Pectoral fin length index—PFLI, Condition factor—K) and hormonal (17β-estradiol—E2, testosterone—T) parameters were measured, and some subject/group were killed for histological and lipid analysis and age determination. Morphometric parameters showed the CM-Group to have highest values of Body weight (BW), Body length (BL), and K, while MG-Group presented highest PFLI and Gonadosomatic index (GSI) values. Regarding hormonal analysis, the CM-Group showed significantly higher T and E2 levels than the MG-Group, both groups showed considerably rapid increase at T5 (5th injection). A positive trend in gonadal development was found through histological evaluation; a more regular maturation was observed in the MG-Group, whereas the CM-Group presented an exponential oocytes development starting from T10 (10th week), which led to an anticipated spawning. Lipid content showed significant differences in T0 (start study), post-ovulation, and Control (30th week) between CM and MG eels. As to zootechnical performances, while MG eels released spontaneously into the water, the CMs were stripped in order to check ovulation. The MG eels were statistically the most productive with 40.1 ± 6.33 % BW of eggs released. Furthermore, CM females ovulated mainly between the 19th and 22nd week (77.8 % spawned eels) instead in the MG’s ovulation goes from the 24th to the 28th week (100 % spawned eels). As fertilization is of concern, in both groups fertilized eggs were obtained with no difference in larvae production. These results seem to indicate that bigger dimensions, higher K, and larger lipid content (Comacchio eels) could fasten gonadic maturation without positively influencing reproductive performance of animals, both in term of quantity and quality of produced eggs. Smaller females with a highest SI (Marano-Grado eels) presented a more regular gonadic development, leading the animals to spontaneous spawning.     

Effects of silvering state on induced maturation and spawning in wild female Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

ABSTRACT:   The effects of silvering state of wild female Japanese eels Anguilla japonica on the success of induced maturation and the following spawning were examined. Thirty-eight females, collected in Mikawa Bay, were divided into four stages based on their silvering state: yellow (Y1), late-yellow (Y2), silver (S1) and late silver eels (S2). Despite injections of salmon pituitary extract (SPE) through the standard technique, Y1 and Y2 eels did not respond to the treatment with undeveloped gonad (gonad-somatic index [GSI]: 0.3–0.9), and all these females died by 5 weeks, probably due to an abnormal physiological condition. Most S1 (81%) and S2 eels (100%) matured completely (GSI: 17.8–51.4), and finally spawned successfully (69% for S1, 89% for S2). S2 eels fully matured with oocytes of over 750 μm in diameter by significantly smaller number of injections of SPE (5–6 times) than the case of S1 eels (6–8 times). The amount of eggs released by S2 eels (0.65 ± 0.11 g/fish per body weight [BW]) was significantly larger than those by S1 eels (0.54 ± 0.09 g/fish per BW). There was no difference in fertilization and hatching rates between eggs released by S1 eels and those of S2 eels. These results indicate that the success of induced maturation and spawning in wild female Japanese eels depends on their silvering state, and matured eggs can be obtained efficiently through the use of S2 eels rather than other stages.     

Metabolic scope, swimming performance and the effects of hypoxia in the mulloway, Argyrosomus japonicus (Pisces: Sciaenidae)

The culture of the mulloway (Argyrosomus japonicus), like many other Sciaenidae fishes, is rapidly growing. However there is no information on their metabolic physiology. In this study, the effects of various hypoxia levels on the swimming performance and metabolic scope of juvenile mulloway (0.34 ± 0.01 kg, mean ± SE, n = 30) was investigated (water temperature = 22 °C). In normoxic conditions (dissolved oxygen = 6.85 mg l^− 1), mulloway oxygen consumption rate (M·o₂) increased exponentially with swimming speed to a maximum velocity (U_crit) of 1.7 ± < 0.1 body lengths s^− 1 (BL s^− 1) (n = 6). Mulloway standard metabolic rate (SMR) was typical for non-tuna fishes (73 ± 8 mg kg^− 1 h^− 1) and they had a moderate scope for aerobic metabolism (5 times the SMR). Mulloway minimum gross cost of transport (GCOT_min, 0.14 ± 0.01 mg kg^− 1 m^− 1) and optimum swimming velocity (U_opt, 1.3 ± 0.2 BL s^− 1) were comparable to many other body and caudal fin swimming fish species. Energy expenditure was minimum when swimming between 0.3 and 0.5 BL s^− 1. The critical dissolved oxygen level was 1.80 mg l^− 1 for mulloway swimming at 0.9 BL s^− 1. This reveals that mulloway are well adapted to hypoxia, which is probably adaptive from their natural early life history within estuaries. In all levels of hypoxia (75% saturation = 5.23, 50% = 3.64, and 25% = 1 .86 mg l^− 1), M·o₂ increased linearly with swimming speed and active metabolic rate (AMR) was reduced (218 ± 17, 202 ± 14 and 175 ± 10 mg kg^− 1 h^− 1 for 75%, 50% and 25% saturation respectively). However, U_crit was only reduced at 50% and 25% saturation (1.4 ± < 0.1 and 1.4 ± < 0.1 BL s^− 1 respectively). This demonstrates that although the metabolic capacity of mulloway is reduced in mild hypoxia (75% saturation) they are able to compensate to maintain swimming performance. GCOT_min (0.09 ± 0.01 mg kg^− 1 m^− 1) and U_opt (0.8 ± 0.1 BL s^− 1) were significantly reduced at 25% dissolved oxygen saturation. As mulloway metabolic scope was significantly reduced at all hypoxia levels, it suggests that even mild hypoxia may reduce growth productivity.     

Breeding and juvenile culture of the lined seahorse, Hippocampus erectus Perry, 1810

All seahorse species worldwide have been placed under CITES Appendix II since 2004, because they have been over-exploited for traditional Chinese medicine and aquarium trades. Aquaculture has been recognized as a long-term solution for sustaining the seahorse trade while minimizing wild collection. In this study, we evaluated the breeding and juvenile culture of an important aquarium seahorse species, the lined seahorse Hippocampus erectus, Perry 1810. Pairing, mating and copulation behavior were observed. Gestation time and brood size were 17.33 ± 2.94 days and 272.33 ± 66.45 individuals/brood, respectively. Growth rates differed among juveniles from different broods. Effects of temperature on the growth rates and survivorship of the juveniles during the first two weeks were compared. The highest growth rate and survivorship of the juveniles occurred at 28–29 °C among the temperatures tested (24–33 °C). Growth rate and survivorship of the juveniles during the first 9 weeks at 28 °C were investigated. The final standard length and survivorship of the juveniles were 6.32 ± 0.52 cm and 71.11 ± 10.18%, respectively, and the relationship between the wet weight and the standard length of the juvenile seahorses can be expressed as: W = 0.0034 L^2.5535 (r² = 0.9903, n = 12, P < 0.01). These findings suggest that H. erectus is a good candidate for commercial aquaculture.     

An improved enzyme preparation for rapid mass production of protoplasts as seed stock for aquaculture of macrophytic marine green algae

Preparation of protoplasts and their subsequent applications for both basic and applied research of marine macroalgae remains largely under developed due to lack of development of reliable methods with consistent yields of viable protoplasts. An improved enzyme preparation with a single commercial enzyme, e.g. 2% Cellulase Onozuka R-10 in 1% NaCl solution, was developed to produce protoplasts rapidly from different green algal genera of Ulva, Enteromorpha and Monostroma. The simple dissolution of enzyme powder in 1% NaCl resulted in exclusion of 2% Macerozyme R-10 from the mixture consisting of 2% Cellulase Onozuka R-10 with 3% NaCl earlier reported as superior for the same algae. Optimal conditions for the isolation of maximum yields of viable protoplasts were found to be with 2% Cellulase Onozuka R-10 incubated at 20 °C for 2 h in 1% NaCl solution with 0.8 M mannitol adjusted to pH 6.0. The protoplast yield with optimized enzyme mixture was as high as 102.8 × 10⁶ cells g^− 1 f. wt for M. oxyspermum while it was in the range of 74.4–88.6 × 10⁶ cells g^− 1 f. wt thallus for seven species of Ulva, and 82.5–95.4 × 10⁶ cells g^− 1 f. wt for three species of Enteromorpha. The regeneration rate of protoplasts isolated using this method ranged from 89 to 92% with normal morphogenesis. The seeding of nylon threads with isolated protoplasts of M. oxyspermum was successful and after 3–4 weeks the entire frame with nylon threads became thick green in color with tiny germlings in laboratory culture. Thus, the method described in the present study allow for rapid mass production of viable protoplasts that could be potentially used as a source for seed material for mariculture and for other applied phycological research.     

Growth and survival of larval and juvenile cobia Rachycentron canadum in a recirculating raceway system

Cobia Rachycentron canadum is a fast-growing, pelagic marine species that has recently attracted aquaculturists in both the research and commercial sectors. The typical method of grow-out for this species is in outdoor systems where production is limited to locations and seasons conducive for adequate growth and survival. Expanding the culture of cobia to indoor recirculating aquaculture systems (RAS) would allow for the production of fingerlings throughout the year and extend production to cooler regions. Two rearing trials were conducted to examine the growth and survival of cobia from hatching through 4 (trial 1, T1) or 35 (trial 2, T2) g in RAS. Cobia larvae were reared in circular tanks placed in a raceway to control water temperature and quality. During early juvenile grow-out, fish were transferred without grading to a second raceway on 29 dph (T1) or over a period of grading from 29–43 dph (T2). Larval growth (1–22 dph) measured as standard length was similar for both trials ranging from  3.9 to 14.7 mm. However, larval growth measured as wet weight (0.033 g, T1; 0.026 g, T2) or dry weight (5.7 mg, T1; 3.9 mg, T2) was significantly greater on 22 dph during T1 as was the ratio between myotome height and standard length. These differences may have resulted from an increase in initial densities from 8.7 larvae l^− 1 (T1) to 14.7 larvae l^− 1 (T2) which apparently caused an increase in food competition and overall aggression. During juvenile grow-out, cobia reached 4.0 g on 43 dph in T1 and 35.4 g on 71 dph in T2 matching weights achieved during grow-out in outdoor ponds. Over the course of both trials, survival was similar to that reported in outdoor ponds. Mean survival (± S.D.) during the early rearing phase (hatching through 29 or 43 dph) averaged 13.2 ± 3.2 % and 10.4 ± 3.2 % corresponding to final densities of 0.9 ± 0.2 and 1.2 ± 0.4 fish/l for T1 and T2, respectively. During the first grow-out phase (29–43 dph), survival of fish moved into the open raceway was 64.5% in T1 and 88.7 % in T2. Survival of cobia during the second grow-out phase (43–71 dph) for T2 was 92.5%. The results of this study indicate that cobia can be successfully cultured in indoor systems from hatching through at least 35 g without negatively affecting growth or survival.     

Welfare aspects of live chilling and freezing of farmed eel (Anguilla anguilla L.): neurological and behavioural assessment

The overall objective of the study was to evaluate a slaughter method of eels, which consisted of chilling until their body temperature was <5 °C for stunning, and subsequently placing them in cold brine at −18 °C for 15 min for killing. Three distinct experiments and a control were performed.

Firstly, 19 eels with an average live weight of 758±44 g were restrained and equipped with EEG, ECG electrodes and a temperature sensor inside the body. Then, they were placed in the ice water. Indices for the induction of unconsciousness and insensibility were the appearance of theta and delta waves and no response on pain stimuli, which disappeared at a body temperature of 8.0±2.1 °C after 12±5 min in 15 eels. The responses to pain stimuli did not disappear in three eels. Within a confidence level of 95%, the percentage of eels that is not effectively stunned during the procedure in ice water of <5 °C was at least 5%. The heart rate decreased from 24±10 beats/min (n=14) to 7±4 (n=11) and became irregular during cooling down. When placed in the brine water of −18 °C, the EEG showed rapid and extreme depolarisation of the membranes, which started after 27±17 s (n=18). The ECG showed fluttering of the heart in all eels. None of the eels recovered after this procedure.

For 10 eels with an average live weight of 128±27 g, it was observed that the body temperature decreased from 17.1±0.6 to 4.0±0.5 °C in the ice water. After 15 min in the brine water of −16.1±2.2 °C, the body temperature decreased to −3.1±2.3 °C.

Finally, three groups of seven eels and eight single eels were placed in ice water of −0.0±0.1 °C. The observation of unrestrained eels revealed four phases. Animals were (1) swimming around in the water, (2) attempting to escape from the ice water, (3) pressing their nose to the wall or corner while showing clonic muscle cramps, and finally (4) breathing only, while all other muscle activity was totally suppressed. Afterwards, they were transferred to cold brine at −18 °C, and none of the eels recovered.

The eight control eels, which were transferred to water at 18 °C, swam around, except for one that was lying in an S-shape position at the bottom. After 570 and 605 s, two eels tried to escape from the box.

The obtained results show that the eels, which were transferred from water at 18 °C to ice water, might be stressed, a specific behaviour and an irregular heart rate were observed. From an animal welfare point of view, it is therefore not recommended to stun eels by live chilling. Moreover, at least 5% of the eels will not be stunned at a body temperature of <5 °C. Placing eels in brine water of −18 °C is an effective method to kill the eels before slaughter. However, it cannot be recommended to place conscious eels in cold brine water, because it takes more than 27 s before unconsciousness may be induced.     

Effect of starvation on the growth and survival of post-larval abalone (Haliotis iris)

The starvation tolerance of post-larval abalone (Haliotis iris) was determined by examining post-larval growth and survival after various periods of starvation. Competent larvae (10 days old at 16°C) were induced to attach and metamorphose with 2 μM GABA. Post-larvae were either fed diatoms (Nitzschia longissima) or starved. In Experiment 1, post-larvae were starved immediately after metamorphosis for periods of 1, 2, 4, 8, 15, 20, 25 and 30 days. Starved post-larvae grew relatively well for several days after metamorphosis despite the absence of food (averages of 10.4 and 17.8 μm shell length (SL) per day after 8 days for two batches). Subsequent growth was minimal, averaging 1.7 and 0.7 μm day⁻¹ over 6–7 days for the two batches. There was no clear relationship between period of starvation and growth rate when fed. Mean daily growth rate over 3 weeks when fed ranged from 15–22 μm day⁻¹. However, the duration of starvation did have a significant effect on survival. Survival of post-larvae fed after 1–2 days of starvation was 90–100% after 3 weeks of feeding. Longer starvation periods gave progressively lower survival and post-larvae starved for 30 days all died within a week of being fed. In Experiment 2, post larvae were fed for 3 weeks after metamorphosis, then starved for 0, 3, 7, 14 or 21 days. Growth rates of starved post-larvae averaged only 5–6 μm day⁻¹ in the first week (vs. 30 μm day⁻¹ in controls), and later declined to zero. Growth resumed within a week following return to food, but the 14- and 21-day starvation treatments took 2 weeks to reach growth rates comparable to controls. The no-starvation controls and the 3- and 7-day starvation treatments all had >70% survival over 4 weeks after return to food. Survival in the 14- and 21-day starvation treatments was 15–20%, with almost all mortalities occurring in the first week after return to food. These data suggest that Haliotis iris post-larvae are relatively tolerant of starvation, so abalone farmers have a week or so to remedy food shortages before major post-larval mortality begins.     

Extenders and cryoprotectants for cooling and freezing of piracanjuba (Brycon orbignyanus) semen, an endangered Brazilian teleost fish

The aim of this study was to develop a protocol for semen storage of piracanjuba (Brycon orbignyanus) by both cool storage at 4 °C and cryopreservation at − 196 °C. Semen was diluted in some fish semen extenders (Exp. 1) or in extenders combined with the antibiotic gentamycin sulfate (Exp. 2) and stored at 4 °C. Sperm motility was estimated every 24 h. Then, the effects of egg yolk (0 and 5%), cryoprotectants (dimethyl sulphoxide — DMSO, methanol, and methylglycol) and extenders (NaCl 154 mM, BTS™ Minitub and M III™ Minitub) on semen cryopreservation were evaluated (Exp. 3). Semen was added to each of eighteen cryosolutions (2 yolk concentrations × 3 cryoprotectants × 3 extenders), aspirated into 0.5-mL straws, frozen in nitrogen vapor (Taylor-Wharton, CP 300, “dry shipper”) and stored at − 196 °C. Sperm motility was evaluated after thawing at 60 °C-water bath for 8 s. The three cryosolutions that produced the highest post-thaw sperm motility were used again to freeze semen. Post-thaw semen quality was then evaluated under three tests: sperm motility, the percentage of live spermatozoa and hatching rate (Exp. 4). Piracanjuba semen diluted (1:10 total volume) in NaCl 200 mM or in Saad solution (NaCl 200 mM, Tris 30 mM) maintained motility above 35% for as long as 7 days, at 4 °C. Motility of only 7% was observed on undiluted semen after 3 days at 4 °C. There was neither beneficial nor detrimental effect of gentamycin on sperm motility at 250 μg/mL. Egg yolk addition to the cryosolution was beneficial in samples cryopreserved in NaCl 154 mM and in M III™, but detrimental for samples cryopreserved in BTS™. Methylglycol was the most effective cryoprotector compared to DMSO and methanol. Motility and percentage of live spermatozoa were similar among semen cryopreserved in NaCl–yolk, M III™–yolk and BTS™, all containing 10% methylglycol, but lower than fresh control. Hatching rates of eggs fertilized with sperm cryopreserved in NaCl–yolk or BTS™ were higher than for eggs fertilized with sperm cryopreserved in M III™–yolk, but lower than control fertilizations. The semen cryopreservation protocols developed here will be used to set up a gene bank for endangered piracanjuba populations.     

Constructed wetlands as a treatment method for effluents from intensive trout farms

This study examined the effects of different hydraulic loading rates on the treatment efficiency of subsurface flow (SSF) constructed wetlands treating effluents from trout farming over a period of 6 months. Six identical wetland cells with a pre-sedimentation zone of 9.6 m² and a root zone of 23.6 m² were loaded with effluents from intensive trout farming (> 2.1 kg feeding stuff per L/s and day). The total runoff of 13.2 L/s was treated in the wetland cells, where two duplicate cells received equal hydraulic loads of 3.9, 1.8 and 0.9 L/s. All examined wetland cells had significant treatment effects on the nutrient fractions containing particulate matter [total nitrogen (TN), total phosphorous (TP), biological oxygen demand in 5 days (BOD₅), chemical oxygen demand (COD), and total suspended solids (TSS)].

Efficiency was between 5.5% for TN and 90.1% for TSS. The SSF wetland also had a high treatment effect on total ammonia nitrogen (TAN), with efficiencies of 61.2 to 87.8%. Nitrate nitrogen (NO₃–N) and phosphate phosphorous (PO₄–P) showed a significant increase in the wetland effluent by 8.4 to 209%. Nitrite nitrogen (NO₂–N), had no significant, or significant effluent increase depending on the inflow rate. Treatment efficiency for particulate nutrients and TAN increased with decreasing hydraulic load, while the differences between 1.8 and 0.9 L/s were not significant. The treatment efficiency for TP was constant for all cells, at around 40%. The wetland receiving 3.9 L/s was over-flooded after 10 to 12 weeks due to colmatation. Nevertheless, the wetland still showed high treatment efficiencies. For commercial trout farms, SSF wetlands are a highly effective method of effluent treatment. A hydraulic load of 1 L/s on 13.3 m² wetland area (1.8 L/s on the examined wetland) seems most suitable. Higher loads lead to accelerated wetland colmatation, while lower loads waste space.     

A soft technology to improve survival and reproductive performance of Litopenaeus stylirostris by counterbalancing physiological disturbances associated with handling stress

The consequences of handling stress (fishing, transfer, eyestalk ablation) on shrimp broodstock are poorly documented. The weakness of farmed shrimp, Litopenaeus stylirostris, during winter is a major problem in New Caledonia, because of seasonal climate (tropical–sub-temperate). The transfer of broodstock in winter from earthen outdoor ponds to indoor maturation tanks in the hatchery (T = 20 °C, Salinity = 35‰, fed shrimp) usually leads, after 48 h, to high mortality (up to 70%). Eyestalk ablation to induce ovarian maturation in females leads to further mortality.

Starting from a background analysis of physiological disturbances (initial osmoregulatory imbalance) associated with handling stress (Wabete, N., Chim, L., Lemaire, P., Massabuau, J.-C., 2004. Caractérisation de problèmes de physiologie respiratoire et d'échanges ioniques associés à la manipulation chez la crevette pénéide Litopenaeus stylirostris à 20 °C. Styli 2003. Trente ans de crevetticulture en Nouvelle-Calédonie. Ed. Ifremer. Actes Colloq. 38, 75-84.), we developed a protocol using a soft technology, based on modifications of water salinity, temperature and feeding regime. The aim was to minimize problems of osmoregulatory imbalance and associated mortalities. The protocol we developed, called the LSD OT protocol (Low Salinity and Diet, Optimal Temperature), was first evaluated on sub-adult shrimp (20–25 g) and then applied to broodstock. Survival after transfer and following eyestalk ablation, as well as reproductive achievement (spawning rate, nauplii number) was considerably improved when shrimps were transferred under “physiological comfort” i.e. warmed isosmotic water (26 °C and 26‰) and unfed for 3 d. This new handling protocol, based on a better control of salinity, temperature and feeding conditions, has been transferred successfully to private hatcheries and already contributes to an increased profitability of New-Caledonian shrimp industry.     

Purification and partial characterization of Cu, Zn superoxide dismutase from haemolymph of Oriental river prawn Macrobrachium nipponense

The copper plus zinc superoxide dismutase (Cu, Zn-SOD) was purified from haemolymph of the Oriental river prawn, Macrobrachium nipponense and partially characterized. Partial protein precipitation in crude extract was affected by using heat treatment and (NH₄)₂SO₄ fractionated precipitation methods. Fractionation of superoxide dismutase was performed by DEAE-cellulose 32 ion-exchange chromatography and followed by CM-cellulose cation-exchange chromatography. The molecular weight of it was about 66.1 kDa, as judged by SDS polyacrylamide gel electrophoresis. The enzyme was sensitive to cyanide and H₂O₂, and contained 1.08 ± 0.14 atom of copper and 0.98 ± 0.11 zinc per subunit shown in atomic absorption spectroscopy, which revealed that purified SOD was Cu, Zn superoxide dismutase. The purified enzyme had an absorption peak of 269 nm in ultraviolet region and the enzyme remained stable at 25–45 °C within 60 min. But it was rapidly inactivated at higher temperature (50 °C). The activity of purified shrimp Cu, Zn-SOD was remained stable over the range pH 5.8–8.3. Treated with 10 mM mercaptoethanol, the enzyme activity significantly increased. However, the enzyme activity was obviously inhibited by 10 mM CaCl₂, ZnCl₂, SDS, EDTA–Na₂ and 1 mM and 10 mM K₂Cr₂O₇. The results showed that it might be a kind of EC-SOD. And it was the first report of some characterizations of this EC-SOD in M. nipponense.