Aquaculture Research, 1996, 27, 405-412'

Vitamin C status and piiysiological response of thegilthead seabream, Sparus aurata L., to stressorsassociated with aquaculture

M M F Henrique, P C Morris & S J DaviesFish Nutrition Unit, Department of Biological Sciences, University of Plymouth, Plymouth, UK

Correspondence: M M F Henrique. Fish Nutrition Unit, Department of Biological Sciences, tJniverslty of Pbnnouth. Drake Circus. PlymouthPL4 8AA. UK

Abstract

Gilthead seabream, Sparus aurata L., fed diets of equalvitamin C content were exposed to three differentstressors—hyposalinity, shallow water and handling—and the effect of each on the internal homeostasis ofthe fish was evaluated. The individuals subjected tohyposaline stress exhibited no significant changes inplasma osmolality, Na*, Cl", K+, glucose, protein andtriglyceride concentrations. Hepatic glycogen contentwas significantly lower in the fish subjected to the lowersalinity (12%o), while the red muscle glycogen wasunaltered. Amongst the challenged individuals, tissueascorbate content was not significantly altered.Shallow water stress significantly diminished the liverand red muscle glycogen content, and lowered the liver,kidney and spleen vitamin C concentrations in thestressed fish. No significant changes were recorded inthe plasma glucose, protein and triglyceride concen-trations. In response to handling stress, no significantdifferences were detected in any of the measuredparameters, with the exception of plasma glucose levelsand the splenic L-ascorbic acid and ascorbate-2-sulphate (vitamin C2) contents. The lack of existingstandardization of both experimental designs andanalytical methods made comparisons of the presentdata with the work of other authors difficult, thushighlighting the necessity of coordinated research inthis field.

Introduction

Ascorbic acid appears to be a component of all living

organisms, which implies that this nutrient plays animportant role in metabolism. All vertebrate bodytissues contain ascorbic acid in different concen-trations according to the tissue and the species inquestion with, in the case of fish, high concentrationsbeing found in certain tissues such as the adrenal andpituitary glands, liver, and spleen (Agrawal & Mahajan1980). However, vitamin C is an essential dietarynutrient for only a limited number of vertebrate species(Bender 1992). Some species of fish have been shownto have the capacity to synthesise ascorbic acid de novo(Yamamoto, Sato & Ikeda 1978; Thomas, Bally & Neff1985), but most teleosts cannot produce sufficientvitamin C to meet their requirements, and therefore,are dependent on a dietary supply

In fish, the functions of ascorbic acid have not beenwidely studied, but some of its functions have beenelucidated (Halver, Smith, Tolbert & Baker 1975;CarragherS Sumpter 1990; Masumoto, Hosokawa &Shimeno 1991), and the deficiency symptoms havealso been described (Halver et a/. 19 7 S; Sato, Yoshinaka&Ikeda 1978; Akand, Sato, Yo.shinaka&lkeda 1987),

One of the currently expanding fields of researchin terms of the role of vitamins in fish nutrition, is therelationship between the intake of certain vitamins andthe resistance to stressors and diseases. Commonaquaculture procedures such as netting, handling,disease treatment, transportation and grading arestressful to fish (Donaldson 1981; Barton & Iwama1991; Sandness 1991), and are often associated withincreased susceptibility to pathogens as well as areduced capacity to maintain homeostasis andwithstand additional stressors (Wedemeyer & Mcl̂ eay1981; Robertson, Thomas, Arnold &Trant 1987).

© 1996 Blacfcwell Science Ltd. 405

Gilthead seabream and stress MMF Henrique et al.Aquaculture Research, 1996, 27, 405-412

When fish are subjected to stress, many mor-phological, biochemical and physiological changestake place enabling the organism to adjust to theunsuitable environmental conditions (Halim. Faisal& Ahmed 1987). It has been proven in mammalsthat there is an increase in ascorbic acid require-ment during exposure to various stresses (Hughes.Jones, Williams & Wright 1971). In fish, there alsoexists a relationship between stress and the metabolismof vitamin C. as has been observed in several studies(Agrawal, Juneja & Mahajan 1978: Thomas, Bally &Neff 1982: Thomas 1984: Navarre & Halver 1989:Hardie, Fletcher & Seccombes 1991). However, thisrelationship has not been clearly established, asSandnes & Waagbo (1991) demonstrated in theirstudy.

The aim of the present investigation was the charac-terization of gilthead seabream, Sparus aurata L., tissuesin terms of Vitamin Cl (ascorbate) and C2 (ascorbyl-2-sulphate) concentrations, and its variation withstress. For this purpose, fish were subjected to threedifferent stressors which are commonly experiencedby cultured fish: hyposalinity, shallow water andhandling. These stressors were appUed for differentperiods of time, in order to allow the observation of theresponses to chronic, semi-acute and acute stresses,respectively.

Materials and methods

Juvenile gilthead seabream were obtained from theSociete D'Ingenierie Aquacole Mediterraneennehatchery, Montpelier, France, and were acclimatedand grown at the aquarium of the University ofPlymouth. A group of 70 individuals was selected, ina weight range between 100 and 16 5 g, and acclimat-ed to the stock holding/experimental system, whichcomprised six square 400-1 fibreglass tanks equippedwith a 1200-1 biofilter in a closed recirculation system.With a paraUel flow through the tanks of 10.8 1 min"'.During the acclimation and holding periods, fishwere maintained in full-strength sea water (33%o) at22.7°C with an artificial photoperiod of 12 h light/12 h dark.

Fish were presented with a commercial troutfeed (Trouw Aquaculture Standard Expanded 40,containing 46% crude protein and 15% lipid and avitamin C concentration of 104 mg kg-\ determinedby assay) at a rate of 1.5% of live body weight perday for a 2-week period until the appUcation of thestressors.

Stress challenges

To account for tank effects and mortalities during theexperimental period, each stressor was applied usingduplicate tanks each containing five fish.

Hyposaline shock

Tanks containing 20 and 12%o sea water were pre-pared by adding dechlorinated city water to isolatedtanks within the stock holding/experimental system.Two groups of gilthead seabream were exposed to 20and 12%o saUne water, respectively, for 24 h. At theend of each experimental period, the fish were bledby cardiac puncture, weighed and kept on ice untilthe excision of tissue samples (liver, kidney, spleen,red muscle and gill) for analysis. The blood wasimmediately centrifuged, and the plasma was storedat-70°C until required.

Stress induced by shallow water

By adjustment of the waste pipe the animals weremaintained in duplicate tanks with a low water depth(approximately 72 mm, i.e. just greater than the bodydepth of the seabream) for a period of 3 h. Fish werethen captured, weighed and representative tissueswere coUected (blood, liver, kidney, spleen and redmuscle), as outlined above.

Acute handling stress

A random group of gilthead bream were individuallynetted out of the stocking tanks and exposed to air for1 min by placing them in an empty container. Theywere then replaced into a tank of the experimentalsystem for a recovery period of 30 min. Again,representative tissues were coUected. At the end of eachexperiment, five control fish were randomly coUectedfrom the stock holding tanks, and tissues samples werecollected as described.

Analytical methods

Plasma osmolality was measured directly in wholeplasma using a Shuco n Osmette (Shuco InstrumentsLtd, London, UK), sodium and potassium ion con-centrations were determined with a IL343 FlamePhotometer (Instrument Laboratories Ltd), and chlo-ride was measured in a Corning 920 Chloride meter

406© 1996 Blackweil Science Ltd./iquiiraJtureResearc/i. 27, 405-412

Aquaculture Research, 1996, 27, 405-412Gilthead seabream and stress M M V Henrique et at.

(Corning Medical and Scientific). Plasma glucose,total protein andTriglycerides were determined usingclinical diagnostic kits (Sigma Diagnostics. St Louis,MO, USA. Procedures No 510a. 541 and 336,respectively), and liver and red muscle glycogen con-tent were determined by the method outlined inPlummer(1987).

Ascorbic acid (vitamin C1) and ascorbate-2-sulphate(vitamin C2) were determined by a modification of thedinitrophenylhydrazine technique (Terada. Watanabe,Kunimoto & Hayashi 1978) described by Thomaset al. (1982) and Carr, Bally, Thomas & Neff (1983),Freshly excised tissues were weighed and imme-diately homogenized in a Potter homogenizer, andhomogenates were frozen at -18°C, as recommendedby Dabrowski & Hinterleitner (1989), until assayedfor vitamin C,

Statistical analysis

Two-group comparisons were made with Student's£-test (P < 0,05 and P < 0,01), more than two-groupcomparisons were made with one-way analysis ofvariance (ANOVA), in which case treatment means wereseparated using Duncan's multiple range test (Duncan1955),

Results

Salinity tolerance

As shown in Table 1, the fish exposed to salinities of12 and 20%o exhibited no significant differences with

respect to the controls for any of the haematologicalparameters measured. However, a decrease in plasmatriglyceride concentration was observed in response todecreasing salinity.

By comparison with the control fish and the groupheld at the salinity of 20%<), liver glycogen was foundto be significantly lower for those fish subjected to asalinity of 12%« (Table 3), No significant differenceswere recorded in the red muscle glycogen content, butthe results show a tendency for a declining glycogencontent in both tissues with the decreasing salinity.

Sudden exposure to a hyposaline environment of 12or 20%) made no significant impact on either theascorbate or ascorbate-2-sulphate concentration inthe liver (Table 4).

Shallow water stress

In the shallow water stress experiment, stressed fishexhibited no significant differences in plasma glucose,protein and triglyceride levels (Table 2), Hepatic and redmuscle glycogen contents (Table 3) were significantlylower in the stressed group (P < 0,01) when comparedto controls.

Shallow water stress also led to a significant de-crease (Table 5) in liver, kidney (P < 0,01) and spleen(P < 0,05) ascorbic acid content, and a decrease in theascorbate-2-sulphate content of the kidney (P < 0,01),

Handling stress

There was a marked increase in plasma glucose(P<0,01) after handling stress, but no significant

Table 1 Effect of salinity on the haematoiogical parameters of the gilthead seabream*

Plasma^

Glucose (mg d|-')

Protein (g dM)Triglycerides (mg dh')Osmolality (mOsmol kg-')Na* (mmol |-')Cl- (mmol I-')K* (mmol I-')

Control:

S = 33%.

42.38 ±1.753.92 ± 0 38

127.85+15.87346.20 ± 4.89149.45 ±9.97158.00 ±11.91

4.06 ± 0.26

48.93 ± 2.003.94 ± 0.23

112.14± 13,18349.8013.42156.70 ±9.07144.57 ±10.36

4.47 ± 0,37

Stress

S = 12%.

42.10 ±2.893.57 ± 0 32

83.59 ± 11 45347.44 ± 4 44

158.13±4.23140.00 ± 1.89

4.26 ± 0.3

* Means significantly different from controls (P < 0,05),• Mean and SE (n > 4),

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Gilthead seabream and stress MMF Henrique et al. Aquaculture Research, 1996, 27, 405-412

Table 2 Changes in the plasma concentrations of glucose, total protein and triglycerides in response to shallow water andhandling stresses

Plasma^

Glucose (mg dh')Protein (g dl-')Triglycerides (mg dl-')

Shallow water stress

Control

53.69 ± 3.703.96 ±0.34

122.58+19.75

Stress

59.05 ±5.764.01 ± 0.24

163.01 ±5.05

Acute handling stress

Control

43.26 ±6.163.49 ± 0.30

120.61 ±29.87

Stress

97.01 ±6.13*3.82 ±0.16

169.55 ±25.39

* Means signilicantly different (P < 0,01) from Controls.' Mean and SE (n > 4).

Table 3 Stress-induced liver and red muscle glycogendepletion in gilthead seabream

Type of stress

HyposalinityControlS = 20%<,S = 12%oShallow waterControlStressHandlingControlStress

Glycogen*

Liver

94.33 ± 9.2978.20 ± 5.33'30.48 ± 6.69*

94.12 ±4.4955.65 ± 3.35*

84.68 ±1.9790.30 ± 5.22

Red muscle

7.46 ± 0.235.17±1.123.35 ± 0.65

7.64 ± 0.234.18 ±0.50*

8.70 ±1.776.33 ±0.49

• Mean glycogen content (mg g"' tissue wet weight) ± SE

* Means significantly different from controls (P < 0,01).* Means significantly different (P < 0,05) from the otherstressed group.

differences were observed with regards plasma proteinand triglycerides (Table 2). Glycogen levels in both liverand red muscle did not exhibit significant changes withthe experimental stress (Table 3).

Acute stress induced a marked increase in splenicascorbic acid (P < 0,05) and ascorbate 2-sulphate(P< 0,01) concentrations, but had no effect in thecontent of these two substances in the other tissuesexamined.

Discussion

Exposure of gilthead seabream to changes of salinity

over a period of 24 h did not reveal any notablechanges in plasma osmolality and electrolyte concen-tration. Rapid transfer of euryhaline fish from seawaterto fresh water leads to a reduction in Na* and Cl"effluxes (Eddy 1981), and thus, in the present case,the normal values observed amongst the challengedanimals suggests that either full adaptation to the newconditions occurred, or that changes in salinity did notact as a stressor for the seabream. Hyperglycaemiais generally used as a measure of the duration andseverity of stress (Mazeaud, Mazeaud & Donaldson1977; Wedemeyer & Yasutake 1977: Wedemeyer &McLeay 1981). Thus, given the normal glycaemiaobserved, no conclusions can be drawn.

Tissue vitamin C depletion, particularly in liver andkidney, is also described as an indicator of physiologicalstress (Wedemeyer & Yasutake 1977; Wedemeyer &McLeay 1981; Thomas et al. 1982; Thomas 1990).Within the current study, no differences were observedamongst the challenged individuals, again implyingeither rapid adaptation or absence of stress. However,the significant depletion of the hepatic glycogen andthe decline in muscle glycogen content suggests thatfish were subjected to chronic stress (Wedemeyer &McLeay 1981). In fish, it is believed that the mobili-zation of the glycogen reserves is very slow (reviewedin Cowey & Sargent 1979; Walton & Cowey 1982),Thus, it is reasonable to postulate that, amongst thechallenged fish, glycogen reserves were used to respondto the stressor and the need for energy was either toogreat to be met by gluconeogenesis alone, or that animmediate demand for energy necessitated the mobili-zation of reserve glucose from glycogen.

The lower plasma triglyceride concentration ob-served amongst the stressed fish also suggests thatenergy derived from lipid oxidation was utilized. Anabsence of increased plasma glucose concentration.

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Aquaculture Research, 1996, 27, 405-412 Gilthead seabream and stress MMF Henrique et al.

Table 4 Effect of exposure tohyposaline environment on theascorbic acid and ascorbate-2-sulphate concentrations in tissues ofgUthead bream

Ascorbic acid'^LiverKidneySpleenRed muscieGiilPlasmaAscorbate-2-suiphate •LiverKidneySpleenRed muscleGillPlasma

Controtr

S = 33%.

79.77 ± 3.2752.85 ±12.0135.59 ± 8.6811.45 ±0.78N.D.'20.24 ±1.47

35.41 ± 7.6239.29 ±11.364.79 ±2.470.81 ± 0.69

N.D.N.D.

69.74 ±2.8072.77 ± 9.0938.29 ± 7.4810,52 ± 1.56N.D.18.01 ±1.50

45.41 ± 6.5962.87 ± 9.7413.31 ±4.341.53 ±1.22

N.D.N.D.

Stress

S = 12%.

82.58 ± 4.2971,88 ±7.7830.97 ± 3.4512.48 ±0.82N.D.18.22 ±2.24

52.08 ±3.6634.97 ±4.166.08 ± 2.470.96 ± 0.58

N.D.N.D.

' Mean ascorbic acid and ascorbate-2-sulphate concentrations ( ^ g"' of tissue wet

weighit or /^ ml ' of plasma) and SE (n > 4).* Means significantly different (P < 0.05) from controls.' N.D. = not determined.

Table 5 Ascorbic acid and ascorbate-2-sulphate fluctuations in tissues of gilthead bream when subjected to shallow water

and handling stresses

Shallow water stress

Control Stress

Acute handling stress

Control Stress

Ascorbic acid'LiverKidneySpleenRed musclePlasmaAscorbate-2-sulphate'LiverKidneySpleenRed musclePlasma

89.44 ± 0.8258.04 ± 5.9469.05 ± 6.839.1610.72

21.04±1.14

40.04 ± 6.4840.95112.869.70 ± 3.231.72 ±0.89

N.D.'

70.71 ± 4.79*"33.01 ± 4.46"41.31 ±5.05*12.71 ±1.7520.21 ±1.78

25.32 ± 3.510.92 ± 0.92"

10.46 ±3.032.33 ±1.04

N.D.

77.04 ± 6.2538.32 ± 4.8549.34 ± 12.4615.76± 1.4619.49 ±2.94

29.86 ± 4.0510.24 ±4.0911.95 ±3.010.63 ± 0.63

N.D.

76.93 ±3.5244.75 ± 6.30

100.72111.4917.7210.8722.33 ± 3.07

23.001 5.343.5811.78

54.041 5.52*0.3710.37

N.D.

" Mean ascorbic acid and ascorbate-2-sulphate concentrations (fig r ' of tissue wet weigbtor /jg ml"' of plasma) and SE (/i > 4).

* Means sigaificantly different (P < 0.05) from controls.

•* Means significantly different (P < 0.01) from controls,

< N.D. = not determined.

the normal kidney vitamin C content, close regulation

of plasma osmolality and electrolyte balance indicate

a rapid response to salinity change in gilthead sea-

bream. Consequently, it is not unreasonable to propose

that, because of the longer period required for glycogen

repletion, the low hepatic glycogen content was the

only measurable indicator of stress remaining at this

point. Other studies (Thomas 1984) suggest that each

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Giltbead seabream and stress M M F Henrique et al. Aquaculture Researcb, 1996, 27, 405-412

species responds over widely varying time periodsto similar stressors, making the direct comparison ofresults and their interpretation difficult.

After a 3-h period, seabream exposed to shallowwater exhibited significant decreases in liver andmuscle glycogen content, declining liver, kidney andspleen ascorbic acid levels, and a fall in the con-centration of ascorhate-2-sulphate in the kidney. Thisimplies the need to utilize ascorbate-2-sulphate(vitamin C2), which was considered hy Halver et al(1975) to be a major storage form of vitamin Cin mostfish tissues, Thomas (1984) after restraining mullet,Mugil cephalus L.Ana dipnet for 30 min, also detecteda decrease in kidney ascorbic acid content one day afterthe experiment. In 1969, Wedemeyer demonstratedthe depletion of the interrenal ascorbic acid contentof coho salmon, Oncorhynchus kisutch (Walbaum),subjected to shallow water stress for 15 min. Thus,depletion of tissue ascorbic acid consistently appearsto he a response to this particular form of stress.

In the present investigation, a slight increase inplasma glucose was observed in response to stressinduced by shallow water. In 1991, Thomas &Robertson found no plasma glucose increase in reddrum, Sciaenops ocellatus L., 15 min or one hour aftershallow water stress. Since a similar type of stresshad been shown to induce a response in salmonids(Donaldson 1981), Thomas & Robertson (1991)suggested that habituation had occurred amongst theirexperimental fish because of the use of shallow waterduring the routine maintenance of the culture facilities.The same argument is not valid for the present study,since the experimental animals had not been regularlysubjected to this stressor prior to the experiment.

In a study carried out hy Carmichael, Tomasso,Simco & Davis (1984), largemouth bass, Micropterussalmoides (Lacepede), subjected to 2 days of confine-ment in a net (resulting in decreased swimming space)exhibited increased plasma glucose concentration andrequired up to 14 days to recover normal plasmaconcentrations. A similar response was not observedin the present experiment, which could be explainedby a rapid recovery of the plasma glucose levels inrelation to all the other parameters, or an absence ofinduction of a significant hyperglycaemic response.In terms of tissue ascorbic acid content, the resultsobtained in the present experiment are supported byWedemeyer's findings (1969), i.e. shallow water stressinduces ascorbic acid depletion in certain tissues, suchas the kidney.

The fmal experiment within this investigationconcerned the effects of an acute stressor on the

physiology of the gilthead seabream. The applicationof handling stress by exposure of the individuals toair for one minute, as expected, induced a significantincrease in the plasma glucose, and hence, corrobo-rated the observations of other authors (Mazeaudet al 1977; Wedemeyer & Yasutake 1977; Thomas,Woodin & Neff 1980; Wedemeyer & McLeay 1981,Morata, Faus, Perez-Palomo & Sanchez-Medina 1982;Halim et al 1987; Barton & Iwama 1991; Thomas &Robertson 1991; Hopkins SCech 1992).

In response to acute stress, no significant changeswere recorded in the liver and red muscle glycogencontent in the present experiment (except for a smalldecrease in the muscle glycogen), despite the fact thatthe fish struggled vigorously during their exposure toair. Working with the rainbow trout, Oncorhynchusmykiss (Walbaum), Morata et al. (1982) obtainedresults not only contrary to those of the present experi-ment, but also those of the remaining experimentswithin the current study. Thus, according to Morataetal (1982). glycogenoly sis takes place under stressfulconditions in the liver during the early stages of stressand the degradation ceases after 30 min of stress.Moreover, the same authors found that glycogenrepletion had hegun after 30 min. Conversely, Vijayan& Leatherland (1989) observed that in coho salmonfitted with slow release cortisol implants (simulatingchronic stress), liver glycogen levels were only signifi-cantly decreased after 30 days of constant exposure tohigh levels of cortisol. Similar results were obtainedby Soengas, Rey, Rozas, Andres & Aldegunde (1992)in rainbow trout, suggesting that acute stress (shortterm) did not induce a mobilization of the liverglycogen, at least immediately, therefore preventing thedetection of significant changes within the presentexperiment.

With regard to the tissue vitamin C content of thefish exposed to acute stress, no significant changeswere recorded with the exception of the spleen. Bothsplenic ascorbic acid and ascorbate-2-sulphateconcentrations exhibited a marked increase amongstthe stressed fish by comparison to the controls.According to Bender (1992), leucocytes possess amarked ability to concentrate ascorbate in humans,and even though no confirmation of the same featurehas been determined in fish, it is not unreasonable toassume that the same process may occur in fish.Additionally, in teleosts, it is known that lymphocytestake up residence in the spleen, among other organs,for a period of time (Ellis & de Sousa 1974), and thatstress results in lymphocytopenia (Ellis 1981). It isalso known that lymphocytopenia results from a

410 © 1996 Blackwell Science Ltd,/lijuacuKure Research. 27, 405-412

Aquaculture Research, 1996. 27, 405-412Gilthead seabream and stress MMF Henrique et ai.

sequestration of circulating T lymphocytes into bone

marrow and lymph nodes in mammals (Pearson

Clements & Yu 19 78), If a similar mechanism exists infish, it is possible that lymphocytes are sequestered intothe spleen, and since these cells possess a high contentof vitamin C, the ascorbate content of this organ maybe elevated.

In conclusion, the present study and the analysis of

several published works in the same field, emphasizes

the need for longer and broader evaluations of

physiological stress responses in fish. It was clear that

the intensity of change of a given parameter varies

with the species studied, the stressor applied to the

individuals, the time of exposure to the stressor and

the capacity of the species to adjust to that particular

stressor (e.g. a euryhafine species has a faster adjust-

ment to salinity changes). Thus, a study of several

parameters should be considered with measurements

taken on a temporal basis, in order to characterize the

stress induced physiological changes within each

species. Also, a knowledge of the variations of the

normal tissue levels of ascorbic acid due to sex, age and

season, and dietary status is essential to allow an

accurate interpretation of the results obtained (Sato

et al. 1978; Agrawal & Mahajan 1980; Thomas et al.

1985: Dabrowski, Lackner & Doblander 1990).

Hence, standard experimental designs and analytical

procedures should be clarified in order to allow the

direct comparison and analysis of the physiology of

stress in fish. Finally, the use of different methods

to measure the same parameter by different authors

(particularly evident in vitamin C content determi-

nation) will continue to make comparisons of results

tenuous.

Acknowledgments

M.M.E Henrique was supported by a grant donated by

the Junta Nacional de Investiga^ao Cientifica e

Technologica QNICT), Portugal.

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