Influence of egg vitamin A status and egg incubation temperature on subsequent development of the...

Influence of egg vitamin A status and egg incubation

temperature on subsequent development of the early

vertebral column in Atlantic salmon fry

R. ØRNSRUD*†, A. WARGELIUS‡, Ø. SÆLE§, K. PITTMAN§ AND

R. WAAGBØ**National Institute of Nutrition and Seafood Research, Strandgt. 229, P. O. Box 176Sentrum, N-5804 Bergen, Norway, ‡Institute of Marine Research, P. O. Box 1870Nordnes, 5817 Bergen, Norway and §Department of Fisheries and Marine Biology,

University of Bergen, P. O. Box 7800, 5020 Bergen, Norway

(Received 1 April 2003, Accepted 27 October 2003)

The effect of egg vitamin A (VA) status and egg incubation temperature on the development of

spinal disorders was investigated in Atlantic salmon Salmo salar fry. Atlantic salmon eggs were

sorted into two groups with high VA (3�3� 0�1mg retinol g�1 dry mass) and low VA (2�2� 0�3mgretinol g�1 dry mass) status before fertilization and incubated at high (14� C) or low (8� C)temperature from 133 day degrees until the onset of feeding. High egg incubation temperatures

increased the concentration of retinol in the eggs: the high VA and high temperature group

displayed a significantly higher retinol concentration than the high VA and low temperature

group (P¼ 0�001). After hatching, all experimental groups increased their retinol concentration.

The source of the increased retinol levels was probably retinal, although astaxanthin may also be

a VA precursor after hatching. Atlantic salmon fry incubated at high temperatures had increased

amounts of notochord tissue. When measuring morphogenic activity in the notochord using the

expression of sonic hedgehog (shh, mRNA), however, no significant difference was found

between the experimental groups. No clear effect of VA status or incubation temperature could

be found on the formation of the early vertebral column although Atlantic salmon fry incubated

at low temperatures had less regular constrictions of the prospective vertebral column than fry

incubated at high temperatures. # 2004 The Fisheries Society of the British Isles

Key words: Atlantic salmon fry; egg incubation temperature; spinal deformity; vitamin A.

INTRODUCTION

The Atlantic salmon Salmo salar L. farming industry has experienced a range offish deformities in recent years. The most significant deformity economically isthat of the ‘short-tailed’ Atlantic salmon, which shows coalesced, and thusshortened, vertebra (Kvellestad et al., 2000). In addition to these spinal deform-ities, other types of bone deformities in notably the gills, fins and jaws havebeen observed and give rise to ethical concerns on animal welfare issues. It hasbeen suggested that high temperature in the egg incubation period is a major

†Author to whom correspondence should be addressed. Tel.: þ47 55 905153; fax: þ47 55 905299;

email: [email protected]

Journal of Fish Biology (2004) 64, 399–417

doi:10.1046/j.1095-8649.2003.00304.x,availableonlineathttp://www.blackwell-synergy.com

399# 2004TheFisheries Society of theBritish Isles

risk factor (Bæverfjord, 1998), but the causal relation has not been elucidated.One possible cause to deformity in fishes may be an imbalance in the metab-olism of vitamin A. Vitamin A (VA) is a generic term comprising all compoundsthat show vitamin A activity, including provitamin A carotenoids such asastaxanthin (Ross, 1999). In addition to being necessary for vision, VA isessential as a key factor in embryonic development through the regulation ofcell differentiation and proliferation (Maden, 1993, 1994). VA exerts its effectthrough retinoic acid, which activates the retinoic acid receptor (RAR) andretinoid X receptor (RXR). The RAR and RXR form a heterodimer that isthe functional unit in transducing the retinoid signal at the gene level (Ross,1999).Many studies have shown that an embryonic VA imbalance, both deficiency

and excess, may have teratogenic effects, i.e. it causes abnormal development ormiscarriage (Nau et al., 1994). In rats, VA deficiency before and during gesta-tion lead to increased mortality and a range of deformities in the live offspring,such as malformation of the eye, urogenital system, respiratory tract and theheart (Maden, 1994). Embryonic VA excess causes a wide spectrum of mal-formations in all species examined, e.g. malformation of craniofacial structures,the central nervous system, vertebra and limbs (Maden, 1994). In fishes,embryological deformities have been induced in the brain, jaw, gills, fins andtail after exposure to different retinoids in the embryonic stage (Herrmann,1995; Ellies et al., 1997; Vandersea et al., 1998; Suzuki et al., 1999, 2000). Inaddition to malformations at the embryonic stage, VA may also induce mal-formations in later life stages, especially in the vertebra. Vitamin A is consideredto be a very potent skeletal toxin (Binkley & Krueger, 2000) and studiesperformed on fishes have shown that feeding with excess amounts of VA canbring about bone deformities (Primbs et al., 1971; Hilton, 1983; Dediet al., 1995, 1997; Estevez & Kanazawa, 1995; Saleh et al., 1995; Takeuchi et al.,1995, 1998, Haga et al., 2002) including changes in the vertebra of Atlanticsalmon (Ørnsrud et al., 2002).Egg nutrient status is affected by broodstock nutrition. In a study performed

on Japanese flounder Paralichthys olivaceus (Temminck & Schlegel) it was shownthat fish egg VA status was influenced by brood fish VA intake (Furuita et al.,2001). It has been suggested that eggs from salmonid species can have anadditional passive accumulation of VA through lipid bulk transport (Irie &Seki, 2002) since such eggs accumulate more lipids than marine teleost eggs.Further, salmonid eggs also contain high concentrations of provitamin Acarotenoids, primarily astaxanthin (Craik & Harvey, 1986; Christiansen &Torrissen, 1997). It has been suggested that temperature is an important factorfor the conversion of carotenoids into retinoids (Poston et al., 1977; Guillouet al., 1989).The concentration of VA in Atlantic salmon broodstock feed depends upon

which fish species is used as raw material for the formulated feed and may reachlevels as high as 80mg VAkg�1 feed while the dietary requirement has been setto 0�75mg VAkg�1 feed (NRC, 1993). The concentration of astaxanthin inAtlantic salmon feeds is strictly controlled through feed regulations and istypically c. 60mg astaxanthin kg�1 feed. The potentially high levels of VA andprovitamin A carotenoid levels in broodstock nutrition may therefore make

400 R. ØRNSRUD ET AL .

# 2004TheFisheries Society of theBritish Isles, Journal of FishBiology 2004, 64, 399–417

salmonid eggs more prone to excess VA. The purpose of this study was toinvestigate whether a high VA and provitamin A carotenoid status in combin-ation with an elevated egg incubation temperature has any effect on the form-ation of the early vertebral column in Atlantic salmon.

MATERIALS AND METHODS

EXPERIMENTAL DESIGN

Egg samples were collected at a commercial production site (Sandvoll, Hardanger,Norway). These were obtained from 60 brood fish, immediately frozen on dry ice andkept frozen at �80� C until analysed for VA and astaxanthin. The remaining egg batcheswere transported to a nearby aquaculture station for fertilization and incubation. The 60egg samples were screened for VA and astaxanthin concentration and sorted intotwo VA status groups with high (high VA) and low (low VA) VA and astaxanthin levels.The High VA group consisted of egg batches from 10 brood fish and the low VA groupconsisted of egg batches from five brood fish. There were no differences in brood fishmass, egg production volume, gonado-somatic index or egg vitamins D, K1 and K2, orfolate concentration in the different egg batches at the start of the trial (unpubl. data)indicating that the differences in egg VA status were not caused by differences in feedintake by the brood fish. At 133 day degrees (d�) after fertilization and incubation at8� C, each of the 15 selected egg batches were separated in two groups, kept in separatecompartments in egg incubation trays and subjected to a continuous flow of heated waterat 14 or 8� C (normal egg incubation temperature). The incubation temperature regimeswere maintained until the start of feeding, except for the low temperature group thatexperienced a temperature drop to c. 4� C for 10 days (from 365 to 408 d�) due to logisticproblems at the hatchery. There were thus four experimental groups; high VA and lowtemperature, high VA and high temperature, low VA and low temperature and low VAand high temperature.

SAMPLING

Eggs were sampled at 0 (after stripping), 133 and at 320 d�, and fry were sampled at509 and 502 d� (for the high and low temperature groups, respectively) and at 800 and838 d� (prior to the start of feeding). The fry were killed using an overdose of tricainemethane-sulphonate (MS-222).

CHEMICAL ANALYSES

Retinol (vitamin A1), 3, 4-didehydro retinol (vitamin A2) and retinal were extractedfrom Atlantic salmon eggs and fry as described in CEN (1999). Briefly, egg and frysamples were dissolved in an ethanolic KOH solution using pyrogallol, ascorbic acid andethylenediaminetetra acetic acid (EDTA) as antioxidants, and saponified for 20min at100� C. The samples were extracted three times with n-hexane, N2 evaporated andredissolved in n-hexane. Retinols were analysed using an HPLC system as described inØrnsrud et al. (2002). Retinals were analysed using a Hypersil H3 column (Hichrom,England, 150mm, 4�6mm i.d., 3mm particle size) and a heptane : tert-butyl methyl ether(92 : 8) solution at 1�5mlmin�1 as the mobile phase. Detection wavelength was set at371 nm for retinal. The HPLC system used was the same as described for carotenoidanalyses. The quantification of retinal was performed using an external standardmethod where standard solutions of all-trans-retinal (Sigma-Aldrich, Oslo, Norway)were measured with a spectrophotometer (UV-260, Shimadzu, Japan) using a molarabsorptivity in hexane of E1 cm

1% ¼ 1690.Carotenoids were extracted from eggs and fry using a procedure modified from Bligh &

Dyer (1959) by Bjerkeng et al. (1997). Briefly, egg samples were dissolved in distilled

VITAMIN A IN ATLANTIC SALMON EGGS AND FRY 401


water, methanol containing 500mg l�1 BHT (2,6-di-t-butyl-p-cresol) and chloroform.The sample was mixed vigorously and stored in the dark for 45min. After remixing,the sample was centrifuged for 15min at 2000 g and 18� C in a cooling centrifuge (JouanCR312, St Mazaire, France). An aliquot of the carotenoid-containing hypophase wasN2-evaporated at 35� C and the dry sample redissolved in n-hexane. The solution wasfiltered through a 0�45mm syringe filter into a HPLC vial and immediately analysed byHPLC. All-trans and 9&13-cis astaxanthin was analysed using a Spherisorb 5 CN nitrilecolumn (Chrompack, The Netherlands; 250mm, 4�6mm internal diameter, i.d., 5mmparticle size), and a heptane : acetone :methanol (80 : 19 : 1) solution at 1�5mlmin�1 as amobile phase. The detection wavelengthwas set at 470nm. The quantification of astaxanthinwas performed using an external standard method where standard solutions prepared fromcrystalline all-trans-astaxanthin (Hoffman LaRoche, Basle, Switzerland) were measuredwith a spectrophotometer (UV-260, Shimadzu, Japan) using a molar absorptivity inhexane of E1 cm

1% ¼ 2100 at lmax¼ 472 nm for astaxanthin.Carotenoids were analysed using a YMC30 carotenoid column (YMC Europe, Tekno-

lab A/S, Drøbak, Norway, 250mm, 4�6mm i.d., 3 mm particle size), and a methanol : tert-butyl methyl ether : water mobile phase gradient (81 : 15 : 4–39 : 57 : 4, run time 50min,equilibration time 5min) at 1�0mlmin�1. Detection wavelength was set at 450 nm.Carotenoids were identified using retention times and spectres of authentic standardsof astaxanthin, lutein, zeaxanthin (Roche, Oslo, Norway) and echinenone (CarotenNature,Lupsingen, Switzerland). The HPLC system consisted of a P4000 TSP pump (ThermoSeparation Products, Instrumentteknikk, Oslo, Norway), an AS3000 TSP autoinjector,an UV1000 TSP detector and an UV6000 TSP photodiode array detector. All sampleswere integrated using the Chromquest software (ver. 2.52, Thermoquest CE Instruments,Milan Italy).

SONIC HEDGEHOG (SHH) EXPRESSION

Atlantic salmon eggs sampled at 320 d� were immediately frozen on dry ice and storedat �80� C until analysed. Total RNA was extracted using Trizol (Invitrogen, Oslo,Norway) and treated with DNAse (Promega, Oslo, Norway). First strand cDNA wasreverse transcribed using Oligo-dT primers (Roche, Oslo, Norway) and MMLV-reversetranscriptase (Promega, Oslo, Norway). The PCR primers used for amplification of shh(accession number AY370830) and ß-actin (accession number AF012125) were purchasedfrom MWG Biotech, Ebersberg, Germany. Real-time PCR was carried out on eachcDNA using the Applied Biosystems 7700 system. Each reaction (25ml) contained 5 mlof cDNA diluted 1 : 1 in ddH20, 12�5ml of Cyber-green PCR master mix (ABI, Oslo,Norway) and 0�9mMF/R primers. The quantification of the target gene was done using astandard-curve for ß-actin compared to target gene curve using triplicate samples andseven cDNA dilutions. No template controls were run together with the samples in anyexperiment.

HISTOLOGY

Atlantic salmon larvae were sampled for histology after hatching (at 509 and 502 d�)and prior to the start of feeding (at 800 and 838 d� for the high and low temperaturegroups, respectively). The samples were fixed in 4% phosphate buffered formalin andstored for c. 6 months. All sections were evaluated prior to identification within atreatment group.

For the sampling at 509 and 502 d�, fry from three selected batches in the high and lowVA groups at both temperature treatments were measured for standard length, LS. Theyolk sac was removed prior to embedding in historesin. Each block of embedded larvawas divided into slices of 1–2mm thickness giving 6–10 slices per fish [Fig. 1(a), step 1].The size and profile of the notochord varies from anterior to posterior, as does the tissuedensity within the notochord. To get a truly representative average volume and densitymeasure of the notochord in each individual, a stereological technique for generating



isotropic uniform random (IUR) sections (Howard & Reed, 1998) was modified (Fig. 1)so that all possible planes of orientation within a block had a chance of being sampled.Notochord area of each cross-section of each individual was measured by randomly

distributing a uniform point grid (Howard & Reed, 1998) over the two-dimensionalobject (Zeiss Axiophot microscope and software calibrated with CAST 2, Olympus,Denmark) to give an initial measure of the surface area of the cross-sections of thenotochord. The distance between any array of points in the grid is known and the area(a� b) is multiplied by the number of points P hitting the object (Pab). The smallest andlargest surface area measurements of the notochord cross-sections in each fish werediscarded, the average surface area was calculated for each fish and multiplied by LS togive an estimate of notochord volume.Notochord tissue density measurements were performed using IUR sections (modified

from Howard & Reed, 1998) and subsequent Cavaleiri measurements (point grid) within

AVentral

Dorsal

1 2

P

PA

x

4

3

43

21

0

98

7654

32

1

0

9

87

6 5

(a)

(b)

FIG. 1. (a) Generation of modified isotropic uniform random (IUR) sections of notochord fractions. 1,

Sawing the embedded larvae in sections and measuring the notochord cross-section area; 2, placing

the sections along the 0-axis and finding the cutting axis; 3, cutting the section parallel to the

cutting axis in the constant distance x; 4, embedding the sections anterior–posterior in a new block.

Grey represents larval tissue. A, anterior; P, Posterior. All diagrams top view. (b) IUR sections

of the notochord of one Atlantic salmon larva for measurement of average notochord tissue

density.

V ITAMIN A IN ATLANTIC SALMON EGGS AND FRY 403


a counting frame of known area. The details are as follows: each cross-section was placedanterior side down on the 0 axis of a uniformly divided clock [Fig. 1(a) step 2]. The firstsection was then cut along the axis indicated by a uniform random number between 0 and9, generated from a random number table [Fig. 1(a), step 3]. The following section wascut along the axis corresponding to the original axisþ 3 [Fig. 1(a)]. The distance from thenotochord to the cutting axis must be constant within each fish. This generated a series ofsections with randomly determined presentations of the notochord. The series of cutsections from each fish was embedded in a new block, ordered anterior–posterior[Fig. 1(a), step 4]. Sections were affixed cut-side down to the bottom of the form withdouble-sided tape, and the form refilled with historesin. The block was then cut into 5 mmthick slices, displaying all portions of the notochord randomly rotated. The slices foreach fish were examined under a light microscope calibrated with stereological softwareand measured with a point grid inside a counting frame of known area. The tissue areawithin 22 144 mm2 in each section of the notochord was measured as the number of pointsoverlapping notochord tissue within the counting frame. The average tissue area perreference area in each individual was calculated for average tissue density. Notochordtissue amount was estimated as the product of the estimated notochord volume and themeasured notochord tissue density.

For the sampling at 800 and 838 d�, the Atlantic salmon larvae were measured, fixed in10% buffered formalin, the hard tissues decalcified by immersion in 100% Decalc(Histolab Products AS, Gøteborg, Sweden) for 10 h, rinsed in phosphate buffer threetimes, dehydrated in a series of ethanol and xylene, embedded in paraffin, and sliced inserial slices of 10mm on a Leica RM 2065 microtome. Two of the central serial sectionswere stained with haematoxylin and eosin and examined under a Zeiss Axioskop lightmicroscope. One hundred and eighty slides with two sections each were examined. Thesections were evaluated for regularity of cell structure within the notochord and forregularity of constriction of the developing vertebra (Fig. 2). Structures considered nor-mal or regular were assigned the value 0 while structures deviating from normal orregular were assigned the value 1.

STATISTICAL ANALYSES

All analyses were performed on Statistica 6.0 (StatSoft Inc. Tulsa, OK, U.S.A.).Differences in retinol, retinal and astaxanthin concentration between experimentalgroups were assessed using one-way ANOVA. Differences in histological measurements(LS, notochord volume, tissue density and total tissue amounts) between experimentalgroups were assessed using nested ANOVA with four fry from each egg batch nestedin their VA and temperature group. The Levene’s test was used to assess homogeneity ofvariance and Tukey’s unequal n HSD post hoc test was used to test for significantdifferences between group means in ANOVA. The Mann–Whitney U-test was used toassess differences in categorical data (regularity of constrictions and regularity ofnotochord cell structure). Differences and effects were considered significant atP< 0�05 for all tests.

RESULTS

EGG VITAMIN STATUS

The total retinol concentration (all-trans retinol; A1þ all-trans 3,4-didehy-droretinol; A2) in eggs at commencement of the trial ranged from 1�4 to4�2 mg g�1 dry mass (Fig. 3). The high VA group had significantly higher(P¼ 0�004) total retinol concentration than the low VA group at the start ofthe trial. Both VA groups subjected to the low temperature treatment showed adecline in retinol concentration between fertilization and hatching. For the highVA and high temperature group, there was an increase in retinol concentration



from the second egg sampling and the sampling prior to hatching: at 320 d�; thehigh VA and high temperature group had significantly higher total retinol thanthe high VA and low temperature group (P¼ 0�001). For the low VA and hightemperature group there was a less pronounced decrease in retinol concentra-tion than the low VA and low temperature group between the second eggsampling and the sampling prior to hatching. After hatching, there was asignificant increase in retinol concentration for all VA groups (P¼ 0�0001).The retinal concentration (all-trans retinal) in eggs at the start of the trial

ranged from 0�6 to 1�1 mg g�1 dry mass and there were no significant differencesin retinal concentration between the low and high VA groups (Fig. 4). Theretinal concentration declined for all groups until hatching and after hatchingthere was a significant decline in retinal concentration for all VA groups(P< 0�03) except the low VA and low temperature group (P¼ 0�09).The total astaxanthin concentration (sum of all-trans, 9- and 13-cis) in eggs at

the trial start ranged from 22 to 49mg g�1 dry mass (Fig. 5) and the high VAgroup was significantly higher than the low VA group at the start of the trial(P¼ 0�004) and at the second egg sampling (P¼ 0�0006). All groups showed adecline in astaxanthin concentration over time. After hatching there was a

nc

nc

ncnc

ys

ys

(a) (b)

(c) (d)

(c)(d)

FIG. 2. Histological sagittal sections of notochord from Atlantic salmon fry incubated at low and high

temperatures. (a) Regular constrictions of the developing vertebrae. (b) Irregular constrictions of

the developing vertebrae. (c) Regular notochord cell structure with cells of comparable size and

shape. (d) Irregular notochord cell structure with varying size and shape of notochord cells. (a) and

(b): ·4 magnification, bars indicate 1mm (c) and (d): ·20 magnification, bars indicate 0�1mm. nc,

notochord; ys, yolk sac.



significant decline in astaxanthin concentration for all VA groups (P¼ 0�0002)except the low VA and low temperature group (P¼ 0�16).Astaxanthin was the main carotenoid species in the Atlantic salmon eggs, but

traces of lutein, zeaxanthin and echinenone were also found (Fig. 6). At the lastsampling point a range of unidentified metabolites appeared in the chromato-gram (Fig. 6).

SHH EXPRESSION

The relative sonic hedgehog (shh) expression in eggs of the VA groupssubjected to high and low temperature treatments at 320 d� is shown in Fig. 7.Relative shh expression in Atlantic salmon eggs was in the range from 1�0 to 3�9units and there were no significant differences between temperature treatmentand VA status.

HISTOLOGY

There were no differences in LS between the groups at 502 and 509 d�

(Table I). The low and high VA groups incubated at high temperatures had

Day degrees

0 100 200 300 400 500 600 700 800 9001

2

3

4

5

6

7

Egg stage

Hat

chin

gFry stage

All-

tran

s re

tin

ol (

A1

+ A

2) in

egg

s an

d fr

y (µ

g g–

1 dr

y m

ass)

FIG. 3. Mean� S.E. retinol (sum of all-trans retinol and all-trans 3,4 didehydroretinol) in eggs and fry of

Atlantic salmon of high vitamin A and low temperature (*), high vitamin A and high temperature

(&), low vitamin A and low temperature ( &) and low vitamin A and high temperature (n) ( n¼ 10

for the high and n¼ 5 for the low VA groups). Since the data are reported as mg g�1 the retinol

concentration apparently increases after shedding of the eggshell.



significantly higher notochord volume than the high VA and low temperaturegroup (P¼ 0�001 and P¼ 0�02, respectively), but not higher than the low VAand low temperature group. The high VA and high temperature group hadsignificantly higher notochord tissue density than the low VA and low tempera-ture group (P¼ 0�03). The low and high VA groups incubated at high tempera-tures had significantly larger notochord tissue amounts than the high VA andlow temperature group (P¼ 0�02 and P¼ 0�03, respectively), but not higher thanthe low VA and low temperature group (P¼ 0�10 and P¼ 0�06, respectively).The data for regularity of constrictions and regularity of notochord cell

structure are reported as the ratio between fry with abnormal development tothe total number of fry examined (Table II). No differences in LS or regularityof notochord cell structure were found between the groups at 800 and 838 d�.The high VA and high temperature group had significantly higher values forregularity of constrictions than the low VA and low temperature group and thehigh VA and low temperature group (P¼ 0�01 and P¼ 0�002, respectively).

Day degrees

0 100 200 300 400 500 600 700 800 9000·4

0·6

0·8

1·0

1·2

1·4

1·6

1·8

2·0

2·2

2·4

Egg stage

Hat

chin

g

Fry stage

All-

tran

s re

tin

al e

ggs

and

fry

(µg

g–1

dry

mas

s)

FIG. 4. Mean� S.E. all-trans retinal in eggs and fry of Atlantic salmon of high vitamin A and low

temperature (*), high vitamin A and high temperature (&), low vitamin A and low temperature

( &) and low vitamin A and high temperature (n), (n¼ 10 for the high and n¼ 5 for the low VA

groups). Since the data are reported as mg g�1 the retinal concentration apparently increases after

shedding of the eggshell.



DISCUSSION

Vitamin A is essential for animals during embryonic development (Ross et al.,2000). Vitamin A as retinoic acid, is for example actively involved in the formationof the anterior–posterior axis and vertebra (Conlon, 1995), and is thus vital forcorrect development of certain key structures. In some cases, however, a com-pound that is usually involved in normal development can have teratogeniceffects if it is present in large amounts at specific times during development. Forthe vascularized embryo of mammals, teratogenetic effects of VA on the embryocan be caused by excessive maternal VA intakes. Fish eggs, however, do not receiveexogenous substances during embryogenesis. An increased formation of retinolas an effect of elevated temperature was found during the egg incubationstage (Fig. 3) in the present study. In teleost eggs, the primary storage formof vitamin A is the aldehyde form, retinal (Rønnestad et al., 1998, Irie & Seki,2002). The reason that fishes use retinal as the preferential VA storage form ineggs instead of retinylesters, which are the most common storage forms in adultfishes and other vertebrates, has not been established. It is possible that thisfacilitates the incorporation of retinal into visual pigment chromophores sinceeye formation occurs before the liver is functional (Gorodilov, 1996) and thus

Day degrees

0 100 200 300 400 500 600 700 800 9005

10

15

20

25

30

35

40

45

Egg stage

Hat

chin

g

Fry stage

Ast

axan

thin

(su

m o

f al

l-tr

ans

and

9&13

-cis

)in

egg

s an

d fr

y (µ

g g–

1 dr

y m

ass)

FIG. 5. Mean� S.E. astaxanthin (sum of all-trans and 9&13-cis astaxanthin) in eggs and fry of Atlantic

salmon of high vitamin A and low temperature (*), high vitamin A and high temperature (&), low

vitamin A and low temperature ( &) and low vitamin A and high temperature (n), ( n¼ 10 for the

high and n¼ 5 for the low VA groups). Since the data are reported as mg g�1 the astaxanthin

concentration apparently increases after shedding of the eggshell.



reduces the necessity of the liver as a site for metabolic transformation of retinolinto retinal. The decrease in retinal in the egg and fry stage (Fig. 4) indicatedthat retinal may have been the source of the increased retinol levels, as was alsosuggested by Rønnestad et al. (1998). The changes in retinal levels, however, didnot correlate stoichiomoetrically with the observed retinol increase. Further-more, the retinal levels in the present study ranged from c. 100 to 120ng egg�1

for unfertilized eggs (unpubl. data) whereas Irie & Seki (2002) showed a retinalcontent of >700ng egg�1 in unfertilized chum salmon Oncorhynchus keta(Walbaum) eggs. These discrepancies in retinal may be explained by differencesin species and habitat, but are most likely due to differences in extractionmethods since the retinol levels were comparable between the two species(T. Irie, pers. comm.).Several pathways for provitamin A conversion of carotenoids have been

suggested in fishes (Gross & Budowski, 1966; Hata et al., 1973; Barua &Goswami, 1977; Del Tito, 1983; Schiedt et al., 1985, 1986; Davies & Davies,1986; Al-Khalifa & Simpson, 1988; Katsuyama & Matsuno; 1988, Guillou et al.,1989, 1992; Matsuno, 1991; Yamashita et al., 1996). The understanding ofcarotenoid provitamin A metabolism in freshwater fishes is complicated bythe presence of two forms of VA, VA1 (e.g. dehydroretinol) and VA2 (e.g.3,4 didehydroretinol). The VA2 may come from oxidative conversion of VA1

(Lambertsen & Brækkan, 1969; Schiedt et al., 1985), or by direct conversionfrom astaxanthin (Katsuyama & Matsuno, 1988; Guillou et al., 1989). Theretinol increase for the high temperature group in the egg and fry stage was

IV

III

II

I

1 24 5

6

3

FIG. 6. HPLC profiles of carotenoid metabolites in eggs and fry of Atlantic salmon from one selected egg

batch. I, Carotenoid composition in eggs before fertilization; II, carotenoid composition in eggs

incubated at 14� C for 320 day degrees; III, carotenoid composition in fry incubated at 14� C for

509 day degrees; IV, carotenoid composition in fry incubated at 14� C for 800 day degrees. 1, 2,

unknown; 3, astaxanthin; 4, lutein; 5, zeaxanthin; 6, echinenone; �, new carotenoid metabolite.



primarily caused by an increase in the VA2 concentration (unpubl. data),implying that the source of the increased retinol levels might be astaxanthin.In the present study, however, there was a general tendency towards a higherastaxanthin concentration in the high temperature groups in the egg incubationphase indicating that astaxanthin was not the source of the increased VA statusseen in the high VA and high temperature group. Furthermore, Fig. 6 showsthat except for the decrease in astaxanthin levels, the carotenoid compositionchanged very little until the last sampling where a range of carotenoid metab-olites appeared in the chromatogram (Fig. 6, IV). This implies that there was littleor no metabolism of carotenoids until the liver became functional, probablysome time between the two last samplings in the fry stage. Thus, carotenoidsare probably not significant contributors to VA status during the egg stage, butmay contribute after hatching. The direct metabolic transformation of astax-anthin into VA, however, cannot be confirmed since radio-labelled egg astax-anthin was not used in the present study. Thus, the retinol increase observed inthe egg stage was probably caused by an increased conversion from retinal toretinol. In the fry stage, both retinal and astaxanthin may be potential precur-sors for the observed increase in retinol.

0

1

2

3

4

5

Rel

ativ

e sh

h e

xpre

ssio

n

FIG. 7. Meanþ S.E. expression of sonic hedgehog (shh) in Atlantic salmon eggs at 320 day degrees

for high vitamin A and low temperature (&; n¼ 3, loss of sample), high vitamin A and high

temperature ( ; n¼ 1, loss of sample), low vitamin A and low temperature ( ; n¼ 4) and low

vitamin A and high temperature ( ; n¼ 2, loss of sample).



TABLEI.

Mean�

S.E.standard

length,notochord

volume,

notochord

tissuedensity

andtotalnotochord

tissueamounts

inAtlanticsalm

on

larvaewithhighandlowvitamin

A(V

A)statusrearedathighorlowtemperaturesandsampledat509and502daydegrees

forthehighand

low

eggincubationtemperature

groups,

respectively.Notochord

volumewascalculatedbyaverageareaofIU

Rslices

andLS,notochord

tissuedensity

wasmeasuredbystereology,andnotochord

tissueamounts

calculatedbydensity

andvolume

Group

LS(m

m)

Notochord

volume(m

m3)

Notochord

density

(tissue)

Notochord

tissueamounts

(mm

3)

HighVA

andlow

temperature

17� 0�0� 2

0� 94�0� 02b

38� 1�1� 1

ab

0� 36�0� 01b

HighVA

andhightemperature

17� 6�0� 4

1� 13�0� 05a

38� 9�2� 1

a0� 44�0� 04a

Low

VA

andlow

temperature

17� 4�0� 3

1� 11�0� 05ab

33� 3�1� 6

b0� 37�0� 02ab

Low

VA

andhightemperature

17� 4�0� 3

1� 18�0� 04a

36� 7�1� 0

ab

0� 43�0� 02a

Differentsuperscriptlettersdenote

significantdifferences(nestedANOVA,n¼3withfourfryin

each

replicate).



TABLEII.Mean�

S.E.standard

length,regularity

ofnotochord

cellstructure

andregularity

ofconstrictionsofAtlanticsalm

onlarvaewith

highandlow

vitamin

A(V

A)statusrearedathighorlow

temperaturesandsampledat800and838daydegrees

forthehighandlow

egg

incubationtemperature

groups,

respectively

Group

LS(m

m)

Regularity

ofnotochord

cellstructure

(%)

Regularity

ofconstrictions(%

)

HighVA

andlow

temperature

25� 9�0� 2

39�7

41�8a

HighVA

andhightemperature

25� 7�0� 2

38�7

82�6b

Low

VA

andlow

temperature

25� 7�0� 2

52�10

44�11a

Low

VA

andhightemperature

26� 0�0� 2

25�9

65�11ab

Differentsuperscriptlettersdenote

significantdifferences(M

ann–Whitney

U-test,n¼10andn¼5withsixfryin

each

replicate

forthehighVA

andlow

VA

groups,

respectively).

412 R . ØRNSRUD ET AL .


The significance of the observed retinol increase in the egg stage is difficultto establish. The retinol increase, however, may be interpreted as a disturbanceof VA homeostasis where more retinal is shuttled into reductive pathwaysaway from the potent gene activator retinoic acid. It has been shown thathuman keratinocytes treated with excess retinoic acid increased retinol ester-ifying activity eight-fold with a subsequent 50% reduction in retinol conver-sion to retinoic acid via retinal (Kurlandsky et al., 1996). The retinol increasemay therefore be a compensatory mechanism aimed at reducing the concen-tration of retinoic acid by reducing the conversion from retinal to retinoic acidin order to avoid unwanted gene expression. Minuscule amounts of VA arerequired to elicit a response on the gene level and under normal conditions;the cellular metabolism of VA is tightly regulated. The gene product of VAinducible genes is often a transcription factor itself that control the expressionof other sets of genes, which may in turn induce the expression of yet anotherset of genes and so on, resulting in a cascade of gene expression whichamplifies the original retinoid signal (Levin, 1995). One such VA inducible(Chang et al., 1997) signalling molecule is sonic hedgehog (Shh). Hedgehogproteins constitute one of the major classes of intercellular signals that controlinductive interactions during animal development, for example by regulatingcell differentiation and proliferation (Ingham & McMahon, 2001). Shh is the mostabundant of the hedgehog proteins and is initially produced by the notochord.Among its functions is induction of sclerotomal tissue in certain parts of thesomite which eventually form the cartilage of the spine and ribs (Fan &Tessier-Lavigne, 1994; Johnson et al., 1994). This protein has also beenshown to act in concert with VA in several processes necessary for correctdevelopment of certain structures, for example digits in the chick wing (Helmset al., 1994; Ogura et al., 1996), asymmetry across the left–right axis (Mercola& Levin, 2001) and growth of the zebrafish Danio rerio (Hamilton) fin budthrough activation of hoxd genes (Neumann et al., 1999). Spatial and temporalrestricted expression of shh, in addition to the absolute level of shh excretion,is essential for orderly patterning of embryonic tissues. In the present study itwas found that high egg incubation temperatures increased notochord volumeand tissue amounts (Table I). Enlargement of the notochord was also seenin the VA-treated regenerating tail of the tadpole, Polypedates maculatusGravenhorst (Das & Mohanty-Hejmadi, 1999) suggesting a direct effect of VAon notochord formation. It has been shown that impaired notochord formationresults in loss of shh expression (Ang & Rossant, 1994), it is thus possible thatthe increase in notochord tissue (Table I) in the high temperature groupsaffects the notochord’s ability to secrete morphogens such as shh. No cleareffect of temperature on shh expression, however, could be found in thepresent study (Fig. 7) although an increased expression of shh was indicated inthe high VA and high temperature group which also experienced a temperature-related increase in VA status. It is possible that shh expression was measuredprematurely since the effects on notochord formation were found c. 180 d�

after shh expression was measured. Further studies are required to concludeif the increased amounts of morphogen producing tissue found in thehigh temperature groups have any effects on the secretion of morphogenssuch as shh.



It is not clear if the observed differences in notochord volume, density andtissue amounts have any impact on the formation of the early vertebral column.Bearing in mind that the notochord plays an important role in the ossificationprocesses of the prospective vertebral column, both as a morphogen secretingtissue inducing sclerotome differentiation (Fan & Tessier-Lavigne, 1994) and insegmental patterning (Fleming et al., 2001), it is likely that the integrity of thenotochord at this early stage is important for normal vertebral development. Itwas not possible, however, to identify any markers of abnormal development inthe prospective vertebral column in the high temperature groups at the 800 and838 d� sampling (Table II). Further, both temperature groups exhibited a sub-stantial degree of what was characterized as deformities, even the group incu-bated under normal conditions. This may be partly explained by the difficultyof assigning a quantitative value to the subtle structural differences observed orpossibly as a result of the unwanted temperature fluctuations in the low tem-perature groups. Furthermore, the differences observed may also be within thenormal variation range. The lack of data describing a normal development ofbone structures in Atlantic salmon larvae complicates the search for abnormal-ities and clearly illustrates the need for further research in this area.

The authors wish to acknowledge R. Brokstad at Marine Harvest Norway for the useof rearing facilities, K. Watanabe, V. Fauskanger, I. Kallestad, M. Rygg and O. Breckfor technical assistance, and S. Davies and G. Ingunn Hemre for reading through themanuscript.

References

Al-Khalifa, A. S. & Simpson, K. L. (1988). Metabolism of astaxanthin in the rainbowtrout (Salmo gairdneri). Comparative Biochemistry and Physiology 91B, 563–568.

Ang, S. L. & Rossant, J. (1994). Hnf-3-beta is essential for node and notochord formationin mouse development. Cell 78, 561–574.

Bæverfjord, G. (1998). Feilutvikling og deformiteter hos laks As: Akvaforsk (in Norwegian).Barua, A. B. & Goswami, U. C. (1977). Formation of vitamin A in a freshwater

fish – isolation of retinoic acid. Journal of Biochemistry 164, 133–136.Binkley, N. & Krueger, D. (2000). Hypervitaminosis A and bone. Nutrition Reviews 58,

138–144.Bjerkeng, B., Følling, M., Lagocki, S., Storebakken, T., Olli, J. J. & Alsted, N. (1997).

Bioavailability of all-E-astaxanthin and Z-isomers of astaxanthin in rainbow trout(Oncorhynchus mykiss). Aquaculture 157, 63–82.

Bligh, E. G. & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification.Canadian Journal of Biochemistry and Physiology 37, 911–917.

CEN (1999). Foodstuffs – Determination of vitamin A by high performance liquidchromatography – Part 1: Measurement of all-trans retinol and 13-cis retinol.Comite Europeen de Normalisation prEN 12823–1.

Chang, B.-E., Blader, P., Fischer, N., Ingham, P. W. & Strahle, U. (1997). Axial (HNF3b)and retinoic acid receptors are regulators of the zebrafish sonic hedgehog promotor.The EMBO Journal 16, 3955–3964.

Christiansen, R. & Torrissen, O. J. (1997). Effects of dietary astaxanthin supplementationon fertilization and egg survival in Atlantic salmon (Salmo salar L). Aquaculture153, 51–62.

Conlon, R. A. (1995). Retinoic acid and pattern-formation in vertebrates. Trends inGenetics 11, 314–319.



Craik, J. C. A. & Harvey, S. M. (1986). The carotenoids of eggs of wild and farmedAtlantic salmon, and their changes during development to start of feeding. Journalof Fish Biology 29, 549–565.

Das, P. & Mohanty-Hejmadi, P. (1999). Histological effects of vitamin A on thetail-amputated tadpoles of Polypedates maculatus with special reference to homeotictransformation. Cells Tissues Organs 164, 90–101.

Davies, B. W. & Davies, B. H. (1986). Retinol and 3,4-dehydroretinol formation fromxanthophylls in the goldfish, Carassius auratus. Biochemical Society Transactions14, 952.

Dedi, J., Takeuchi, T., Seikai, T. &Watanabe, T. (1995). Hypervitaminosis and safe levelsof vitamin A for larval flounder (Paralichthys olivaceus) fed Artemia nauplii.Aquaculture 133, 135–146.

Dedi, J., Takeuchi, T., Seikai, T., Watanabe, T. & Hosoya, K. (1997). Hypervitaminosis Aduring vertebral morphogenesis in larval Japanese flounder. Fisheries Science 63,466–473.

Del Tito, B. J. (1983). Role of b-carotene and lutein in the synthesis of vitamin A ingoldfish. Progressive Fish-Culturist 45, 94–97.

Ellies, D. L., Langille, R. M., Martin, C. C., Akimenko, M. A. & Ekker, M. (1997).Specific craniofacial cartilage dysmorphogenesis coincides with a loss of dlx geneexpression in retinoic acid-treated zebrafish embryos. Mechanisms of Development61, 23–36.

Estevez, A. & Kanazawa, A. (1995). Effect of (n-3) PUFA and vitamin A Artemiaenrichment on pigmentation success of turbot, Scopthalmus maiximus (L).Aquaculture Nutrition 1, 159–168.

Fan, C.-M. & Tessier-Lavigne, M. (1994). Patterning of mammalian somites by thesurface ectoderm and the notochord: evidence for sclerotome induction by Sonichedgehog/Vhh-1. Cell 79, 1175–1186.

Fleming, A., Keynes, R. J. & Tannahill, D. (2001). The role of the notochord in vertebralcolumn formation. Journal of Anatomy 199, 177–180.

Furuita, H., Tanaka, H., Yamamoto, T., Shiraishi, M. & Takeuchi, T. (2001). Effects ofhigh dose of vitamin A on reproduction and egg quality of Japanese flounderParalichthys olivaceus. Fisheries Science 67, 606–613.

Gorodilov, Y. N. (1996). Description of the early ontogeny of the Atlantic salmon, Salmosalar, with a novel system of interval (state) identification. Environmental Biologyof Fishes 47, 109–127.

Gross, J. & Budowski, P. (1966). Conversion of carotenoids into vitamin A1 and A2 intwo species of freshwater fish. Journal of Biochemistry 101, 747–754.

Guillou, A., Choubert, G., Storebakken, T., de la Noue, J. & Kaushik, S. (1989).Bioconversion pathway of astaxanthin into retinol2 in mature rainbow trout(Salmo gairdnieri rich.). Comparative Biochemistry and Physiology 94B, 481–485.

Guillou, A., Choubert, G. & De La Noue, J. (1992). Absorption and blood clearance oflabelled carotenoids ([14C]astaxanthin, [3H]canthaxanthin and [3H]zeaxanthin inmature female rainbow trout (Oncorhynchus mykiss). Comparative Biochemistryand Physiology 103A, 301–306.

Haga, Y., Takeuchi, T. & Seikai, T. (2002). Influence of all-trans retinoic acid onpigmentation and skeletal formation in larval Japanese flounder. Fisheries Science68, 560–570.

Hata, M., Hata, M. & Onishi, T. (1973). Conversion of �-carotene and retinol1 to retinol2in freshwater fish. Tohoku Journal of Agricultural Research 24, 197–204.

Helms, J., Thaller, C. & Eichele, G. (1994). Relationship between retinoic acid andsonic hedgehog, two polarizing signals in the chick wing bud. Development 120,3267–3274.

Herrmann, K. (1995). Teratogenic effect of retinoic acid and related substances on theearly development of the Zebrafish (Brachydanio rerio) as assessed by a novelscoring system. Toxicology in Vitro 9, 267–283.

Hilton, J. W. (1983). Hypervitaminosis A in rainbow trout (Salmo gairdneri): Toxicitysigns and maximum tolerable level. Journal of Nutrition 113, 1737–1745.



Howard, C. V. & Reed, M. G. (1998). Unbiased Stereology. New York: Springer-Verlag.Ingham, P. W. & McMahon, A. P. (2001). Hedgehog signaling in animal development:

paradigms and principles. Genes and Development 15, 3059–3087.Irie, T. & Seki, T. (2002). Retinoid composition and retinal localization in the eggs of

teleost fishes. Comparative Biochemistry and Physiology 131, 209–219.Johnson, R. L., Laufer, E., Riddle, R. D. & Tabin, C. (1994). Ectopic expression of Sonic

hedgehog alters dorsal–ventral patterning of somites. Cell 79, 1165–1173.Katsuyama, M. & Matsuno, T. (1988). Carotenoid and vitamin A, and metabolism of

carotenoids, b-carotene, canthaxanthin, astaxanthin, zeaxanthin, lutein andtunaxanthin in tilapia Tilapia nilotica. Comparative Biochemistry and Physiology90B, 131–139.

Kurlandsky, S. B., Duell, E. A., Kang, S., Voorhees, J. J. & Fisher, G. J. (1996). Auto-regulation of retinoic acid biosynthesis through regulation of retinol esterificationin human keratinocytes. Journal of Biological Chemistry 271, 15 346–15 352.

Kvellestad, A., Høie, S., Thorud, K., Tørud, B. & Lyngøy, A. (2000). Platyspondylyand shortness of vertebral column in farmed Atlantic salmon Salmo salar inNorway – description and interpretation of pathologic changes. Diseases ofAquatic Organisms 39, 97–108.

Lambertsen, G. & Brækkan, O. R. (1969). In vivo conversion of vitamin A1 to vitamin A2.Acta Chemica Scandinavica 23, 1063–1064.

Levin, A. A. (1995). Receptors as tools for understanding the toxicity of retinoids.Toxicology Letters 82–3, 91–97.

Maden, M. (1993). The effect of vitamin A (retinoids) on pattern formation implies auniformity of developmental mechanisms throughout the animal kingdom. ActaBiotheoretica 41, 425–445.

Maden, M. (1994). Role of Retinoids in Embryonic Development. New York: MarcelDekker Inc.

Matsuno, T. (1991). Xanthophylls as precursors of retinoids. Pure and Applied Chemistry63, 81–88.

Mercola, M. & Levin, M. (2001). Left–right asymmetry determination in vertebrates.Annual Review of Cell and Developmental Biology 17, 779–805.

Nau, H., Chahoud, I., Dencker, L., Lammer, E. J. & Scott, W. J. (1994). Teratogenicity ofVitamin A and retinoids. In Vitamin A in Health and Disease (Blomhoff, R., ed.),pp. 615–665. New York: Marcel Dekker, Inc.

Neumann, C. J., Grandel, H., Gaffield, W., Schulte-Merker, S. & Nusslein-Volhard, C.(1999). Transient establishment of anteroposterior polarity in the zebrafish pectoralfin bud in the absence of sonic hedgehog activity. Development 126, 4817–4826.

NRC (1993). Nutrient Requirements of Fish. Washington, DC: National Academy Press.Ogura, T., Alvarez, I. S., Vogel, A., Rodrıguez, C., Evans, R. M. & Belmonte, J. C. I.

(1996). Evidence that Shh cooperates with a retinoic acid inducible co-factor toestablish ZPA-like activity. Development 122, 537–542.

Ørnsrud, R., Graff, I. E., Høie, S., Totland, G. K. & Hemre, G.-I. (2002). HypervitaminosisA in first-feeding fry of the Atlantic salmon (Salmo salar L.). Aquaculture Nutrition8, 7–13.

Poston, H. A., Riis, R. C., Rumsey, G. L. & Ketola, H. G. (1977). The effect ofsupplemental dietary amino acids, minerals and vitamins on salmonids fedcataractogenic diets. The Cornell Veterinarian 67, 472–509.

Primbs, E. R. J., Sinnhuber, R. O. & Warren, C. E. (1971). Hypervitaminosis A in therainbow trout: Counteraction by vitamin C. International Journal of Vitamin andNutrition Research 41, 331–338.

Rønnestad, I., Hemre, G.-I., Finn, R. N. & Lie, Ø. (1998). Alternate sources anddynamics of vitamin A and its incorporation into the eyes during the earlyendotrophic and exotrophic larval stages of Atlantic halibut (HippoglossusHippoglossus L.). Comparative Biochemistry and Physiology 119A, 787–793.

Ross, A. C. (1999). Vitamin A and retinoids. In Modern Nutrition in Health and Disease(Shils, M. D., Olson, J. A., Shike, M. & Ross, A. C., eds), pp. 305–327. Baltimore,MD: Williams & Wilkins.



Ross, S. A., McCaffery, J., Drager, U. C. & De Luca, L. M. (2000). Retinoids inembryonal development. Physiological Reviews 80, 1021–1053.

Saleh, G., Eleraky, W. & Gropp, J. M. (1995). A short note on the effects of vitamin Ahypervitaminosis and hypovitaminosis on health and growth of Tilapia nilotica(Oreochromis niloticus). Journal of Applied Ichthyology 11, 382–385.

Schiedt, K., Leuenberger, F. J., Vecchi, M. & Glinz, E. (1985). Absorption, retention andmetabolic transformation of carotenoids in rainbow trout, salmon and chicken.Pure & Applied Chemistry 57, 685–692.

Schiedt, K., Vecchi, M. & Glinz, E. (1986). Astaxanthin and its metabolites in wildrainbow trout (Salmo gairdneri R.). Comparative Biochemistry and Physiology83B, 9–12.

Suzuki, T., Oohara, I. & Kurokawa, T. (1999). Retinoic acid given at late embryonic stagedepresses sonic hedgehog and Hoxd-4 expression in the pharyngeal area andinduces skeletal malformation in flounder (Paralichthys olivaceus) embryos.Development Growth & Differentiation 41, 143–152.

Suzuki, T., Srivastava, A. S. & Kurokawa, T. (2000). Experimental induction of jaw, gilland pectoral fin malformations in Japanese flounder, Paralichthys olivaceus,larvae. Aquaculture 185, 175–187.

Takeuchi, T., Dedi, J., Ebisawa, C., Watanabe, T., Seikai, T., Hosoya, K. & Nakazoe, J.-I.(1995). The effect of b-carotene and vitamin A enriched Artemia Nauplii on themalformation and color abnormality of larval Japanese flounder. Fisheries Science61, 141–148.

Takeuchi, T., Dedi, J., Haga, Y., Seikai, T. & Watanabe, T. (1998). Effect of vitamin Acompounds on bone deformity in larval Japanese flounder (Paralichthys olivaceus).Aquaculture 169, 155–165.

Vandersea, M. W., McCarthy, R. A., Fleming, P. & Smith, D. (1998). Exogenous retinoicacid during gastrulation induces cartilaginous and other craniofacial defects inFundulus heteroclitus. Biological Bulletin 194, 281–296.

Yamashita, E., Arai, S. & Matsuno, T. (1996). Metabolism of xantophylls to vitamin Aand new apocarotenoids in liver and skin of black bass, Micropterus salmoides.Comparative Biochemistry and Physiology 113B, 485–489.



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