© 2005 International Council for the Exploration of the Sea
Growth, survival, and development of Atlantic cod (Gadus morhua L.) weaned onto diets containing various sources of marine protein
a Institute of Marine Research Austevoll, N-5392 Storebø, Norway
b Institute of Marine Research Matre, 5984 Matredal, Norway
c Norwegian Institute of Fisheries and Aquaculture Research Kjerreidviken 16, 5141 Fyllingsdalen, Norway
*Correspondence to I. Opstad: tel: +47 56182267; fax: +47 56182222. e-mail: ingegjerd.opstad{at}imr.no.
We studied the effects of partial or complete substitution of fishmeal with alternative sources of marine protein (amphipod or krill meal) on growth, survival, liver index, and deformities of juvenile cod (Gadus morhua L.). The diets contained either 100% fishmeal or fishmeal that was replaced with 25%, 50%, or 100% amphipod meal or 50% or 100% Antarctic krill meal. Cod larvae were start-fed on rotifers and weaned directly to one of the six formulated feeds at a mean wet weight of 28 mg, 40 days post first-feeding. The mean weight of fish reared on different diets at the end of the experiment ranged from 0.92 to 2.52 g. The best growth was obtained by cod fed 100% fishmeal and 50% krill meal. There was a trend in the direction of slower growth with increasing levels of amphipod meal in the diets. Survival ranged from 87% to 79%, and there was a tendency for higher mortality with increasing content of amphipod meal. The liver index varied between 11.7% and 9.9%. The composition of the diets also had a significant influence on the occurrence of skeletal deformities. The highest proportion of deformities (16% of all fish) was in fish fed 100% amphipod meal, decreasing as the proportion of amphipod meal in the diets declined. A similar effect was not seen with Antarctic krill meal. The amphipod meal had a high content of ash, fluoride, cadmium, and mercury, which may have caused the deformities.
Keywords: alternative protein sources, cod, weaning
Received 21 September 2004; accepted 14 November 2005.
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Introduction |
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Recently, there has been a growing interest in intensive farming of Atlantic cod (Gadus morhua L.). Juveniles can be produced on cultured rotifers or fed natural zooplankton in pond systems and then weaned onto formulated feed (Kvenseth and Øiestad, 1983; Opstad et al., 1989; Baskerville-Bridges and Kling, 2000).
Cod is one of the most valuable commercial species of the northern Atlantic region (Kurlansky, 1997) and a promising species for aquaculture (Tilseth, 1990; Tilseth et al., 1992). Production volumes of farmed cod have risen steadily during the past few years. The massive increase in marine aquaculture, including salmon production, suggests that within a few years there will be shortages of fishmeal and oil, raw materials traditionally used in aquaculture feeds. According to the UN Food and Agriculture Organization (FAO, 1997), 70% of the world's stocks of fish (including fish used for meal and oil production) are either overfished or wholly depleted, and it will be impossible to increase the volume of catches in the foreseeable future. Further growth of the aquaculture industry will require alternative feed sources. One possibility is to harvest from lower trophic levels of the marine community, utilizing species like krill, amphipods, and copepods. These are also natural items of food for young cod. Several hundred million tonnes of zooplankton are produced each year, and only a few per cent are harvested.
The purpose of this experiment was to investigate whether alternative marine protein sources (krill and amphipod meal) influence the survival, growth, and development of cod during weaning.
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Material and methods |
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TopIntroductionMaterial and methodsResultsDiscussionReferences |
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Larvae
The experiment was carried out at the Institute of Marine Research, Austevoll, Storebø, Norway. Source of broodstock was coastal cod, maintained at Austevoll in 25 000-l tanks. They were light manipulated to spawn in autumn (September/October), when they spawned naturally. A single batch of eggs was collected from the spawning tank and incubated in black 70-l flow-through tanks at about 8°C. The cod larvae were start-fed in 1500-l tanks at 12°C from four days post-hatch, using rotifers (Brachionus plicatilis) enriched with RotimacTM (BioMarine AquaFauna Inc., Hawthorne, California, USA). The larvae were fed three times a day. After 40 days of feeding, they were transferred to 24 green 50-l polyethylene tanks, each holding 100 fish. Mean start wet weight was 0.028 ± 0.012 g. Water flow (2 l min–1) entered at the surface of the tank and exited through a cylinder covered with 500-µm mesh located in the centre of the tank.
Triplicate tanks with larvae were weaned onto the six experimental diets. The larvae were fed by hand twice a day and by an automatic feeder every hour. Water temperature was kept at 12°C, and light intensity was 400 µW cm2, with 24-Lux photoperiod. Bottoms of the tanks were cleaned, and dead larvae were removed and counted daily. Salinity was 34 ± 0.5, and oxygen varied between 96% and 100%.
Diets
The experimental meals of frozen Antarctic krill (Euphausia superba) and Norwegian Sea amphipod (Themisto libellula) were produced in the pilot plant at the Norwegian Institute of Fisheries and Aquaculture Research in Bergen. The fish protein was 999 Con-Kix, TripleNine Fish Protein. The attractant was from Primex, Norway, fishmeal and herring (Clupea harengus) oil from Norsildmel, soya lecithin from Denofa, starch from SFK, Norway, vitamins from Norsk Mineralnæring, G. O. Johnsen and Likan AS, and minerals from Norsk Mineralnæring.
The commercial fishmeal and the plankton meals were micronized. The soluble parts were included in the meals, and the diets were agglomerated at 400–800 µm. The content of amphipod meal was increased successively to replace 25%, 50%, and 100% of the fishmeal, while 50% and 100% of the fishmeal was replaced by krill meal. The components and composition of the diets are shown in Table 1.
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Chemical analysis
Total lipid was extracted from a sample of feed, using chloroform/methanol (2:1, by volume) according to Folch et al. (1957). After evaporation to dryness in vacuum at room temperature, total lipid was re-dissolved in chloroform/methanol (2:1, by volume) and stored under nitrogen at –80°C until analysis (Olsen et al., 2004). To determine the fatty acid composition of total lipid, an aliquot of known weight was subjected to acid-catalysed transesterification using 1% H2SO4 in methanol. The resulting fatty acid methyl esters (FAME) were separated and quantified by gas liquid chromatography as described previously (Olsen et al., 2004). Protein was determined by the combustion method (ISO/DIS 16634, 2004). Water-soluble protein in the sample was extracted in boiling water for 30 min, and the protein content of the aqueous phase was determined by the Kjeldahl method (ISO 5983, 1997). Ash was determined after incineration at 550°C (ISO 5984, 1978). Water content was determined after drying at 103 ± 1°C (ISO 6496, 1999). Lipid in the meals was determined by the Soxhlet method (AOCS Ba 3-38). Cadmium was determined by inductively coupled plasma atomic emission spectrometry (ICP) after total digestion (ISO/DIS 11885). Mercury was determined by the AAS cold vapour technique (NS 4768-1). Fluoride was determined by an ion selective electrode. For determination of chitin, the sample was demineralized with 5% HCl at 50°C for 30 min and then deproteinized with 4% NaOH at 80°C for 90 min. Nitrogen in the insoluble residue was determined by the Kjeldahl method and chitin = N x 14.51.
Growth, survival, liver index, and deformities
The experiment lasted for 50 days. Survival was calculated by counting total mortality in each tank at the end of the experiment. Percentage survival is given as the mean value of three tanks. Growth was measured by wet weight to the nearest 0.001 g at the end of the experiment following anaesthesia with Benzocainum (Unikem, DK-1503 København). Specific growth rate (SGR) was calculated according to Houde and Schekter (1981) as SGR = 100(ec – 1) where c = (ln WB – ln WA)
t–1, where WA is the mean weight at the start of the experiment, and WB is the mean weight at the end of the experiment. After weighing, the number of fish with external deformities was counted visually before the liver was excised from seven fish from each tank and weighed to the nearest 0.001 mg. The liver index was calculated as the ratio of liver weight to the total body weight.
Statistics
Statistical analyses were performed using Statistica (StatSoft Inc., Tulsa, Oklahoma, USA). Data were subjected to one-way ANOVA and Tukey's HSD post hoc tests. The deformities, survival, and SGR data were transformed before testing. Survival and deformities data were tested with LSD test. Differences were considered significant at p < 0.05.
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Results |
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The highest survival rate (87%) was in the group of cod fed the 100% fishmeal diet, and the lowest rates in the groups fed 100% and 50% amphipod meal, with 70% and 73% survivorship, respectively (Table 2). Among the other groups, there were only minor differences in survival.
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The highest mean weights at the end of the experiment were in cod fed 100% fishmeal and 50% krill meal (2.5 g). In general, the average fish weight appeared to decrease to the lowest value (0.8 g) as the content of amphipod meal rose to 100%. SGRs were high and varied between 9.0% and 9.4%, except in the groups fed 50% and 100% amphipod meal, they were 8.5% and 6.8%, respectively (Table 2). The liver index was fairly stable and ranged from 9.6% to 11.7%. There was a tendency towards larger livers in fish fed 50% and 100% amphipod meal. The group fed 100% krill meal had the lowest liver index (Table 2).
At the end of the experiment, some of the fish exhibited spinal deformities (Figure 1). The number of deformities was significantly influenced by dietary composition (Table 2). There appeared to be a dose–response relationship with the percentage of amphipod meal, in that the lowest level of deformities (0.2%) was in fish fed 100% fishmeal and the highest (16%) in cod fed 100% amphipod meal. The trend was not observed in fish fed Antarctic krill.
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An amphipod has a thick shell and provides the meal with a high content of ash (Table 3) and fluorine and a low content of protein (Table 4). Antarctic krill displays high post-mortem autolytic activity and provides the meal with a high content of water-soluble protein (Table 3).
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The highest content of protein was in the feed of 100% fishmeal (54.3%), and the lowest was in the feed of 100% amphipod meal (38.4%). The four other diets had nearly identical contents of protein (46.3–48.8%) (Table 4).
The contents of docosahexaenoic acid (DHA, 22:6 n-3) and arachidonic acid (ARA, 20:4 n-6) were not significantly different among diets. Eicosapentaenoic acid (EPA, 20:5 n-3) was significantly higher in the 100% krill group (8.4%) than in the other groups (5.4–6.6%). The content of PUFA was highest in the 50% and 100% amph groups at 38.0% and 38.8%, respectively. In the other groups it ranged from 31.2% to 36.1% (Table 4).
Feed consisting of 100% amphipod meal had the highest contents of mercury, cadmium, and fluoride (Table 4). The amphipod meal had the highest contents of ash (22.2%) and chitin (10.4%) (Table 3), and fishmeal the lowest contents (11.6% and 0%, respectively).
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Discussion |
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TopIntroductionMaterial and methodsResultsDiscussionReferences |
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The various groups of cod, except those on the diet containing 100% amphipod meal, displayed relatively good growth and high rates of survival in comparison with, or better than, those obtained in previous experiments on cod of similar size and developmental stages (Otterå and Lie, 1991; Baskerville-Bridges and Kling, 2000; Leifson, 2003).
However, we found some striking differences among the groups of fish that could only be attributed to dietary composition. Growth rate was lower, and mortality, liver index, and skeletal deformities were higher following the substitution of higher proportions of amphipod meal for fishmeal. Cahu et al. (2003) have reviewed the connection between skeletal deformities and nutrient components (peptides, phospholipids, amino acid, ascorbic acid, and retonic acid). The protein level decreased with increasing amount of amphipod meal. However, the content of protein in the feed containing 100% krill meal was almost the same as in the feed containing 50% amphipod meal, and the deformities in the two groups were 0.3% and 8%, respectively. Thus, protein content does not appear to be the cause of deformities. DHA, EPA, and ARA are essential for growth, development, and survival (Sargent et al., 1997). Low contents of unsaturated fatty acids could cause deformities according to Cahu et al. (2003). In our experiment, there were no significant differences in the contents of DHA, EPA, and ARA, except in the diet of 100% krill, which was higher than the other groups. This group experienced a very low rate of spinal deformities.
The most striking difference, however, was the rise in the levels of mercury, cadmium, fluoride, ash, and chitin as the proportion of amphipod meal increased. It has been reported that fish living in areas with a high content of mercury in water bio-accumulate mercury, although no effects on growth and deformities have been reported (Southworth et al., 2000; Latif et al., 2001). Aqueous cadmium has been reported to cause spinal deformities and to retard growth, particularly of young fish (Cheng et al., 2000; Williams and Holdway, 2000; Calta, 2001; Asagba et al., 2004). Then again, cadmium in feed may act differently than aqueous cadmium. No physiological process has been reported to be dependent on cadmium, and it is regarded, therefore, as a non-essential element and known mostly as an environmental pollutant and toxic agent (Gill and Pant, 1981). Feeding cadmium to sheep caused mineral disturbance (Phillips et al., 2004). In fish, the retention of dietary cadmium in intestinal tissue is high, and the intestine seems to form an important barrier to the absorption of dietary cadmium (Harrison and Kloverkamp, 1989; Handy, 1992). Maximum permitted concentration for cadmium in animal feed has been set at 0.5 mg kg–1 dry weight (European Commission 1999 Directive 2002/32 EC). Berntssen (2000) reported that excess dietary cadmium retards the digestive process and causes depletion of energy storage at a level of 25 mg kg–1 in feed. Although dietary cadmium caused a clear reduction in calculated energy stores, there were no effects on growth in Atlantic salmon (Salmo salar). Berntssen (2000) suggests an upper limit of cadmium in feed of 7–11 mg kg–1. The highest amount in this experiment (6.5 mg kg–1) was in the diet containing 100% amphipod meal, but this was still below the suggested upper limit.
Fluoride is essential for optimal growth, reproduction, and mineralization of bones and teeth. In animals, naturally occurring fluoride is usually found in hard tissue such as bone or exoskeleton. High doses of fluoride can be toxic to human beings, mainly as a result of fluoride's harmful effects on tooth development and skeletal growth (Dean and Elvove, 1935). Unlike warm-blooded animals, marine fish seem able to tolerate extraordinarily large amounts of fluoride in their diet without suffering any ill effects (Gulbrandsen, 1979; Grave, 1981; Tiews et al., 1981, 1982). In a feeding experiment with Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), the amount of fluoride in fish fillets did not increase markedly when fish had been fed frozen krill and krill meal (Grave, 1981; Tiews et al., 1982). On the other hand, the bone fluoride content increased considerably. The EU sets the upper limit for fluoride in animal feed, including feed for farmed fish, at 150 mg kg–1 (Council Directive 2002/32 EC). Julshamn et al. (2004) studied how fluoride from krill meal enriched muscle, whole fish, and bone of adult salmon (Salmo salar) reared in seawater. The amount of fluoride in the feed ranged from 18 to 358 mg kg–1. They found that salmon are highly tolerant of dietary fluoride and that these doses did not lead to accumulation of fluoride in the fish. They concluded that the biological availability of fluoride from krill was low. The feed in our experiment contained higher levels of fluoride (420–840 mg kg–1 in the diets with 50% and 100% krill meal), but this did not seem to have any negative effect on the juvenile cod. It is also interesting that no spinal disorders were observed when larger cod, salmon, and halibut (Hippoglossus hippoglossus) were fed diets containing amphipods and Antarctic krill (R. E. Olsen, pers. comm.). Then again, there may be a difference in absorbance rates between small and large fish. At a length of 20 mm, all the vertebrae in cod are visible and fully developed (Pedersen and Falk Pedersen, 1992). According to Otterå and Lie (1991), a cod larva at 18.6-mm length has a wet weight of 49.2 mg. The start weight in this experiment was 28 mg, a weight at which the vertebrae were not yet fully developed, suggesting that high fluoride content might have caused the deformities.
The high ash content is another possible cause of the deformities observed. The amount and balance of minerals are important, as several minerals use the same uptake sites in the gut. Amphipod meal has a very high content of ash.
The conclusion we can draw from this experiment is that fishmeal may be replaced with 100% krill meal or 25% amphipod meal without any influence on growth, survival, or development. Further study is needed to identify the reason for the deformities in the groups fed high proportions (100% and 50%) of amphipod meal.
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Acknowledgements |
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I am grateful to the technical staff at the Institute of Marine Research, Austevoll for making this experiment possible. This work was supported by funds from the Norwegian Ministry of Fisheries.
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References |
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