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A 90-day experiment was conducted to evaluate the effects of practical diets with different lipid contents on survival, growth performance and whole-body composition of juvenile tench ( Tinca tinca ). Five-month-old juveniles (34.48 ± 0.77 mm total length, 0.382 ± 0.08 g weight) were fed practical diets with different lipid contents: 6.5%, 8.5%, 10%, 12%, 14%, 15.5% or 18%. Survival rates ranged from 96.7% to 100%. The highest growth was obtained with diets containing 8.5, 10 and 12% lipid. The five intermediate lipid contents (between 8.5% and 15.5%) caused lower FCRs (1.27–1.38). The percentages of fish with externally visible deformities were around 12%, with no significant differences due to the dietary lipid level. The relation among fatty acid profiles of the diets, body composition, growth performance of juveniles and requirements of other fish species is discussed.

The aim of this study was to evaluate different replacement levels of fish meal (FM) by poultry by-product meal (PBM) on survival and growth of juvenile crayfish (Pacifastacus leniusculus). An 80-day experiment was conducted with stage 2 juveniles from the onset of exogenous feeding. Six practical diets (50% crude protein) differing in the level of replacement of FM protein by PBM protein were prepared: 0% (control), 15%, 25%, 35%, 45% or 55%, corresponding to 0%, 10.5%, 17.7%, 24.4%, 31.3% or 38.2% dietary PBM, respectively. Each diet was tested on grouped or individually isolated crayfish. There were no significant differences in survival among grouped crayfish fed the control diet (0% replacement) and those fed replacement levels up to 45% (31.3% dietary PBM) (average: 74.9%). Survival of grouped crayfish fed the highest replacement level (55%) was significantly lower (63.0%). In the case of isolated crayfish, diets tested had no significant effects on survival (average: 98.3%). Diets with 15%, 25%, 35% or 45% replacement of FM protein by PBM protein resulted in growth values (grouped and isolated: 13.2 mm carapace length, 555 mg weight) similar to those obtained with the control diet. Final growth of isolated crayfish was significantly higher than that of grouped crayfish with all diets. FCRs (average of grouped and isolated crayfish) ranged from 0.98 to 1.32. The relations among the amino acid profile of the diets, the amino acid requirements of other crustacean species, the amino acid composition of whole body of crayfish and the performance of juveniles are discussed. The present study provides the first data on the substitution possibilities of FM by PBM in diets for freshwater crayfish. Up to 31.3% PBM (45% replacement of FM protein) can be included in formulated diets (50% crude protein) for juvenile P. leniusculus during the first 80 days of intensive rearing without impairing growth or feed conversion.

The aim of this study was to evaluate different replacement levels of fish meal (FM) with pea protein concentrate (PPC) on survival and growth of juvenile crayfish (Pacifastacus leniusculus). A 100-day experiment was conducted with stage 2 juveniles from the onset of exogenous feeding. Four practical diets (50% crude protein) differing in the level of replacement of FM protein by PPC protein were prepared: 0% (control diet), 25% (20.2% dietary PPC), 35% (28.2% dietary PPC) or 45% (35.5% dietary PPC). Each diet was tested on grouped or individually isolated crayfish. The 25% or 35% replacement of FM protein by PPC protein resulted in survival (average of grouped and isolated: 83.6%) and growth (grouped and isolated: 14.44 mm carapace length, 674.9 mg weight) similar to those obtained with the control diet. Final growth of isolated crayfish was significantly higher than that of grouped crayfish with all diets. The relation among the amino acid profile of the diets, the performance of juveniles and the amino acid requirements determined for other crustacean species is discussed. The present study provides the first data on the substitution possibilities of FM by PPC in diets for freshwater crayfish. A 28.2% PPC (35% replacement of FM protein) can be included in formulated diets (50% crude protein) for juvenile P. leniusculus during the first 100 days of intensive rearing without impairing growth or feed conversion.

Two 100-day experiments were conducted to evaluate effects of practical diets with different protein content and substitution possibilities of fish meal (FM) protein with soybean meal (SBM) protein on survival and growth of juvenile crayfish ( Pacifastacus leniusculus ) from the onset of exogenous feeding. In experiment 1, four practical diets were prepared to contain 35, 40, 45 or 50% crude protein. Each diet was fed to crayfish housed individually or as a group. Survival rates of isolated crayfish fed 45 or 50% dietary protein were the highest (100%). There were no significant effects on survival of grouped crayfish (average: 75.09%). Weight gain plateaued at 45% crude protein (grouped and isolated crayfish: 14.56 mm CL, 691 mg W). Final growth values of isolated crayfish were significantly higher than those of grouped crayfish for all protein levels tested. In experiment 2, four practical diets (50% crude protein content) were prepared to test replacement levels of FM protein by SBM protein of 0% (control group), 25%, 35% or 45%, in crayfish housed in groups. Replacement levels of 0% and 25% SBM enabled the highest survival (average: 74%) and growth (average: 14.49 mm CL, 651.9 mg W). The amino acids profile of all diets is presented and compared with the requirements determined for other crustacean species. In conclusion, a 45–50% dietary protein with 25% replacement of FM protein by SBM protein can be recommended for juvenile P. leniusculus during the first period of intensive rearing.

Considering that the use of decapsulated Artemia cysts as direct food for juvenile crayfish could be an alternative to live nauplii, a 100-day experiment was carried out under controlled conditions to evaluate the effects of cysts, comparing with nauplii, as supplement to a dry diet for salmonids on the survival and growth of juvenile signal crayfish (Pacifastacus leniusculus) from the onset of exogenous feeding (stage 2). The reduction of feeding frequency was also tested. According to a bifactorial design, six treatments, differing in the supplement and feeding frequency, were tested: the dry diet supplemented with Artemia nauplii or decapsulated cysts was supplied once a day, once every two days and once every three days. Survival rates ranged from 56.7% to 81.7%, rising significantly with increasing the feeding frequency. The highest growth (12.94 mm carapace length and 593.08 mg weight) was reached by the crayfish that received the dry diet supplemented with cysts once a day, with significant differences from the rest of the treatments. Considering the supplement, the cysts supported significantly higher growth than the nauplii. Regarding the feeding frequency, growth was higher when the food was supplied once a day, showing significant differences from the other two frequencies (once every two days and once every three days). This study shows that decapsulated cysts are better dietary supplement than live nauplii. In crustacean culture, this is the first report of successful use of Artemia cysts from the onset of exogenous feeding.

The aim of this study was to determine the possibilities of increasing egg density in artificial incubation using low flow rates. Eggs of signal crayfish ( Pacifastacus leniusculus ) were incubated at three densities: 6.6, 20 and 42 eggs cm − 2 and two flow rates: 0.5 and 1 l min − 1 (0.0095 l cm − 2 min − 1 and 0.019 l cm − 2 min − 1 , respectively). Formaldehyde doses of 3000 and 2500 ppm were administered as antifungal treatment. In experiment 1, the best survival to stage 2 juvenile (84.3%) was obtained with the administration of 3000 ppm of formaldehyde at a density of 6.6 eggs cm − 2 , but differences were not significant either with a higher density (20 eggs cm − 2 ) or with a lower formaldehyde concentration (2500 ppm). In experiment 2, the best final survival (88.8%) was obtained with the administration of 2500 ppm of formaldehyde at a density of 20 eggs cm − 2 , and a flow rate of 0.5 l min − 1 (0.0095 l cm − 2 min − 1 ), without significant differences either with the same density and a flow of 1 l min − 1 (0.019 l cm − 2 min − 1 ), or with the density of 42 eggs cm − 2 (around 86%). The progressive reduction of formaldehyde concentration we have tested may have found its limit at 2500 ppm since, although was shown to be effective, a light fungal growth could be observed. Nevertheless, with 3000 ppm the growth of fungi was kept out completely with densities up to 20 eggs cm − 2 . Antifungal treatment seems to be a key factor to make feasible artificial incubation of crayfish eggs at high densities such as 42 eggs cm − 2 , even with a limited water flow.

Two 100-day experiments were carried out under controlled conditions in order to evaluate the effects of different combinations of a dry diet supplemented with live feed on survival and growth of juvenile Pacifastacus leniusculus Dana juveniles from the onset of external feeding. In the first experiment, juveniles were subjected to five feeding and density conditions: a dry diet for rainbow trout (D) with a density of 50 animals m− 2, the same dry diet supplemented with Artemia nauplii for 20, 60 or 100 days (50 m− 2) and D supplemented with Artemia nauplii for 100 days at a density of 200 m−2. When juvenile crayfish did not receive live feed supplement, mean survival rate (11.33%) and growth were significantly lower. The highest survival rates were obtained when nauplii were added for 100 days (57.83% and 57.33%). Survival rates in the two densities were similar but growth was significantly higher at the lowest one. When the period of supplementation was reduced to 20 or 60 days, survival rates decreased significantly. In experiment 2, amount and frequency of Artemia supply were increased and freshwater zooplankton (Daphnia pulex) was used as supplement of D. An increased frequency (three times a week) and amount of live food supply led to high survival rates (up to 82%) and growth (Carapace length: 11.79 mm, Weight: 360.16 mg). From these results it can be deduced that live feed can be the source of some nutritional factors needed for astacid juvenile crayfish from the onset of external feeding to guarantee the viability during the first months of life.

In artificial incubation of astacid crayfish eggs, the use of effective antifungal treatments is advisable for controlling the spread of fungi from dead to healthy eggs and increasing final efficiency rates. The aim of this study was to evaluate the effectiveness of formaldehyde, malachite green, hydrogen peroxide, isopropyl alcohol, copper sulfate, potassium permanganate and iodine (as polyvidone iodine) on signal crayfish ( Pacifastacus leniusculus Dana) eggs incubated at 6.6 eggs/cm 2 for long periods (up to 71 days). The administration of 3000 ppm formaldehyde for 15 min every other day up to the beginning of hatching allowed a stage 2 juvenile survival rate of 74.5%, without significant differences with 15 ppm malachite green (81.5%). These treatments did not show differences with the 150,000 ppm isopropyl alcohol dosage (65.5%). Formaldehyde at 3000 ppm can be administered up to the beginning of moulting to stage 2, without affecting the survival rates. Formaldehyde and malachite green treatments effectively controlled fungi. However, isopropyl alcohol and copper sulfate weakly inhibited mycelial growth only at the highest concentrations (150,000 and 18 ppm, respectively). Hydrogen peroxide below to 1500 ppm was insufficient to control fungi resulting in low survival rates to juvenile stage 2 (

Studies carried out to evaluate reproductive efficiency from maturing up to juvenile stage 2 production in astacid crayfish are scarce. Research with a view to determining total losses over the whole reproductive cycle is required. Two experiments were carried out with Pacifastacus leniusculus to obtain data on reproductive efficiency in earthen ponds and under laboratory conditions, in each case with two different densities of animals. All the parameters recorded were related to the initial number of females. In earthen ponds, numbers of egg-bearing females close to hatching (April) showed no significant differences between the two densities (76.1% with 3.4 breeders/m 2 and 72.5% with 6.6 breeders/m 2 ), with an estimated mean of 228 eggs per female. In the laboratory, 81.3% and 75% of females yielded stage 2 juveniles at densities of 6 and 12 breeders/m 2 , respectively, with an average of 135.4 juveniles per initial female, achieving an efficiency from first to final count around 70%. Productivity results are discussed with relation to those attained in other astacid species.

In astacid crayfish breeding, a sex ratio of one male to two females (1M:2F) is usually recommended. In light of the fact that an increase in the proportion of females in the broodstock would be desirable, two experiments were carried out with Pacifastacus leniusculus Dana to test a proportion of females with a ratio as high as 1M:4F under different conditions of animal density and group size, with mating and spawning rates being evaluated in addition to the production and further viability of the eggs. In both experiments, no breeder mortality was recorded and all the females mated. No significant differences were noticed between groups of 5 and 125 animals nor between densities of 5 and 25 animals per square metre. Likewise, no differences were detected between the sex ratios 1M:2F and 1M:4F. With regard to overall results, some 10 days after spawning 98% of egg-bearing females was achieved, with a mean of 323 pleopodal eggs. When the eggs reached phases VII–IX (embryo with blastopore in closing process-embryo with naupliar appendages), the percentage of berried females was 93.6% with 245 eggs per female. At that point, eggs were stripped, pooled and artificially incubated by methods developed by this team. Finally, 165 stage 2 juveniles were obtained per female. This allows, at least in this species, a considerable increase in the proportion of females in the broodstock, and better use of farming facilities.

During the artificial incubation, fungi can grow over nonviable eggs and invade the healthy ones causing a decrease of efficiency rates. Thus, the use of antifungal substances must be considered. Different doses and treatment frequencies of formaldehyde, hydrogen peroxide, sodium chloride and malachite green were tested on eggs of signal crayfish (Pacifastacus leniusculus Dana). Eggs were incubated at two densities: 2.2 and 6.6 eggs/cm2. The highest efficiency to stage 2 juvenile (67.3%) was obtained with formaldehyde (4500 ppm) applied for 15 min three times a week up to the beginning of hatchings, without significant differences with the same treatment applied twice a week neither with malachite green (15 ppm). In both cases, the fungal growth was effectively inhibited. The formaldehyde dose of 1500 ppm administered every 3 days, as well as hydrogen peroxide and sodium chloride treatments, were insufficient to control fungi. It was detected a positive effect when density was reduced, so that with 2.2 eggs/cm2, a final efficiency of 71% was obtained even without antifungal use; while with 6.6 eggs/cm2, it was 22.6%.

The critical phase that would limit duration of egg storage in the astacid crayfish Pacifastacus leniusculus was investigated. Weekly monitoring was conducted during an 84-day period of storage at three different constant temperatures (15.5±1, 10.5±1.5 and 4±0.5 °C). After artificial incubation, stage 2 juveniles were only obtained from the lowest storage temperature, the sole treatment in which eggs did not reach the critical embryonic stage by the end of the storage. This limiting stage occurs at phase XII (embryo with pulsating heart) when embryogenesis of stored eggs ceased and phase XIII (eyed stage) was not reached. Eggs were also stored at a constant refrigeration temperature (4±0.5 °C) for periods ranging from 84 to 126 days. Mean survival rates to juvenile stage 2 were acceptable (approximately 50%) and different batches of juveniles were obtained over a 3-month period, depending on the duration of storage. The combined use of storage and artificial incubation techniques could have important applications to the development of astacid crayfish culture.

The development of artificial incubation techniques for freshwater crayfish eggs and the incorporation of these practices to the working scheme in production centres would have numerous advantages. This study evaluates the efficacy of one particular device, designed by this research group, for the artificial incubation of white-clawed crayfish ( Austropotamobius pallipes ) eggs. Comparisons between maternal and artificial incubation demonstrated the suitability of the tested system for artificial incubation for eggs of this species, during a period of 42% of the total duration of the embryonic development. Overall survival rate to juvenile stage 2 was significantly higher ( P

Four experiments were carried out in order to evaluate survival and growth rates of Stage 2 juvenile Pacifastacus leniusculus under different management conditions: effects of offspring origin (artificial incubation, maternal incubation under laboratory conditions and maternal incubation under natural conditions), of different shelter availability, of water circulation systems (partially re-circulated and full flow-through), and effects of certain flow rates. Juveniles kept singly, rather than in groups, had higher survival rates. However, when juveniles were in groups, they showed faster growth. Offspring from artificial incubation did not show significant differences in survival and growth rates from those from maternal incubation. The addition of pebbles as shelter did not improve survival rates. The use of a full flow-through system allowed significantly higher survival and growth rates. A flow rate of 1 l/min improved the survival rate in comparison with 6 l/min, but not growth. In general, the highest mortality occurred during the first 40–60 days of the experiments, although a great variation in the final results was observed.

Three experiments on artificial incubation of crayfish eggs ( Pacifastacus leniusculus Dana) were carried out under different thermal regimes using a specially designed incubator tested in previous trials. Each treatment included a period of low temperature of different duration. Three flow rates were used in each experiment: 3.0, 1.5 and 0.5 l/min. In all cases the eggs were detached from females at three phases of embryonic development: beginning of gastrulation (mean degree days 230, 18 days after spawning); embryo between closing of the blastopore and appearance of mandibular rudiments (mean degree days 365); and embryo with naupliar appendages (mean degree days 480). The results showed that it is possible to auain acceptable juvenile stage 2 survival rates (58.9%) even though eggs were detached at the earliest embryonic phase (beginning of gastrulation). In the experimental incubator, flow rates could be reduced to 0.5 l/min without decreasing survival. The highest survival to juvenile stage 2 (72.1%) was obtained by delaying egg removal until the appearance of naupliar appendages, and using a thermal treatment of 15.5 ° ± 1 °C preceded by a 3-week period of low temperature (5 ° ± 1 °C) commencing at stripping time.

Three artificially compounded diets (purified diet, compound feed, and crab protein crustacean reference diet) and several fresh diets were tested under laboratory conditions in an attempt to improve survival and growth rates of juvenile freshwater crayfish ( Pacifastacus leniusculus ). The highest survival obtained was 44.44% after 80 days of test. Fresh aquatic plant material, when added to artificially compouned diets, improved growth and slightly increased survival, and the animals became more pigmented. Juveniles held singly, rather than in groups, achieved higher survival rates. The results with juvenile P. leniusculus have been related to those obtained in research with other crustaceans.