ANNEX D (Contd.)

EIFAC SESSION 5/CARBOHYDRATES/E/28

CARBOHYDRATE NUTRITION IN ATLANTIC SALMON (SALMO SALAR)

Gro-Ingunn Hemre*, K.Sandnes, Ø. Lie, O. Torrisen¹ and R. Waagbø

Institute of Nutrition, Directorate of Fisheries, P.O.Box 1900, N-5024 Bergen, Norway
¹ Matre Aquaculture Research Station, N-5198 Matredal, Norway

Five groups of 300 Atlantic salmon were fed diets containing increasing levels of starch (from 0% to 30%) and concomitant decreasing levels of protein. The fish were fed the experimental diets in duplicate for 3 months. The results indicate that a diet for young salmon should contain dietary starch in the range from 5% to 20%, while no inclusion or above 20% seemed to exert negative effects on growth and feed utilization. Blood haematocrit and haemoglobin showed significant reduced levels as dietary starch increased, while no effect was found on red blood cell count. Plasma glucose levels ranged above reference levels only when the feed starch content was higher than 20%. Low levels of glycogen were found in kidneys, heart and gills, and the levels were significantly correlated to feed carbohydrate content. No variations were found in proximate or glycogen compositions of fillet, while the liver composition reflected the diet composition.

EIFAC SESSION 5/CARBOHYDRATES/E/29

EFFECTS OF DIETARY CHROMIC OXIDE LEVEL AS THE APPARENT DIGESTIBILITY MEASURING INDEX ON CARBOHYDRATE UTILIZATION IN TILAPIA

Shi-Yen Shiau* and Hong-Shiang Liang

Department of Marine Food Science, National Taiwan Ocean University,
Keelung, Taiwan 202, ROC

A feeding trial was conducted to investigate the influence of adding chromic oxide (Cr₂O₃) in the diet as the digestibility measuring index on carbohydrate utilization in tilapia, Oreochromis niloticus X O. aureus. Two levels of Cr₂O₃ (0.5 and 2%) were incorporated into the basal diet containing either glucose or starch as the carbohydrate source. The experimental diets were fed in three replicates to groups of tilapia weighing 1.11 ± 0.05 g in a recirculating, double-filtered rearing system for eight weeks. Results indicated that fish fed the starch diet had significantly (p < 0.05) higher body weight gain, feed conversion ratio (FCR), protein deposition, energy deposition and body lipid content than fish fed the glucose diet in both the 0.5 % and 2 % Cr₂O₃ supplementation levels. Fish fed the glucose diet supplemented with 0.5 % Cr₂O₃ had significantly higher body weight gain, FCR, protein digestibility, lipid digestibility, carbohydrate digestibility, dry matter digestibility, intestinal amylase activity and significantly lower liver glucose-6-phosphatase activity than those fed glucose diet supplemented with 2% Cr₂O₃. Delayed plasma sugar peak reach time was observed in fish fed the glucose diet supplemented with 0.5 % Cr₂O₃ compared with fish fed the glucose diet supplemented with 2 % Cr₂O₃. These data suggest that tilapia utilize starch better than glucose and that different dietary Cr₂O₃ inclusion levels affect glucose utilization.

EIFAC SESSION 6/MINERALS AND TRACE ELEMENTS/R/5

MINERALS AND TRACE ELEMENTS, REQUIREMENT AND DETERMINATION

F.J. Schwarz

Institut für Ernährungsphysiologie, Technische Universität München-Weihenstephan,
8050 Freising, Germany

A controlled nutrition of fishes implies an adequate dietary supply of minerals (major elements: Ca, P, Mg, Na, K, Cl; and trace elements: Zn, Cu, Fe, Mn, Se, I). According to the mineral status of the fish there can be distinguished a deficient supply (which is characterized by a low growth rate, clinical symptoms or high mortality), a suboptimal supply (which is described by biochemical changes or other indicators), an optimal supply (which gives best performance and health) and a subtoxic and a toxic supply. An optimal dietary supply should also consider the possible pollution of the water with minerals (see phosphorus). However, the measurements of the optimal dietary mineral requirement are considerably rendered, because nearly all elements are absorbed across the gills and skin from the external environment. To some extent a deficient status can be compensated or a subtoxic or toxic status can be produced. Therefore, balance studies are not quite well qualified to measure the dietary requirement. A factorial approach, which means a calculation of the net requirement by retention and endogenous loss and taking into consideration the availability (utilization) of the minerals, would be desirable. However, there are a lot of methodical difficulties and only a few data can be given. A further possibility is to use the dose response technique. The response of the dietary mineral, which is graduated over an extensive supply, is measured by qualified criteria (indicators) and analyzed by regressions. Therefore, it is necessary to acquire the best indicators for each mineral.

Preliminary results are presented. However, there are many factors which influence the data, including the weight or age of the fish, the performance status (weight gain rate), the composition of the diet (i.e. vitamins), the chemical composition of the mineral, possible interactions with other minerals, and the amount of water borne elements. Experimental designs and methodologies should consider these factors. The respective mineral requirement is specific for each fish species. In this review data on rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio L.) are produced, and the experimental results presented for nearly all elements confirm the above-mentioned statements.

EIFAC SESSION 6/MINERALS AND TRACE ELEMENTS/E/30

EFFECTS OF PHOSPHORUS-SUPPLY ON GROWTH AND MINERALIZATION IN CARP

Arndt Schafer*, W.M Koppe, K.-H. Meyer-Burgdorff and K.D. Günther

Institut für Tierphysiologie und Tierernährung,
Kellner Weg 6, 3400 Göttingen, Germany

The effect of phosphorus(P)-supply on growth and mineralization of whole body and defatted body fractions such as backbone, opercula and dorsal scales was investigated in mirror carp (Cyprinus carpio L.). The diet used in this experiment was based on soybean meal, fishmeal and wheat starch resulting in a total P-content of 7.2 g/kg DM. This diet (RO) was supplemented at two different levels with mono calcium phosphate. The total P-content of these diets was 9.2 g (RI) and 11.6 g (R2) per kg DM. Triplicate groups of fish (10 carp/tank) with an initial weight of 40 g were fed at a level of 20 g/kgW^0.8d for nine weeks at a water temperature of 23°C.

The different levels of dietary phosphorus affected growth, crude ash-content and the P content of all fractions. The results of the mineralization (% of DM) and the total weight gain (g/fish) are presented in the following table (p≤0.05).

The results suggested that between the diets there was an enhancement in weight gain, crude ash and P-content in whole body, backbone, scales and opercula. Weight gain increased linearly with P-content in the diet. P-content and crude ash-content of the scales and whole body were influenced in the same way. Backbone and opercula were only slightly affected by the highest level of phosphorus in the diet. Best correlation to weight gain was found in the P-content of scales and so they may be useful as a marker to describe the P-status of carp even when fish are still alive, and thus could be of practical importance.

EIFAC SESSION 6/MINERALS AND TRACE ELEMENTS/E/31

THE USE OF PHOSPHORUS BUDGET STUDIES TO DETERMINE THE PHOSPHORUS REQUIREMENTS OF THE EUROPEAN EEL (ANGUILLA ANGUILLA L.)

L.T.N. Heinsbroekl¹,²*, P.A.T. Tijssen¹ and A.M. Hariati¹,³

¹ Department of Fish Culture and Fisheries, Agricultural University, Wageningen,
P.O.Box 338, 6700 AH Wageningen, The Netherlands
² R & D Department Fishfeeds, Coppens International B.V., Waaldijk 35,
5305 CB Zuilichem, The Netherlands
³ Faculty of Fisheries, Brawijaya University, Jalan Mayjen Haryono 161, Indonesia

Minimization of the environmental impact of aquaculture, by discharge of wastes, necessitates optimization of dietary nutrient levels and their utilization. Nutrient budget studies, which are helpful in determination of nutrient requirements and nutrient utilization, are frequently published for nitrogen but not for phosphorus. In this study European eel, Anguilla anguilla, of 50 – 60 g were fed four experimental diets, with phosphorus levels of 0.44, 0.57, 0.78 and 1.09 %, and one commercial diet containing 1.30 % phosphorus, for four weeks. Digestibility and excretion of phosphorus were determined twice a week. Intake and retention of phosphorus were also determined.

The digestibility of the phosphorus source used to create different phosphorus levels in the experimental diets (NaCaPO₄) proved to be only about 15 % for eels. The dietary levels of available phosphorus in the experimental diets were therefore 0.14, 0.16, 0.19 and 0.25 %. The commercial diet contained 0.46 % available phosphorus. The dietary phosphorus level had no effect on phosphorus retention. The phosphorus retention efficiency therefore decreased with increasing dietary phosphorus level, from 52.9 to about 15 % of the phosphorus intake. Based on these data the requirement for available phosphorus of European eel of 50 – 90 g weight is estimated to be 0.14 % of the diet. The excretion of ortho-phosphate increased with dietary phosphorus level, from 0.9 to 17.4 % of the phosphorus intake, or from 0.05 to 3.06 mg/fish/day. The total phosphorus recovery decreased with increasing dietary phosphorus level, from 121.1 to 98.3 % of the phosphorus intake. The possibility of phosphorus uptake from the water at low dietary phosphorus levels is not substantiated by the phosphorus excretion data. It is therefore concluded that the presented methodology harbours a small and constant overestimation, of 0.5 – 1.0 mg/fish/day in this study, of either faecal, excreted or retained phosphorus.

EIFAC SESSION 6/MINERALS AND TRACE ELEMENTS /E/32

TRACE ELEMENTS IN ATLANTIC SALMON NUTRITION; FEED CONCENTRATIONS, REQUIREMENTS AND INTERACTIONS

Amund Maage*, Mette Lorentzen, Friede Andersen, Marit Bjørnevik and Kåre Julshamn

Institute of Nutrition, Directorate of Fisheries,
P.O.Box 1900, Nordnes, N-5024 Bergen, Norway

The requirements for trace elements in fish, including Atlantic salmon (Salmo salar), are scarcely known. The supplementation employed in the feed industry has therefore been largely based on experiments on other animals.

With the growing importance of the salmon industry it has been necessary to give a scientific basis for the recommendation of trace element concentrations in salmon feed. We have therefore in our Institute performed a series of experiments aimed at establishing the requirements of trace elements by the use of semi-synthetic diets. Further, we have performed experiments to evaluate whether it is necessary to supplement feeds when they are based on fish meal.

Special problems in establishing requirements of trace elements for Atlantic salmon have been to establish acceptable fish growth on semi-synthetic diets. However, by using cod muscle meal as a protein source this has been satisfactory solved.

Most of the experiments have been performed on salmon in the fresh water phase. We have established the requirement for zinc to be in the range of 37–67 mg/kg dry feed and of iron to be in the range of 48–59 mg/kg dry feed. Further it seems prudent to supplement also fish meal based diets with zinc and manganese and perhaps also iron, depending on the form and amount of iron in the fish meal. However, it does not seem necessary to supplement fish meal based diets with selenium.

This project has received continued support from the Norwegian Fisheries Research Council and new experiments are being performed.

EIFAC SESSION 6/MINERALS AND TRACE ELEMENTS/E/33

EFFECTS OF EXTRA DIETARY IRON SUPPLY ON SEA BREAM (SPARUS AURATA) GROWTH RATE, REARED UNDER REDUCED WATER OXYGEN LEVELS

Sofronios E. Papoutsoglou*, G. Tziha, D. Barkas and X. Brettos

Agricultural University of Athens, Laboratory of Applied Hydrobiology,
lera Odos 75, Votanikos 118 55 Athens, Greece

The effect of extra dietary iron on the growth rate of gilthead sea bream (Sparus aurata) of mean initial body weight 18.1 ± 3.9 g, under low dissolved oxygen conditions, was investigated. A factorial experimental design was used for two dissolved oxygen levels (6.51 ± 0.7 and 3.76 ± 1.5 ppm) and for three feed iron (50.1 ± 0.8, 65.9 ± 1.4 and 81.2 ± 1.6 mg Fe/100 g of food) levels. The experiment lasted 12 weeks and each one of the six experimental populations consisted of 15 specimens.

The results obtained showed that there is no need for more dietary Fe than that contained in the commercial diet used (50.1 mg Fe/100g of food) under high dissolved oxygen conditions (6.5 ppm). On the contrary, under low water dissolved oxygen conditions (3.76 ppm), an extra dietary Fe supply of about 16 mg Fe/100 g of food (total 65.9 mg Fe/100 g of food) showed a clear improvement of FCR (food conversion ratio), PER (protein efficiency ratio), SGR (specific growth rate) and % body weight increase levels. Furthermore, according to the economical analyses made, the same amount of the extra dietary Fe showed a considerable beneficial effect in terms of both the cost of gained body weight (1 kg) and the cost of final body weight (1 kg) per 1 mg of extra supplied Fe used. The possible explanations of the results obtained are discussed.

EIFAC SESSION 7/VITAMINS/R/6

VITAMIN REQUIREMENT STUDY TECHNIQUES

John E. Halver

School of Fisheries HF-15, University of Washington, Seattle, WA 98195, USA

Vitamin requirement studies have been conducted for 50 years using growth and clinical signs of deficiency for quantitative studies. Histological confirmation of apparent signs confirm and establish subclinical measurements for deficiency. Test diets for these studies with positive experimental control over the test vitamin, have been developed using varying degrees of vitamin free ingredients in the formulations. Haematology values and microanatomical changes reflect failures of a metabolic system and provide convincing data for requirements. Quantitative studies have used specific test diets plus increasing aliquots of the test vitamin, coupled with growth response, tissue storage analysis, and specific enzyme system activity. Results have been reported for levels which support normal growth, clinical enzyme saturation, and maximum liver or other tissue storage levels. Megavitamin intake studies have been correlated with improved resistance to stress and certain fish diseases. Statistical measurement of response to various parameters used become essential for quantitative vitamin requirement determinations. Early studies using vitamin analysis of feedstuffs mixtures coupled with growth response, absence of deficiency signs, and liver or tissue storage have been superseded by diets with more positive control of all nutrients, including the vitamin to be tested. Sparing effects of one vitamin upon another can be demonstrated. Various vitamin forms may have different activity to supply physiological requirements for the vitamin. Most water-soluble vitamins act as co-enzymes in metabolic systems. Some fat-soluble vitamins have molecular functions acting as hormones, free radical traps, intracellular reducing agents, pigments, antioxidants, etc.. Appropriate techniques for the water-soluble vitamins and fat-soluble vitamins will be discussed.

EIFAC SESSION 7/VITAMINS/E/34

MAXIMAL GROWTH IN VITAMIN REQUIREMENTS DETERMINATION - FISH OR HATCHERY REQUIREMENT?

M. Kuczynski

Institute of Ichthyobiology and Aquaculture, Polish Academy of Sciences,
Golysz, 43-422 Chybie, Poland

The experiment was conducted in plastic aquaria to determine optimal dosage of “VITASOL EC” preparate in African catfish (Clarias gariepinus Burchell 1822) starter diet. African catfish fry (2.3 mg average initial weight) were fed commercial starter-type diet supplemented with 0.25, 0.64, 1.60, 4.00, 10.00 and 25.00 g/kg of VITASOL EC. Calculated vitamin dosage was 25, 64, 160, 400, 1000 and 2 500 mg of L-ascorbic acid and 5, 13, 52, 80, 200 and 500 mg of α-tocopherol acetate in one kilogram of feed. After four weeks, weight gain was the best in the group fed the diet supplemented with VITASOL EC at a dosage of 1.60 g/kg (737.07 mg average final weight), but survival rate was 31%. The best survival rate was in the group fed the diet supplemented with 10.00 g/kg VITASOL EC, although weight gain was smaller (596.39 mg average final weight). There were no gross signs of vitamin C deficiency in any of the fish.

During the first two weeks of the experiment, weight gain was not very different for all fish. However, survival rate depended on the level of VITASOL EC supplementation in the diet. Survival rate was best (over 80 %) in the group fed the diet containing 25.00 g VITASOL EC/kg. The lowest survival rate was in the group fed the diet containing 25.00 g VITASOL EC/kg. Regression analysis of survival data from every 5-day period showed that the activity of vitamin C was not in growth, but in survival rate improvement. The growth of African catfish larvae was similar to that reported in the literature. The fact that no deficiency signs were detected in any of the fish show that the minimum requirement for both vitamin C and E was covered by feed intake. Dramatically declined survival rate was directly connected with VITASOL E-C supplementation in the diet. This can be an effect of the improved disease resistance after feeding with high doses of vitamins. The first growth differences between groups detected during the first two weeks of fish life could be a result of the high vitamin needs of rapidly growing young fish. The ascorbic acid concentration of eggs decline rapidly during development and because of this, hatched larvae are extremely sensitive to vitamin deficient diets. The maximum growth obtained after 30 days in group number 3 fed the diet supplemented with 4.00 g of VITASOL E-C per 1 kg was the result of high mortality. Growth of African catfish larvae strongly depends on stocking densities. Regression analysis of survival data from every 5-day period showed that activity of the VITASOL E-C was not in growth, but in survival rate improvement during the first 15 days of the experiment.

EIFAC SESSION 7/VITAMINS/E/35

CARNITINE, A VITAMIN FOR TROUT ?

Annette Schuhmacher

Institut für Tierernährung, Gustav-Kühn-Str. 8, 0-7022 Leipzig, Germany

L-carnitine - the quaternary amine β-hydroxy- γ-trimethyl amino butyric acid - mediates the transport of middle- and long-chain fatty acids from cytosol into mitochondria. In contrast to newborns, adults possess the capability for endogenous synthesis of l-carnitine from lysine and methionine, respectively. Based on its function in intermediary metabolism, l-carnitine improves lipid utilization and may accordingly increase body gain and feed efficiency; nevertheless, a potential effect of carnitine supplementation seems to depend on the presence of its precursors lysine and methionine. Therefore, in a trial on 480 juvenile rainbow trout (20 g BM at start) the influence of l-carnitine supplementation (450 mg/kg feed) was studied with diets (50 % CP, 18 % CL) containing different levels of lysine and methionine, respectively. Growth, feed consumption and feed efficiency was investigated over a period of 84 days. The results are given in the table below.

Carnitine supplementation improved body mass gain (BMG), feed conversion ratio (FCR) and specific growth rate α, but not survival rate, in trout fed diets marginal in lysine and sulphur amino acids. Increasing lysine or sulphur amino acids lead to an improvement of experimental criteria comparable to the effect of carnitine.

It is concluded that l-carnitine, besides of its effects on lipid metabolism, exerts a certain balance for marginally insufficient dietary amino acid pattern because of its compensatory effect on low lysine and sulphur amino acid levels. Studies on pigs and Japanese quail (unpublished results) would support this view. Further findings are necessary to finally decide on the vitamin character of carnitine for trout.

¹ This study was performed with the financial support of Lonza Ltd., Basle

EIFAC SESSION 7/VITAMINS/E/36

EFFECTS OF SUPPLEMENTAL BIOTIN ON GROWTH AND STRESS RESPONSE IN
RAINBOW TROUT (ONCORHYNCHUS MYKISS W.)

Koppe, W.M.*, Frigg. M.¹ and Günther, K.D.

Institute for Animal Physiology and Animal Nutrition, Kellnerweg 6,
W-3400 Goettingen, Germany
¹ Hoffmann - La Roche AG, CH-4002 Basel, Switzerland

A wide range of dietary biotin contents (0.1 to 1.2 mg/kg diet) in fish feeds is recommended for maximum performance of salmonids reflecting either different experimental conditions or the use of different criteria. To evaluate the usefulness of biotin supplementation to feed mixtures two experiments were conducted.

In experiment 1 a semipurified biotin-free casein/gelatine-based diet was gradually supplemented with D-biotin levels ranging from 100 to 2000 mcg/kg diet. Duplicate groups of rainbow trout (12 g) were fed on the experimental diets for 12 weeks. The results confirmed a positive effect of the addition of 100 mcg biotin/kg diet on growth, nutrient and energy retention of trout. Furthermore behaviour of trout fed on the unsupplemented diet showed signs of deficiency primarily affecting feed intake. Biotin supplementation exceeding 100 mcg/kg diet raised plasma biotin levels gradually.

In experiment 2 two fishmeal-based diets either unsupplemented (110 mcg biotin/kg diet) or supplemented (2260 mcg/kg) were compared. No differences were observed in growth and feed conversion between experimental groups. No deficiency symptoms occurred. After a 24 weeks feeding period trout were subjected to a 30 second handling stress and subsequent changes in plasma levels of glucose, lactate and lysozyme were recorded at different times post stress. While lactate and lysozyme were not affected by supplemental biotin the raise in plasma glucose in the supplemented group was significantly higher 1 hour poststress (p <0.01) indicating an improved ability of fish to counteract the stressor. This effect was possibly due to an enhanced activity of biotin-dependent enzymes involved in gluconeogenesis (e.g. hepatic pyruvate carboxylase).

Conclusively it must be recommended to supplement trout feeds with biotin even if the native biotin content seems to satisfy the requirement for maximum growth. The importance of additional criteria like stress response or immune response to evaluate experimental results in requirement studies becomes evident especially when suboptimal conditions in practical fish farming are considered.

EIFAC SESSION 7/VITAMINS/E/37

REQUIREMENTS FOR CAROTENOIDS IN FISH DIETS

O.J. Torrissen and R. Christiansen

Institute of Marine Research, Matre Aquaculture Station
N-5198 Matredal, Norway

Carotenoids are among the most widespread and important pigment classes in living organisms, and in marine animals, astaxanthin is the most common occurring red carotenoid. Carotenoids are Vitamin A precursors and are fundamental in photosynthesis and light protection in plants. Increasing attention has been drawn to a possible light protection, cancer prevention and immune enhancement by carotenoids in mammals. Reported functions in fish range from a general enhancement of performance to specific functions in reproduction and metabolism.

In this paper, we show that astaxanthin is essential for growth and survival of Atlantic salmon (Salmo salar), and discuss this fact in relation to the use of purified and semipurified diets in nutritional studies. The similarity in action of astaxanthin and canthaxanthin compared to α-tocopherol (Vitamin E) and retinol (Vitamin A) suggests that these two carotenoids should be listed among the fat soluble vitamins.

EIFAC SESSION 7/VITAMINS/E/38

NUTRITIONAL STUDIES ON VITAMIN B₆ IN ATLANTIC SALMON (SALMO SALAR)

Sissel Albrektsen* and Kjartan Sandnes

Institute of Nutrition, Directorate of Fisheries
P.O.Box 1900-Nordnes, N-5024 Bergen, Norway

Dietary vitamin B₆ affects growth, tissue vitamin B₆ retention and activity of the vitamin B₆ dependent enzyme aspartate aminotransaminase (ASAT) in muscle, liver and serum. The requirement for vitamin B₆ in Atlantic salmon fry as judged by these criteria will be discussed (table).

The presentation will further include results on tissue vitamin B₆ retention, ASAT activity and body fat contents in juveniles fed graded dietary vitamin B₆ levels.

Table: Dietary vitamin B₆ requirement in Atlantic salmon as evaluated on the basis of growth, tissue vitamin B₆ concentration and aspartate aminotransaminase (ASAT) activity

EIFAC SESSION 7/VITAMINS/E/39

STABILITY AND UTILIZATION OF VITAMIN C DERIVATIVES IN RAINBOW TROUT (ONCORHYNCHUS MYKISS)

Jacques Gabaudan*, Viviane Verlhac, Martin Gadient and Peter Hofmann

Vitamins and Fine Chemicals Research, F. Hoffmann-La Roche Ltd.,
CH-4002 Basel, Switzerland

The stability of vitamin C derivatives during feed processing and storage was determined. Ascorbate-2-phosphate and ascorbate-2-sulfate were shown to be highly stable during extrusion and storage.

Bioavailability of ascorbic acid (AA) from its derivatives was assessed using three approaches: AA plasma kinetics after oral administration of a single dose of the substance, determination of apparent digestibility coefficients using chromic oxide as an indicator and AA tissue concentrations during feeding trials. In the kinetic study, the area under the plasma AA concentration curve of ascorbate 2-polyphosphate was 3101 mg.h/l compared to 357 mg.h/l for ascorbate-2-sulfate. The apparent digestibility coefficient of ascorbate-2-polyphosphate and ascorbate-2-sulfate was 99.5% and 23.1 %, respectively. Results consistently indicated a very high bioavailability of AA from its phosphate derivatives as opposed to the sulfate derivative.

Recent results in the literature indicate that the minimum AA requirement for growth of fish fed a stable form of ascorbic acid under controlled culture conditions is lower than previously thought. However, we have found that parameters of the nonspecific immune response such as lympho proliferation and natural killer cell activity were significantly higher with increased intake of ascorbate-2-phosphate. This results in improved resistance against infectious diseases.

EIFAC SESSION 7/VITAMINS/P/1

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH ION PAIRING AND ENZYME PEAK SHIFTING FOR THE SIMULTANEOUS DETERMINATION OF THREE FORMS OF VITAMIN C

P.D. Maugle

P.D.M. & Associates, 88 Central Avenue, UK-Norwich, CT 06360, USA

A variety of stable vitamers of ascorbic acid (C₁) have been introduced as sources of vitamin C supplements for fish feeds. The assessment of these more stable analogues of ascorbic acid, (dipotassium ascorbyl 2-sulfate dihydrate (C₂), l-ascorbyl 2-mono phosphate sodium (C₃-M), and l-ascorbyl 2-polyphosphate (C₃-P)) require the development of improved analytical methods.

Using an improved High Performance Liquid Chromatographic method the relative stability of ascorbate 2-sulfate and ascorbate 2-polyphosphate in salmon feed, and the simultaneous determination of ascorbic acid, ascorbate 2-monophosphate and ascorbate 2-sulfate was studied.

The effects of the manufacture process, time and temperature during storage, and the extraction procedure used to extract the vitamers from feed and in salmon tissue were investigated.

EIFAC SESSION 7/VITAMINS/P/2

COMPARATIVE STUDIES ON THE EFFECT OF VITAMIN C FEEDING OF EUROPEAN CATFISH
(SILURUS GLANIS L.) AND STURGEON HYBRID
(ACIPENSER RUTHENUS X ACIPENSER BAERI)

Gy. Papp, Zs.*, Jeney, Zs. and G. Jeney

Fish Culture Research Institute, Szarvas, P.O. Box 47, H-5541 Hungary

Most cultured fish require vitamin C (L-ascorbic acid) because they are unable to synthesis it. At the same time they can store this micro nutrient in chemically stable form (Tucker and Halver, 1984). On the way to determine the vitamin C requirement of a fish species the first step has to be the assay of it's effect on the growth, physiological status and storage capacity. The effects of feeding vitamin C on some fish species like rainbow trout (Sato et al., 1991), common carp (Dabrowska at al.. 1991), channel catfish (Wilson et al., 1989) are well known but only limited information is available concerning the vitamin C requirements of some new species for aquaculture, such as the European catfish and sturgeon hybrids.

Eight weeks old fingerling European catfish (5.35 ± 0.27 g) and sturgeon hybrids (11.9 ± 2.1 g) were fed in duplicate with diets containing graded levels of dietary vitamin C (vitamin C free-diet, AA < 5 mg/kg as control, 100 mg/kg, 1000 mg/kg and 2000 mg/kg AA). After eight weeks feeding period no significant differences were found in the growth among the experimental groups of the catfish, while a slight positive effect of vitamin C supplementation on growth of sturgeon hybrids was observed, although the differences were not significant. The level of vitamin C in muscle tissue were about 10–20 μg/g in both species. No correlation was found between the levels of vitamin C in food and in muscle. The storage capacity in the liver of the sturgeon hybrids was two times higher than that of catfish. The liver of the European catfish has limited capacity to store l-ascorbic acid (120–160 μg/g AA), appearing only above 1000 mg/kg vitamin C supplementation in the diet. Enhanced haemopoiesis in both fish species fed vitamin C levels above 1000 mg/kg was reflected by significantly higher values for haematocrit and leucocrit, as well as by the number of erythrocytes and leucocytes. In these groups the number of NBT positive cells were significantly higher. Vitamin C feeding did not affect the haemoglobin and glucose concentration.

EIFAC SESSION 7/VITAMINS/P/3

NEEDS FOR VITAMIN C IN FOOD FOR SEA BASS (DICENTRARCHUS LABRAX, L.) INTENSIVELY REARED IN MARINE WATER

M. Saroglia*¹, G. Scarnano², Maria R. Bosco¹ and Genciana Terova³

¹ Dipartimento Scienze delle Produzioni Animale, Università della Basilicata, Via Nazzario Sauro
85, Potenza, Italy
² Consiglio Nazionale delle Ricerche, Istituto per lo Sfruttamento Biologico delle Lagune, Lesina
(FG), Italy
³ Agriculture University of Tirana, Albania

The need for vitamin C has been studied on sea bass (Dicentrarchus Labrax, L.) reared with intensive technology.

In particular the minimum amount of ascorbic acid required in the diet (sufficient to give a plateau concentration in the liver of fish) and the decay of ascorbate in pelleted food during storage and on contact with seawater was studied. In addition, the description of artificially induced vitamin C deficiency syndrome in sea bass and the protective effect of ascorbic acid versus fish intoxication induced by nitrite are reported.

EIFAC SESSION 7/VITAMINS/P/4

LIVE FOOD MEDIATED VITAMIN C TRANSFER TO DICENTRARCHUS LABRAX
AND CLARIAS GARIEPINUS

G. Merchie¹, P. Lavens¹, Ph. Dhert¹, R. Pector², A.F. Mai Soni¹, M. Abbes¹, H. Nelis³, F. Ollevier², A. De Leenheer⁴ & P. Sorgeloos¹

¹ Laboratory of Aquaculture & Artemia Reference Center, University of Gent,
Rozier 44, B-9000 Gent, Belgium
² Laboratory of Ecology and Aquaculture, Catholic University of Leuven, Naamsestraat 59,
B-3000 Leuven, Belgium
³ Laboratory of Pharmaceutical Microbiology, University of Gent, Harelbekestraat 72, B-9000
Gent, Belgium
⁴ Laboratory of Medical Biochemistry and Clinical Analysis, University of Gent, Harelbekestraat
72, B-9000 Gent, Belgium

The live food enrichment technique, using artificial diets and emulsions for improving the nutritional quality of Brachionus and Artemia respectively, was further investigated as a tool for transferring vitamin C to fish larvae through the food chain. In previous experiments with the crustacean Macrobrachium rosenbergii feeding vitamin C-boosted live food resulted in a beneficial effect on the stress resistance of the larvae and a higher incorporation of ascorbic acid (AA) into the body tissue.

The effect of feeding a high concentration of vitamin C on larviculture success has now been verified for seabass (D. labrax) and African catfish (C. gariepinus) using three different enrichment levels in the live food (0 %, 10 % and 20 %).

Artemia nauplii enriched for 24 h with an experimental emulsion containing 20 % HUFA and 0 %, 10 % and 20 % ascorbyl palmitate (AP; a chemically stable ester of AA) were administered to catfish larvae in a 20 day feeding trial. Survival was not affected by the dietary AA, but from day 7 onwards a significantly positive effect of supplemental AA (20 % AP compared to 0 % AP compared to 0 % and 10 % AP) on dry weight was demonstrated, while on the final day of the experiment the 20 % AP group weighed 30 % more than the control (0 % AP), respectively 9.5 and 6.4 mg DW. Evaluation of the physiological condition by means of salinity tests demonstrated a significantly higher resistance of the larvae according to the dietary vitamin C level. As for all three treatments the larval growth was relatively low, it still has to be verified if extra vitamin C in the diet really promotes growth or only slows down a growth retardation caused in suboptimal conditions.

Seabass larvae were fed successively rotifers (day 4–12) and enriched Artemia nauplii (days 13–46). Rotifers were cultured on an artificial diet (Culture Selco, Artemia Systems N.V., Baasrode, Belgium) supplemented with 0 %, 10 % and 20 % AP respectively. Artemia nauplii were enriched for 24 h with an experimental emulsion containing 50 % HUFA in which 0 % 10 % and 20 % AP respectively was included. No significant differences in production characteristics (survival, dry weight, length) nor in stress resistance of the fish larvae could be observed; however, for all salinity stress tests the 20 % AP group performed better. AA was incorporated into the predator larvae for the Artemia phase (10 % and 20 % AP group compared to control group), although no differences between the 10 % and the 20 % AP treatment could be detected.

Comparing the results for the two aquaculture fish species, together with those reported earlier for the prawn larvae of Macrobrachium, the requirements for vitamin C are probably not only species dependent, but might differ also according to the culture conditions.

EIFAC SESSION 7/VITAMINS/P/5

STUDIES ON VITAMIN C IN ATLANTIC SALMON (SALMO SALAR)

Kjartan Sandnes* and Rune Waagbø

Institute of Nutrition, Directorate of Fisheries
P.O. Box 1900, N-5024 Bergen, Norway

The presentation comprises studies on vitamin C in Atlantic salmon carried out at our institute. Various aspects of supplementing vitamin C through the diet in order to optimize intensive production of salmon have been evaluated, including:

1. Chemical derivatives of ascorbic acid
2. Stage of salmon development

3. Environmental factors

It has been shown that the bioactivity of ascorbate-2-sulfate is inferior to (approximately 25 % of) L-ascorbic acid and phosphate derivatives of ascorbic acid. The dietary requirement ranges from 10–20 mg ascorbic acid equivalents/kg feed to support growth, to more than thousand times this level for optimum disease resistance.

Consequently, the term “dietary requirement of vitamin C” is of no value if used without further definitions and/or precisions. In view of the topics of this workshop, data from feeding studies with vitamin C in Atlantic salmon will be presented and discussed. Suggestions for alternatives to denote feed levels will be given.

EIFAC SESSION 7/VITAMINS/P/6

EFFECTS OF VITAMIN A AVITAMINOSIS AND HYPERVITAMINOSIS ON HEALTH AND GROWTH OF TILAPIA NILOTICA (OREOCHROMIS NILOTICUS)

G. Saleh¹, Wafa Eleraky² and J. M. Gropp*³

¹ Department of Poultry and Fish Diseases,
² Department of Hygiene and Preventive Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt
³ Institut für Tierernährung, Gustav-Kühn-Straβe 8, D-04159 Leipzig, Germany

The Vitamin A requirement of tilapia is estimated in the range of 1 000 to 2 500 IU/kg diet with highest values up to 5 500 IU. An experiment with tilapia (O. niloticus) was performed to study vitamin deficiency symptoms and to evaluate the vitamin A tolerance above the assumed requirement. 120 tilapia fingerlings were held in glass aquaria (92-l) with dechlorinated, aerated water (25 ± 1°C, 4.8 mg dissolved O₂/l, pH-value: 7.2) for 18 weeks. Pelleted diets (for composition see table) were supplemented with 0, 5,000, 10,000 and 40,000 IU vitamin A/kg, respectively.

Vitamin A deficient tilapia showed restlessness, abnormal movement, blindness combined with haemorrhages and exophthalmia and anaemia. Fish showed haemorrhages on the skin and at the bases of the fins. Skin mucus secretion stopped. Post mortem examination showed serous fluid in the abdominal cavity, haemorrhages in the kidneys and clubbed gills.

The tolerance to higher vitamin A doses was rather small.

5,000 IU vitamin A resulted in the best performance of tilapia, and further increasing dietary vitamin A level resulted in reduced growth and finally (at 40,000 IU vitamin A) to increased mortality. Hypervitaminosis A provoked abnormal bone formation, skin haemorrhages and necrosis of the caudal fin. Post mortem necropsy revealed enlarged liver and spleen and a fusion of the vertebrae.

EIFAC SESSION 7/VITAMINS/P/7

STUDIES ON THE VITAMIN E REQUIREMENT OF TILAPIA NILOTICA (OREOCHROMIS NILOTICUS), EFFECTS ON HEALTH AND GROWTH

Wafa Eleraky¹ G. Saleh² and J.M. Gropp³

¹ Department of Hygiene and Preventive Medicine,
² Department of Poultry and Fish Diseases, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt
³ Institut für Tierernährung, Gustav-Kühn-Straβe 8, D-04159 Leipzig, Germany

The vitamin E requirement of tilapia is commonly deduced from other warm water fish like carp. For example, the reported vitamin E requirement data for carp is 80–100 mg/kg diet (Halver, 1979) and 100 mg/kg (Bryant et al., 1980). Interactions between the tocopherols and Se, as well as polyunsatured fatty acids, are generally well known and also accepted for fish. However, a dietary level of 100 mg vitamin E/kg diet seems relatively high in comparison to other fish species and also to homeothermic animals. Therefore an experiment was conducted with tilapia nilotica fingerlings in order get an approximation of the potential range of the vitamin E requirement. 144 tilapia fingerlings were held in glass aquaria (92-l) with dechlorinated, aerated water (26 ± 1°C, 4.8 mg dissolved O₂/1, pH-value: 7.2) for 16 weeks. Diets (for composition see table) without added Se were supplemented at levels of 0, 10, 20 and 100 mg vitamin E/kg, respectively.

Fish deficient in vitamin E showed abnormal movement, depigmentation, sometimes protrusion of the eyes with haemorrhages, oedema and ascites, and they became anaemic. The liver was pale, and ceroids occurred in the liver and spleen.

Mortality decreased with increasing dietary vitamin E concentration. The results show that 10 mg vitamin E is obviously too low; the requirement may be between 20 and 100 mg/kg diet, but for the conditions tested probably nearer to 20 mg than to 100 mg.

EIFAC SESSION 8/FEED ADDITIVES/R/7

EVALUATION OF ADDITIVES IN FISH FEEDS

T. Storebakken, E. Austreng, G. Beverfjord and M. Rye.

AKVAFORSK, Institute of Aquaculture Research, Ltd., N-6600 Sundalsøra, Norway

Additives are quantitatively minor ingredients in the feed, and they are not mainly included as a source of nutrients. Examples of additives are antioxidants, attractants, absorbants, binders, carotenoids, growth stimulants, nutrient partitioners, softeners and other components useful to improve the technical or nutritional quality of the feed. There are two main aims of routine experimental work with additives in fish feeds: to document whether the additive has the desired effect, and to identify positive or negative side effects. This necessitates careful consideration of the variation in all methods employed, including the statistical tests. A testing procedure for a feed additive requires the use of large resources in order to fulfil the formal requirements for functionality and safety:

1. Technical tests of the additive and the feed includes detailed chemical analyses and evaluation of the technical properties of the feed during production and use. 2. Biological short-term tests may include digestibility and feed preference studies in order to document if the additive affects the absorption and acceptability of the feed. 3. Biological long-term tests may be designed as dose-response studies with increasing amounts of the additive. It is very important that such tests are carried out in systems which gives growth comparable to that obtained in commercial production since fish from the long-term tests also may supply material for several of the subsequent tests. Important criteria which are evaluated in long-term tests are: growth, body composition, survival, feed conversion (necessitates long term feed waste collection), and eventually reproduction. 4. Tests concerning the health of the fish should be performed as an integrated part of the biological long-term studies. A thorough morphological examination of major organs involved in absorption, metabolism and excretion of the additive should be carried out, with special reference to objective judgement of the material. 5. Tests concerning the health of the consumer are mainly limited to the analysis of residuals and metabolites of the additive in material from long-term tests. Methods for studies concerning toxicity, carcinogenity and allergenity of food additives and the authorization of laboratories for such studies are regulated by detailed legislation. 6. Tests on the sensory aspects of the product may be carried out on material from the long-term tests by means of trained panellists or consumer panels. 7. Environmental tests. The impact of agriculture and aquaculture on the environment has received increased attention during the last few years. The evaluation of environmental effects of fish feeds may mainly be based on the interpretation of results obtained from the previously described tests 1 Does the feed particle leach nutrients or does the technical quality of the feed impair feed acceptability by the fish? Does the quality of the feed facilitate removal from the effluent water?, 2 Are the nutrients well absorbed or does low digestibility represent a potential source of pollution? Do the fish like the taste of the feed, or may rejected feed pollute? and 3 Is the absorbed feed retained in growth, or is it a source of water soluble nutrients through metabolic losses?.

Astaxanthin is used as an example of an additive. Astaxanthin is present in the ppm-range in feed and tissues, and is easily destroyed by oxidation. Utilization of astaxanthin is life stanza-, fish size- and species-dependent and it is metabolized by the fish. This necessitates a careful selection of analytical and biological methods.

EIFAC SESSION 8/FEED ADDITIVES/E/40

STRATEGIES FOR SALMONID PIGMENTATION

Ole J. Torrissen

Institute of Marine Research, Matre Aquaculture Station
N-5198 Matredal, Norway

The market demands a pigmented flesh of farmed salmonids and, second after freshness, a good pigmentation is regarded as the most important quality criteria for farmed Atlantic salmon (Salmo salar). The required redness of salmonid flesh has steadily increased from an astaxanthin concentration in salmonid flesh of about 2–3 mg/kg during the 1970s to above 6 mg/kg today.

The rate of carotenoid deposition in the flesh of salmonids, and the visual pigmentation response, depends on multiple factors including; the specific carotenoid fed, the dietary concentration of the carotenoid, energy density of the diet, fish size, the physiological stage of the fish, environmental factors, diseases, genetical background and time of carotenoid feeding. A measurable and comparable difference among treatments in pigmentation studies require attention to the above listed factors.

An individual variation in flesh pigmentation of more than 100 % is experienced under practical as well as experimental conditions. An individual variation of this magnitude makes the precision of carotenoid estimations low, and impairs comparisons of treatments.

The present paper discusses the importance of standardization of the experimental conditions in relation to the level of flesh pigmentation and within population variation. The main focus will be the effect of fish size, flesh carotenoid concentration and environmental factors.

EIFAC SESSION 8/FEED ADDITIVES/E/41

A BIOASSAY FOR STUDIES ON FEEDING STIMULANTS USING RADIOGRAPHY

Hilde Toften*, Even Jørgensen and Malcolm Jobling

Norwegian Institute of Fisheries and Aquaculture,
P.O. Box 2511, N-9002 Tromsø, Norway

To achieve optimal production of fish in intensive aquaculture it is necessary to understand the factors affecting their appetite. Extensive studies on diet formulation have led to improved nutritional quality of commercial feeds. However, the feed will not be accepted if palatability is poor.

Considerable attention has been directed towards the examination of different chemical mixtures stimulating the senses of olfaction and taste in fish. In the majority of the studies involving potential attractants or stimulants the tests have involved either direct introduction of chemicals to the holding water followed by observation of any changes in the behaviour of the fish, or the feeding of neutral-tasting gels to which chemical mixtures have been added. The results obtained in studies of this type are obviously of limited practical application since they give no information about the direct effects of the “stimulant-mixtures” on rates of ingestion, long-term feed intake or about influences on the palatability of complete feed formulations.

Recently, radiography has been used for the quantitative determination of the gastrointestinal content of fish, and this technique may also have application in studies of both dietary preferences and in experiments designed to examine the effects of potential stimulants on feeding behaviour and feed intake. The use of radiography in feeding studies involves the incorporation of a low concentration of particulate X-ray dense marker into the feed. By marking feed with different sizes of X-ray dense glass beads it is possible to measure feed preference.

In the present study we examined the responses of juvenile Atlantic salmon, Salmo salar, (initial weight 30 g) to feeds of different flavours. In our study, groups of salmon were presented with a choice of two different feed types; medicated feed (commercial feed plus antibiotics) and medicated feed to which a squid extract had been added. Antibiotics were included in the feed to make it less palatable. Our earlier studies have shown that it is difficult to make a tasteless test feed with the nutritional quality required to support good growth. Consequently, we chose to lower the palatability of our basic test diet by addition of antibiotics.

The results showed that the addition of feeding stimulants to the medicated feed made it more palatable, indicating that the squid extract has taste properties that can mask the antibiotics. The feed intake measurements showed a significant preference for squid supplemented feed and the numbers of fish that ate this feed type were much higher on all sampling occasions. This suggests that the addition of stimulants to aquafeeds may have great importance, at least on occasions when fish are presented with medicated diets. Furthermore, our method can be an effective way to identify stimulants and to confirm their efficacy for use in commercial aquafeeds.

EIFAC SESSION 9/APPLICATION OF NUTRIENT REQUIREMENT DATA/R/8

APPLICATION OF NUTRIENT REQUIREMENT DATA - SPECIAL PROBLEMS OF INTENSIVE AND SEMI-INTENSIVE FISH FARMING SYSTEMS

Albert G.J. Tacon

Fisheries Department, Food and Agriculture Organization of the United Nations (FAO), 00100 Rome, Italy

Methodological approaches for undertaking research on the dietary nutrient requirements of farmed fish should ensure that the studies are designed and conducted in such a manner that the ensuring results can be applied under practical farming conditions. Sadly, the majority of studies to date, and in particular those for omnivorous warm water fish species, have had little or no practical applicability; the bulk of nutrient requirement studies having been conducted under controlled artificial laboratory conditions.

Despite the fact that silver carp (Hypophthalmichthys molitrix), common carp (Cyprinus carpio), grass carp (Ctenopharyngodon idella), bighead carp (Hypophthalmichthys nobilis) and milkfish (Chanos chanos) are the top five most cultivated fish species in the world (total aquaculture production of these fish species in 1990 totalled 4.8 million metric tonnes or 57 % of the total world farmed finfish production), little or no information exists concerning their dietary nutrient requirements under practical semi-intensive pond farming conditions. To a large extent this has been due to the reluctance of the conventional laboratory-based fish nutritionist to work under applied field conditions and the difficulty of quantifying the contribution of natural food organisms in the overall nutritional budget of pond raised fish.

If meaningful conclusions are to be drawn from nutrient requirement studies it is essential that the experimental fish be reared under conditions mimicking as far as possible those of the intended farm production unit and environment, including holding facility (indoor or outdoor tank, cage or pond), feed preparation technique (grinding, pelleting, drying; diet texture, form, shape, size, buoyancy and water stability), feeding method (hand, demand or automatic feeding; feeding frequency and feeding rate - fixed or satiation feeding), water quality (temperature, turbidity, salinity, oxygen and mineral concentration; water exchange rate, water circulation pattern and artificial aeration), photoperiod (artificial or natural) and fish stocking density. Finally, but not least, it is essential that the growth performance of the experimental fish be at least equal to or greater than that of the target fish species under practical farming conditions so that dietary nutrient requirements can be ascertained under conditions of maximum attainable growth.

EIFAC SESSION 9/APPLICATION OF NUTRIENT REQUIREMENT DATA/E/42

MONITORING VOLUNTARY FEED INTAKE UNDER PRACTICAL CONDITIONS-METHODS AND APPLICATIONS

M. Jobling*, E.H. Jørgensen, B.M. Baardvik, J.S. Christiansen & A.M. Arnesen

NFH, University of Tromsø, 9037 Tromsø, Norway

Within fish farming it is the desire of the commercial producer that the fish grow rapidly, efficient use is made of the feed provided and wastage is kept to a minimum. From the marketing viewpoint it is also desirable that the harvested fish are of uniform size and the nutritional value of the saleable product is maintained within clearly defined limits. Whilst this requires that the fish be provided with correctly formulated feeds, it will be impossible to achieve these goals if feeding and management practices are sub-optimal. There will, for example, be little benefit to be gained by feeding highly palatable diets that are known to fulfil all the nutritional requirements of the fish, if feed presentation is inadequate or the propensity of the fish to feed is reduced due to a poor rearing environment.

Thus, food consumption may be reduced if food particles are of the incorrect size or texture, presentation is incorrect in terms of either time or space, or feeding responses are depressed as a result of the fish being stressed either due to the presence of conspecifics or by deteriorations in water quality. In order to provide the fish with a rearing environment that ensures optimal growth and effective production it is essential to have information about the ways in which the feeding behaviour, voluntary feed intakes and growth performances of individual fish are influenced by changes in various biotic and abiotic factors. Information of this type has been obtained for individuals held in small groups, either by direct observation or by the use of video recording, but until recently it was not possible to study voluntary feed intake and growth performance of individual fish held under conditions mimicking those found in commercial production facilities.

Recent improvements in on-demand feeding devices and monitoring systems, the development of sophisticated marking and tagging techniques (e.g. PIT tags) and the introduction of X radiography for the quantitative determination of the gastrointestinal content of fish has enabled the collection of data relating to the feeding behaviour, food consumption and growth performance of individual fish held in large groups. Data collected using these methods will be presented and discussed, and examples will be given as to how these methods can be applied in order to improve the rearing environments of farmed fish.

EIFAC SESSION 9/APPLICATION OF NUTRIENT REQUIREMENT DATA/E/43

PRACTICAL EXPERIENCE WITH HIGH ENERGY DIETS - GROWTH, FCR AND QUALITY

Niels Alsted and Terkel Due

Dansk Ørredfoder A/S, Danish Aqua Feed International,
P.O. Box 39, DK-7330 Brande, Denmark

Extrusion has made it possible to make diets containing an oil level at 24–30 % for portion sized trout grown in fresh water. These diets have shown a remarkable positive effect on discharge control. Danish trout farmers have, due to strict discharge control, been using these diets for some years. However, the high fat content in diets has been proposed to be the reason of the reduced quality of the fish.

A commercial trial was conducted involving 23 fresh water farms and 100 ponds. Portion sized trout were farmed under different but controlled situations. Diets containing 20–26 % oil were employed. The effect was measured as FCR, growth rate, yield (percent of entrails) and chemical composition of entrails, fillet and backbone. The observed effects were adjusted for size and temperature.

The results showed a remarkably high variation due to the effect of the farms. General results will be presented.

EIFAC SESSION 9/APPLICATION OF NUTRIENT REQUIREMENT DATA/E/44

EFFECTS OF ESSENTIAL FATTY ACID DEFICIENT DIETS ON THE CARCASS FATTY ACIDS AND MEMBRANE VISCOSITY IN THE COMMON CARP

I. Csengeri*¹, and T. Farkas²

*¹ Fish Culture Research Institute, Szarvas, Hungary
² Inst. Biochem., Biol.Re. Centre, MTA, Szeged, Hungary

Changes in fatty acid composition of carcass total lipids had been monitored for two groups of common carp raised on essential fatty acid (EFA) deficient rations: fat-free diet (FF) and lauric acid diet (LA). Membrane lipid composition and membrane physical state had been studied in other groups of fish, raised on EFA-sufficient (+EFA) diet and EFA-deficient (-EFA) diets with subsequent in vivo and in vitro incubation at low and high environmental temperature.

The fish had grown reasonably fast on the deficient diets. A continuous increase had been observed in the oleic acid content in the lipids of the fish in FF and LA groups referring to a rapid usage of polyunsaturated fatty acids (PUFA) and/or to an enhanced de novo synthesis of fatty acids. Analyses of the fatty acid composition revealed adverse progression in the 6, 9, 12- eicostrienoic acid to polyunsaturated fatty aids (PUFA) ratios.

EFA sufficient carp rapidly adjusted the physical state of their membranes to the prevailing temperature as assessed by fluorescence polarization techniques of temperature up- and down shifted erythrocytes and liver slices. This reaction was preceded by adjustment of fatty acid synthesizing machinery to the new temperature. The response was dependent on the EFA supply. EFA deficient carp were unable to increase the synthesis of PUFA when exposed to cold temperatures.

EIFAC SESSION 9/APPLICATION OF NUTRIENT REQUIREMENT DATA/E/45

THE MEASUREMENT AND APPLICATION OF INDIVIDUAL CONSUMPTION RATES OF FISH HELD IN GROUPS FOR THE STUDY OF FISH NUTRITION

Chris G. Carter, Ian D. McCarthy* and Dominic F. Houlihan

Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen, AB9 2TN, UK

The measurement of individual consumption rates of fish held in groups using a radiographic method has enabled a new approach to fish nutrition trials to be developed (Carter et al., 1992). This technique, developed by Talbot & Higgins (1983), involves the addition of a radio-opaque particulate marker, such as ballotini glass beads, to the food. The labelled diet is fed to fish which are then X-rayed and the number of ballotini in the gastro-intestinal tract used to calculate the amount of food consumed by individual fish. In order to compare diets, groups of individually numbered fish are fed different experimental diets over extended periods of time (similar to standard nutrition trials). However, X-rays are taken at regular intervals over the course of the experiment and this allows the response of each fish to the test diet to be analysed. The relationship between consumption and growth can be described using simple linear or curvilinear models and analysis of covariance used to compare regression coefficients. This approach has been successfully used to compare the consumption-growth response of fish fed diets with or without supplementary enzymes, with different protein/energy ratios and with different amounts of single cell protein. The advantages of this approach are several, (1) fewer fish are needed to establish ration-growth curves over a large range of consumption rates, (2) consumption rates are measured so that ‘true’ growth efficiencies can be calculated for each fish and (3) individual differences between fish in the same tank such as differences in absorption efficiencies can be analysed and differences between individual fish are highlighted.

Carter, C.G., Houlihan, D.F. and McCarthy, I.D. (1992). Feed Utilization efficiencies of Atlantic salmon (Salmo salar L.) parr: effect of a single supplementary enzyme. Comparative Biochemistry and Physiology 101A, 369–374.

Talbot, C. and Higgins, P.J. (1983). A radiographic method for feeding studies using metallic iron powder as a marker. Journal of Fish Biology 23, 211–220.

EIFAC SESSION 9/APPLICATION OF NUTRIENT REQUIREMENT DATA/E/46

REPORT ABOUT THE MEETING OF THE ICES WORKING GROUP ON MARINE FISH LARVAE CULTURE

P. Lavens

Laboratory for Aquaculture and Artemia Reference Center,
State University of Gent, Rozer 44, B-9000 Gent, Belgium

At the occasion of the ICES Symposium on Mass Rearing of Juvenile Fish a two-day discussion on larviculture bottlenecks was organized by the ICES Working Group on Marine Fish Larviculture. This presentation reports especially on the workshop items related to nutrition methodologies.

To date, nutrition research of marine fish larvae has focused mainly on lipid requirements, more specifically Highly Unsaturated Fatty Acids (HUFAs). Test diets used for these studies are based on live food organisms (Brachionus and Artemia) of which the nutritional composition is adapted by means of bioencapsulation techniques. This does not only complicate the study of a sole nutritional factor (other components may also vary), but there is also a high variability in the results of current, so-called standard, enrichment procedures which may have an effect on the interpretation of results from lipid research. It is therefore of utmost importance that analytical information is available of the food at the moment of feeding (and this at several time intervals during the trial) rather than of the enrichment substances used. Moreover, the biochemical composition of Artemia cysts, even of the same geographical origin, varies from batch to batch. It is therefore recommended to use selected batches of known biochemical composition which would furthermore enable more detailed comparison of nutritional data from different groups.

Quantifying nutritional requirements furthermore implies that studies should be undertaken on the quantitative and qualitative assessment of live food ingestion by fish larvae. The impact of environmental and behaviour factors should not be neglected.

It is also obvious that nutritional requirements may change radically during the larval development. For example, for several marine fish the DHA requirement is much higher during the first-feeding period than for the post-metamorphosis phase. Especially for this first period there may also be an impact of egg and larval quality as the nutrient requirement may be affected by what is endogenously supplied through the fertilized egg.

Potential interferences from unknown ‘live food factors’ should be eliminated whenever possible. This means that as soon as the larvae accepts artificial particles nutrition studies should be used selecting weaning diets. These test diets should have an open formulation using semi-purified ingredients to facilitate their application, and a good performance (i.e. acceptable growth and survival). This would furthermore allow comparison of results from inter-laboratory studies.

Last but not least, analytical procedures should be further standardized and critically evaluated. Two HUFA intercalibration studies revealed important inter-laboratory as well as intra-laboratory differences, irrespective if a prescribed analytical method or the own ‘in-house’ method was applied.

EIFAC SESSION 9/APPLICATION OF NUTRIENT REQUIREMENT DATA/E/47

QUALITY CONTROL OF FOOD REQUIRES QUALITY CONTROL OF TEST FISH

R.W. Hoffmann

Institut für Zoologie, Fischereibiologie und Fischkrankheiten der Universität München Kaulbachstraβ 37, D-80539 München, Germany

Testing the quality of fish food is regularly done with target fish species both in experimental as well as in field studies. The test parameters are mostly only the effects on growth as well as on transformation-rate. In contrast to most homiothermic farm animals, the test design is not standardized and test animals are not defined. The experiences of our institute however show that the results are influenced by the health status of fish itself before the test starts. Chemically inapparent bacterial or parasitic infections may become apparent if the food is of suboptimal quality. But even if the fish health seems to be not affected by the test food, pathomorphological as well as histological examination of at least liver and kidney should supplement the present used test parameters to detect early stages of malnutrition not readible on the growth.

In summary, both the test fish should be examined before testing a food as well as the test parameters themselves after the test period, should be supplemented by methods of pathomorphology.

EIFAC SESSION 9/APPLICATION OF NUTRIENT REQUIREMENT DATA/P/8

ACTUAL TECHNOLOGY AND NEW DIETS FOR WEANING OF MARINE FISH LARVAE

A. Roncarati¹, L. Gennari¹, P. Melotti¹, O. Mordenti² and F. Loro²

¹ Centro Universitario di Ricerca e Didattica in Aquacoltura e Maricoltura, Camerino University, Lungomare Europa, 63039 San Benedetto del Tronto (AP) Italy
² Instituto di Zoocolture, Bologna University, Via San Giacomo 9, 40126 Bologna, Italy

Using the current weaning technology of seabass (Dicentrarchus labrax) and seabream (Sparus aurata), we are confronted with different choices which can allow a higher quality and lower cost in fingerling production. Final results will be evaluated looking at growth rate, survival rate, metabolic deficiency, and size homogeneity.

The first problem is the weaning diet quality and the age which allows a good digestibility in relation to the digestive tract development and to the efficiency of certain metabolic pathways. The second problem in the weaning diet physical properties in relation to the behaviour of larvae towards the inert diet, where the properties we have to consider are particle size, attractivity, buoyancy and sedimentation speed, colour and stability in water. In production conditions, with 150/800 μm weaning commercial diets, starting weaning from larvae of 30 mg (live weight) we had lower growth and survival rates and higher size heterogeneity than starting with 40 mg larvae. Such a result has to be verified with new 80–100 μm starting diets available this year that should allow further anticipation of weaning starting from 20 mg larvae. The third problem is the weaning duration of the weaning period. From experiments carried out with Japanese feed the possibility of direct passage from live food to artificial diet in 24 hours was demonstrated. In conclusion, the answer to these different problems will allow a further standardization of weaning methodology and could contribute to solve, at least in part, the problems of metabolic deficiency that is supposed to be at the origin of some common malformations.

EIFAC SESSION 9/APPLICATION OF NUTRIENT REQUIREMENT DATA/P/9

THE ANALYSIS OF INDIVIDUAL DIFFERENCES IN FEEDING BEHAVIOUR AND GROWTH PERFORMANCE OF FISH AND ITS IMPORTANCE IN FISH NUTRITION

lan D. McCarthy, Chris G. Carter* and Dominic F. Houlihan

Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen, AB9 2TN, UK

Within groups of fish used for nutritional trials considerable individual variation in consumption and growth can be present, however in many studies the mean group response has been the sole focus of analysis and comparison. Few studies have investigated the factors that could explain the range of growth rates, or growth depensation, recorded by individuals within a group. This poster will examine how differences in feeding behaviour, consumption and protein turnover influence the growth rates and food conversion efficiencies of individual fish held in groups using commercially important species (Atlantic salmon, rainbow trout, sea bass, tilapia and grass carp). The importance of analyzing individual variability in fish nutrition studies will be discussed.

Using radiographic estimates of consumption we have examined the distribution of food between fish held in groups and analyzed how social rank and food availability influences food acquisition and growth of individual fish. Once consumption rates of individual fish are known it is possible to:

1. correlate individual consumption and growth rates of fish and to measure individual food conversion efficiencies
2. correlate individual differences in growth and food conversion efficiency with differences in protein turnover
3. compare rates of protein synthesis and growth and the concentration of amino acids in the plasma and tissue free pools of individual fish with known dietary intake

4. construct nitrogen budgets for individual fish.

Therefore the assessment of individual variability has valuable applications to the study of nutritional requirements in fish. The advantage of this approach is that it may now be possible to select fish for commercial aquaculture on the basis of efficient utilization of ingested protein.

EIFAC SESSION 9/APPLICATION OF NUTRIENT REQUIREMENT DATA/P/10

QUALITY TRIALS WITH CAPELIN MEAL BY FEEDING EXPERIMENTS WITH SALMON FRY 1988–1992

Soffia Vala Tryggvadottir*, Jonas Bjarnason and Olafur Guomundsson

Iceland Fisheries Laboratories, Skulagata 4, IS-Reykjavik, Iceland

This project was intended to investigate, by chemical analysis and feeding trials with salmon fry, the effects of freshness of raw material on the feeding value of capelin meal in salmon diets in order to provide quality control criteria. The effect of biogenic amines (tyramine, putescine, cadaverine, histamine) content in the meal was evaluated. Four feeding experiments have been conducted in the years 1988–1992. For all the feeding trials the initial weight of the salmon fry was abut 2 g and the growth period was about 6 months.

The experimental procedures for the different meals in the test diets were as follows:

Capelin meal was processed from different freshness of raw material; TVN 40, 90, 130 and 175 mg N/100 g

Biogenic amines as free bases and as hydrochlorides were added to high quality meals (feeds) in different concentrations

Experimental feeds were made from capelin meal of three different TVN-values in raw material, i.e. 30, 90 and 120 mgN/100 g.

The fish growth decreased with increasing TVN-value of the raw material. Close to a linear correlation was found between TVN value of raw material and the concentration of biogenic amines in the meal. The biogenic amines analysed in the trial feeds were: 20, 105, 150 and 260 mg/100 g. There was no growth difference for the feed groups fed the three lowest concentrations of biogenic amines, but there was a significant decline in growth for the fish group fed the feed which contained total biogenic amines of 260 mg/100 g adding a bad smell to the feed. This amount of biogenic amines corresponds to the concentration expected in meal from TVN 90 in raw material. No growth reduction and no decaying smell was observed when biogenic amine hydrochlorides were added to the feed. No difference in growth was detected for fish fed the acidified feed (pH 5.70 instead 6.20) in the cases of TVN values 30 and 120 in the raw material. But there was a growth improving effect found after acidification in the TVN 90 feed.

It can be hypothesized from these trial series, that the four biogenic amines up to a total concentration of 280 mg/100 g in feed, have possible growth reducing effects on salmon fry because of lowered palatability rather than toxicity.

EIFAC SESSION 9/APPLICATION OF NUTRIENT REQUIREMENT DATA/P/11

METHODOLOGY IN THE STUDY OF SEABASS NUTRITION REQUIREMENTS IN SINGAPORE

H.B. Lee*¹, J. Guillaume*², R. Chou*¹, G. Cuzon*³, H.H. Heng*¹ and J. Fuchs*⁴

*¹ Marine Aquaculture Section, Primary Production Department, 300-C Nicoll Drive, Singapore 1749
*² Institut Francais de Recherche pour l'Exploitation de la Mer, Centre du Brest, B.P. 70 219263 Plouzane, France
*³ Institut Francais de Recherche pour l'Exploitation de la Mer, Centre Oceanologique du Pacifique, B.P. 7006 Taravao, Tahiti, French Polynesia
*⁴ Institut Francais de Recherche pour l'Exploitation de la Mer, 155, Rue Jean-Jacques Rousseau, 92138, Issy-les Moulineaux Cedex, France

The Marine Aquaculture Section, Primary Production Department of Singapore, twins with the Institut Francais de Recherche pour l'Exploitation de la Mer (IFREMER) and the Institut National de la Recherche Agronomique (INRA) of France on a 5-year project under the ASEAN-EEC Aquaculture Development and Coordination Programme (AADCP) to develop marine fish feeds. One of the prime considerations of the project is to establish standardized, accepted methodology in fsh nutrition.

This paper describes the methodology and interpretation of results for blank tests to demonstrate homogeneity in fish response in newly established tank systems. The purpose of standardization is also to align results with those obtained in France from similar studies. The results of blank tests are discussed in relation to the experimental design. The method employed in the next experiment on optimum dietary protein requirement of seabass using the same tank system is also reviewed.