Assessment of magnesium chloride as an anaesthetic for adult sea urchins (Paracentrotus lividus): incidence on mortality and spawning

During this study different concentrations of magnesium chloride (MgCl₂) were tested on adult wild‐caught sea urchins (Paracentrotus lividus) immediately after their collection in the field in order to assess whether MgCl₂ can act as an anaesthetic during their transport and prevent unplanned spawning. Such accidental spawning is a stress response associated with transport of this species. The mortality and gonadal indices were recorded after a 1‐h submersion in different strength solutions of MgCl₂ (5, 20, 40, 80 and 100 mmol L⁻¹ MgCl₂), followed by 5 h of dry transport under two different temperatures (19 and 25 °C). The effective concentration of MgCl₂ was defined as one that does not induce mortality and gonad mass loss following sea urchin transport and laboratory conditioning. Treatment of sea urchins with 5 mmol L⁻¹ MgCl₂ prevented mortality and unplanned spawning; however, elevated concentrations of magnesium (40, 80 and 100 mM) were toxic to sea urchins, inducing a massive mortality and gonad mass decrease during the acclimatization period, essentially when sea urchins were transported under an elevated temperature (25 °C). It is concluded that MgCl₂ is a suitable non‐toxic anaesthetic, easily available for adult sea urchins (P. lividus) when it is used at its optimal anaesthetizing concentration.     

Protein and energy digestibility and gonad development of the European sea urchin Paracentrotus lividus (Lamarck) fed algal and prepared diets during spring and fall

Protein and energy are two of the main limiting factors for sea urchin growth. However, the requirement of daily protein and energy to maximize gonadal production is still unknown. Paracentrotus lividus were fed three experimental diets: Ulva lactuca, Gracilaria conferta and a prepared diet for 2 months in the fall of 1999 and spring of 2000. Sea urchins from a laboratory‐cultured population of equal age, weight and test diameter were used. Apparent digestibility coefficients (ADC%) for protein and energy, using acid‐insoluble ash as a marker, were measured for all experimental diets. Apparent digestibility coefficients for protein was high (>75%) for all diets. Energy digestibility varied among the diets and was lowest for G. conferta (50–62%). The three diets contained varying digestible protein (DP) to digestible energy (DE) ratios of 25, 26 and 12 mg kJ^?1 for U. lactuca, G. conferta and the prepared diet respectively. Digestible protein intake was similar for all treatments, but DE intake was greater for sea urchins fed the prepared diet in both seasons. As a result, the gonad production was significantly higher for urchins fed the prepared diet, suggesting that energy was limiting in the algal diets. Paracentrotus lividus spawned during the spring experiment, resulting in protein loss in all treatments. Protein loss was lowest in the sea urchins fed the prepared diet. Enhanced gonadal growth and gamete development of P. lividus resulted from the higher dietary energy content of the prepared diet.     

Gonad quality of sea urchin Paracentrotus lividus cultured in an offshore pilot‐scale trial on the south‐east Italian coast

A pilot project aimed at testing roe enhancement strategies based on offshore Paracentrotus lividus cultures was conducted off the south‐east coast of Italy (Apulia Region). Adult sea urchins were reared in sea cages located 700 m offshore at a depth of 12 m for 3 months. The animals were fed once a week on two formulated diets, prepared mixing nutrients with agar 20 g/Kg and differing only in terms of the protein source: anchovy flour (Diet A) or krill flour (Diet K). At the end of the rearing trial, the gonad somatic index of sea urchins fed on formulated diets significantly exceeded that of wild sea urchins. Total FAA content in the gonads of wild sea urchins and Diet A‐fed sea urchins was similar, whereas in Diet K‐fed sea urchins it was significantly higher. In terms of fatty acids, the gonads contained SFAs, MUFAs and PUFAs. In visual and sensory assessment of gonads by panel test and electronic nose, the gonads of reared sea urchins were rated as being of better size, while no differences were recorded for coloration, taste and odour. This study shows that under these experimental conditions, commercial‐grade Paracentrotus lividus roe enhancement can be achieved after 3 months in sea cages.     

An experimental assessment on the effects of photoperiod treatments on the somatic and gonadal growth of the juvenile European purple sea urchin Paracentrotus lividus

Determining the optimum light conditions for sea urchins reared in land‐based systems is vital for the future use and assessment of possible commercial systems of sea urchin farming. The effects of two different light regimes, complete darkness and a long day photoperiod of 16 h light:8 h darkness, on the somatic and gonadal growth of the European sea urchin Paracentrotus lividus (19.5–23.0 mm) was investigated using the commercial UrchinPlatter^? System over a 6‐month period (5 March to 5 September). Hatchery‐produced P. lividus were transported to the Aquaculture Fisheries Development Centre (AFDC, University College, Cork UCC). Before arrival at the AFDC, sea urchins were reared on a diet of Laminaria digitata. Females were the predominant species of the animal group, displaying a reproductive Stage III (growing stage) where gametogenesis was commencing. Results show that darkness supports higher somatic growth than the photoperiod treatment. Feeding rates were higher for sea urchins reared under darkness with gonadal growth increasing for both experimental treatments. Individuals reared under darkness had a higher per cent change in gonad index from the initial sample taken at the beginning of the experiment.     

Evaluation of dried macrophytes as an alternative diet for the rearing of the sea urchin Paracentrotus lividus (Lamarck, 1816)

The aim of this work was to assess the potential use of different dried macroalgae as food in the rearing of Paracentrotus lividus. Growth, consumption and food conversions were compared in adult sea urchins fed with fresh or dried thalli of four macroalgae species. Six experimental diets were tested: (a) fresh Palmaria palmata; (b) fresh Saccharina latissima; (c) dry P. palmata; (d) dry S. latissima; (e) dry Laminaria digitata and (f) dry Grateloupia turuturu. Linear growth rates were similar for all treatments. Specific growth rate was higher in sea urchins fed with fresh P. palmata, but no difference was found between animals fed with fresh S. latissima and those fed with dried diets. Regarding daily food consumption (DFC), sea urchins consumed the same amount of dried macroalgae as fresh but exhibited a higher food conversion efficiency (FCE) when fed with fresh P. palmata. However, this FCE was only significantly higher when compared to sea urchins fed with dry L. digitata. Dried G. turuturu is not a suitable diet due to its rapid degradation after rehydration. The results suggest that P. lividus adults can be reared on dried macroalgae thalli without detriment to their somatic growth, especially over short periods. The low cost of feeding sea urchins with this diet could help small shellfish farmers to diversify their production into echinoculture.     

Can sea urchins benefit from an artificial diet? Physiological and histological assessment for echinoculture feasibility evaluation

In the Mediterranean sea, since the time of ancient Greece, the sea urchin Paracentrotus lividus has been considered a delicacy, and as a consequence, the market demand has increased since the early 1970s, significantly causing a depletion of wild stock. For this reason, the interest in breeding sea urchins has widened over the last two decades and several formulated diets were developed. In order to identify a low‐cost diet, which is able to promote both the maintenance and the gonadal growth of organisms in recirculating aquaculture system (RAS), we evaluated the food intake of three different diets: the first composed of macroalgae naturally present in the sea urchins collection site, a second diet based on maize and spinach and finally a diet based on pellet normally used in aquaculture. The results presented in this study demonstrated the good feasibility of a low‐cost and easy‐to‐standardize diet, such as that based on maize and spinach, in rearing vulnerable species such as P. lividus in RAS.     

Enhanced production of the sea urchin Paracentrotus lividus in integrated open-water cultivation with Atlantic salmon Salmo salar

Survivorship and performance was investigated for two size classes of the sea urchin Paracentrotus lividus grown adjacent to open-water Atlantic salmon (Salmo salar) cultivation on the north-west coast of Scotland. Juvenile P. lividus were maintained for 12 months at 0 m, 50 m and 2.5 km from the mariculture activity. The sea urchins at the 0 m station showed higher survivorship than at the 50 m and 2.5 km stations and had significantly greater test diameter than at the 50 m station. Only urchins at 0 m developed gonads and, although small, these were of acceptable or excellent colouration in terms of their marketability. Adult P. lividus were maintained for 3 months at 0 m and 15 m from the mariculture activity, either with or without additional macroalgae Laminaria spp. Sea urchins at the 0 m station and fed additional macroalgae showed higher gonadal growth than sea urchins at the 15 m station held without additional food. Gonad colouration in the adult urchins, irrespective of the treatment, was acceptable or excellent. The 0 m station also received significantly greater quantities of particulate organic matter (POM) than the other stations in both the juvenile and adult experiments. At the 0 m station, the presence of the fatty acid 22:1n-11 and elevated levels of the long chain n-3 fatty acid DHA 22:6n-3 in the gonadal tissue of the urchins confirmed the consumption of fish farm derived POM and highlighted the potential human health benefits from consuming sea urchin roe.The results show that P. lividus can thrive in the salmon culture environment and suggests that the integration of P. lividus with Atlantic salmon can provide a viable means to culture this species, even at this northerly latitude. Salmon-sea urchin co-culture would enable fish farms to diversify into producing a second highly valuable product and would reduce the increasing worldwide pressure of sea urchin fisheries on wild stocks.     

Effect of three diets on the gametogenic development and fatty acid profile of Paracentrotus lividus (Lamarck, 1816) gonads

In this study, the effects of three diets were investigated to enhance Paracentrotus lividus production for commercial purposes. P. lividus were fed ad libitum for 80 days with: diet A—fresh Codium tomentosum Stackhouse, 1797; diet B—formulated using a jellified mix of macroalgae and vegetables, including C. tomentosum (20%), Coralina sp. Linnaeus, 1758 (17%), cabbage Brassica oleracea var. capitata Linnaeus, 1753 (30%), carrot Daucus carota Linnaeus, 1753 (30%) and agar (3%) as a gelling agent. Diet C consisted of maize Zea mays Linnaeus, 1753 (56%) and New Zealand spinach Tetragonia tetragonoides (Pallas, 1781) Kuntze, 1891 (44%). Their effects on the gonadal and somatic growths, gonadosomatic index (GI) and gametogenesis were evaluated, as well as on the total lipid content and fatty acid composition of sea urchin's gonads. Diet A provided high values of eicosapentaenoic acid (EPA). Gonads of sea urchins fed with diet A were found mostly in growth and maturation stages of gametogenesis and showed the lowest lipid content. Sea urchins fed with diet B presented their gonads in the reabsorption stage and had the highest values of omega‐3 polyunsaturated fatty acids (PUFAs). Sea urchins fed with diet C were in the early stages of gametogenesis and had the highest values of lipid content, plus omega‐6 PUFAs. Once as an ingredient in a balanced mix with vegetables, C. tomentosum can be a key factor to the development of new promising high‐quality and low‐cost feed for P. lividus roe enhancement.     

Assessment of commercial and recreational fishing effects on trophic interactions in the Cap Roux area (north‐western Mediterranean)

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Agar‐based pellets as feed for sea urchins (Paracentrotus lividus): rheological behaviour,digestive enzymes and gonad growth

The algal polysaccharide agar has long been used as a food binder due to its structure, rheological behaviour, stability and interactions – properties that help to generate firm, round, disk‐shaped pellets that may be used in recirculating sea urchin‐rearing systems. Three algae‐based diets (Ulva lactuca, Gracilaria gracilis, Cystoseira sp.) containing 3% and 6% agar were tested on the sea urchin Paracentrotus lividus in order to examine the effect of varying percentages of agar on pellet stability in water and sea urchin gonad growth. The kinetics of water absorption and solute leaching of pellets were measured by immersing quadruplicate samples of the pellets in water for 1, 2, 3, 4, 5 and 6 days. Our results show that the pellets had good water stability, were readily consumed by sea urchins and the presence of agar did not hamper sea urchin gonad growth. Animals fed Ulva‐containing pellets reached a more advanced gametogenic stage with respect to animals fed Cystoseira‐ and Gracilaria‐containing pellets. Moreover, the presence of agarase activity in the digestive system indicated that agar may be an energy source. Pellets are relatively low cost and easy to prepare and store. They may represent a useful resource for rearing sea urchins under intensive conditions.     

Effect of formulated diets on the proximate composition and fatty acid profiles of sea urchin <Emphasis Type="Italic">Paracentrotus lividus</Emphasis> gonad

Three formulated diets were tested to evaluate their effects on gonad quality in Paracentrotus lividus. Experiments were conducted in parallel by the Consiglio Nazionale delle Ricerche (CNR) of Taranto (trial 1) and the University of Genoa (trial 2), in land-based systems. In both trials, somatic and gonadsomatic index (GSI) were measured and the nutritional profile of the sea urchins has determined significant variations in the biochemical composition. Sea urchins fed the experimental diets contained higher levels of nutrients (protein and lipid and carbohydrate) compared to wild sea urchins. However, total polyunsaturated fatty acids (PUFAs), especially EPA and DHA, and the n-3/n-6 ratio were lower in urchins fed with formulated diets. In both trials, sea urchins fed with diet 2 (SABS) showed a similar profile with PUFAs higher than SAFAs and MUFAs, the highest UNS/SAT ratio, although the highest n3/n6 ratio was observed in the group fed diet 3 (CNR). Atherogenicity, thrombogenicity, and hypocholesterolemic/hypercholesterolemic indices showed the best values in sea urchins fed diet 2 in both trials.     

Effect of Protein Source and Concentration on Somatic Growth of Juvenile Green Sea Urchins Strongylocentrotus droebachiensis

Abstract— An important aspect in the development of any aquaculture industry is the maximization of juvenile somatic growth (i.e., body growth) to reduce production time and increase the size of the final product. In this study, green sea urchins Strongylocentrotus clroebachiensis were fed a prepared diet from 4 December 1998 to 10 September 1999 (i.e., 280 d) in a laboratory to investigate the effect of protein source (soybean andlor fish), protein concentration (20,30,40, and 50% dry mass) and juvenile size (4‐8 mm and 12‐20 mm initial test diameter) on somatic growth. A natural diet of Laminwia longicrurus (i.e., kelp) was used as a reference. There was no difference in initial size among the treatments for either the smaller cohort 1 or the larger cohort 2 sea urchins (6.3 mm and 13.8 mm initial average test diameter, respectively) (P > 0.05 for all tests). After 280 d, the sea urchins fed kelp had an average size of 20.7 mm and 24.5 mm (cohort 1 and cohort 2, respectively). The final average sizes of the sea urchins fed the prepared diets, which did not relate to dietary protein concentrations andlor protein source, ranged from 13.2 mm to 16.2 mm (cohort 1) and from 20.4 mm to 22.9 mm (cohort 2), and were significantly smaller than the kelpfed sea urchins (P < 0.05 and P < 0.001, cohort 1 and cohort 2, respectively). All treatments experienced 95% survivorship or greater. Sea urchin size appears to affect growth rate when optimal conditions for growth are available (i.e., diet and water temperature). As water temperatures increased during the summer of 1999, the sea urchins in cohort 1 fed kelp had a significantly higher growth rate (0.069 dd) than the cohort 2 kelpfed sea urchins (0.052 dd) (P < 0.05). However, within each cohort, there were no significant difference in growth rate (P > 0.05) among the sea urchins fed prepared diets, suggesting sea urchins do not require high concentrations of dietary protein for superior growth, and that plant protein can substitute fish protein in sea urchin diets. Furthermore, the sea urchins fed the prepared diets had poorer test quality and larger gonad yields (13‐22%) compared to the kelpfed sea urchins (4.2%) and a wild sample of sea urchins of similar size (4.0%). The results suggest that the sea urchins fed the prepared diets allocated more energy to gonad production, whereas those fed a natural diet allocated more energy toward test production. To address this gap, more research is required to identify the nutritional components required for test growth that were present in kelp, but appeared to be deficient in the prepared diets.     

Comparison of spawning induction techniques on <Emphasis Type="Italic">Paracentrotus lividus</Emphasis> (Echinodermata: Echinoidea) broodstock

The performance of different and expedite spawning induction methods was compared on samples of both mature wild and captive Paracentrotus lividus sea urchin populations. Thermal, saline and mechanical shocks, emersion during different periods of time, addition of con-specific gametes and different KCl concentrations were tested. Percentage of spawned sea urchins, mean number of released eggs, percentage of males that released more than 200 million spermatozoa, and survival after 5 days were the variables analysed. Results indicate that both thermal and saline shocks were ineffective methods to trigger spawning. Mechanical shock and addition of con-specific gametes were able to promote spawning but with a reduced number of released gametes. Emersion for a period of 3 h induced spawning with 100% broodstock survival but longer periods up to 12 h, although increasing the number of spawners can cause significant broodstock mortality. An injection of 1-mL intra-peristomial KCl was an expedite method to obtain P. lividus gametes, but high mortalities were always recorded and was related with excessive KCl concentrations. When there is need for a small number of gametes, the mechanical shock technique can be considered since led to 100% survival. When large spawnings are required, the emersion procedure can be a viable method, but further investigation must be carried out to assess the best time period to obtain broodstock full survival.     

Long‐term Growth Study of Male and Female Green Sea Urchins,Strongylocentrotus droebachiensis,under Constant Light and Temperature Regime

Juvenile green sea urchins, Strongylocentrotus droebachiensis, with an initial mean test diameter of 9.5 mm (±1.2 SD) were held in the laboratory, in individual compartments and at constant temperatures (8.5 C) (n = 90) for a period of 99 wk. The animals were supplied with flow‐through sea water, and fed the Nofima sea urchin manufactured feed ad libitum. Growth, survival, feed intake, feed conversion ratio (FCR), and gonad index (GI) of the sea urchins were monitored during this period. The mortality was 11% throughout the experimental period. The test diameter (TD) increased significantly with time, best described by a polynomial equation (TD = 0.8756 + 0.584Time − 0.002Time², R² = 0.995). Sex was included as dummy variable, but did not contribute significantly; hence, there were no differences in growth trajectories between males and females. The study showed there were no significant differences in body wet weight or FCR between males and females. However at the end of the experiment the female urchins had a significantly higher GI of 31.1% (±4.4), compared with the male urchins which had an average GI of 26.8% (±5.4). In conclusion, the present study showed that male and female green sea urchins have the same growth capacity and feed convention ratio but by the end of the experiment the gonad index was significantly higher in female than male urchins.     

Cloning,characterization and transcription analysis of fatty acyl desaturase (Δ6Fad‐like) and elongase (Elovl4‐like,Elove5‐like) during embryos development of Strongylocentrotus intermedius

Strongylocentrotus intermedius has high nutritional value because it is rich in proteins, amino acids and long‐chain polyunsaturated fatty acids (LC‐PUFA). LC‐PUFA are essential nutrients that not only determine the nutritional value of sea urchins but also guarantee normal growth and reproduction performance. To better understand the molecular basis of LC‐PUFA biosynthesis in S. intermedius, the Δ6Fad‐like, Elovl4‐like and Elovl5‐like genes were cloned and fatty acid compositions during the early developmental stages of sea urchins were detected. The full‐length of Δ6Fad‐like was 2,199 bp, with a putative open reading frame of 1,248 bp encoding a polypeptide of 415 amino acid (AA). The Elovl4‐like and Elovl5‐like genes encoded 310 and 234 AA, respectively, which exhibited all of the characteristics of the Elovl family, such as a histidine box motif and putative transmembrane‐spanning domains. Tissue distribution analysis revealed that Δ6Fad‐like, Elovl4‐like and Elovl5‐like genes were expressed at the highest levels in the gonads and intestine, and the expression levels gradually increased in embryos during development. These results can help to understand the role of the Δ6Fad‐like, Elovl4‐like and Elovl5‐like genes in the different developmental stages of the sea urchin and to clarify the biosynthetic pathways of LC‐PUFA during the development of the sea urchin and provide a theoretical basis for improving the quality and breeding of excellent traits in sea urchins.     

Effect of different feed formulations on feed efficiency, gonad yield and gonad quality in the purple sea urchin Heliocidaris erythrogramma

The nutritive values of three pelleted prepared diets, based on animal (AP), vegetable (VP) and yeast protein (YP) were studied for the sea urchin Heliocidaris erythrogramma (Val.). Fresh macroalga Ulva australis was used as a natural control diet. Triplicate groups of five animals were fed one of the four diets ad libitum every second day for 85 days. Sea urchins fed pelleted feeds had significantly higher food consumption rates (dry basis) and significantly lower total and protein absorption efficiencies compared with the algal diet. The gonad yield and gonad production efficiency in sea urchins fed the natural diet were significantly higher compared with initial group (gonad yield only) and urchins fed animal and vegetable diets, but did not differ significantly from those of the animals fed the yeast diet. Percent protein and lipid in the gonads were not affected by the dietary source. The taste and smell of gonads were generally better in sea urchins collected from the wild or fed yeast and natural diets than in sea urchins fed animal and vegetable diets. The animal protein diet was the most stable in seawater while the yeast protein diet had the poorest water stability. The results of this study suggest that development of a more stable, single‐cell, protein‐based diet has a potential to promote gonad production of H. erythrogramma.     

The effects of tank system,water velocity and water movement on survival,somatic and gonad growth of juvenile and adult green sea urchin,Strongylocentrotus droebachiensis

The effects of water velocity and tipping frequency (water movement) on survival, somatic and gonad growth of juvenile and adult green sea urchin, Strongylocentrotus droebachiensis were investigated. Juvenile and adult urchins were held in ‘laminar’ or ‘tipper’ tanks. Both were supplied with three inlet water flows (1.5, 3.0 and 7.5 L min^?1) which converted to water velocities of 0.28, 0.57 and 1.43 cm s^?1 in the ‘laminar’ tanks and tipping frequencies of 30, 10 and 5 s in the ‘tipper’ tanks. Juvenile sea urchins had significantly lower mortality and greater somatic growth when held in ‘laminar’ flow tanks compared with ‘tipper’ tanks. The varying water velocities tested in the ‘laminar’ flow treatment had no effect on the growth of juvenile sea urchins. The juvenile sea urchins in the ‘tipping’ treatment held at high tipping frequency had significantly slower growth than those in the medium and low tipping frequency treatments. There were no differences in mortality or gonad growth in the adult sea urchins regardless of treatment. The ‘laminar’ flow tanks retained significantly more organic material compared with the ‘tipper’ tanks. The authors discuss the effects of water velocity and tipping frequencies in system design for aquaculture of sea urchins.     

Experimental rearing of the sea urchin Paracentrotus lividus fed with discards of the lettuce Lactuca sativa in a sea‐based system

Small‐sized specimens (test diameter: 16.64 ± 0.93 mm, mean ± SE) of the sea urchin Paracentrotus lividus were fed with fresh discards of the lettuce Lactuca sativa during a 24‐week experiment in a sea‐based system. At the end of the experiment, stock survival was high (88.8 ± 6.6%). Sea urchin test diameter and total weight grew by 35% and 56%, respectively, while gonad somatic index, after an initial decrease, increased by 3.2%. Moreover, more than 90% of specimens achieved the gonad colour that the market demands. These results support the exploitation of L. sativa as the main ingredient in a manufactured diet for echinoculture of P. lividus. Employing diets formulated with discard ingredients, combined with a low‐cost sea‐based rearing system, could be a suitable approach for sustainable management of echinoculture.     

Effect of Lipid Source and Concentration on Somatic Growth of Juvenile Green Sea Urchins, Strongylocentrotus droebachiensis

Supplying juvenile sea urchins with an abundant supply of resources and essential nutrients for growth will facilitate somatic growth and, hence, improve the success of the sea urchin aquaculture industry. Lipids are essential in physical processes such as membrane production and are a concentrated source of energy. This study, using prepared diets, tested the effects of lipid sources containing different major fatty acids (i.e., n‐3 and/or n‐6) (Part 1) and lipid concentration (i.e., 1, 3, 7, and 10%) (Part 2) on the somatic (i.e., test or shell) growth of two size cohorts (7.0‐ and 15.3‐mm average initial test diameter [TD]) of juvenile green sea urchins, Strongylocentrotus droebachiensis. The growth of the sea urchins fed prepared diets was compared to the growth of sea urchins fed a kelp reference diet, Laminaria longicruris. After both feeding trials, the kelp‐fed sea urchins had superior test growth and were more similar in physical appearance to wild sea urchins (i.e., test color, spine length, and gonad color). The sea urchins fed the prepared diets had pale test color, short, stubby spines, and large, pale‐colored gonads compared to wild sea urchins. The smaller cohort of sea urchins grew at a faster rate, but growth patterns for both cohorts were similar. The juveniles fed the prepared diets (in both feeding trials) had high initial growth rates that decreased after approximately 100 d compared to the kelp‐fed juveniles. Differences in test growth were not shown to be affected by sea urchin size (i.e., similar results for both cohorts) or by differences in dietary lipid sources (i.e., the presence of n‐3 and/or n‐6 fatty acids). However, the sea urchins fed diets with lower lipid concentration (≤3%) had larger average TDs than those fed diets with higher lipid concentrations (≥7%). Differences in test growth and physical appearance among those fed the prepared diets and kelp may have been because of nutritional deficiencies in the prepared diets.