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Enhanced pyridoxine supplementation of diets for gilthead seabream ( Sparus aurata L.)

P. C. Morris and S. J. Davies

Animal Science / Volume 61 / Issue 02 / October 1995, pp 445 ­ 452DOI: 10.1017/S1357729800013990, Published online: 02 September 2010

Link to this article: http://journals.cambridge.org/abstract_S1357729800013990

How to cite this article:P. C. Morris and S. J. Davies (1995). Enhanced pyridoxine supplementation of diets for gilthead seabream ( Sparus aurata L.). Animal Science, 61, pp 445­452 doi:10.1017/S1357729800013990

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Animal Science 1995, 61: 445-452© 1995 British Society of Animal Science

13577298/95/56510445S20-00

Enhanced pyridoxine supplementation of diets for giltheadseabream (Spams aurata L.)

P. C. Morris and S. J. Davies

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

Abstract

Using a high protein diet (483 g/kg), two trials were carried out to assess any potential benefit which may resultfrom the supplementation of diets for fingerling and grower class gilthead seabream with pyridoxine at levels whichwere below, matched and far exceeded the minimum dietary requirement. At the level of supplementation below theminimal requirement (lowest level) the responses from the practical diet almost matched the responses from the dietcontaining an amount of pyridoxine corresponding to the published minimum requirement for the species and nosignificant improvement in performance was recorded in response to increasing supplement level. However, despitethe absence of a marked effect on overall performance, a small potential for increased activity of alanineaminotransferase was recorded amongst grower class fish given diets containing the lowest supplement. Theproximate composition of the grower class fish was unaffected by the level of pyridoxine supplementation thoughmarginal increases in the lipid content of the fingerlings were observed. At the haematological level, haematocrit,total haemoglobin and the plasma concentrations of glucose and protein were also unaffected. However, on theapplication of an acute stressor (repeated netting), significant alterations in haematocrit and plasma glucoseconcentration reflecting dietary pyridoxine supplement were recorded.

Keywords: protein utilization, pyridoxine, seabream, Spams aurata, stress, vitamin B complex.

IntroductionRecent investigations in this laboratory (Morris,Davies and Lowe, 1995) have emphasized thequalitative requirement of the gilthead seabream,Sparus aurata for pyridoxine (vitamin B6). Thus, incommon with the observations of Kissil, Cowey,Adron and Richards (1981), a deficiency ofpyridoxine resulted in significant growthretardation, anorexia, food refusal with emaciation,highly inefficient nutrient utilization and increasedmortality. In keeping with its central role inintermediary metabolism, pyridoxine is recognizedas an essential micronutrient in the diets of culturedfinfish (Halver, 1989; Steffens, 1989; Tacon, 1991).

Given the role of pyridoxine in the non-oxidativedegradation of amino acids some attention has beenafforded to the relationship between vitamin B6 andaspects of dietary protein supply in fish (Hardy,

Halver and Brannon, 1979; Baker and Davies 1995).Hardy et al. (1979) demonstrated that elevation ofdietary protein resulted in an increased pyridoxinerequirement for chinook salmon (Oncorhynchustshawytscha) and it would appear that increasedsupplementation with this vitamin may improveprotein utilization. On the basis of normal growthand optimum activity of alanine amino transferase,Kissil et al. (1981) estimated the dietary pyridoxinerequirement of Sparus aurata to be 1-97 mg B6 per kgof diet. Therefore, more recently, Baker and Davies(1995) investigated the relationship between proteinto energy ratios and pyridoxine requirements injuvenile Sparus aurata. In summary, increasing thepyridoxine content of the diet up to 50 mg/kgresulted in incremental improvements in foodconversion efficiency (FCE) and apparent net proteinutilization (ANPU) amongst fish given diets of equalprotein to energy ratio. Incremental improvements in

445

446 Morris and Davies

growth proportional to dietary B6 content were alsoobserved amongst the fish given diets where theprotein to energy ratio was high.

Further to the association between pyridoxine andprotein, recent investigations have examined therelationship between pharmacological applicationsof B6 and health. Thus, pyridoxine has been shown toprevent liver lipid peroxidation in rats (Selvam andRavichandran, 1991), is closely involved with avariety of both cellular and humoral immunefunctions (Chandra and Sudhakaran, 1990; Lakshimi,Lakshimi, Divan and Bamji, 1991) and has been usedto regulate serum lipid and plasma homocysteineconcentrations amongst dialysis patients (Arnadottir,Brattstrom, Simonsen, Thysell, Hultbert, Anderssonand Nilsson-Ehle, 1993).

Intensive aquaculture is an environment in whichfish are subjected to a multitude of stressors such ashigh stocking density and handling. Therefore, thedevelopment of dietary treatments to minimize theeffects of stress and promote disease resistance

Table 1 Experimental test diet

Component g/kg

Fish meal (Provimi 66)Cod liver oilWheatfeedMolassesMineralstFat soluble vitamins^B complex vitamins (B6 free)§Macro vitamins||Pyridoxine in a-cellulosei

712-063-0

188-05-0

15-02-04-2

10-00-8

+ Mineral pre-mix (mg/kg diet): CaHPO4, 2756-3; CaCO3,375-0; NaCl, 1875-0; K2SO3, 2500-0; MgSO4.7H2O, 2578-4;FeSO4.7H2O, 87-5; MnSO4.4H2O, 28-4; ZnSO4, 64-5;CuSO4.5H2O, 6-4; CoCl2.6H2O, 3-3; KI, 1-9; Na2SeO3, 0-3;a-cellulose, 4723-0.X Fat-soluble vitamin pre-mix (mg/kg diet): vitamin Apalmitate, 4-2; vitamin D3 0-046; vitamin E acetate, 439-8;menadione sodium bi-sulphine, 78-0; maize gluten meal,1,477-95.§ Pyridoxine-free B vitamin pre-mix (mg/kg diet): biotin 2%,300-0; folic acid 88-8%, 16-9; niacin, 48-6; calcium pantothenate,305-3; riboflavin 96%, 208-3; thiamine hydrochloride, 69-9;cyanocobalamin 95%, 0-01; a-cellulose; 3250-99.|| Macro vitamin pre-mix (mg/kg diet): polyphosphorylatedascorbic acid, 2000; choline chloride, 5000; inositol, 2000;maize gluten meal, 1000.1 Pyridoxine in a-cellulose represents the space within theformulation allowed for the pyridoxine supplement. Diets 1,2 and 3 were supplemented with pyridoxine hydrochloride at0-5,5-0 and 100-0 mg/kg respectively and the remaining spacewithin the formulation filled with a-cellulose.

would be beneficial. Owing to its recentlydiscovered role in steroid modulation (Leklem,1991) and the recent clinical applications of thisvitamin, elevation of dietary pyridoxine represents asuitable candidate for further study.

The principle aim of the current study was to assessthe potential for improvements in growth, proteinutilization and carcass composition which mayresult from the supply of pyridoxine at levels inexcess of the published minimum dietaryrequirement of Spams aurata. In addition, the effectof super supplementation on aspects of fish healthbefore and after exposure to an acute stress wasassessed.

Material and methodsThe Spams aurata used in the present experimentwere supplied as juveniles by the SIAM hatchery(Montpellier, France). The fish were transported toPlymouth and acclimated to the aquariumconditions in the 6 months prior to thecommencement of the study. Duplicate batches of 15seabream growers (initial weight 39-13 (s.e. 0-05) g)were stocked into 400-1, self cleaning fibreglasstanks within a closed recirculation system thatprovided a parallel flow of 10-8 1/min of sea waterthrough the tanks. Additionally, duplicate groups of20 fingerlings (initial weight 2-80 (s.e. 0-01) g) werestocked into 15-1 round, polyethylene tanks within asecond closed recirculation system that provided aparallel flow of 2-3 1/min'of seawater to the tanks.Water temperature and salinity were maintained at24°C and 33 to 36%o saline respectively. Daylightbalanced fluorescent discharge lamps were used tomaintain a 12 h light/dark photoperiod.

The basal diet was formulated using fish meal andcod liver oil to provide, on a dry-matter (DM) basis,approximately 480 g crude protein and 130 g lipidper kg. Based on this formulation (Table 1), threetest diets were designed differing only with respectto the level of pyridoxine supplementation. Diets 1,2 and 3 were supplemented with 0-5, 5-0 and100-0 mg B6HC1 per kg of diet respectively(pyridoxine hydrochloride, Roche Products Ltd).The diet was manufactured by mixing the dryingredients, oil and water in a Hobart A120 foodprocessor and the mash was then cold extrudedthrough a 2-mm die. The resultant pellets were airdried at 45°C for 24 h and stored at ambienttemperature in air-tight bags. Proximate analysisshowed that on an air-dry basis, the diets contained929-9 g DM, 483-0 g crude protein, 134-3 g lipid and179-6 g ash per kg. Additional samples were assayedby high performance liquid chromatography for B6content courtesy of F. Hoffmann La Roche AG

Pyridoxine for seabream 447

(Basel). The pyridoxine content of the diets was 1-75,5-40 and 103-00 mg/kg.

The test diets were presented to the fish three timesdaily (09.00, 13.00 and 16.00 h). For the growers thediet was supplied at proportionally 002 body weightper day (0-02M) for the first 16 weeks followed by aration of 0-018M for the remaining 10 weeks. Thefingerlings were presented with proportionally 0-05live weight per day (0-05M) for the first 8 weeks andwith 0-045M during weeks 9 to 12 inclusive. For theremaining 6 weeks of the study, the fingerlings weregiven food to appetite up to 0-035M and the foodsupplied was recorded. The fish were individuallyweighed twice monthly in order to re-determine theappropriate allowance, with the growers starved inthe 24 h prior to weighing. The change in weight,specific growth rate (SGR), food consumption andFCE were determined as outlined by Baker andDavies (1995).

After 26 weeks of feeding, five growers from eachtank were selected for individual proximate analysis.Due to the small size of the fingerlings, fourindividuals from each tank were pooled forproximate analysis after 18 weeks. Crude proteinwas determined by the Kjeldahl (N X 6-25) methodand lipid by a method derived from that of Barnesand Blackstock (1973). Moisture and ash wereassayed according to the Association of OfficialAnalytical Chemists (1990). Based on the growthdata, nutrient analysis of the test diet and theproximate composition of fish subsampled at thebeginning of the trial, the ANPU was calculated asoutlined by Baker and Davies (1995).

At the end of the trial involving the growers, fiveindividuals were selected from each tank and bloodwas withdrawn into heparinized syringes from thecaudal vein. A second group of five fish from eachtank was subjected to a short-term, acute stress bynetting each fish five times followed by a 30-minrecovery period. At the end of this time the fish wereagain recaptured and exsanguinated from the caudalvein. For both control and stressed fish, the packedcell volume of the blood was determined by microhaematocrit while the haemoglobin content wasdetermined using the cyanmethemoglobin technique(Sigma Total Haemoglobin Test, Sigma No. 525 A).Following centrifugation the plasma was decantedinto micro-centrifuge tubes and stored at -70°C untilrequired. Plasma protein was determined by theBiuret method (Sigma No. 541-2) and plasma glucoseby the glucose oxidase method (Sigma no. 510 A).

At this point the hepatopancreas was excised fromfive fish per tank and the weight of this organ as aproportion of the body weight (hepatosomatic index)

Table 2 Growth and dietary performance criteria for growers givendiets containing varied pyridoxine supplements over 26 weeks

Pyridoxine HC1 content(mg/kg)

0-5 5-0 100 s.e.

Initial mean weight (g)tFinal weight (g)tSGR(%/day)tTotal food intake(g per fish)t

FCEtANPUJ

3913 39-11 39-15 0-05252-22 250-29 248-07 3-87

1-02 1-02 1-02 0-01

347-17 346-98 338-55 4-660-614 0-609 0-617 0-0120-237 0-234 0-240 0-003

t Food conversion efficiency based on the mean data forreplicate tanks.% Apparent net protein utilization based on the proximatecomposition of five fish per replicate tank.

determined. In addition, these liver samples wereused as part of a parallel investigation of the effect ofpyridoxine supplementation on the activity ofalanine amino transferase (AAT) in the growers asdescribed by Brooksbank (1993). In summary, theactivity of AAT was first estimated relying only onthe pyridoxal-5-phosphate (PLP) contained in thetissue itself (baseline activity). The potential forincreased AAT activity, i.e. activity over and abovethe baseline level, was then determined by pre-incubating the tissue homogenate with excess PLP.This potential for increased activity was used toindicate the concentration of pyridoxine in the liveras outlined by Kissil et al. (1981).

The data were subjected to analysis of variance(ANOVA) where P < 0-05 was judged to be indicative

Table 3 Growth and dietary performance criteria for seabreamfingerlings given diets containing varied pyridoxine supplementsover 18 weeks

Initial mean weight (g)+Final mean weight (g)tSGR(%/day)tTotal food intake(g per fish)t

FCEtANPUt

Pyridoxine HC1 content

0-5

2-8049-45

2-30

61-270-7620-269

(mg/kg)

5-0

2-8049-45

2-28

59-230-7840-267

100

2-8149-91

2-29

61-700-7640-270

s.e.

0-014-750-09

2-900-0570-001

t Food conversion efficiency based on mean data for replicatetanks.\ Apparent net protein utilization based on proximatecomposition of a single pool of fish per replicate tank.

448 Morris and Davies

Table 4 Proximate composition of grower carcasses expressed relative to live weight (no. = 10 per dietary treatment)

Dietary pyridoxineHC1 (mg/kg)

0-55-0

100-0Initial fish

Moisture(g/kg)

Mean

615-0632-0632-4698-0

s.e.

10-06-05-38-7

Crude protein(g/kg)

Mean

183-218241844164-7

s.e.

2-52-92-111

Lipid(g/kg)

Mean

176-0150-5159-2104-8

s.e.

8-67-6947-8

Ash(g/kg)

Mean

35-235-436-441-8

s.e.

0-6100-71-6

Table 5 Proximate composition of two groups of pooled fingerling carcasses expressed as a proportion of the live weight

Dietary pyridoxineHC1 (mg/kg diet)

Moisture(g/kg)

Crude protein(g/kg)

Lipid(g/kg)

Mean s.d. Mean s.d. Mean s.d.

Ash(g/kg)

Mean s.d.

0-55-0

100-0Initial fish

738-4719-3717-3728-4

11-94-68-75-4

1704173-5170-1159-0

0-22-02-11-6

65-569-575-283-2

4-314-19-55-1

36-034-534-637-3

211-41-31-0

of a significant difference. Where the ANOVA revealedsignificant differences, Duncan's multiple range test(Duncan, 1955) was applied in order to characterizeand quantify the differences between treatments.

ResultsIn terms of growth, no significant differences wereobserved between the dietary regimes i.e. weightgain and SGR amongst both the fingerlings andgrowers were independent of the level of dietarypyridoxine supplementation (Tables 2 and 3). Theremaining performance criteria i.e. FCE and ANPUwere not significantly altered in response topyridoxine supplementation for either of the sizeclasses (Tables 2 and 3).

With regard to all components, the carcasscomposition of the growers showed no significantdifferences between dietary treatments (Table 4). Anincrease in carcass lipid and decrease in carcassmoisture in response to elevated dietary pyridoxinecontent was observed amongst the fingerlings.However, insufficient replicates were available toallow statistical analysis of these data (Table 5).

The haematological profile of the growers given eachdiet prior to and after repeated netting is shown inTable 6. The haematocrit of the unstressed fishshowed no significant differences betweentreatments. However, after being subjected torepeated netting, those fish given the diet

supplemented with the lowest level of B6 (0-5 mg/kg) exhibited a packed cell volume significantlygreater than that of the remaining dietary regimes(P < 0-001). The haemoglobin concentration recordedin the blood of all fish showed no significantdifferences between dietary regimes and wasunaffected by stress. Due to the small samplevolumes obtained from the stressed fish, the plasmaprotein assay could not be performed; however, inthe absence of a stressor, the plasma proteinconcentration showed no significant variationbetween the three dietary groups.

The plasma glucose concentration of the test fishshowed a series of significant differences (P < 0-001).Prior to stress, there were no significant differencesbetween treatments with regards plasma glucoseconcentration. Where the fish had been presentedwith diets fortified at either the 5-0 or the 100-0 mg/kg level, the plasma glucose concentration observedin the stressed fish was significantly higher than thatin the control individuals. The post-stress plasmaglucose concentration of the fish given the dietsfortified with the lowest dietary B6 level was notsignificantly higher than that of the control group.Those fish given the diets fortified with pyridoxine atthe 5-0 and 100-0 mg/kg had a plasma glucoseconcentration which was significantly higher thanthat of the control individuals under both regimes.With respect to dietary supplement level, the post-stress glucose concentrations were significantlydifferent from each other, i.e. the 0-5 and 50 mg/kg

Pyridoxine for seabream

Table 6 Haematological measures of control and stressed growers

449

Dietary ]pyridoxineHC1 (mg/kg)

0-5

50

100

Control(No.)

Stressed(No.)

Control(No.)

Stressed(No.)

Control(No.)

Stressed(No.)

Haematocrit

Mean

35-54a

52-60b

38-45a

40-43a

34-40a

41-06a

(%)

7

7

7

7

9

6

s.e.

3-42

2-03

2-50

2-58

2-07

4-61

[Haemoglobin!(mg/cm3)

Mean

75-00

77-00

74-40

71-10

68-00

56-00

s.e.

5-0010

8-3210

3-379

7-539

5-7210

6-805

[Plasma glucose](mg/crr

Mean

0-658ab

0-91 lbc

0-527a

l-034c

0-529a

l-534d

10

7

10

7

9

8

i3)

s.e.

0-019

0-115

0-049

0191

0-035

0-163

[Plasma protein](mg/cm3)

Mean s.e.

30-09 1-5510

25-21 3-4510

30-01 2-3610

ab Values within parameter columns bearing common superscripts are not significantly different from each other (P < 0-01).

supplemented groups were not significantlydifferent from each other. However, plasma glucoseconcentration in the fish given diets containing the100 mg/kg supplement was significantly higher thanthat observed amongst fish given diets containingthe smaller supplement (Table 6).

The hepatosomatic indices of all groups showed nosignificant differences between the dietary regimes(Table 7). Analysis of the data recorded byBrooksbank (1993) (Table 7) shows that for thegrowers, there was potential for increased AATactivity at all dietary levels of pyridoxine when theassay was pre-incubated with PLP. This potential foran increase in activity was higher for those fishwhich had been given B6 at the lowest level (0-5 mg/

Table 7 Hepatosomatic index (HSI) and relative activity ofalanineaminotransferase (AAT) after pre-incubation with pyridoxal-5-phosphate (PLP) in the livers of seabream growers (Brooksbank,1993)

Pyridoxine HC1 content(mg/kg)

HSI (X lO"2)Mean (±s.e.)No.

Relative activity!of AATMean (± s.d.)No.

0-5

1-01 (0-09)10

1-442(0-101)4

50

1-10(0-07)10

1-330 (0-085)4

100

1-04(0-08)10

1-316 (0-190)4

t Activity of AAT after pre-incubation with PLP. Valueexpressed as a proportion of AAT activity in absence of addedPLP.

kg) while diets 2 and 3 demonstrated changes inactivity which were not similar to each other butlower than that of the fish given diet 1. However,these differences were shown not to be significant(P > 0-05).

DiscussionThe minimum pyridoxine requirement of giltheadseabream, i.e. that which supplies sufficient vitaminfor growth and the prevention of mortality is1-97 mg/kg diet (Kissil et ah, 1981). Analysis of thetest diet gave a minimum B6 content of 1-75 mg/kgdiet, almost sufficient pyridoxine to satisfy theminimum requirement of the species. This isprobably responsible for the maintained growth andhigh food conversion observed amongst the fishgiven the diets containing the supplement of 0-5 mgB6 HC1 per kg diet.

In the study of Baker and Davies (1995) optimalprotein utilization was achieved by Sparus auratapresented with diets containing 50 mg pyridoxineper kg semi-purified diet. Both Baker and Davies(1995) and Kissil et ah (1981) utilized semi-purifieddiets of comparable protein content. However, thediets used by Baker and Davies (1995) contained asupplemental amino acid pre-mix whilst the diets ofKissil et ah (1981) relied solely on casein as a sourceof amino acids. The efficacy of amino acidsupplements for fish has been questioned (Coweyand Sargent, 1979; Wilson, 1989) and it would appearthat the supplementation of both purified andpractical diets with crystalline amino acids mayresult in poorer growth than the dietary amino acidprofile would suggest. Several hypotheses exist forthis phenomenon including an amino acid imbalance

450 Morris and Davies

caused by preferential and more rapid uptake of thecrystalline supplement when compared with protein-bound amino acids (Tacon and Cowey, 1985). It istherefore suggested that the diets of Baker andDavies (1995) raised the pyridoxine requirement ofthe seabream to service the greater demand fortransformed amino acids in order to restore thebalance of absorbed amino acids.

Hardy et al. (1979) working with chinook salmon, didnot observe a pyridoxine dose dependency forgrowth within diets of equal protein content.However, food conversion and protein efficiencyratios did show improvements with respect todietary pyridoxine supplement. Additionally, at lowlevels of protein the activity of serum glutamic-oxaloacetic transaminase (SGOT) was dependent onthe availability of the protein. Where protein wassupplied to excess, the rate determining factor wasthe availability of B6 supplied by the diet. Finally,Hardy et al. (1979) observed that the requirement forpyridoxine was elevated at higher dietary proteinlevels. It is quite possible that the protein content ofthe diet offered during the present study wasinsufficient to command a higher pyridoxinerequirement. Increasing the protein content ofseabream diets from 480 g/kg to 600 g/kg or abovemay enforce a greater B6 requirement but theprovision of surplus energy from protein may notnecessarily result in improved growth or proteindeposition. Instead detrimental changes in carcasscomposition may result.

Furthermore, with respect to AAT, all treatmentsshowed a potential for increased activity when theassay was spiked with PLP. With respect to maximalAAT activity, Kissil et al. (1981) recommendeddietary supplements of 3-0 and 5-06 mg/kg diet forfry and fingerlings respectively. These levelsexceeded the pyridoxine content of the present dietwhen supplemented with pyridoxine at 0-5 mg/kg.For the present study, it may be postulated that thesmall potential for PLP mediated elevation in AATactivity amongst the growers showed thatpyridoxine was close to that value required for thesaturation of the transaminase system. Hence, incontradiction of the results of Kissil et al. (1981), thesupply of increased supplemental vitamin B6 did noteffect a change in AAT activity.

In the present study, the deposition of protein in thecarcasses of the fish was independent of dietarypyridoxine content. Additionally, given that ANPUdid not show a dose dependent effect, it may beassumed that sufficient pyridoxine was available forefficient protein utilization. The lipid content withinthe carcasses of the fingerlings showed a dose-dependent effect with those fish maintained on the

lowest dietary level of pyridoxine exhibiting thelowest levels of carcass lipid. However, the use ofpooled samples precluded a statistical analysis. Thediets employed in this investigation were allisocalorific and isonitrogenous and hence thedeposition of fat can only be related to the B6 intake.On the basis that, under optimal conditions proteincontent is determined by fish size (Shearer, 1994),then the products of the metabolism of excess dietaryamino acids must be catabolized. Thus, afterdeamination in the liver, excess amino acids aremetabolized to form a-keto acids which areconverted either into glycogen or lipid (Cowey andWalton, 1989). Observation of the carcasscomposition of the fry shows that as a consequenceof pyridoxine mediated deamination and subsequenta-keto acid formation, the potential for improvedprotein utilization via transamination was notrealized. Instead, excess calories were deposited asfat and not as protein.

With respect to haematocrit, only those individualsgiven the lowest dietary level of pyridoxine scored apacked cell volume which was significantly higherthan that recorded for all other groups. It is welldocumented that stress in fish may result inelevation of haematocrit (Wedemeyer and Yasutake,1977; Hunn and Greer, 1991) and that handlingstress may result in altered plasma osmolarity(Fletcher, 1975; Davis and Parker, 1983; Avella,Young, Prunet and Schreck, 1990). Given these twohypotheses it is fair to asume that handling stresswas the cause of the increased packed cell volumeobserved in those fish given the diet fortified withthe lowest B6 level.

Elevations of both plasma glucose concentration andthe rate of glycogenolysis are frequently associatedwith stress in fish (Flos, Reig, Torres and Tort, 1988;Thomas and Robertson, 1991; Hopkins and Cech,1992; Soengas, Rey, Rozas, Andres and Aldegunde,1992; McDonald, Goldstein and Mitton, 1993). Undercontrol conditions, the lack of differences betweenthe treatments may indicate that high levels of B6 didnot cause hyperglycaemia. However, under acutestress, the dietary pyridoxine made a significantimpact upon the response of the fish. The resultsobserved may be attributed to the role of pyridoxineas a co-factor in the action of glycogenphosphorylase (Palm, Klein, Schinzel, Buehner andHelmreich, 1990). Thus, the provision of largeamounts of pyridoxine may facilitate high rates ofglucose release by allowing high rates ofphosphorylase activity. Additionally, as statedabove, the a-keto acids formed as a consequence ofexcess amino acid availability, may be stored asglycogen in the livers of those fish given diets with ahigh B6 content. If catabolized, during or after stress

Pyridoxine for seabream 451

this glycogen reserve may provide a large source ofglucose.

In the light of the present study and given theimportance of pyridoxine in human health (Chandraand Sudhakaran, 1990; Lakshimi et ai., 1991; Bender,1992), a considerable scope for further work remains.With respect to fish immunology, little work apartfrom the study by Hardy et at, (1979) has beencarried out concerning the role of pyridoxine indisease resistance in fish. Hence, the relationshipbetween pyridoxine and cellular immunity should befurther investigated.

Although the relationship between the steroidhormones and stress in fish have been studiedextensively (Davis and Parker, 1983; Avella et al.,1990; Foo and Lam, 1993), little research has beencarried out to investigate the role of B6 in theregulation of the steroid hormones (Bender, 1992).Given the stressors implicit in aquaculture,pyridoxine may provide a dietary mechanism bywhich the effects of stress in fish may be moderated.

AcknowledgementsThe authors would like to acknowledge the technical andfinancial support given by the Vitamins and Fine Chemicalsdivision of F. Hoffmann La Roche A. G. (Basel,Switzerland). We would also like to thank Matt Brooksbankfor the use of the aminotransferase activity data.

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(Received 16 March 1995—Accepted 22 April 1995)