Efficacy, tissue distribution, and residue depletion of oxytetracycline in WS-RLP infected California red abalone Haliotis rufescens

Oxytetracycline (OTC) was recently demonstrated to be an effective therapeutant for California red abalone (Haliotis rufescens) infected with the agent of withering syndrome (WS), a Rickettsia-like prokaryote (WS-RLP). This study examined the influence of temperature on the efficacy and pharmacokinetics of oral OTC treatments on WS-RLP infected red abalone, H. rufescens. Medication was administered in an artificial diet containing 1.85% active OTC at a rate of 103.4 mg/kg abalone for 10, 20 and 30 days at both 13.4 °C and 17.3 °C. Drug residue concentrations, WS-RLP burden and WS-associated pathological changes within foot muscle and digestive gland were measured 3, 17, 23, 42, 63, 81, 102, 122, and 160 days after OTC treatment. In all samples the highest concentrations of OTC were detected on the first sample day. Furthermore, all sampling periods showed significantly less OTC in foot muscle samples relative to corresponding digestive gland samples (p < 0.001). While drug depuration occurred rapidly from foot muscle, unusually high concentrations and long depuration times were measured within the digestive gland. The pharmacokinetic trends in both the foot muscle and digestive gland at 13.4 °C were adequately described by a one-compartment model, while the digestive gland depuration models within the 17.3 °C treatments conformed to a two-compartment model. At both temperatures treatment duration gave rise to significant differences in OTC accumulation and depletion (p < 0.001). Despite this, terminal elimination rate constants were similar. Estimated half-lives were similar among all treatments for digestive gland (23.8–27.5 d) and all treatments for the foot muscle at 13.4 °C (10.7–13.4 d; calculated only for the 13.4 °C treatments). At both temperatures all three durations of drug treatment led to significant reductions in WS-RLP prevalence, foot muscle atrophy, and mortality. WS-RLP re-challenge trials initiated 44, 88, and 122 days following the 10-day treatment at 17.3 °C showed reduced susceptibility to re-infection for up to 88 days after treatment, suggesting that digestive gland OTC residues confer long-term resistance to pathogen. Atomic absorption spectroscopy conducted on both foot muscle and digestive gland found significantly higher concentrations of iron, zinc, and manganese in the digestive gland, offering an explanation behind the underlying mechanism of OTC retention for extended periods within this tissue.     

Effects of starvation and recovery on the survival, growth and RNA/DNA ratio in loliginid squid paralarvae

The ability of Loligo opalescens paralarvae to resist and recover from starvation was examined by measuring their survival, growth rate and RNA/DNA ratios during starvation and refeeding. Paralarvae were fed Artemia sp. nauplii, zooplankton and mysid shrimp. Fourteen days after hatching they were separated into five feeding treatments: a control treatment (food was always available) and treatments starved for 2, 3, 4 and 5 days, and then refed. Each day, 5–7 paralarvae from each treatment were anesthetized to measure mantle length and wet weight (WW), and then RNA and DNA were extracted using an ethidium bromide fluorometric technique. Paralarvae did not survive 4 and 5 days of starvation, showing that at 15 days of age and at 16 °C the limit to recovery was 3 days of starvation. Paralarvae starved for 2 and 3 days showed compensatory growth that mitigated the effects of starvation, in that at the end of the experiment (10 days), they attained mean final body weights similar to the control treatment. Differences in the RNA/DNA ratios between control and starved paralarvae were detected within 2 days of food deprivation. For paralarvae starved 2 and 3 days, it took 1 day after refeeding to attain RNA/DNA ratios not significantly different from the control treatment. Additionally, RNA/DNA ratios were highest during the day (0800, 1200, 1600 h) and lowest at night (0000, 0400 h), suggesting daytime feeding activity. Growth rates ranged from − 14% to 21% WW day^− 1 and the resulting equation between RNA/DNA ratio and growth rate (GR) of paralarvae was GR = 1.74 RNA/DNA − 11.79 (R² = 0.70). After starvation, there was a reduction in growth variability in all starved treatments, while growth variability remained high in the control treatment. Findings from the present study indicate that nucleic acids are a valid indicator of nutritional condition and growth in squid paralarvae.     

Ontogeny of the digestive tract in shi drum (Umbrina cirrosa L.) reared using the mesocosm larval rearing system

Histological changes of the digestive tract were studied in shi drum (Umbrina cirrosa) from hatching until 41 days post hatching (dph), when the fry had a mean (±S.D.) total length (TL) of 32 ± 2 mm and wet weight (WW) of 0.42 ± 0.07 g. Larvae were reared using the mesocosm technique, the most natural among commercially employed rearing methods for marine larvae. Shi drum opened their mouth at 2 dph (2.78 ± 0.09 mm TL), at which time 90% of the larvae already had an inflated swim bladder. The differentiation of the digestive tract into buccopharynx, esophagus, and anterior and posterior intestine was completed by 3 dph (2.82 ± 0.07 mm TL), 1 day after the onset of exogenous feeding. The alimentary canal started coiling and formed its first loop at 2 dph, while the pancreas and liver were differentiated at 3 dph. Yolk sac reserves lasted until 7 dph (4.3 ± 0.1 mm TL), suggesting a brief period of endogenous and exogenous feeding. The first esophageal goblet cells appeared at 7 dph containing acid mucins and at 8 dph taste buds appeared on the buccopharyngeal epithelium. The stomach was morphologically differentiated at 9 dph (5.5 ± 0.1 mm TL) when gastric glands became abundant in the cardiac region, and the first pyloric caeca appeared at 14 dph (10.1 ± 0.9 mm TL). Supranuclear eosinophilic vacuoles were observed in the posterior intestine between 3 and 11 dph (6.3 ± 0.9 mm TL). Their number decreased as the stomach differentiated, suggesting a change in the protein digestion mechanism. The results of the study suggest a rapid development of shi drum and its digestive system and underline the possibility of weaning larvae to artificial feed even earlier than the 12 dph employed in the present study.     

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.     

Effect of low water temperature on viral replication of white spot syndrome virus in Procambarus clarkii

This study evaluated the effect of low water temperature (10 ± 1 °C) on viral infection and replication of white spot syndrome virus (WSSV) in crayfish, Procambarus clarkii, under standardized conditions. Crayfish were (i) maintained at 24 ± 1 °C before challenge and 10 ± 1 °C afterwards, or (ii) maintained at 10 ± 1 °C before challenge and 24 ± 1 °C afterwards. No mortality was observed when crayfish were held at 10 ± 1 °C after challenge, but mortality reached 100% when they were transferred to 24 ± 1 °C. Competitive PCR showed that viral levels at 10 ± 1 °C rose from 10⁶ to 10⁸ copies/mg of gill tissues, while at 24 ± 1 °C levels increased from 10⁶ to 10¹⁰ copies/mg of gill tissues during the same time interval. These results showed that a low water temperature of 10 ± 1 °C could reduce viral replication when compared to 24 ± 1 °C but could not prevent it.     

Oogenesis and relevant changes in egg quality of abalone Haliotis discus hannai during a single spawning season

Oogenesis of abalone Haliotis discus hannai was examined histologically during a single spawning season using broodstock of various maturation conditions, which were controlled by effective accumulative temperature (EAT). The quality of eggs spawned was determined in relation to oogenesis. For histological examinations, three to five females were sacrificed at 300, 600, 850, 1050, and 1150 °C days EAT, without induction of artificial spawning. Other females were successfully induced to spawn at 700 °C days EAT and were reared following spawning. Three of these females were then sacrificed every 200 °C days EAT until 1300 °C days EAT. Gonad histology showed that two oocyte cohorts matured in H. discus hannai ovaries during a single spawning season. One mature oocyte cohort could be spawned in multiple times. The second oocyte cohort started developing after the first oocyte cohort had been spawned or reabsorbed, and became fully mature 400 °C days EAT after the first cohort was depleted. For egg quality measurements, three to five females were successfully induced to spawn at 850, 1050, 1150, 1900, and 2350 °C days EAT (Experiment 1). Three females were induced to spawn twice, at 700 and 1500 °C days EAT, resulting in two batches of eggs from the same individuals (Experiment 2). Total lipid and protein content of eggs were measured and were greater in eggs from the second cohort than in eggs from the first cohort. No carbohydrates were detected in eggs and there was no difference in cytoplasm volume between the two cohorts. In hatcheries producing H. discus hannai, it is important to increase post-larval starvation tolerance by increasing the quality of eggs, to yield higher and more consistent survival. The results of this study suggest that H. discus hannai hatcheries should use eggs from the second oocyte cohort, which are of higher quality, rather than eggs from the first oocyte cohort.     

Impact of starvation and handling stress on rigor development and quality of Atlantic salmon (Salmon salar L)

Farmed Atlantic salmon (Salmo salar L) were fed to satiation or starved for 35 d, and subjected to acute pre-slaughter stress or careful handling before slaughtering. Rigor development, post-mortem energy metabolism and quality variations were analysed during 72 h cold storage. The body weight averaged 2.9 and 3.5 kg of the fed and starved salmon, respectively. The starved salmon had lower condition factor (0.99 vs. 1.11) and higher slaughter yield (92.6 vs. 87.5%), but the fat content was similar (11.8–12.9%) in the fed and starved salmon. The initial glycogen level was lower in the starved salmon (21 vs. 29 μmol g^− 1), whereas initial ATP levels (6.4–6.6 μmol g^− 1) were not affected by starvation. Pre-slaughter stress accelerated rigor development, stimulated lactate formation through post-mortem glycolysis, and increased the breakdown of ATP and CP. The effect of stress on rigor development was less pronounced in starved salmon, and a significant interaction was observed between nutritional status and stress. Rigor development was closely correlated to ATP breakdown, especially in fillets. Acute stress accelerated flesh softening during ice storage. After 72 h storage, the fillets of the starved salmon exposed to careful slaughter handling had the significantly firmest texture, whereas the most intense fillet colour was observed in the starved/stressed salmon. Liquid leakage from the muscle during cold storage was lower in the starved salmon, but pre-slaughter stress had no significant impact. It is concluded that a starvation period of five weeks can improve the resistant to acute stress prior to slaughtering of Atlantic salmon that in turn hampers rigor development.     

Physiological changes in rainbow trout held under crowded conditions and fed diets with different levels of vitamins E and C and highly unsaturated fatty acids (HUFA)

Rainbow trout (Oncorhynchus mykiss) maintained in crowded (100 kg m^− 3) and uncrowded (20 kg m^− 3) conditions were fed 42 days with five experimental diets having different levels of vitamin E (25.6 and 275.6 mg kg diet^− 1), C (0 and 1000 mg kg diet^− 1) and HUFA (highly unsaturated fatty acids, 12.5 and 320.5 g kg diet^− 1): −E−HUFA, −E+HUFA, +E−HUFA, +E+HUFA, −C+E+HUFA. Cortisol, plasma metabolites, tissue glycogen, fish composition, and tissue fatty-acid profile were evaluated at the end of the experimental period. In general, no changes in cortisol levels were associated with crowding, although +E+HUFA and −C+E+HUFA fish showed higher levels (mean ± SE, 55.5 ± 11.1 and 78.0 ± 11.3 ng ml^− 1) as a consequence of a possible interaction between chronic crowding and diet composition. Protein and glucose con-centration in plasma displayed no effect of crowding, but liver glycogen showed a general tendency to decrease in −E−HUFA, −E+HUFA, +E−HUFA, +E+HUFA, −C+E+HUFA crowded groups (70.2 ± 2.1, 52.1 ± 2.5, 73.4 ± 7.4, 91.7 ± 3.3, 74.2 ± 8.4 mg g^− 1 tissue, respectively) compared to uncrowded groups (108.9 ± 14.2, 82.7 ± 8.8, 92.4 ± 10.7, 99.1 ± 10.0, 103.5 ± 15.6 mg g^− 1 tissue, respectively), thus proving significant in −E+HUFA fish. Variations in total lipids, triglycerides, total cholesterol and HDL as well as LDL cholesterol in plasma were manifested under crowding conditions, displaying a certain influence of vitamin E and HUFA dietary content. Final body composition, in general, showed no change attributable to fish density, but some differences associated with diet composition were found in lipid and moisture percentages of crowded fish. Liver and muscle fatty-acid profile revealed a clear effect of the dietary lipid source that was more evident in muscle than in liver at normal fish density, and in some cases this effect was modulated by dietary vitamin E and C content and fish-culture conditions.     

Spawning, early development and first feeding in the gobiid fish Priolepis nocturna

The reproductive behavior, embryonic development and early larvae of Priolepis nocturna are described. Three pairs of P. nocturna began spawning 41 days after acquisition and maintained a 5–10 day spawning cycle lasting beyond several months. Spawning was initiated by the female who signaled her readiness to spawn by displaying to the male. Egg clutch size averaged 1578 ± 51.23 eggs and ranged from 268 to 3121. Egg length averaged 0.82 ± 0.01 mm total length (TL) and ranged from 0.75 to 0.90 mm. Egg width averaged 0.51 ± 0.51 mm total width (TW) and ranged from 0.49 to 0.52 mm. Fertilized eggs were ovoid in shape and attached to the ceiling of provided shelters via adhesive filaments at the proximal end. Hatching rates averaged 97.3 ± 0.51% and ranged from 91.9 to 99.8%. Larvae measuring 1.89 ± 0.04 mm TL hatched 121 ± 0.5 h post fertilization and did not rotate position prior to hatching. Skeletal elements of the chondrocranium were simplistic and dominated by the hyoid, hyomandibulosymplectic cartilage, ethmoid and Meckel's cartilage in first feeding larvae. No elements were added to the cranial architecture by 5 days post hatch (DPH) when larvae measured 2.05 ± 0.04 mm TL. First feeding larvae consumed only dinoflagellates and tintinnids suggesting that feeding was constrained by a poorly developed feeding mechanism. Embryology and larval development are described to 5 DPH.     

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.     

Optimal conditions for egg storage, incubation and post-hatching growth for the freshwater turtle, Chelodina rugosa: Science in support of an indigenous enterprise

Incubation of northern snake-necked turtle (Chelodina rugosa) eggs and subsequent sale of hatchlings for the pet industry has the potential to provide culturally suitable employment for indigenous communities in northern Australia. Developmental arrest in response to egg inundation is unique to C. rugosa. Eggs can be stored under water for up to 10 weeks without appreciable impact on egg or embryo survival, allowing the transport and sale of eggs into niche markets without high levels of mortality, and permitting eggs to accumulate in diapause until there are sufficient numbers to incubate as batches. Eggs that are not inundated or inundated for short periods experience similar survival rates to eggs inundated for lengthy periods. Incubation temperature influences embryo survival and development period in C. rugosa. Embryonic survival is greatest at 26 °C, steadily declining as temperature increases to 32 °C. A similar increase in incubation temperature decreases incubation period by approximately 40 days, however almost half of this variation is attributed to the increase in incubation temperature from 26 to 28 °C. Hatchling growth in C. rugosa is characterized by two phases. There is an initial phase of relatively slow growth under the partial influence of initial egg size and incubation duration, followed by a second phase of relatively rapid growth under the partial influence of water temperature and mass at hatching. Post-hatching survival is negatively correlated with duration of egg inundation and water temperature. Evidence suggests that inundation of C. rugosa eggs for 6 weeks, incubation of embryos at 28 °C and raising hatchlings in 28 °C water will yield the best overall outcomes.     

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.     

Inhibition of sexual maturation in tank reared haddock (Melanogrammus aeglefinus) through the use of constant light photoperiods

The haddock (Melanogrammus aeglefinus) is believed to be a potential candidate for aquaculture in the Atlantic coastal countries including the UK, Norway and Canada. However, under culture conditions, haddock will sexually mature prior to the attainment of a suitable harvest weight. Therefore, a long term tank based experiment was performed where three populations of haddock (hatched spring 2002, approximately 150 fish per population) were exposed to either a simulated natural photoperiod (SNP) or SNP until January or July 2003 and thereafter continuous illumination (Jan LL or July LL respectively) with individual growth rate and maturation status being recorded until July 2004 (27 months post-hatch). While the SNP treated population matured in the spring of 2004 (two years post-hatch) with 88% of the population being observed to release gametes, no mature individuals were observed in either of the LL treated populations and furthermore there was no evidence of gonadal development or elevation in sex steroids (testosterone, estradiol-17β or calcium). The application of LL appeared to directly stimulate the growth rate of haddock (e.g. 14 to 27% increase in weight thermal growth coefficient) and improve food conversion rates. At the end of the trial there was an approximate 50% increase in wet weight (SNP: 647 ± 53 g, Jan LL: 982 ± 34 g and July LL 985 ± 33 g), however no significant difference in weight in relation to the length of LL exposure was observed. Plasma melatonin analysis revealed that the natural diel rhythm had been inhibited in both LL treated populations. Along with highlighting the similarity in reproductive entrainment between haddock and other gadoids, these results demonstrate how photoperiod manipulation could be used as a management strategy to improve growth performance in farmed haddock stocks.     

Energy and nitrogen partitioning in 250 g Atlantic cod (Gadus morhua L.) given graded levels of feed with different protein and lipid content

The effects of feed intake level on energy and nitrogen partitioning were studied in juvenile Atlantic cod (250 g) fed two fish meal based diets differing in protein and lipid content (54:31 and 65:16) at 10 °C. Replicate groups of cod were feed deprived for 32 days or fed one of the two diets at 25, 50, 75 or 100% of group satiation for 60 days. Feed intake and oxygen consumption were measured daily and weights and chemical composition of carcass, liver, viscera and whole body were measured at start and end. Diet digestibilities were assessed in a separate experiment.

The whole body and carcass growth rates at a given feed intake did not differ between dietary groups, but the liver grew faster in the fish fed the low protein diet, resulting in higher hepatosomatic indices at the end of the experiment in the groups fed this diet.

The efficiency of utilisation of digestible nitrogen for growth (k_DNg) was higher for the low protein diet (0.73 ± 0.02) than for the high protein diet (0.53 ± 0.05), resulting in higher nitrogen retention at a given nitrogen intake. No difference in percentage nitrogen retention was seen in full-fed fish however (31.2 ± 2.5 and 28.4 ± 1.6% for the low protein and high protein diets, respectively). This can be explained by higher nitrogen intake in the fish fed the high protein diet, resulting in a smaller proportion of the intake being used for maintenance.

There was no difference in energy utilisation between dietary groups. The digestible energy requirement for maintenance (DE_maint) was 53.8 ± 0.9 kJ kg^− 1 d^− 1 (42.3 ± 0.7 kJ kg^− 0.8 d^− 1) and the utilisation efficiency for growth (k_DEg) was 0.80 ± 0.02. The energy retention in full-fed fish was 31.3 ± 3.5 and 31.7 ± 1.0% for the low protein and high protein diets, respectively. The deposited energy was distributed in approximately equal proportions in the liver and carcass, whereas viscera accounted for a minor proportion. At a given energy intake, the fish fed the high protein diet deposited more energy in the carcass and less in the liver than did those fed the low protein diet.     

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.     

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.     

Influence of different food sources on the initial development of sexual recruits of reefbuilding corals in aquaculture

In coral aquaculture, sexual reproduction increasingly plays an important role for serving trade and reef restoration purposes. However, until coral juveniles reach a semi-stable size which makes them less vulnerable against algal growth and sedimentation, high mortality rates may occur in the first several weeks to months after settlement. In the present study, the influence of several food sources on the growth and survival of newly settled primary polyps was studied under laboratory conditions for 5 months. In order to estimate effects on specimens of both reproductive modes, experiments were carried out with the brooder, Favia fragum, and with the broadcast spawner, Acropora tenuis. Primary polyps kept in 2-liter aquaria were daily fed with freshly hatched Artemia salina, the micro algae Phaeodactylum tricornutum and a commercially available dry food (Nori Micro, Zoolife®), respectively, at various concentrations. Growth rates in both species were significantly higher in the Artemia treatment with maximum rates of 9.4 ± 4.9 mm² (mean ± S.D.; d = 5 months) for F. fragum and 26.8 ± 10.3 mm² (d = 5 months) for A. tenuis compared to all other treatments and the control (no additional food). Survival in F. fragum was overall higher than 60% in all treatments with maximum rates of 85.0 ± 12.6% at the highest Artemia concentration. Survival rates in A. tenuis ranged from 28.9 ± 4.7% (lowest Nori concentration) to 86.2 ± 5.9% (medium Artemia concentration). The present study shows that Artemia nauplii may greatly enhance the growth and partly enhance the survival of early sexual recruits which may significantly help to more rapidly overcome the early and most fragile post-settlement stages. As a consequence, the residence time for sexual coral recruits in cost- and labour-intensive hatcheries may be greatly reduced.     

Diurnal variation of tryptic activity in larval stage and development of proteolytic enzyme activities of malabar grouper (Epinephelus malabaricus) after hatching

Diurnal variation in tryptic activity and developmental changes in proteolytic enzyme activities of malabar grouper larvae (Epinephelus malabaricus) were examined. Five different groups were prepared for the experiment of diurnal variation of tryptic activity in larvae: larvae were fed Thai-type rotifers Brachionus rotundiformis from the time of mouth opening, fed rotifers from 6 h after mouth opening, 12 h, 24 h and not fed rotifers (starved control). The experimental tanks were placed in temperature-controlled baths at 28 °C under 24 h light. Developmental changes in proteolytic activity of trypsin and pepsin-like enzyme were measured from hatching to 57 days after hatching (DAH).

The tryptic activity of all fed groups showed the same pattern, and the diurnal variation of tryptic activity was clearly observed from 3 to 6 DAH. The highest tryptic activities were found at 19:00, and the activities were lowest from 01:00 to 07:00. In contrast, that of non-fed larvae was low compared to the fed groups, however the diurnal variation of tryptic activity was shown same tendency to the fed groups. Interestingly, both groups (fed and non-fed) were exhibited a circadian rhythm under the 24 h light conditions and delaying of first-feeding. Tryptic activity of larvae notably increased from 40 to 45 DAH and markedly decreased at 52 DAH. In contrast to the tryptic activity, that of pepsin-like enzyme clearly increased from 47 to 51 DAH. The results suggest that a functional change of protein digestion occurs from 40 to 50 DAH related with metamorphosis in malabar grouper. These results could contribute to determining appropriate feeding schedules, such as feeding time, frequency and optimal time to change food items, in mass-scale production of the present species.     

Effect of increasing salinity on physiological response in juvenile scallops Argopecten purpuratus at two rearing temperatures

Argopecten purpuratus can be cultivated using Recirculating Aquaculture Systems (RAS) as a method to increase production. In order to determine physiological response of A. purpuratus under different salinities and temperature conditions, two groups of juvenile scallops (small: h = 6.5 mm and large: h = 25.5 mm) were acclimated and close-cultured at salinities of 34, 38, and 42 g/l, at 16 and 22 °C and fed on Isochrysis galbana and Chaetoceros calcitrans. Survival, shell growth and scope for growth were determined at the end of the trials. Survival showed an inverse relationship with temperature and ammonia levels. In small scallops an increase in salinity at 16 °C increased survival. However, this relationship was not evident at 22 °C. On the other hand, salinity did not affect survival of large juveniles. Small juveniles had a lower survival (approximately 40%) than larger scallops (up to 85%) throughout the trials. Oxygen consumption was not affected by salinity. Small scallops showed similar oxygen consumption at 16 and 22 °C but in large juveniles higher values were registered at 22 °C. In large juveniles routine consumption at 16 °C was higher (up to 35%) than standard consumption. This pattern was not evident at 22 °C, suggesting that oxygen demand is higher regardless of feeding condition. NH₄⁺–N excretion rate is inversely related to salinity. Only small juveniles showed a higher NH₄⁺–N excretion rate at 22 °C. Scope for growth was positive in all treatments, although the upper limit of salinity should not be based only in this index. Higher scope for growth values at 38 and 42 g/l was related with a reduction in ammonia excretion and high absorption efficiency. In addition, an increase in salinity produced a reduction in NH₃–N proportion and under hypersaline conditions scallops tended to decrease excretion as a way of osmoconformation. This explains our findings of higher survival rates at higher salinities. Even though the scope for growth is positive at 42 g/l, the osmotic stress reduces the survival chances. The data obtained can be considered useful information for A. purpuratus culture under controlled conditions.     

Efficacy of bithionol as an oral treatment for amoebic gill disease in Atlantic salmon Salmo salar (L.)

This study examined the efficacy of bithionol as an oral treatment for Atlantic salmon Salmo salar affected by amoebic gill disease (AGD). The current commercial management strategy of AGD is a costly 3 h freshwater bath. It is labour intensive and the number of baths needed appears to be increasing; hence, there is an effort to identify alternative treatments. Efficacy was examined by feeding AGD-affected Atlantic salmon twice daily to satiation with bithionol, an antiprotozoal, at 25 mg kg^− 1 feed. Three seawater (35‰, 17 °C) re-circulation systems were used each consisting of three tanks containing 32 Atlantic salmon smolts with an average (± SEM) mass of 90.4 g (± 5.2). Three feeds were examined in the trial including bithionol, plain commercial control and oil coated commercial control. Feeding commenced 2 weeks prior to exposure to Neoparamoeba spp. at 300 cells L^− 1 and continued for 28 days post-exposure. Efficacy was determined by examining gross gill score and identifying percent lesioned gill filaments twice weekly for 4 weeks post-exposure. Bithionol when fed as a two-week prophylactic treatment at 25 mg kg^− 1 feed delayed the onset of AGD pathology and reduced the percent lesioned gill filaments by 53% and halved the gill score from 2 to 1 when compared with both the plain and oil controls during an experimental challenge. There were no palatability problems observed with mean feed intake of bithionol over the trial duration with fish consuming higher levels of the bithionol diet compared to both the oil and plain controls. This study demonstrated that bithionol at 25 mg kg^− 1 feed, when fed as a two-week prophylactic treatment for Neoparamoeba spp. exposure, delayed and reduced the intensity of AGD pathology and warrants further investigation as an alternative to the current freshwater bath treatment for AGD-affected Atlantic salmon.