The effects of supplemental dietary cholesterol on growth, development and survival of mud crab, Scylla serrata, megalopa fed semi-purified diets

The effects of varying levels of dietary cholesterol on growth, development time and survival of mud crab, Scylla serrata megalopa were investigated using semi-purified microbound diets (MBD). Five iso-energetic diets containing different level of cholesterol ranging from 0.14% to 1% of dry weight of the diet were tested. Fifteen megalopa were reared individually for each dietary treatment, and development time and survival were recorded on a daily basis. More than 25% of megalopa from all treatments were able to metamorphose into the first crab stage, suggesting that the endogenous level of cholesterol in the basal diet (0.14%) was sufficient to support development of the megalopa stage of this species. Widest mean carapace width (3.53 ± 0.08 mm) and highest mean dry weight (2.11 ± 0.22 mg) were recorded for juveniles that molted from megalopa fed live Artemia, whereas no megalopa in the unfed control treatment metamorphosed into crabs. The average development time from megalopa to the juvenile crab stage varied between the treatments, where megalopa fed live Artemia or MBD containing 0.2%, 0.4% or 0.8% total cholesterol showed the most synchronized molting (between 8.0 and 9.9 days). Longest development time was recorded for the megalopa fed diets containing 0.14% or 1% total cholesterol (both 11 days). Highest survival (74.3%) was recorded for the megalopa fed a diet containing 0.8% cholesterol. The results of this study are valuable in research to develop formulated diets for mud crab larvae as a replacement for live food in hatchery culture.     

The effects of fish hydrolysate (CPSP) level on Octopus maya (Voss and Solis) diet: Digestive enzyme activity, blood metabolites, and energy balance

As has been demonstrated in previous studies, Octopus maya can be fed on artificial diets. In the present study six different diets were assayed. Five diets were designed to test the effect of percentage of inclusion of fish protein concentrate (CPSP: 0, 5, 10, 15, and 20%) and were offered to octopuses as a specifically designed artificial diet. The sixth diet consisted of frozen crab (Callinectes spp) and was used as control diet. Blood metabolites and energy budget of octopuses were evaluated to determine how CPSP levels modulate the digestive capacity and allow retaining energy for growth. Wild animals (316.4 ± 9.8 g) were used in the study. Results showed that CPSP produced a positive specific growth rate (SGR, % day^− 1) with high value in octopuses fed 15% CPSP level. A maximum growth rate of 0.86% day^− 1 was recorded in these animals, a value that is extremely low when compared with the SGR obtained when animals were fed fresh crab (3.7% day^− 1). In general, blood metabolites were affected by diet composition, indicating that some metabolites could reflect the nutritional and/or physiological status of octopus. Preliminary reference values for O. maya fed crab were found for glucose (0.09 ±0.02 mg/ml), lactate (0.004 ± 0.002 mg/ml), cholesterol (0.16 ± 0.02 mg/ml), acylglycerol (0.14 ± 0.01 mg/ml), protein (0.37 ± 0.04 mg/ml), hemocyanin (1.85 ± 0.04 mmol/l), and digestive gland glycogen (1.86 ± 0.3 mg/g). Total energy content can be used as an indicator of tissue metabolic reserves. In the present study, higher energy content in the digestive gland and muscle was observed in octopuses fed crab, followed by animals fed 15% CPSP. Results from the digestive gland indicated that the retained energy derived from glycogen, suggesting that lipids and protein were the main sources of variation linked with energy content. In general, digestive gland proteases activity and trypsin were induced in octopuses fed 15% CPSP. The capacity of O. maya juveniles to adjust their digestive enzymes to different types of food was evidenced. Essential amino acid content (EAA) of the diet was not a limiting factor. When dietary EAA profiles were compared with O. maya EAA profiles, all dietary EAA resulted in a higher concentration than whole body octopus composition. In the present study, all experimental groups ingested between 3300 and 4106 kJ wk^− 1 kg^− 1 without statistical differences among treatments, indicating that experimental diets were as attractive as crab. Differences were recorded in the proportion of absorbed energy (Ab, %) between CPSP-based and crab meat diets, suggesting digestion limitations associated with artificial diets. The present results indicate that the 15% CPSP diet had characteristics that stimulate digestive enzymes and reduce energetic costs associated with its digestion (HiE or SDA), channeling more biomass production than the other experimental diets.     

Dietary influence on growth and development of flat oyster,Ostrea angasi (Sowerby, 1871), larvae

The influence of algal diet on survival, growth and development of hatchery reared flat oyster, Ostrea angasi, larvae was investigated in a series of uni, binary and ternary algal diet trials. Early stage larvae (140–230 μm shell length) generally grew faster than late‐stage larvae (230–340 μm shell length) when fed unialgal diets. Of the 24 algal diets evaluated, larvae fed unialgal diets of Isochrysis sp. (T. Iso), Nannochloropsis oculata, Tetraselmis chuii or Pavlova lutheri; a binary diet of T. chuii+T. Iso; or ternary diets of T. chuii+T. Iso combined with P. lutheri or N. oculata had the greatest larval growth, survival, development and metamorphosis, in the respective trials. The correlation between growth rate and spat produced in late‐stage larvae was stronger when fed unialgal diets (r = 0.75) than when larvae were fed either binary or ternary diets (r = 0.44 and r = 0.45 respectively). Marked differences in proportion of spat produced (24 h post metamorphosis) were evident among diets producing similar growth rates. For hatchery production of O. angasi larvae, ternary diets of T. chuii+T. Iso combined with either P. lutheri or N. oculata were the best diets to maximize larval growth rate, development and survival 24 h post metamorphosis.     

Performance of different types of diets on experimental larval rearing of endangered Chitala chitala (Hamilton) in recirculatory system

This is the first report on the successful larval rearing of captive bred population of Chitala chitala (Hamilton). C. chitala is one of the endangered fresh water fish species in India for which the development of controlled larval rearing procedures are needed for stock enhancement. Fifteen days old post-hatchlings were stocked for 28 d in a 30 L recirculatory tanks using eight different diets i.e. live feed (tubifex worms, chironomous larvae, zooplanktons,), dry feed (dry tubifex, spirulina, daphnia) and other non-conventional feed (fish eggs and boiled egg-yolk). Fishes accepted all types of diets. The study revealed that specific growth rate (SGR) was higher in post-hatchlings fed on live tubifex worms (2.40 ± 0.72) followed by fish eggs (2.15 ± 0.71), dry tubifex (2.12 ± 0.40), chironomous larvae (1.91 ± 0.44), spirulina (1.79 ± 0.38), daphnia (1.42 ± 0.79) and planktons (1.37 ± 0.77) whereas minimum SGR was recorded with boiled egg-yolk (0.63 ± 0.5). A highly significant difference (p < 0.01) in SGR was observed in fish fed on live feed (tubifex worms, chironomous larvae, planktons, spirulina), dry tubifex and fish eggs whereas for daphnia and boiled egg-yolk it was only significant (p < 0.05). The final mean weight and weight gain showed highly significant difference (p < 0.01) in live tubifex, zooplanktons, spirulina, chironomous larvae, dry tubifex and fish eggs, whereas daphnia and boiled egg-yolk fed larvae showed significant difference (p < 0.05). Highest mean survival rate on day 28 was observed in live tubifex worms (94%) and chironomous larvae (92%). The post-hatchlings reared with spirulina and daphnia showed same survival rate of 88% whereas the lowest mean survival of 66% was recorded in boiled egg-yolk. The experiments showed that captive bred post-hatchlings of C.chitala could be reared in experimental recirculatory system for attaining higher growth and survival during early life stages. However, methods to improve the larval rearing have to be improved further for commercial farming of the species.     

Successful culture of larvae of Litopenaeus vannamei fed a microbound formulated diet exclusively from either stage PZ2 or M1 to PL1

In three separate experiments, harpaticoid copepods Tisbe monozota (alive and dead) and a microparticulate microbound diet were evaluated as alternatives to live Artemia nauplii as food, beginning at either stage PZ2 or M1, in the larval culture of Litopenaeus vannamei. Larvae were cultured in 2 L round bottom flasks at a density of 150 L^− 1 (Experiment 1) and 100 L^− 1 ( 3.2 and 3.3) at 28 °C, 35‰ salinity and 12:12 LD photoperiod, and fed 4×/day^- 1. Larvae were initially fed a mixture of phytoplankton to stages PZ2 or M1 and then fed either live Artemia, live or dead copepods, or a microparticulate microbound diet. The experiments were terminated and all larvae were harvested when more than 80% of larvae had molted to postlarvae 1 (PL1) within any flask representing any of the treatments. The comparative value of the different diets and feeding regimes was determined by mean survival, mean dry weight and total length of individual larva, and percentage of surviving larvae that were PL1. Trypsin activity of samples of larvae from each treatment was also determined. The microparticulate microbound diet effectively served as a complete substitute for Artemia nauplii when fed beginning at stage M1. When fed at the beginning of the PZ2 stage, survival was comparable to that of larvae fed Artemia, but mean dry weight, mean total length, and percent of surviving larvae that were PL1 generally were significantly less. Responses to the feeding of copepods, whether fed dead or live, as a substitute were generally significantly less than those of larvae fed either the Artemia nauplii or the microparticulate diet. Values of trypsin activity (10^− 5 IU/μg^- 1 dry weight) corresponded to the relative proportions of the different larval stages within a treatment, with higher activity being characteristic of early stages. Previously demonstrated successful results with another species of crustacean suggest that the microparticulate microbound diet has characteristics that should be effective in the culture of the carnivorous stages of other crustacean and fish larvae that are currently fed live Artemia nauplii.     

Activity of digestive enzymes along the gut of juvenile red abalone, Haliotis rufescens, fed natural and balanced diets

Two carbohydrases (cellulase, lysozyme), three proteases (trypsin, aminopeptidase and non-specific protease), a non-specific lipase, and semiquantitative tests of 19 digestive enzymes were assayed in different gut sections of juvenile red abalone, Haliotis rufescens, in order to identify the regions where digestion takes place and investigate the extent to which diet composition can modify the digestive capacity of abalone. The abalone were fed either fresh kelp (K) or balanced diets containing 25 or 38% crude protein for 6 months. Enzyme assays were carried out on different sections of the abalone's gut at the end of this period. On a weight-specific basis, the digestive gland was the site containing most of the enzymes. On a protein-specific basis, two main digestion regions were identified: the digestive gland-stomach region that is characterized by high activities of cellulase and lysozyme, chymotrypsin and protease, and the mouth-intestine region with a typically high activity of lipase and amino peptidase. Significant dietary effects were observed on the activity of enzymes, especially in the digestive gland. Abalone fed with 25 and 38% crude protein diets exhibited higher cellulase (39.8 ± 4.6 and 14.2 ± 0.8 mU mg^− 1 protein, respectively) and lysozyme activities (88.0 ± 20.4 and 56.6 ± 15.7 U, respectively) than those fed with fresh kelp (5.5 ± 0.7 mU mg^− 1 protein and 17.1 ± 1.8 U). In contrast, higher protease activity was found in kelp-fed organisms (234.1 ± 20.4 μg product/mg protein) than those fed the 25 and 38% crude protein diets (109.5 ± 20.7 and 119.5 ± 20.5 μg product/mg protein, respectively). Semiquantitative API ZYM assays resulted in no clear food-specific effects on the activity of carbohydrases, proteases, ester hydrolases or phosphohydrolases, yet organ-specific differences were conspicuous in various cases, and generally agreed with quantitative results. It is suggested that the increased carbohydrase activity exhibited by organisms fed the balanced diets resulted from a combination of an increased number of resident bacteria in the abalone's gut and facilitated contact between dietary substrates and digestive cells. The present results indicate that H. rufescens can adjust their enzyme levels in order to maximize the acquisition of dietary protein and carbohydrates. This characteristic can be advantageously used to search for suitable diets in abalone aquaculture.     

Dietary copper requirements of juvenile grouper, Epinephelus malabaricus

To quantify dietary copper (Cu) requirements in grouper, Epinephelus malabaricus, copper sulfate was added to the basal diet at 0, 1, 2, 4, 6, 8, 10 and 20 mg Cu/kg diet providing the actual dietary value of 0.11, 1.66, 2.41, 4.37, 6.56, 8.97, 11.03 and 20.05 mg Cu/kg diet, respectively. Each diet was fed to triplicate groups of grouper (initial body weight 13.35 ± 0.01 g) in a recirculated rearing system for 8 weeks. The Cu concentration in rearing water was monitored during the feeding period and ranged from 1.0-1.5 μg/l. Weight gain (WG) and feed efficiency (FE) were higher (P < 0.05) in fish fed diets with 4.37 and 6.56 mg Cu/kg diet than those in fish fed diets with ≥ 11.03 and ≤ 1.66 mg Cu/kg diet. Hepatic thiobarbituric acid reactive substances (TBARS) value was lowest in fish fed diets with 4.37 and 6.56 mg Cu/kg diet, intermediate in fish fed diets with 11.03 and ≤ 1.66 mg Cu/kg diet, and highest in fish fed the diet with 20.05 mg Cu/kg diet. Differences between each of the three groups were statistically significant (P < 0.05). Both hepatic copper-zinc superoxide dismutase (Cu-Zn SOD) and plasma ceruloplasmin activities in fish fell into four groups. Cu-Zn SOD value was highest in fish fed the diet with 6.56 mg Cu/kg diet, second highest in fish fed the diet with 8.97 mg Cu/kg diet, intermediate in fish fed the diet with 20.05 mg Cu/kg diet, and lowest in fish fed the diet with 0.11 mg Cu/kg diet; plasma ceruloplasmin was highest in fish fed the diet with 6.56 mg Cu/kg diet, second highest in fish fed diets with 2.41, 4.37 and 8.97 mg Cu/kg diet, intermediate in fish fed diets with ≥ 11.03 mg Cu/kg diet, and lowest in fish fed diets with ≤ 1.66 mg Cu/kg diet. All values in each of the four groups were significantly different from the values of three other groups (P < 0.05). Analysis by broken-line regression of WG, hepatic SOD activity and TBARS value and linear regression of whole-body Cu retention of the fish indicate that the adequate dietary Cu concentration in growing grouper is about 4-6 mg Cu/kg diet.     

Influence of dietary soybean meal levels on growth, feed utilization and gut histology of Egyptian sole (Solea aegyptiaca) juveniles

The present study was undertaken in order to determine the effect of a dietary incorporation of soybean meal (SBM) on growth performance, nutrient utilization and gut histology of Egyptian sole Solea aegyptiaca. This species, which is genetically and morphologically close to Solea solea, is currently present in the Mediterranean Sea and reared in some Italian farms, using the same techniques for S. solea production and it could represent a promising new species for Mediterranean aquaculture.Three isoproteic and isolipidic extruded diets (crude protein, 47%, crude fat, 20%) were formulated containing 0 (diet 1), 18 (diet 2) and 30% SBM (diet 3). 540 juveniles (initial body weight of 6.9 ± 0.2 g) were equally distributed into nine 150 l squared tanks (bottom surface: 520 cm²) connected with a recirculation system (temperature 20 ± 1 °C and dissolved oxygen above 6 ppm). Animals have been hand-fed twice a day (at 9.00 a.m. and 5.00 p.m.) at a daily ratio of 1.5% body weight^− 1. Diets were tested in triplicate for 87 days. The fish were weighed at days 28, 57 and 87, and they were sampled at days 0, 57 and 87 for nutrient retention determination. One-way ANOVA with Newman-Keuls' post-test were used to analyze data (P ≤ 0.05).No differences in terms of palatability were observed and any diet has been consumed completely all throughout the experiment. At day 87, fish fed diet 1, 2 and 3 reached weights of 25.8 ± 0.8 g, 27.5 ± 0.9 g and 26.1 ± 1.5 g, respectively and no differences in performances or nutrient retention were found.Histopathological gut examinations have revealed no noticeable differences in the appearances of the intestines between any of diet groups. Intestinal mucosal cells presented well vacuolated upper, with well defined microvillar brush borders. On the basis of the results of this trial, SBM seems to be a good protein source for Egyptian sole and can be added in the diet up to 30% without any reduction in growth rate and no adverse effect on gut histology.     

Effect of dietary supplementation of phospholipids and highly unsaturated fatty acids on reproductive performance and offspring quality of Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards), female broodstock

This study was conducted to determine the optimum phospholipids: highly unsaturated fatty acids (PL/HUFA) ratio in maturation diets for female Chinese mitten crab, Eriocheir sinensis broodstock. Five isolipidtic and isonitrogenous diets were formulated to contain different amounts of pork lard, fish oil and soy lecithin with resulting PL/HUFA levels (%dry weight) of 1.4/0.3 (Diet 1), 1.6/2.5 (Diet 2), 2.3/2.0 (Diet 3), 3.8/1.5 (Diet 4) and 5.1/0.8 (Diet 5). Diet 1 lacked supplemental fish oil and soy lecithin and served as the control. Each of the five formulated diets was fed to a total of 150 female crabs for a period of 7 months. All diet treatments were triplicated with 50 crabs stocked in a pond as a replicate. After 6 months, male crabs were introduced into the ponds where females were kept, mating, spawning and egg hatching (in seawater) occurred in the following month. The nutritional value of various formulated diets was assessed based on survival, gonadosomatic index (GSI), egg production per female, fecundity (eggs/g female weight) of the females fed those diets and egg and larval (newly hatched zoea I larvae) quality.Females fed Diet 1 had the lowest GSI, egg production per female and fecundity while no significant difference were found for survival, hepatosomatic index (HSI) and the percentage of female spawned among the diet treatments (P > 0.05). Although there were no significant differences in egg diameter, egg wet or dry weights among all treatments (P > 0.05), the highest and the second highest proportions of essential fatty acids (EFA), i.e. 20:5n-3 (EPA), 22:6n-3 (DHA) and HUFA were found in the eggs produced by the females fed Diet 3 and Diet 2, respectively. Statistical analysis showed that EPA, DHA and HUFA in eggs produced by females fed Diet 3 were significantly higher than those from the other treatments (P < 0.05). Meanwhile, zoea I larvae from crabs fed Diet 3 had significantly larger carapace length than those from the other four treatments (P < 0.05). The zoea I larvae from crabs fed Diet 2 and Diet 3 also showed generally better tolerance to starvation and osmotic shock.In conclusion, our results indicated that diet included levels of PL/HUFA around 1.6/2.5 or 2.3/2.0 (% dry weight) fed female E. sinensis broodstock supported increased fecundity and elevated level of HUFA in egg, which in turn resulted in improved overall quality of newly hatched larvae.     

Enrichment of channel catfish (Ictalurus punctatus) fillets with conjugated linoleic acid and omega-3 fatty acids by dietary manipulation

An experiment was conducted in aquaria with channel catfish (Ictalurus punctatus) to determine the efficacy of augmenting fillets with conjugated linoleic acid (CLA) and omega-3 highly unsaturated fatty acids (n − 3 HUFA) by feeding diets amended with products containing high levels of these nutrients. Refined menhaden fish oil at 1.5% of diet supplied the n − 3 HUFA. CLA was used at dietary levels of 0.5% and 1% with a preparation that contained approximately 65% isomers of CLA. Corn oil was added to the basal diet at maximum inclusion level for added lipids of 3% for the control diet and to adjust total added lipid content of the other diets to 3%. Average initial body weight was 57.39 ± 0.25 g/fish. Six experimental diets were fed twice daily to four replicate aquaria for six weeks. At that time, fish were group weighed for determination of weight gain and feed conversion. Fillets of six fish per aquarium were recovered and stored at − 80 °C for moisture and total lipid analyses, fatty acid analysis, and sensory evaluation. Results showed feed consumption and feed conversion did not differ (P > 0.05). Significantly (P < 0.05) greater body weight gains were observed only for fish fed the diets with two combinations of CLA and 1.5% fish oil compared to fish fed the diet containing 0.5% CLA and corn oil. Fillet n − 3 HUFA levels were significantly (P < 0.05) elevated for fish fed diets containing fish oil when compared to n − 3 HUFA of fillets of fish fed diets containing either corn oil or CLA and corn oil. Similarly, fillets from fish fed diets amended with CLA contained substantial amounts of CLA of up to 6.4% of total lipids. Fillets from fish fed corn oil or fish oil diets had no CLA. Taste panel evaluation indicated that fillets containing n − 3 HUFA and CLA were essentially without fishy off-flavor and had excellent sensory qualities. Catfish fillets produced by amending diets with sources of n − 3 HUFA and CLA at the levels used in this study would contain elevated levels of these nutraceuticals and could be an important human food source for these healthful fatty acids.     

Evaluation of microalgal diets for the intensive cultivation of the tropical calanoid copepod,Parvocalanus crassirostris

As their natural prey, copepods are considered the ideal diets for hatchery culture of fish larvae. However, intensive cultivation of copepods has proven difficult and low culture productivity is the norm, which severely limits their use in aquaculture. This study investigated the effects of algal diets on a range of productivity‐related parameters, including egg production, hatching success, naupliar and copepodite survival, post‐embryonic development time, population growth and sex ratio of a small‐sized tropical copepod, Parvocalanus crassirostris, which has been identified as a high potential species for aquaculture. The diets tested included three live algal diets: Isochrysis sp. (T‐Iso), Pavlova 50 (Pav) and the diatom Chaetoceros muelleri (Cht); two algal paste diets: Isochrysis sp. (P‐Iso) and Thalassiosira weissflogii (P‐TW), as well as four mixed algae treatments composed of three binary algae diets (T‐Iso + Pav, T‐Iso + Cht and Pav + Cht) and a tri‐algal diet (T‐Iso + Pav + Cht). All parameters examined except sex ratio were significantly affected by the diets. The binary algal diet T‐Iso + Cht produced significant higher total egg production over female lifespan (121.0 ± 5.9 eggs) than all other treatments (P < 0.05). Survival during naupliar and copepodite stage was significantly superior when P. crassirostris were fed either the mixed diet of T‐Iso + Cht or the monoalgal diets of Cht or T‐Iso (P < 0.05) than other diets. These three treatments also had the fastest time of development to adults (ranging from 6.2 to 6.7 days). With an initial 100 adults, the T‐Iso + Cht treatment produced the highest population growth over a 15‐day cultivation period, which was significantly higher than other treatments (P < 0.001). In contrast, the algal paste treatments, including (P‐Iso) produced overall the worst results for various parameters examined. However, the sex ratio of P. crassirostris was not significantly affected by diets and was always heavily skewed towards females (>84% were females) across all treatments. Based on the results of this study, T‐Iso + Cht is recommended for intensive cultivation of P. crassirostris.     

Effects of dietary vitamin C on survival, growth, and immunity of large yellow croaker, Pseudosciaena crocea

An 8 weeks growth experiment was conducted to determine the effects of dietary vitamin C on the survival, growth, tissue ascorbic acid contents and immunity of large yellow croaker (Pseudosciaena crocea) with initial weight of 17.82 ± 0.68 g. Seven practical diets were formulated to contain 0.1, 12.2, 23.8, 47.6, 89.7, 188.5 and 489.0 mg ascorbic acid equivalent kg^− 1 diet, supplied as l-ascorbyl-2-polyphosphate (LAPP). Each diet was fed to triplicate groups of fish in seawater floating cages (1.5 × 1.5 × 2.0 m), and each cage was stocked with 120 fish. Fish were fed twice daily (05:00 and 17:00) to apparent satiation for 8 weeks. The water temperature fluctuated from 19.5 to 25.5 °C, the salinity from 25 to 28‰ and dissolved oxygen content was approximately 7 mg l^− 1 during the experimental period. Results showed that the specific growth rate (SGR) (from 1.80 to 1.96% d^− 1) had an increasing trend with the increase of dietary vitamin C, but no significant difference was observed among dietary treatments. No gross deficiency signs were observed in any of the experimental fish. Survival rate, however, significantly increased with increasing dietary vitamin C (P < 0.05). The vitamin C contents in liver and muscle correlated positively with the vitamin C in diets. The vitamin C requirement was estimated to be 28.2 mg kg^− 1 based on survival, and 87.0 mg kg^− 1 on liver content of vitamin C. The activities of serum lysozyme and alternative complement pathway (ACP), phagocytosis percentage (PP) and respiratory burst activity of head kidney significantly increased with increasing dietary vitamin C. The challenge experiment with Vibrio harveyi showed that fish fed the diets with supplementation of vitamin C had significantly lower cumulative mortality compared to the control group (66.7%), and the cumulative mortality (16.7%) in fish with 489.0 mg kg^− 1 ascorbic acid was significantly lower than that (41.7%) in fish with 23.8 mg kg^− 1 ascorbic acid. These results suggested that vitamin C significantly influenced the immune response and disease resistance of large yellow croaker.     

The role of fatty acids enrichments in the larviculture of false percula clownfish Amphiprion ocellaris

At present, clownfishes are the best example of successfully captive bred ornamental specimens but little is known about the relationship between food enrichment and both larval growth and development. In fact, it is well known that a certain percentage of these fishes cultured in captive conditions show a miss-band pigmentation. In the present study, the effects of live prey enrichment on growth and pigmentation in false percula clownfish (Amphiprion ocellaris) larvae were tested.Newly hatched A. ocellaris larvae were divided in three different groups and fed as follows: group A fed on enriched (Algamac 2000) B. plicatilis (10 ind./mL) from day 1 to day 5 post-hatch (ph); group B fed on enriched (Algamac 2000) B. plicatilis (10 ind./mL) followed by Artemia nauplii (5 ind./mL;) and group C fed on Algamac 2000 enriched B. plicatilis and Algamac 2000 enriched Artemia nauplii.Samples of the larvae were collected on day 5 from group A and on day 11 ph in group B and C for morphometric and molecular analysis. On day 11 ph food enrichment resulted in a better growth of group C larvae respect to those of group B fed on not enriched Artemia nauplii (15.8 ± 0.2 mg and 8.78 ± 0.02 mm vs. 6.8 ± 0.2 mg and 6.93 ± 0.01 mm). Moreover, 36 ± 2% of the juveniles obtained from group B showed a miss-band pigmentation as compared to 29 ± 1% of the juveniles obtained from group C. At molecular level, the results obtained by Real-Time PCR are in agreement with the morphometric ones: a positive induction of the Insulin-like Growth Factor II (IGFII) and Peroxisome Proliferator Activated Receptor α and β (PPARα and PPARβ) gene expression and a reduction of Myostatin (MSTN) was observed in group C larvae fed on enriched live prey. IGFI gene expression was higher in group B.The present study provides clear evidences of the positive role of Algamac 2000 on growth and pigmentation of captive cultured false percula clownfish.     

Effect of dietary taurine and cystine on growth performance of juvenile red sea bream Pagrus major

Two experiments were conducted to investigate the effect of dietary taurine and cystine on growth and body composition of juvenile red sea bream Pagrus major. In Experiment I, a casein-based semi-purified diet included a small amount of fish meal were supplemented with taurine at the levels of 0 (control) and 1.0%. The experimental diets in Experiment II were without fishmeal and supplemented with taurine at 0 (control), 0.5, 1.0 and 2.0% or cystine at 1.0 and 2.0%. These diets were fed three times a day for 6 weeks to fish (average body weight: 2.3 g in Experiment I and 2.5 g in Experiment II). In Experiment I, fish fed the taurine-supplemented diet showed significantly (P < 0.05) improved growth, feed efficiency and feed consumption relative to fish fed the unsupplemental diet. The whole body taurine content increased, whereas the non-essential amino acid contents decreased, in fish fed the taurine-supplemental diet compared to fish fed the unsupplemented diet. In Experiment II, the growth, feed efficiency and feed consumption of fish fed the taurine-supplemented diets, irrespective of the dietary taurine levels, were significantly higher than those of fish fed the control diet and the cystine-supplemented diets. Taurine content in the whole body increased with the dietary taurine level, while the taurine contents did not increase by the supplemental cystine. Other free amino acid contents in the taurine-supplemented diet groups followed similar trends to those in Experiment I. These results indicate that supplemental taurine to a casein-based semi-purified diet at more than 0.5% improved the growth and feed performance of juvenile red sea bream. It is also suggested that juvenile red sea bream cannot metabolize cystine into taurine.     

First feeding of Octopus vulgaris Cuvier, 1797 paralarvae using Artemia: Effect of prey size, prey density and feeding frequency

Different assays related to the first feeding of Octopus vulgaris Cuvier, 1797 are compiled in this paper. They include: age at initial feeding age, prey size selection and optimal density, attack timing after feeding, and effect of dose number on the number of captures. Prey capture and ingestion processes were also analysed. Food supplied was cultured Artemia sp. Each assay lasted 15 min.Although paralarvae already start to feed on the hatching day (day 0), it is during day 2 when a greater number of attacks is recorded (81.7 ± 14.7% paralarvae attack). They mainly prefer (significance level α = 0.05) large Artemia, 1.4 ± 0.4 mm (77.0 ± 5.6% of the total attacks) than small Artemia, 0.8 ± 0.1 mm (23.0 ± 5.6%). There is also a slight predilection for the lowest Artemia concentration (33.3 ±12.6% paralarvae attack in a 0.1 Artemia ml^− 1 density, opposite 16.7 ± 7.6 and 18.3 ± 7.6% in densities of 0.5 and 1 Artemia ml^− 1 respectively). The greatest predatory activity is recorded during the first 5 min after food is supplied (72.2 ± 25.5%). An increase in the predatory activity was also observed when food was distributed in several doses instead of a single dose (75.0 ± 10.0% and 46.7 ± 17.6% respectively). It was proved for the first time that paralarvae completely ingest their preys (including their exoskeletons), in this case Artemia. Time needed for their total ingestion ranges between 4 and 10 min.     

Selective particle feeding by the Chilean oyster, Ostrea chilensis; implications for nursery culture and broodstock conditioning

Hatchery broodstock conditioning and nursery culture of the Chilean flat oyster Ostrea chilensis have been hampered by the poor performance of oysters fed typical microalgal hatchery diets. To determine the feeding capabilities of this species the selective removal and consumption of natural planktonic assemblages and artificial inert particles (polystyrene beads) by juvenile and adult oysters were examined experimentally. The arrangement of the eulaterofrontal cirri of the ctenidia was also examined to infer their potential efficiency of particle selection for feeding. Polystyrene beads of 45 and 15 μm in diameter had high rates of removal from suspensions by both juvenile (45 μm = 70%, 15 μm = 73%) and adult (45 μm = 88%, 15 μm = 87%) oysters. In contrast, beads of 6 μm diameter had lower rates of removal (adults = 68%, juveniles = 53%), while 1 μm beads were not removed at all. Both adult and juvenile oysters feeding upon natural plankton assemblages removed only microphytoplankton (20-75 μm in size) despite the presence of nanophytoplankton (2-20 μm), picophytoplankton (< 2 μm), cyanobacterium Synechoccocus spp. (< 2 μm), and bacterial cells (< 75 μm) in the experimental suspensions. Eulaterofrontal cirri of both juvenile (15.2 μm ± 0.9 SE) and adult oysters (18.9 μm ± 0.3 SE) are the shortest that have been reported for any ostreid species which helps to explain the inability of this species to retain small food particles. The clearance rates for oysters feeding on microphytoplankton only were 1.49 (± 0.05 SE) and 7.1 (± 1.2 SE) l h^− 1g^− 1 for juveniles and adults respectively. These values are much higher than previously reported for this species being fed smaller sized cultured microalgae. Our results strongly suggest that the difficulties in the nursery and broodstock hatchery culture of this oyster may be due to inappropriate phytoplankton diets. We recommend the provision of cultured microalgae of 20-75 μm in diameter for improving the performance of hatchery maintained juvenile and adult O. chilensis.     

Growth, survival and biochemical composition of spider crab Maja brachydactyla (Balss, 1922) (Decapoda: Majidae) larvae reared under different stocking densities, prey:larva ratios and diets

The spider crab Maja brachydactyla is overexploited on the NW coast of Spain. Aquaculture of this species can be the solution to the problem, and consequently, several attempts of intensive larval rearing have been conducted. However, most of the studies already published do not provide enough zoo technical data, especially in terms of larval and prey densities or the nutritional quality of diets used for rearing.Three experiments were carried out to evaluate the conditions for intensive larval rearing of M. brachydactyla. Larval stocking density (10, 50 and 100 larvae L^− 1), prey:larva ratio (15, 30 and 60 enriched Artemia larva^− 1) and diet (enriched Artemia, non-enriched Artemia and polychaete supplement) effects on growth and survival of this species were studied. For larval culture nine, 35 L, 150 μm mesh-bottomed PVC cylinders (triplicates for each treatment and larval stage) connected to a recirculation unit, were used. Temperature and salinity were kept constant at 18 °C and 36‰ respectively. A 12 to 18 day trial was conducted for each experiment and samples of larvae were collected at each larval stage (zoea I, zoea II, megalopa) in the inter-molt phase and at first juvenile. Survival, carapace length and width, dry weight (DW), and proximate biochemical content (protein, carbohydrates and total lipid) as well as lipid class composition were determined.Stocking densities of 100 larvae L^− 1 resulted in higher growth in DW and higher content in lipids and protein for zoea I (ZI) and zoea II (ZII) than 10 larvae L^− 1. However, survival decreased with increasing stocking density.The use of 60 preys larva^− 1 produced larvae with significantly higher DW and protein content, especially at ZII stage, than lower prey densities. Survival rate obtained feeding 60 preys larva^− 1 up to the megalopa stage was almost two-fold (42.2%) the rate obtained using 15 preys larva^− 1 (24.8%).Larvae fed on enriched Artemia (EA) showed an increase in weight up to megalopa (518.9 ± 26.5 μg) in contrast to larvae fed on non-enriched prey (A) (467.9 ± 6.9 μg). Variation in DW correlated with the total lipid content (L) of the larvae (L_EA = 70.1 ± 37.5 μg ind^− 1; L_A = 28.9 ± 3.2 μg ind^− 1) especially in terms of neutral lipids. The use of an initial density of at least 50 larvae L^− 1 and 60 enriched Artemia larva^− 1 can be considered the most adequate rearing parameters in order to obtain good results in growth and survival of M. brachydactyla.     

Growth, immune function, and disease and stress resistance of juvenile Nile tilapia (Oreochromis niloticus) fed graded levels of bovine lactoferrin

Juvenile Nile tilapia (Oreochromis niloticus) were fed nutritionally complete, practical basal diets supplemented with bovine lactoferrin (Lf) at 0, 200, 400, 800, or 1600 mg/kg diet to apparent satiation twice daily for 8 weeks. After the feeding trial, the effect of dietary Lf on growth performance, immune function, and resistance to Streptococcus iniae challenge and low-water stress was determined. Dietary Lf did not affect growth performance (weight gain, feed intake, feed efficiency ratio, or survival) or haematological parameters (haemoglobin, white and red blood cell counts, or haematocrit) (P > 0.05). Crowding stress produced significant increases in plasma cortisol, glucose, lactate, and osmolality from baseline values (P ≤ 0.001), but dietary Lf did not affect plasma glucose, osmolality, or cortisol concentrations (P > 0.05). The level of Lf in diet had a significant impact on survival following S. iniae challenge with fish fed the 800 mg/kg Lf diet having significantly higher survival than control fish (P ≤ 0.05). There was not a corresponding increase in activity of non-specific or specific immune parameters (plasma lysozyme and spontaneous haemolytic complement activities or agglutination antibody titer against S. iniae) with addition of Lf to diets (P > 0.05), but plasma iron decreased and total iron binding capacity (TIBC) increased significantly with increasing concentration of Lf in diet (P ≤ 0.05). The ability of Lf to sequester iron, an essential nutrient required for the growth of bacteria, is regarded as one of its key antibacterial properties. The increased survival with increasing dietary concentration of Lf seemed to correspond with a decrease in plasma iron concentration and not enhancement of non-specific or specific immune functions.     

Replacement of fish meal with poultry by-product meal in diets formulated for the humpback grouper, Cromileptes altivelis

An eight-week feeding trial was conducted to examine the possibility of replacing fish meal with poultry by-product meal (PBM) at high inclusion levels in the diets of the humpback grouper, Cromileptes altivelis, a carnivorous marine tropical fish. Six isolipidic (12%) and isoproteic (50%), experimental diets were formulated to contain graded levels of PBM. Fish meal protein was replaced with a feed-grade PBM at 50, 75 or 100% level (FPBM50, FPBM75, FPBM100, respectively), or a pet food grade PBM at 75 or 100% replacement level (PPBM75 and PPBM100, respectively). The control diet contained Danish fish meal as the sole protein source. The experimental diets were fed close to apparent satiation, twice a day to triplicate groups of humpback grouper fingerlings (12.4 ± 0.2 g). The grouper fingerlings were randomly distributed into groups of 15 fish in cylindrical cages (61 cm depth and 43 cm diameter) and placed in a 150-ton seawater polyethylene tank. Except for fish fed the FPBM100 diet, growth performance, survival, and feed utilization efficiency for fish fed PBM-based diets were not significantly lower (P > 0.05) compared to fish fed the control diet. The PBM source and dietary level did not significantly affect (P > 0.05) the hepato- and visero-somatic indices or the condition factor of fish. Dry matter and protein apparent digestibility coefficients (ADC) of the diets decreased with increasing dietary PBM, and ranged from 64.3-71.5% and 86.2 to 91.2%, respectively. High values (91.7 to 96.7%) for lipid ADC were observed in all diets, with no significant differences among dietary treatments. Whole-body moisture and lipid contents of the fish were not affected by the inclusion of PBM in the diets. With the exception of fish fed the FPBM100 diet, whole-body protein of fish fed the PBM-based diets was slightly higher than that of fish fed the control diet. There was a trend of increased whole-body ash with the increase in dietary levels of PBM. The results from this study indicate that good quality terrestrial PBM can successfully replace more than half the protein from marine fish meal in the diets for humpback grouper. However, total replacement of fish meal with PBM might be constrained by lowered nutrient digestibility and limiting essential amino acids, especially lysine and methionine.     

Effects of the partial substitution of dietary fish meal by differently processed soybean meals on growth performance, nutrient digestibility and activity of intestinal brush border enzymes in the European sea bass (Dicentrarchus labrax)

This study evaluated the effects of replacing fish meal (FM) in the diet of European sea bass (Dicentrarchus labrax) with differently processed soybean meals on growth performance; apparent digestibility coefficients (ADCs); the activity of alkaline phosphatase and the brush border digestive enzymes: leucine amino peptidase, maltase and γ-glutamyl transpeptidase; and the fish morphometric and flesh quality. European sea bass with an initial average body weight of 187.8 ± 1.4 g, were fed either a FM based (control) diet or a soy replacement isoproteic, isolipidic and isocaloric diet. The treatments consisted replacing FM protein by: 25% toasted, dehulled and solvent-extracted soybean meal (SE25); 50% dehulled and toasted soy seeds subjected to dry extrusion and mechanical oil extraction (ME50); 50% enzyme-treated soybean meal (ET50) and an inclusion of 60% composed of 30% toasted, dehulled and solvent-extracted soybean meal and 30% enzyme-treated soybean meal (SE + ET/60). ADCs values of dry matter, crude protein, crude lipid and gross energy did not significantly differ between ME50 and SE + ET/60 but both of them were inferior to the other treatments (P < 0.05). Compared with the control FM-based diet, only the group that consumed the ME50 diet had lower specific growth rate, feed conversion ratio and gross protein retention efficiency values (P < 0.05). The protein efficiency ratio did not differ between the FM-based control group and all other soy derivate groups (P > 0.05). In order to gain more insight on the effects of the diet on fish growth, activity of the brush border enzymes was measured. No significant differences were found in leucine amino peptidase and maltase activities, between the control group and the other soy derivate groups. The activity of γ-glutamyl transpeptidase was significantly higher in the upper section in fish that consumed the control diet compared with fish that consumed the soy derivate diets. Yet, the main activity of this enzyme was found in the lower intestinal section. In all the soy derivate diets except for the SE25 diet, the activity of alkaline phosphatase was significantly lower than its activity in the control diet. Feeding diets that include various types and levels of soybean derivatives did not affect the whole body composition and slaughter yield but significantly reduced liver weight. The results show that in methionine supplemented diets, FM can be replaced in terms of protein by 25% SE, 50% ET or 60% soy composed of 30% SE and 30% ET without hampering fish performance. The inclusion of 50% protein from ME resulted in significantly lower growth performance and feed utilization.