The requirement of the gilthead seabream ( Sparus aurata L.) for nicotinic acid

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The requirement of the gilthead seabream ( Sparus aurata L.) for nicotinic acid

P. C. Morris and S. J. Davies

Animal Science / Volume 61 / Issue 02 / October 1995, pp 437 443DOI: 10.1017/S1357729800013989, Published online: 02 September 2010

Link to this article: http://journals.cambridge.org/abstract_S1357729800013989

How to cite this article:P. C. Morris and S. J. Davies (1995). The requirement of the gilthead seabream ( Sparus aurata L.) for nicotinic acid. Animal Science, 61, pp 437443 doi:10.1017/S1357729800013989

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The requirement of the gilthead seabream (Sparus aurata L.) fornicotinic acid

P. C. Morris and S. J. Davies

Fish Nutrition Unit, Department of Biological Sciences, University of Plymouth, Drake Circus, Plymouth,Devon PL4 8AA

Abstract

Semi-purified diets were used to determine the nicotinic acid requirement of the gilthead seabream, Sparus aurata.Vitamin losses during food manufacture were minimal resulting in dietary levels which were close to the declaredcontent. Dietary nicotinic acid contents of 52-21 mg/kg and below resulted in sub-optimum growth, reduced foodefficiency and poor values for apparent net protein utilization. Diets containing less than 28-67 mg/kg gave a lowproportion of carcass lipid concomitant with an elevation in the relative content of carcass moisture. Although grossdeficiency symptoms were not observed, a reduced hepatosomatic index and a slightly lower plasma glucoseconcentration were detected amongst the fish given diets supplemented with nicotinic acid at the level of 25 mg/kgor less. Based upon the results of broken line analysis and data derived by modelling the weight gain of the fish, thenicotinic acid requirement o/Sparus aurata fingerlings lies between 63 and 83 mg/kg of diet or 1-57 to 2-body weight per day.

Keywords: nicotinic acid, seabream, Sparus aurata, vitamin B complex-

IntroductionRecent investigations have indicated a qualitativerequirement for thiamin, riboflavin, pyridoxine,nicotinic acid and pantothenic acid in the nutrition ofthe gilthead seabream, Sparus aurata (Morris, 1994).An absence of nicotinic acid (one of the two niacinvitamers) in semi-purified diets given to giltheadseabream was associated with reduced weight gain,a lowering of the haematocrit, poor nutrientutilization and eventual mortality.

The effects of nicotinic acid deficiency have beenwell characterized for several species of finfish(McLaren, Keller, O'Donnel and Elvehjem, 1947;Halver, 1957; Aoe, Masuda and Takeda, 1967; Yoneand Fujii, 1974; Andrews and Murai, 1978; Butthep,Sitasit and Boonyaratpalin, 1983) especially withrespect to the role of this vitamin in protection of theskin from ultra-violet irradiation (Allinson, 1960;Poston and Wolfe, 1985).

manufacture and storage (Blum, 1991). Additionallythere may be synthesis of nicotinamide from dietarytryptophan but this pathway may represent only aminor source of the vitamin in fish (Poston andDiLorenzo, 1973).

Little information exists about the nicotinic acidrequirements of cultured aquatic species, and thedata are often contradictory in that markedlydifferent requirements have been reported for thesame or similar species (McLaren et ah, 1947; Phillipsand Brockway, 1947; Poston and Wolfe, 1985).

Given the absence of an established quantitativedietary requirement for nicotinic acid in diets for thegilthead seabream, an experiment was designed todetermine the optimum dietary inclusion level of thisvitamin for the promotion of good growth andefficient nutrient utilization.

Due to its stability when subjected to high Material and methodstemperatures, light and humidity, nicotinic acid is The gilthead seabream supplied as juveniles by theregarded as one of the more stable vitamins and SIAM hatchery (Montpellier, France) werethere is usually little loss of activity during food transported to Plymouth and acclimatized to

437

438 Morris and Davies

aquarium conditions for 6 months. Batches of 25seabream fingerlings (initial weight 21-68 (s.e. 0-05)g) were stocked into duplicate 80-1, self cleaning,polyethylene tanks within a closed recirculationsystem. There was a parallel flow of 2-41/min ofseawater through the tanks and water temperatureand salinity were maintained at 24°C and 33 to 36%respectively. Daylight balanced fluorescent striplamps were used to maintain an 8 :16 h light: darkregime.

The basal diet was formulated to provideapproximately 500 g/kg crude protein (CP) and110 g/kg lipid on a dry-matter (DM) basis.Desiccated squid mantle tissue was included toimprove the palatability of the diet (Table 1). Six test

Table 1 Semi-purified test diet used to determine the nicotinic acidrequirement of the pithead seabream

IngredientInclusion

Casein (vitamin free)Gelatin (225 bloom)Maize starch/dextrin (2 : 3)Cod liver oilMineral pre-mixtFat soluble vitamin pre-mix^:B vitamin pre-mix§Macro vitamin pre-mix||Amino acid pre-mixiSquid mealDibasic calcium phosphateNicotinic acid in a-cellulose#

319-50150-001970011840100-00

5-004-50

200051-80

5-0028-300-50

t Composition of the mineral pre-mix (mg/kg diet): CaHPO4,22050; CaCO3,3000; NaCl, 15000; K2SO3,20000; MgSO4.7H2O,20627; FeSO4.7H2O, 700-0; MnSO4.4H2O, 227-3; ZnSO4,515-6;CuSO4.5H20,160-0;CoCl2.6H20,26-0; KI, 150; Na2SeO3,2-5;a-cellulose, 17676-6.% Composition of the fat-soluble vitamin pre-mix (mg/kgdiet): vitamin A palmitate, 4-20; Rovimix D-50 SD, 3-68;rovimix E-50 SD, 100000; Rovimix menadione sodium bi-sulphite, 78000; a-cellulose, 321212.§ Composition of the water-soluble vitamins pre-mix (mg/kg diet) (niacin free B vitamin pre-mix): thiamin HC1, 69-90;riboflavin (96%), 208-30; pyridoxine HC1, 48-60; calciumpantothenate, 305-30; biotin (2%), 300-00; folic acid (88-8%),16-90; cyanocobalamin (95%), 0-01; a-cellulose, 3550-99.|| Composition of the macro vitamin pre-mix (mg/kg diet):ascorbic acid (Rovimix Stay C), 2000; choline chloride, 5000;myo-inositol, 2000; a-cellulose, 11000.f Contribution of amino acids to the diet (g/kg diet): D-L-methionine, 4-80; L-tryptophan, 4-70; L-threonine, 8-70; L-phenylalanine, 5-80; L-histidine, 1-40; L-arginine, 700; L-isoleucine,4-80; L-leucine, 5-00; L-valine, 9-10.# Nicotinic acid in a-cellulose represents the total spacewithin the formulation allocated for nicotinic acid. Nicotinicacid included at 5,25,50,100,150 and 200 mg/kg diet and theremaining space in the formulation filled with a-cellulose.

diets differing only with respect to nicotinic acidcontent were then manufactured. Diets 1 to 6 weresupplemented with nicotinic acid (Rovimix niacin,Roche Products Ltd) in the range 50 to 200 mg/kg.The diets were manufactured by mixing the dryingredients, cod liver oil and water in a Hobart A120food processor. The mash was then extrudedthrough a 3-mm die and the resultant moist pelletswere stored at -18°C. On a DM basis, the dietcontained 537-5 g/kg CP, 107-8 g/kg lipid and90-2 g/kg ash.

The nicotinic acid content of the test diets wasdetermined by microbiological assay usingLactobacilhis plantarum ATCC 8014. Samples of foodwere homogenized in 0025 mol/1 HC1, diluted andsterilized to yield an estimated nicotinic acid contentof 0-05 ng/cm3. Using a stock solution of nicotinicacid in 0-025 mol/1 HC1 (0-1 |ag/cm3), a series ofstandards in the range 0-025 to 0-10 (ig/cm3 wassimilarly prepared. Two and a half cm3 of the extractor nicotinic acid standard was then added to anequal volume of sterile niacin assay medium (Bactoniacin assay medium, Difco Laboratories, Detroit,Michigan, USA) to which 50 ̂ 1 of a suspension ofPBS (phosphate buffered saline) washed overnight L.plantarum was added. All tubes were then incubatedfor 48 h at 37°C and the nicotinic acid content of thefoods determined by turbidometric measurement at540 nm. The actual nicotinic acid contents of diets 1to 6 were, 8-96, 28-67, 52-21, 102-62, 134-44 and173-92 mg/kg respectively.

Each test diet was presented to duplicate tanks offish three times daily (09.00, 13.00 and 16.00 h). Onthe basis of DM (moisture content of the diet as fedwas 345-5 g/kg), the diet was supplied at 0-025 of thelive weight per day (0-025 M) for 6 weeks. Thereafter,due to a decline in appetite amongst the deficientanimals, the fish were then fed to satiation up to0025 M and the amount of food supplied wasrecorded.

The fish were weighed twice monthly in order to re-determine the appropriate ration size and followgrowth parameters. The weight gain, specific growthrate (SGR), food intake, food conversion efficiency(FCE) and cumulative percentage mortality weremonitored throughout the 84-day trial as outlined byBaker and Davies (1995).

After 12 weeks of feeding, five individuals from eachdiet and 10 fish selected at the start of the experimentwere subjected to proximate analysis. After drying(Association of Official Analytical Chemists (AOAC),1990) quadruplicate samples were taken from eachfor individual analysis of protein, lipid and ash. CPwas determined by the Kjeldahl (N X 6-25) method

Nicotinic acid requirement of seabream 439

and ash was assayed according to the methodsoutlined by the AOAC (1990). Total lipid wasdetermined by the method outlined by Barnes andBlackstock (1973). Based on the growth data, nutrientanalysis of the test diet and the proximatecomposition of the carcasses, the apparent netprotein utilization (ANPU) was calculated (Bakerand Da vies, 1995).

At the end of the growth study, five individuals wereselected from each diet and blood was withdrawninto heparinized syringes by cardiac puncture.Following centrifugation, the plasma was decantedinto micro centrifuge tubes and stored at -70°C untilanalysed. Subsequently, total plasma protein wasdetermined by the Biuret method (total proteinreagent, Sigma No. 541-2) and plasma glucose by theglucose oxidase method (Sigma, procedure no.510A). The hepatopancreas was also excised fromthese individuals and the weight of the liver as aproportion of the fish weight (hepatosomatic index,HSI) determined where HSI = weight liver/weightfish.

The growing performance, carcass composition andhaematological data were subjected to analysis ofvariance where P < 005 was judged to be indicativeof a significant difference. Where the analysis ofvariance revealed significant differences Duncan'smultiple range test (Duncan, 1955) was applied inorder to characterize and quantify the differencesbetween dietary regimes. Growth data were used toestimate the nicotinic acid requirement by using bothbroken line analysis and a growth model.

ResultsAfter 12 weeks of feeding, the fish given the dietcontaining 8-96 mg of nicotinic acid per kg of diet

were significantly smaller than fish given any of theremaining diets, while those on the diet containing28-67 mg/kg were significantly smaller than thosefish on diets containing a nicotinic acid content of102-62 mg/kg or greater. The fish given the dietsincorporating nicotinic acid at 52-21 mg/kg wereneither significantly smaller than those given anicotinic acid at 102-62 mg/kg nor larger than thosegiven diets containing 28-67 mg/kg (Table 2).

Mortalities were recorded amongst all dietarytreatments apart from those given diets with anicotinic acid content of 173-92 mg/kg (Table 2).There was no apparent cause of death. Apart fromthe reduced food consumption of the fish given dietscontaining nicotinic acid at less than 102-62 mg/kgthere were no apparent behavioural changes andvisible pathological changes were not observed inthe fish given diets of low nicotinic acid content.

The lipid and moisture contents of the fish givendiets containing the lowest level of nicotinic acid(8-96 mg/kg) were significantly different from thoseof the fish given the remaining diets (P < 0-05; Table3). Thus, in these fish, carcass moisture content wassignificantly elevated in response to a decliningcarcass lipid component. The protein and ash contentof these fish were also slightly increased whencompared with the fish given the remaining diets.

The FCE and ANPU values recorded for the fishgiven diets containing 8-96 and 28-67 mg nicotinicacid per kg of diet were poor and reflected theirgrowth (Table 2). Consequently, the fish given thediets containing the lowest level of nicotinic acid hadFCE and ANPU values that were significantly poorerthan those of the remaining groups (P < 0-05; Table2).

Table 2 Performance characteristics ofgilthead seabream given diets of varied nicotinic acid content

Initial mean weight (g)Final mean weight (g)tSpecific growth rate (%/day)^:Total food intake (g per fish)JFood conversion efficiency:):Apparent net protein utilization§Cumulative mortality (%)

8-96

21-6634-85a

0-5744-64a

0-296a

0-101a

12-00

Nicotinic acid content of diet (mg/kg)

28-67

21-6545-78b

0-8951-09a

0-472b

0-156ab

20-00

52-21

21-5851-42bc

1-0350-25a

0-594b

0-200b

12-00

102-62

21-6857-37c

11664-84b

0-550b

0-187b

12-00

134-44

21-6655-85c

1-1359-81b

0-571b

0-194b

12-00

173-92

21-7356-96c

1-1560-78"0-580b

0-203b

0-00

s.e.

0-053-580-093150-0470-016

t Based on mean performance data of 25 fish per tank. Values on this row carrying common superscripts are not significantlydifferent from each other (P < 0001).£ Based on the performance of replicate tanks. Means carrying common superscripts are not significantly different from eachother (P < 005).§ Mean values based on the proximate composition of five fish per dietary treatment. Means carrying common superscripts arenot significantly different from each other (P < 0-05).


Table 3 Proximate composition of carcasses of gilthead seabream given diets of varied nicotinic acid content

Initial fish

Dietary nicotinicacid (mg/kg)

8-9628-6752-21

102-62134-44173-92

Moisture(g/kg)

Mean

696-3

719.7a683-7b

683-2b

666-lb

674-3b

670-4b

s.e.

4-78-5

14-116-616-67-8

Protein(g/kg)

Mean

195-7

191-3185-9187-0187-9187-5191-2

s.e.

3-02 12-50-72-93-5

Lipid(g/kg)

Mean

107-8

67-6a

110-9b

108-3b

126-2b

114-4b

116-6b

s.e.

4.41-1

15-05-7

16-07-5

Mean

46-945-143-243-044-943-7

Ash(g/kg)

39-9

s.e.

1-00-81-00-31-70-4

a-b Composition of five individuals is expressed as a proportion of the live weight. Means in each column with commonsuperscripts are not significantly different from each other (P > 0-05).

The HSI of the fish given the diet containing nicotinicacid at 8-96 mg/kg was significantly lower than thatof the fish given diets with a nicotinic acid content of102-62 mg/kg or more, while the HSI of theseabream maintained on diet 2 (28-67 mg/kg) wasnot significantly different from that recorded for thefish given any of the remaining diets (Table 4).

The plasma glucose concentration of the fishmaintained on the diet with a nicotinic acid contentof 8-96 mg/kg was the lowest recorded (Table 4) butthere was considerable variation in plasma glucoseconcentration among individuals in all treatmentgroups. Similarly, the plasma protein concentrationstended to be variable and rarely reached levels ofsignificance between treatments (Table 4).

The six dietary treatments could be subdivided intotwo groups based on weight gains for regressionanalysis (Figure 1). Using broken line analysis thedietary nicotinic acid requirement was estimated to

be 63-5 mg/kg. Additionally, a curve was fitted tothe growth data (Figure 1). Using this model twofurther estimates of the nicotinic acid requirementwere made. Proportionately 0-9 of the maximumgrowth could be achieved by feeding diets with anicotinic acid content of 62-6 mg/kg while 82-6 mgnicotinic acid per kg was required forproportionately 0-95 of optimum growth.

DiscussionThe best weight gain and specific growth rate weredisplayed by the seabream given diets containing102-62 mg nicotinic acid per kg or above and thegrowth rate indicated that 52-21 mg/kg wasinsufficient for the maintenance of growth. This wassupported by the requirement values determined byboth the broken line analysis (63-5 mg/kg) and thosegenerated from the growth model (62-6 to 82-6 mg/kg) (Figure 1).

Table 4 Hepatosomatic index (HSI), plasma protein and glucose concentration of gilthead seabream given diets of varied nitocinic acidcontent

Nicotinic acid content of diet (mg/kg)

8-96 28-67 52-21 102-62 134-44 173-92

Mean s.e. Mean s.e. Mean s.e. Mean s.e. Mean s.e. Mean s.e.

HSKX10"2) 0-76a 0-07 l-01ab 0-08 l-22b 0-12 l-10b 0-09 l-30b 014 l-17b 0-08Plasma glucose (ng/cm3) 470-5 74-0 602-7 40-2 527-7 43-5 629-5 62-4 605-4 47-7 622-3 66-2Plasma protein (mg/cm3) 22-41a 2-79 29-84b 1-73 24-86ab 3-11 31-37b 1-51 30-00b 0-97 28-27ab 1-88

a'b Values in each row with common superscripts are not significantly different from each other (P > 0-05). Mean ±s.e. for no.five fish per diet.


1/ = 164-3874 - 0-0191* (R2 = 0-0379)

y = 50-5880 + 1-7683* (R2 = C

6011iiI I I I 11• iii1111• 11• i • 111II111ii1

10 20 30 40 50 60 70 80

Dietary nicotinic acid content (mg/kg)

i • • 11• • II | i iu 11 n i | mi 11 II • 111 II | II II |iiII11111111II|90 100 110 120 130 140 150 160 170 180 190 200

Figure 1 Determination of the nicotinic acid requirement of Sparus aurata by broken line analysis and by the use of a growthmodel. (In equation N = dietary nicotinic acid concentration).

Given the key role of nicotinic acid and nicotinamidein metabolism, it is not surprising that foodutilization should be severely suppressed when thisvitamin is deficient. Appetite was lowest for the fishgiven diet 1 (8-96 mg/kg) and the FCE and ANPUgenerally reflected the level of nicotinic acidsupplementation and growth of the fish.

Food conversion in rainbow trout (Oncorhynchusmykiss) was shown by Poston and Wolfe (1985) to beproportional to nicotinic acid supplementation up tolevels of 10 mg/kg, while Aoe et ah (1967) recordedpoor food conversion in carp (Cyprinus carpio) givendiets with a nicotinic acid content below 28 mg/kg.Food: gain ratio in channel catfish (lctaluruspunctatus) was shown to be suboptimal when fed anicotinic acid supplement of less than 25 mg/kg(Andrews and Murai, 1978).

In the present study, the fish given the dietcontaining the least nicotinic acid had a lower carcass

lipid content than fish in all other groups. A dietarynicotinic acid content of 28-67 mg/kg or aboveappeared to be sufficient to prevent modifications tothe carcass composition (Table 3). Hence, theimprovements in ANPU observed at higher levels ofnicotinic acid were probably a consequence ofchanges in growth and appetite and not the result ofmodified carcass composition. Contrasting with thepresent experiment, Poston and Wolfe (1985)observed no alteration in any aspect of carcasscomposition in rainbow trout given diets that variedin nicotinic acid content.

In an earlier study, Morris (1994) recorded amortality of 13% but did not observe distinctdeficiency symptoms in nicotinic acid deficientSparus aurata. Thus, the observations made in thepresent study are in broad agreement with thisprevious study. Nicotinic acid deficiency wasassociated with a reduction in the HSI (Table 4) andthe values recorded in the present experiment were


similar to those recorded previously (Morris, 1994).The low HSI may be a reflexion of the carcasscomposition since the deficient fish had a lowercarcass lipid content than those given dietscontaining levels of 28-67 mg/kg or more. This mayhave resulted in lower lipid retention in liver and aconsequent reduction in the liver weight. Theobservations of Poston and Wolfe (1985) wouldsupport this argument since rainbow trout given5-0 mg/kg or less exhibited a significantly decreasedhepatic lipid content when compared with thosegiven diets containing an adequate nicotinic acidsupplement.

The requirement of animals for dietary niacin relatesto the ability of each species to synthesize thisvitamin from tryptophan. The niacin synthesizingcapacity of an animal may be determined bymeasuring the relative activities of the enzymes 3-hydroxyanthranilic acid oxygenase (3HAA) andpicolinic acid carboxylase (PC). On the basis of the3HAA/PC ratio, Poston and DiLorenzo (1973)concluded that brook trout (Salvelinus fontinalis) wereunable to synthesize sufficient niacin to meet theirrequirement. Chuang (1991) recorded 3HAA/PCratios which indicated that carp, tilapia (Tilapia spp.),red seabream (Chrysophrys major), black seabream(Mylio macrocephalus), and the milkfish (Chanoschanos) have a capacity for niacin synthesis.However, the extent to which these species may relyon tryptophan as a niacin source was notdetermined. Despite the high protein content andready supply of crystalline L-tryptophan (4-7 g/kg)in the diets used in the present study, it wasdemonstrated that the gilthead seabream has adefinite requirement for preformed niacin. Thiswould indicate that if any niacin is produced de novoby the fish from tryptophan, it is insufficient tomaintain optimal growth.

The minimum nicotinic acid requirement of Spamsaurata appears to lie within the range 63 to 83 mg/kgfood (Figure 1). This value is higher than the 28 mg/kg determined by Aoe et al. (1967) for the carp andthe 14 mg/kg recommended by Andrews and Murai(1978) for the channel catfish, though theobservations of Chuang (1991) would indicate someniacin synthesizing capability in the former species.For the rainbow trout the requirement for nicotinicacid was determined to lie between 1 and 5 mg/kg(McLaren et al, 1947) and 10 mg/kg (Poston andWolf, 1985). The requirements defined by McLaren etal. (1947) and Poston and Wolfe (1985) are muchlower than the 95 mg/kg determined by Phillips andBrockway (1947) as the requirement of brook(Salvelinus fontinalis), brown (Salmo trutta) andrainbow trout, though the study of Phillips andBrockway used maximal liver storage to define the

nicotinic acid requirement. Halver (1989) places therequirement of trout and salmon (all species)somewhere between 120 and 150 and 150 to 200 mgof nicotinic acid per kg of diet respectively. Thesevalues represent the highest published requirements.

Although broken line analysis has been a much usedtool in defining the nutritional requirements of fish,the requirement delineated by this method (63-5 mg/kg) in the present study, would be expected tosupport proportionately only 0-9 of the growthachieved with supplements of 100 mg/kg or more,as determined using the growth model. Althoughnicotinic acid supplements of 100 mg/kg and abovemay provide small additional benefit with regard tofood utilization and growth, the additional cost ofsuch supplements may be prohibitive.

In conclusion, the results of the present studyindicate that the nicotinic acid requirement ofgilthead seabream fingerlings given diets containing537-5 g/kg of protein and 107-8 g/kg lipid lies in theregion of 63 to 83 mg/kg of diet or between 1-57 and207 Hg/kg body weight per day.

AcknowledgementsThe authors would like to acknowledge the efforts of DrChris Ricketts of the Department of Maths and Statistics atthe University of Plymouth for his help in the constructionof the growth model.

ReferencesAllinson, L. N. 1960. 'Sunburning' fingerling lake troutwith ultra-violet light and the effect of a niacin fortifieddiet. Progressive Fish-Culturist 22:114-116.Andrews, J. W. and Murai, T. 1978. Dietary niacinrequirements for channel catfish, journal of Nutrition 108:1508-1511.Aoe, H., Masuda, I. and Takeda, T. 1967. Water-solublevitamin requirements of carp — III. Requirement for niacin.Bulletin of the Japanese Society of Scientific Fisheries 33:681-685.Association of Official Analytical Chemists. 1990. Officialmethods of analysis. 15th ed. (ed. K. Herlich). Association ofOfficial Analytical Chemists, Arlington, Virginia.Baker, R. T. M. and Davies, S. J. 1995. The effect ofpyridoxine supplementation on dietary protein utilisationin gilthead seabream fry. Animal Science 60:157-162.Barnes, H. and Blackstock, J. 1973. Estimation of lipids inmarine animals and tissues: detailed investigation of thesulphophosophvanillin method for 'total' lipids. journal ofExperimental Marine Biology and Ecology 12:103-118.Blum, R. A. 1991. Metabolism and biochemical function. InNiacin in animal nutrition (ed. R. Fenster and R. A. Blum),pp. 8-9. Roche index No. 2288, Basel, Switzerland.Butthep, C, Sitasit, P. and Boonyaratpalin, M. 1983.Water-soluble vitamins essential for the growth of Clarias.


In Finfisli nutrition in Asia. Proceedings of the Asian finfishnutrition icorksliop, Singapore (ed. C. Y. Cho, C. B. Coweyand T. Watanabe), pp. 118-129. IDRC, Ottawa, Canada.

Chuang, J-L. 1991. Fish and shrimp. In Niacin in animalnutrition (ed. R. Fenster and R. A. Blum), pp. 34-37. Rocheindex No. 2288, Basel, Switzerland.

Duncan, D. 1955. Multiple range tests and multiple F tests.Biometrics 11:1-42.

Halver, J. E. 1957. Nutrition of salmonid fishes. III. Water-soluble vitamin requirements of chinook salmon. Journal ofNutrition 62: 225-243.

Halver, J. E. 1989. The vitamins. In Fish nutrition (ed. J. E.Halver), pp. 32-111. Academic Press, London.

McLaren, B. A., Keller, D., O'Donnel, D. J. and Elvehjem,C. A. 1947. The nutrition of rainbow trout. I. Studies onvitamin requirements. Archives of Biochemistry and Biophysics15:169-185.

Morris, P. C. 1994. The water-soluble vitamin nutrition ofthe gilthead seabream, Sparus aurata L. Ph.D. thesis,University of Plymouth.

Phillips, A. M. and Brockway, D. R. 1947. The niacin andbiotin requirement of trout. Transactions of the AmericanFisheries Society 77: 152-159.

Poston, H. A. 1969. The effect of excess levels of niacin onthe lipid metabolism of fingerling brook trout. Fish researchbulletin, State of New York Conservation Department, Albany,no. 31, pp. 9-12.

Poston, H. A. and DiLorenzo, R. N. 1973. Tryptophanconversion to niacin in the brook trout (Salvelinus fontinalis).Proceedings of the Society for Experimental Biology and Medicine114:110-112.

Poston, H. A. and Wolfe, M. J. 1985. Niacin requirement foroptimum growth, feed conversion and protection ofrainbow trout (Salmo gairdneri) from ultraviolet-Birradiation, journal of Fish Diseases 8: 451-460.

Yone, Y. and Fujii, M. 1974. Studies on the nutrition of theRed Seabream. X. Qualitative requirements for watersoluble vitamins. Reports of Fish Research Laboratory, KyushuUniversity 2: 22-32.

(Received 2 March 1995—Accepted 13 April 1995)

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The requirement of the gilthead seabream ( Sparus aurata L.) for nicotinic acid