Water-soluble vitamins in fish ontogeny

REVIEW ARTICLE

Water-soluble vitamins in fish ontogeny

RuneWaagb�National Institute of Nutrition and Seafood Research (NIFES), Bergen, Norway

Correspondence: Dr RWaagb�, NIFES, P.O. Box 2029 Nordnes, N-5817 Bergen, Norway. E-mail: [email protected]

Abstract

Studies on vitamin requirement at early stages aredi⁄cult and vary in quality, both due to the scienti¢capproach and vitamin analysis. Focus has been onwater-soluble vitamins that cause dramatic losses of theo¡spring in practical farming situations or in wild life,like vitamin C and thiamine de¢ciencies respectively.Practical solutions including vitamin administrationthrough brood stock and larvae diets have con¢rmedand corrected the vitamin de¢ciencies. For the otherwater-soluble vitamins, the situation is not so obvious.Descriptive studies of folate and vitamin B6 during ¢shontogeny have shown a net loss of vitamin duringendogenous feeding and a steady transfer of vitaminfrom the yolk sac into the body compartment, and¢nally, dramatic increases in body vitamin levels afterthe start of feeding. The kinetics of mass transfer withontogeny appears, however, to di¡er between vita-mins. Start of feeding of ¢sh larvae with live or formu-lated feeds includes several challenges with respect towater-soluble vitamins, including aspects of live feedenrichment and stability, micro-diet leaching, variablefeed intakes, immature gastrointestinal tract, variablebioavailability of vitamins and larvae vitamin storagecapacity. Consequently, the exact minimum require-ments are di⁄cult to estimate and vitamin recom-mendations need to consider such conditions.

Keywords: ¢sh, vitamin, ontogeny, endogenousfeeding, start feeding, live feed

Introduction

The traditionally de¢ned water-soluble vitaminsseem to be essential to all examined farmed ¢sh spe-cies so far.The basic roles and de¢ciency symptoms ofthe single vitamins are detailed in the literature (NRC

1993;Woodward1994; Dabrowski 2001; Halver 2002;Webster & Lim 2002; Koshio 2007). Among thisgroup of eight vitamins, vitamin C or ascorbic acid isby far the most studied (Dabrowski 2001). This is dueto the rapid and dramatic consequences at de¢ciency,and the high risk for de¢ciency using traditional andpractical ingredients in ¢sh feed production (Sandnes1991). Further, the essentiality of ascorbic acid is ver-i¢ed in several farmed ¢sh species, demonstrated bythe lack of the enzyme gulonolactone oxidase,needed for ascorbic acid biosynthesis (M�land &Waagb� 1998; Moreau & Dabrowski 2001). Finally,the need for a stable and bioavailable ascorbic acidderivative in feed for all commercial farmed ¢sh spe-cies has beena driving force invitamin C research fora long period.For other water-soluble vitamins far less informa-

tion is available, and we like to think that live feed,practical feed ingredients and safety supplementa-tions are su⁄cient supplies for these vitamins to pre-vent suboptimal conditions inall farmed species at allstages of development. For salmonids, Woodward(1994) suggested that the listed minimum require-ments at that time were extremely high comparedwith that for other species, probably re£ecting practi-cal recommendations rather thanminimum require-ments for optimal growth and health. The latter isindeed a safer approach for a rapidly growing aqua-culture industry (Hardy 2001). The focus of the mar-ine ¢sh farming research has also been severelyin£uenced by the commercial interests and pro-gresses that have been made side by side. Practicaland basic researches on larvae nutrition have facedchallenges with feed processing and storage stability,live feed enrichment, leaching, bioavailability, nutri-ent interactions and a situation-dependent increasein requirements. Such conditions may lead to subop-timal vitamin nutrition with potential consequences

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for the larvae quality, and later growth andwelfare ofthe ¢sh (Cahu, Infante & Takeuchi 2003).The aim of the present communication is to dis-

cuss the requirement for water-soluble vitamins for¢sh species during early ontogeny in light of updatedliterature. However, the technical approaches and ex-periments vary in scienti¢c strength, depending onthe experimental design, ¢sh species, developmentalstage of the ¢sh and not the least the feed type.

Experimental approaches and analyticalmethods

Experimental designs and methods used in vitaminstudies on ¢sh larvae and juveniles have mostly beentraditional, including start of feeding experimentswith live feed and dose^response studies with formu-lated diets (Boonyaratpalin 1989; Halver 1995). Thetechnical nature of the diets to ¢sh larvae does not al-ways allowexact determinationof requirements in thesame manner as formulated puri¢ed diets for vitaminstudies in larger ¢sh, in terms of feed levels of vitaminsand intakes (Boonyaratpalin 1989; Woodward 1994).Firstly, there were uncertainties in the suitability ofthe live feed organism for the ¢sh species and thegiven developmental stage. Further, the overall livepray production procedure is a compromise for bothmacro- and micro-nutrient enrichments, with aminor focus on the chemical form and the technicalquality of the water-soluble vitamin. Finally, ownmetabolism of the live prey in the period from feedingto being ingested by the ¢sh larvae may result in re-duced live pray quality, as illustrated for essential fattyacids (Evjemo, Cotteau, Olsen & Sorgeloos1997).Some studies include selective vitamin feeding of

brood stocks and subsequent evaluation of the o¡-spring quality and performance (Fig. 1), like a studyon vitamin C in rainbow trout (Blom & Dabrowski1995). More descriptive studies on the mass transferof vitamins between the yolk sac and the larval bodycompartments during endogenous feeding have beenused as an estimate of the requirement of vitaminsfor growth until exogenous feeding (R�nnestad, Lie& Waagb� 1997; R�nnestad, Hamre, Lie & Waagb�1999; M�land, R�nnestad &Waagb� 2003;Fig.1). Im-portantly, the larvae examined in these studies origi-nated from high-quality o¡spring.At the start of feeding (Fig. 1), minimum require-

ments are determined on the basis of selectedmarkers at di¡erent biological organization levels inthe ¢sh, like mortality, growth, body/tissue vitaminsaturation (maintenance of steady-state tissue con-

centration), retention of nutrients, speci¢c biochem-ical measures like enzyme activities, histopathologyand lately also vitamin speci¢c molecular markers.Using more speci¢c markers, vitamin recommenda-tion levels have generally been reduced.Gouillou-Coustans, Bergot and Kaushik (1998) de-

termined the ascorbic acid requirement of commoncarp (Cyprinio carpio) larvae with semi-puri¢ed dietsadded with graded levels of a stable and bioavailableascorbyl phosphate (AP) form, and demonstrated thatthe level for tissue saturationwas six times higher thanthat of maximumgrowth (45mgAAequiv. kg�1).Thisshows that body vitamin saturation may not necessa-rily be a useful requirement indicator in ¢sh larvae,due to the limited storage capacity for water-solublevitamins and thereby low retention e⁄cacies. In juve-nileAtlantic salmon fed graded levels of AP, tissue vita-min saturation varied both by dose and by time offeeding, illustrating the importance of both feed leveland feed intake (Waagb�, Glette, Raa-Nilsen & Sandnes1993). For ¢sh larvae, the e⁄cacy of intestinal phos-phate hydrolysis of AP and subsequently AA uptakemay increase the insecurity of the requirement esti-mates (Dabrowski, Moreau & El-Saidy1996).The speci¢city, reliability and accuracy of analyti-

cal methods for determination of water-soluble vita-mins comprise a part of the discussion of vitaminrequirements. Normally, one would expect con¢rm-ing feed and tissue vitamin analyses re£ecting thedietary input. For ascorbic acid, the discussion hasfocused on assay techniques and vitamer forms ofAA in the tissues (AA, dehydroAAandascorbate-sul-phate) (Dabrowski, Matusiewicz & Blom1994; Halver& Felton 2001). Feed levels and chemical forms (AA,coated AA, polymereAA, AA-palmitate, AA-sulphateand AA-phosphate salts) have varied, as well as theirrespective stability and bioavailability. In enrichmentprocedures to boost live feed with vitamin C for ¢shlarvae, microalgae (Lie, Haaland, Hemre, Maage,Lied, Rosenlund, Sandnes & Olsen 1997), AA-palmi-tate (Merchie, Lavens, Dhert, Garcia Ulloa Go¤ mez,Nelis, De Leenheer & Sorgeloos 1996) and AA-phos-phate have been used successfully.Although HPLC analytical techniques are increas-

ingly used for vitamin analysis, many of the B vita-mins are analysed using standardized microbiologicalassays (M�land, R�nnestad, Fyhn, Berg & Waagb�2000). These methods include growth of vitamin-sen-sitive microorganisms in tissue or ¢sh larvae homoge-nates. Because the microbial growth and therebyanalytical results can be in£uenced by growth factorswithin the sample homogenates, the results need to be

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carefully evaluated by the technicians. As for ascorbicacid, most di⁄culties in vitamin analytical work werein proper homogenization and extraction of live feedand larvae, but also measurement of di¡erent vitamerforms versus the total vitamin concentration. Somespecies, like carp and herring, contain various activ-ities of thiaminase (Wistbacka & Bylund 2008), whichmay rapidly reduce thiamine in homogenized samplesfrom these species, before a potential heat-treatmentstep in the analysis inactivates the enzymes (own un-published data). In such cases, heat treatment beforehomogenizationwill be necessary.

Discussion

The water-soluble vitamins may be roughly classi¢edaccording to their main biochemical roles into thosetaking part in molecular chemical modi¢cations,coenzyme function in energy metabolism and inredox reactions. However, novel roles of vitaminsand more sensitive markers for requirements arecontinuously being discovered in human and animalnutrition research and these aspects should also beconsidered in future evaluation of requirements for¢sh. Suggested ‘beyond de¢ciency requirements’maydi¡er considerably from the estimates arising fromclassical studies. Again, ascorbic acid (AA) serves asa good example. Besides its classical essential role in

post-translatory hydroxylation of proline and lysinemoieties in collagen, covered by 10^20mg AA kg�1

in several ¢sh species (Sandnes 1991), it has beenshown to interfere with mineral metabolism(Sandnes 1991), the stress response (Fletcher 1997),immunity (Sealy & Gatlin 2001; Waagb� 2006),wound repair (Wahli, Verlhac, Girling, Gabaudan &Aebischer 2003) and detoxi¢cation reactions (Norrg-ren, B˛rjeson, F˛rlin &—kerblom 2001) in ¢sh speciesat considerably higher concentrations than the mini-mum requirement for good growth and survival.

Ascorbic acid

The importance of vitamin C during ontogeny caneasily be demonstrated by high mortalities in o¡-spring from vitamin C-de¢cient brood ¢sh (Sandnes1984, 1991; Mangor-Jensen, Holm, Rosenlund, Lie &Sandnes 1994; Blom & Dabrowski 1995). This ismainly related to its well-de¢ned role in collagensynthesis in the formation of connective tissues likecartilage and bone in the developing ¢sh larvae(Terova, Saroglia, Papp & Cecchini 1998), but also toother essential functions related to the role of AA inthe integrated antioxidant system, constituting animportant part of the cellular water-soluble antioxi-dant capacity together with GSH. Recently, Fontagne,Lataillade, Breque and Kaushik (2008) demonstrated

Spawning fertilisation

Hatching First feeding

Yolk sac larvaeOocyte

Atlantic salmon

Egg

Vitamin consumption

Final maturation

Turbot

Atlantic halibut 50 days

10 days

100 days5 -7 mm

3-3.5 mm

1-1.2 mm

Endogenous feedingstudies

Live & formulated feed experiments

Vitamin accumulation

Brood stock feeding experiments

Fed larvae

Vitamin feeding

Figure 1 Vitamin research in ¢sh ontogeny includes brood stock feeding experiments, studies on endogenous feeding,and start of feeding experiments. These re£ect vitamin status at start of the new life, early consumption and externalvitamin supply, respectively. The ¢gure illustrates that farmed ¢sh species have di¡erent starting points and development(modi¢ed fromTonheim 2004).

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an active antioxidant system in the developing rain-bow trout until swim-up fry, recorded as changes ingene expression of major antioxidant enzymes. Sincethe mRNA expressions could be in£uenced byoxidized lipids, it would not be surprising if the statusof antioxidants could impact the enzyme activitiesand thereby the overall antioxidant capacity.In the brood ¢sh, ascorbic acid has been investigated

for its roles in steroid synthesis and vitellogenesis(Waagb�, Thorsen & Sandnes 1989). Similarly, eggAAdeposition levels may easily be tailored by feedingbrood ¢sh elevated AA before and during vitellogen-esis (Waagb� et al. 1989; Mangor-Jensen et al. 1994;Nortvedt, Mangor-Jensen, Waagb� & Norberg 2001,2003).The fate of AAduring embryogenesis has beenstudied in several ¢sh species and theweight concen-tration has been shown to decline until start of feed-ing. Maternally caused AA de¢ciency results in highmortalities and severely reduced quality of the o¡-spring. Surprisingly, there is not much detailed docu-mentation on AAde¢ciency symptoms inmarine ¢shlarvae during endogenous feeding, except for lowgross performance and survival.The dynamics of col-lagen synthesis, as evaluated by egg and larva hydro-xyl-proline was, however, studied in sea bass andgilthead sea bream o¡spring originating from brood¢sh fed normal or excess levels of AA. The respectiveegg and larvae re£ected the maternal AA feeding,and higher and earlier collagen synthesis wereobserved in the o¡spring fed excess AA comparedwith the control (Terova et al.1998).Themass transferand loss of vitamin C from the yolk sac to the larvalbodyduring developmentwas studied ina populationof well-performing Atlantic halibut larvae (R�nnes-tad et al. 1999). A constant AA content in the larvaefrom hatching and until ¢rst feeding indicated minorloss of vitamin in this period, in contrast to lossesfound in other species like white¢sh (Dabrowski1990). Depending on the overall antioxidant andenergy status, oxidized AA (dehydro AA) may beregenerated to AA and thereby recycled. A start offeeding study on juvenile Atlantic salmon (0.2 g)showed that vitamins C and E interact and therequirement levels of both growth, mortality andcollagen synthesis are in£uenced in vivo (Hamre,Waagb�, Berge & Lie 1997). After start of feedingthe halibut larvae with zooplankton containing600^1000 mg AAg�1dw, thewhole bodyAAcontentincreased rapidlyalongwith the growth, while a con-stant concentration of bodyAA in larvae410mg in-dicated a state of regulation of excess AA (R�nnestadet al. 1999). A similar observation was also made in

striped trumpeter, Latris lineata (Brown, Battaglene,Morehead & Brock 2005).The minimum requirement established in salmo-

nids that were fed on formulated feed with gradedlevels of the stable and bioavailable AP derivative is10^20mg kg�1 (Sandnes,Torrissen & Waagb�1992),which also seems to be adequate for Europeansea bass and turbot (Merchie, Lavens, Dhert, MaiSoni, Nelis, Ollevier, De Leenheer & Sorgeloos1995). Updated knowledge has shown that commoncarp (Cyprinus carpio) also need AA through thediet (Gouillou-Coustans et al. 1998), despite earlierpublished evidences for the opposite (Sato,Yoshinaka,Yamamoto & Ikeda 1978; Yamamoto, Sato & Ikeda1978). Interestingly, Atlantic salmon fry fed dietsdevoid of vitamin C from the start of feeding didnot show mortality or develop skeletal deformities(classical scoliosis and lordosis) until as late as 11weeks of feeding, in accordance with no detectableliver AA levels (Sandnes et al. 1992; Hamre et al.1997). On the other hand, Gapasin, Bombeo, Lavens,Sorgeloos and Nelis (1998) demonstrated bene¢ciale¡ects on the occurrence of opercular malformationsin milk¢sk larvae fed rotifers or Artemia enrichedwith AA and highly unsaturated fatty acids, as com-pared with the same live feed enriched with the fattyacids alone. Cat¢sh (Clarias gariepinus) larvae fedAAenriched Artemia as high as 2300 mg g�1showedhigher growth and stress resistance than larvaefed lower AA levels (Merchie et al. 1995). The sameobservations were made for sea bass, althoughat lowerArtemia AA concentrations (Terova, Cecchi-ni, Saroglia, Caricato & Jeney 2001). The docu-mented e¡ects of elevated AA on growth, collagensynthesis and resistance to stress are mainlyobserved in larvae fed AA far above any assessedminimum requirements. Tissue or whole larvaere£ect the live feed AA concentrations, demonstrat-ing the bioavailability of this form of AA, but theretention e⁄cacy is di⁄cult to estimate. Weaningof Atlantic halibut with formulated feed withincreasing doses of AA in the form of ascorbic phos-phate suggested that AA from Artemia (control)retainedmore e⁄ciently thanAP from the formulatedfeeds, but this changed during the course of theexperiment (M�land, Rosenlund, Stoss & Waagb�1999). Similar observations with available AA fromArtemia were made for white¢sh (Dabrowski 1990;Merchie, Lavens,Verreth, Ollevier, Nelis, De Leenheer,Storch & Sorgeloos 1997). The seemingly elevatedrequirements in for example carp and milk¢sh larvaemay therefore re£ect immaturity with respect to



intestinal development, vitamin absorption e⁄cacyand tissue vitamin storage.

B vitamins

B vitamins participate in biochemical reactions in theintermediate energy metabolism (Table 1), and highconcentrations of the vitamins are found in metaboli-cally active ¢sh tissues. The roles of the single B vita-mins in the ¢sh body are not fully known, and thereis a large gap between historical ¢sh requirementexperiments (NRC 1993; Halver 2002;Webster & Lim2002) and experiments with other species conductedunder modern intensive incubation and farmingconditions. Uncertainties in water-soluble vitaminrequirement estimates are illustrated by the wideranges reported in the literature. Cold water carnivor-ous ¢sh may not have a regular intestinal micro-£orathat supplies additional vitamins. The exogenous sup-ply of B vitamins are therefore the natural content ofthe live feed, feed ingredients and additional enrich-ment or supplementation of the diet through vitaminpremixes. While vitamin C de¢ciency causes drasticchanges, lack of B vitamins is less obvious to reveal,especially at larvae and juvenile stages of farmedspecies that normally experience high mortalitiesearly in the larvae production chain.Fish seem to require B vitamins according to their

growth and metabolic rate. Accordingly, the require-ments have been suggested to be higher at larvalstages compared with juvenile and adult stages(Dabrowski 1986). Analysis of vitamins in ¢sh eggsand natural live pray supports this assumption

(Br�kkan1959; De Roeck-Holtzhauer, Quere & Claire1991; M�land et al. 2000). The developing ¢sh larvaeseem to oxidize the energy-yielding substrates in thesequence glycogen, amino acids and lipids (Finn1994), which may in£uence the need for selected Bvitamins di¡erently during development.Descriptive studies of vitamin B6 (R�nnestad et al.

1997) and folate (M�land et al. 2003) in developingAtlantic halibut larvae showed a net loss of vitaminduring endogenous feeding and a steady transfer ofvitamin from the yolk sac into the body compart-ment. The requirement of folate is related to growthrates and cellular proliferation, which may be espe-cially high during rapid maternal and larvae growth.Indeed, for humans, increased preconceptional folicacid intake is recommended in several countries(Scott, Weir & Kirke 1995). In the hatched halibutegg, there was a 50% net loss of folate from the yolkcompartment during endogenous feeding, of whichhalf was retained in the larval body (M�land et al.2003).The data suggest a need for folate for metabolicand growth purposes during embryogenesis of2 mg g�1 weight gain, which is in agreementwith the requirement suggested by (NRC 1993) forcold-water ¢sh (calculations based on a feed conver-sion factor of 1g feed g�1 weight). According to thisrequirement, the authors expressed concerns regard-ing the critical low folate status observed in selectedbatches of egg from Atlantic halibut brood ¢sh(M�land et al. 2003). Studies on commerciallyhalibut farming in Norway and Iceland during1998^2000 con¢rmed a large variation in egg folateconcentration (Table 3). A trend towards increasedegg folate concentration with time was observed, in-

Table 1 Trivial names, importance in metabolism, sensitive organ at de¢ciency in ¢sh andmain biochemical function of thesingle B vitamins

Vitamin Familiar name Essential for Sensitive organs Biochemical function

Vitamin B1 Thiamin Carbohydrates Nerve system, muscle tissue Carboxylation,

decarboxylation

Vitamin B2 Riboflavin Lipid, protein, carbohydrate,

energy

Skin, epithelium, nerves,

eye tissues

Redox reactions

Niacin Nicotinic acid,

Vitamin B3

Lipid, protein, carbohydrate,

energy

Skin, epithelium, nerves,

muscle cell

Redox reactions

Vitamin B6 Pyridoxine Protein, amino acids, lipid Muscle cell, nerves, blood cell Transaminations/deaminations

Biotin Vitamin H Lipid, carbohydrates Skin, gill, liver tissues Carboxylation, decarboxylations

Pantothenic acid Vitamin B5 Carbohydrates, proteins,

fatty acids

Gills, skin, nerve system Acetylations, acylations

Folate Folic acid Amino acids, nucleic acids Blood cells, nerves, skin,

cell division

1C transfers

Vitamin B12 Cyano-cobalamin Amino acids, nucleic acids,

fatty acids

Blood cells, nerves, cell division Methylations



£uenced by the increasing focus on overall maternalvitamin nutrition. Feeding halibut brood stocks withgraded levels of folate, however, did not a¡ect fertili-zation, egg survival or hatchability in eggs rangingbetween 0.18 and 0.71 mg g�1wet weight (ww) (Nort-vedt et al. 2001, 2003). Unfortunately, the larvae werenot followed during further development, so laterperformance of the o¡spring was not assessed. Criti-cal low levels of folate may constitute a potential pro-blem until start of feeding, after which therequirement can be met by folate from several liveprey alternatives (M�land et al. 2000; Hamre, Srivas-tava, R�nnestad, Mangor-Jensen & Stoss 2008).The whole halibut larvae content of vitamin B6

started to decline10 days post hatch and resulted in anet 25% reduction until ¢rst feeding (R�nnestad et al.1997). This expenditure of whole larvae vitamin B6was attributed to as a need for vitamin B6 for proteinsynthesis and growth (15 nmol vitamin B6 g

�1wwgain), according to the hypothesis of Woodward(1994). Similarly, Albrektsen, Sandnes, Lie andWaagb�(1994) showed a stable egg vitamin B6 of approxi-mately 1mg B6 g

�1 until hatching at 500 day degreesin Atlantic salmon, with a subsequent decline in thebody compartment from1.0 to 0.6mg B6 g

�1 until thestart if feeding 6 weeks post hatch. The kinetics ofvitamin B6 transfer from the yolk to the body wasslower than the main bulk dry matter. This mayindicate need for vitamin B6 according to proteindegradation and increasing transaminase acitivity(Sato,Yoshinaka, Kuroshima, Morimoto & Ikeda 1987;R�nnestad, Groot & Fyhn1993) or simply that the yolkhas excess vitamin relative to the need of the develop-ing larvae, and where the vitamin is mediated by aspeci¢c transporter mechanism.Thiamine requirements are estimated to range

between 0.5 and 1mg kg�1 in several ¢sh speciesbased on weight gain, as summarized byWoodward(1994), and enzyme saturation (erythrocyte transke-tolase) was achieved at higher concentrations.Whilethere are few recent studies on thiamine in farmedspecies, there is a substantial bodyof recent literatureon thiamine de¢ciency in yolk-sac fry of wild salmo-nid species su¡ering from the M74 syndrome (M74)in the Baltic sea, early mortality syndrome (EMS) inthe Great Lakes in North America and the Cayugasyndrome (CS) in the New York State Finger Lakes(reviewed byAmco¡ 2000).Mortality rates of M74 yolksac fry may be as high as 40^95%. The developmentof the disorders has been related to environmentalpollution, change in prey species (to species expres-sing thiaminase; Wistbacka, Heinonen & Bylund

2002; Wistbacka & Bylund 2008) or oxidative stress(Pickova, Kiessling, Petterson & Dutta 1998; Vuori &Nikinmaa 2007). Such maternally transmitted thia-mine de¢ciency manifests in the fry as abnormalswimming behaviour, general weakness and poor re-sponse to physical stimuli, darker skin pigmentation,whitened liver, pale gills, reduced tissue glycogenlevels, extremely low thiamine levels (6% of healthyfry values) and necrotic changes in the brain areas(Amco¡, B˛rjeson, Norrgren & Pesonen 1998b). Atthe terminal stages, the yolk-sac fry are lethargicand have convulsions and bradycardia (Lundstrom,B˛rjeson & Norrgren 1998). The development of theM74 disease occurs normally midway in the yolk-sac phase and has been categorized in severity intopreclinical, clinical and terminal stages, where thestages depend on the egg and hatched larvae thia-mine status. Recently, the development of the M74syndrome was studied using a cDNA micro-arrayand real-time PCR to explore the transcriptionalresponses behind the typical neurological, cardio-vascular and pathological symptoms. Most of thegene expressions could be related to reactions asso-ciated with environmental stress, and terminallyto hypoxia (Vuori, Koskinen, Krasnov, Koivumaki,Afanasyev,Vuorinen & Nikinmaa 2006).The M74 syndrome serves as a good example of a

classical vitamin de¢ciency that can be completelyprevented by thiamine injection of the brood ¢sh or bybathing the egg and fry in thiamine (4500mgL�1)solutions (Amco¡, B˛rjeson, Eriksson & Norrgren1998a). The thiamine-dependent enzymes transketo-lase and a-ketoglutarate dehydrogenase were lower inM74 diseased yolk-sac fry compared with healthy con-trols andmay be part of theM74 pathogenesis (Amco¡2000;Amco¡,—kerman, B˛rjeson,Tjarnlund,Norrgren& Balk 2000). The enzyme activities were restoredwith thiamine treatment, with activities correlatingwith the thiamine status obtained (Amco¡, Akerman,Tjarnlund, Borjeson, Norrgren & Balk 2002a). Finally,yolk-sac fry exposed to the thiamine antagonists pyr-ithiamine and oxythiamine developedM74-like symp-toms, although not the complete array of pathology(Amco¡, Lundstr˛m, Teimert, B˛rjeson & Norrgren1999; Amco¡, —kerman, Tj�rnlund, B˛rjeson, Norrg-ren & Balk 2002b). Eyed eggs with a thiamine statusbelow a certain threshold (0.4^0.8 nmol g�1) had ahigher risk of developing M74, while healthy feraland farmed eggs had thiamine concentrations41nmol g�1 (Amco¡ et al. 1998b). The respectivelow thiamine threshold for yolk-sac fry was 0.3^0.5 nmol g�1 (Amco¡ et al. 1998a). With a relatively



short half-life of thiamine during development, a nor-mal reduction in thiamine during endogenous feedingmaybeas highas 75% (Sato et al.1987).The occurrenceof the discussed thiamine de¢ciency-related syn-dromes shows the importance of a single vitaminduring ontogeny, with interacting environmental anddietary factors contributing to the state of de¢ciency.As formost of the Bvitamins, theminimum require-

ment of ribo£avin is relatively low, estimated to berelativelyequal, between3 and11mg kg�1fordi¡erentspecies. De¢ciency symptoms of ribo£avin areobserved in feeding trials with unsupplemented dietswith a low natural content of ribo£avin. Besides show-ing weight reduction as the ¢rst sign observed, de¢-ciency symptoms include eye disorders (corneallesions and cataracts), nervousness and abnormal skincoloration. Poorly vascularized tissues such as the cor-nea and eye lens are often a¡ected (Tacon 1992; NRC1993). Little is known about ribo£avin during onto-geny. According to historical data, the ovaries showedthe highest values of ribo£avin concentration amongselected organs of several wild-caught ¢sh species(Br�kkan1959), indicating importance in embryogen-esis and for the developing larvae. Live feed ribo£avinconcentrations seem to be in several fold excess ofknown requirements for ¢sh species, which indicateno problems with ribo£avin after start of feeding(Brown, Je¡rey, Volkman & Dunstan 1997; Hamreet al. 2008; Van der Meeren, Olsen, Hamre & Fyhn2008). Pantothenic acid is a part of coenzymeA that isrequired in energy-yielding reactions of glucose, fattyacids and amino acids. Few requirement studies havebeen conducted in ¢sh. Sandnes, Rosenlund, Mangor-Jensen and Lie (1998) showed, however, pantothenicacid accumulation in the ovaries of turbot at a muchlower rate than for example vitamin B6. Sato et al.(1987) showed that the pantothenic acid concentrationdeclined by 50% in developing rainbow trout eggs,from fertilization to start of feeding ready fry. In thesame study, biotin showed a similar declinewith devel-opment (Sato et al.1987).

Vitamin requirement estimates from the startof feeding trials

Start of feeding of altrical ¢sh larvae is mostly per-formed with live feed organisms, but often enrichedto complement the requirement of the larvae. Naturalmarine zooplankton (copepods) has been regarded asthe gold standard feed organisms because theysupport successfully the growthand normal develop-ment of marine ¢sh larvae (Hamre, R�nnestad, Rain-

uzzo, Barr & Harboe 2007). Recently, the nutritionalcomposition of rotifers was examined with respectthe ful¢llment of the nutritional requirements ofmarine larvae like Atlantic cod, Gadus morhua(Hamre et al. 2008). The choice of feed organism (sizeand composition) and the enrichment procedure isalways a compromise in optimizing macro andmicro-nutrient composition, and for use of enrichedArtemia and rotifers to ¢sh larvae, suboptimal levelsof vitamin C, thiamine and vitamin B6 have been infocus among the water-soluble vitamins.There are several challenges with respect towater-

soluble vitamins in live or formulated feeds, besidesaspects of live feed enrichment, like vitamin stabilityand leaching. Micro bound and encapsulated dietsmay undergo severe leaching of up to 90% of water-soluble vitamins during feeding (�nal & Langdon2005; Hamre et al.2007).They reported ona newgen-eration of encapsulated complex diets, with use ofmarine phospholipids or polymerization agents thatseem to cope better with the leaching problems. Bio-logical aspects of requirement assessment from startof feeding include variable individual feed intakes,maturation of the gastrointestinal tract, bioavailabil-ity of inherent and added vitamins and the vitaminstorage capacity in the developing larvae (Dabrowski1986; Segner, R˛sch, Verreth & Witt 1993). Conse-quently, exact minimum requirements are di⁄cultto estimate, and established and future recommenda-tions need to consider such imperfect conditions.Some newer studies on water-soluble vitamin re-quirements in ¢sh species have recently been con-ducted, that adds to and are in accordance with theexisting literature (Table 2).

External interfering conditions

Growth and metabolic rate of ¢sh larvae are highlyin£uenced by water temperature, and other externalrearing conditions (Lein 1996). Generally, elevatedwater temperatures lead to increased growth andintermediary metabolism, and possibly vitaminrequirement. Elevated water temperature or geneticdi¡erences in the growth rate did not, however, a¡ectthe requirement of ribo£avin, and this has been usedas a support for the fairly equal requirement among¢sh species (Woodward 1985; NRC 1993). Uncon-trolled temperature conditions, however, like criti-cally elevated or £uctuating temperatures duringegg development, cause heat shock and developmen-tal errors in some studied ¢sh species, and an asso-



ciation with selected vitamins has been suggested(Waagb� 2008).With respect to salinity, there seems to be minor

di¡erences in requirement between fresh water andmarine species in for example AA requirement(Gouillou-Coustans & Kaushik 2001).Changes in external farming conditions, use of

novel feed ingredients and exposure to contaminants

may increase the relative requirement for selectedvitamins in brood ¢sh, and start fed larvae and fry.The discussed thiamine de¢ciency syndromes in sal-monid species have been related to environmentalpollution, resulting in a low thiamine status in brood¢sh and o¡spring (Vuorinen, Paasivirta, Kein�nen,Koistinen, Rantio, Hy˛tyl�inen & Welling 1997).Whether this is entirely due to the lowmaternal input

Table 2 Recently published requirement studies for the single B vitamins mostly con¢rm existing requirements (NRC1993;Halver 2002;Webster & Lim 2002) for similar species

VitaminRequirement(mgkg� 1) Species, weight Analysedmarkers Reference

Vitamin B1 0.5–1 Woodward (1994)

Vitamin B2 4.1–5.0 Sunshine bass, 2 g Growth, pathology, cataract inspection, hepatic

D-amino acid oxidase activity

Deng & Wilson (2003)

6 Atlantic salmon, 17.5 g Growth performance, pathology, eye lens histology;

body composition; tissue riboflavin

Brønstad, Bjerkas &

Waagbø (2002)

Niacin 25 Indian catfish, 9.4 g Growth performance, pathology, body composition;

tissue niacin

Mohamed &

Ibrahim (2001)

33 African catfish, 17 g Growth performance, pathology, body composition Morris, Baker &

Davies (1998)

7.4 Channel catfish, 5.6 g Growth performance, pathology, body composition;

liver NAD; haematology

Ng, Serrini, Zhang &

Wilson (1997)

Vitamin B6 28% protein:

1.7–9.5

36% protein:

15–16.5

Tilapia, 0.73 g Growth performance, feed efficiency; liver tissue

enzyme activity; haematology

Shiau & Hsieh (1997)

1.75–2.05 Grouper, 14.8 g Growth performance, pathology, body composition;

tissue enzyme activity; haematology

Huang, Tian, Du,

Yang & Liu (2005)

Biotin 0.25 Indian catfish, 3.6 g Maximal weight gain, body biotin content, liver

pyruvate carboxylase and acetyl CoA carboxylase

Mohamed (2001)

0.06 Juvenile hybrid tilapia,

0.98 g

Maximal weight gain; body composition;

body biotin; hepatic pyruvate carboxylase

and acetyl CoA carboxylase activities

Shiau & Chin (1999)

Pantothenic

acid

10 Blue tilapia, 0.7 g Maximal weight gain; prevention of deficiency

signs

Soliman &

Wilson (1992)

Folate 0.3–0.6 Rainbow trout 1.4 and

2.8 g

Growth indices; tissue folate; hematology Cowey &

Woodward (1993)

1.1 Channel catfish Weight gain, hematocrit, erythrocyte and leukocyte

numbers, and plasma and liver folate

Duncan & Lovell

(1994)

Vitamin B12 0.014 Atlantic salmon, 0.2 g Prevention of anaemia with immature erythrocytes (Sandnes, Mæland &

Waagbø 1993,

unpublished data)

Table 3 Selected range (minimum and maximum concentrations) of water-soluble vitamins (wet weight) in egg batchesfrom Atlantic halibut (Hippoglossus hippoglossus) brood stocks during the period1997^2001

Drymatter (g kg� 1) Thiamine (lgg� 1) Folate (lgg� 1) Ascorbic acid (lgg� 1)

Range min–max 44–139 o0.1–4.4 0.02–0.71 5–55

1998 0.6–2.2 0.05–0.09 1–20

1999 2.1–2.3 0.05–0.06 24–29

2000 1.4–3.0 0.03–0.13 31–40

The vitamin concentration increased during the period 1998^2000, re£ecting a general increase in vitamin levels in the brood stockdiets (data from Nortvedt et al. 2003).



of thiamine to the eggs during vitellogenesis or alsoincreased consumption during yolk-sac fry develop-ment is not completely clear. Low maternal statushas, however, been associated with environmentallyinduced changes in the relative biomasses of salmonprey species towards more thiaminase-rich pelagic¢sh species (Wistbacka & Bylund 2008).

Conclusions

Evaluating the progress in vitamin research in ¢shduring the last decade is easy, relative to the substan-tial body of published literature within other areas ofaquaculture nutrition research. However, any pro-gress in other ¢elds is likely to in£uence the vitaminnutrition of ¢sh, and especially in the sensitive juve-nile stages. Regular reevaluations of vitamin require-ments at di¡erent stages of ¢sh development aretherefore needed, including brood stock and larvaenutrition. Strict requirements for water soluble vita-mins are probably relatively similar among ¢sh spe-cies and in metabolically active tissues. There are,however, elevated needs during ontogeny due to lar-vae immaturity and imperfect diets. Because ¢sh lar-vae are more susceptible to de¢ciency, larvae dietsshould contain safe and surplus vitamin levels tocompensate for varying biological and technical con-ditions.

Acknowledgments

I wish to thank Dr. Konrad Dabrowski (The OHIOState University, USA) for arranging this Specialsession on Basic and applied aspects of AquacultureNutrition: Healthy ¢sh for healthy consumers at Aqua-culture Europe 008 conference in Krakow, Polen, andthe OECD for the economical support.

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Water-soluble vitamins in fish ontogeny