The International Journal of Biochemistry & Cell Biology 33 (2001) 1227–1238

Growth, protein-turnover rates and nucleic-acidconcentrations in the white muscle of rainbow trout during

development�

Juan Peragon a, Juan B. Barroso a, Leticia Garcıa-Salguero b,Manuel de la Higuera c, Jose A. Lupianez b,*

a Department of Experimental Biology, Area of Biochemistry and Molecular Biology, Centre for Experimental Sciences,Uni�ersity of Jaen, E23071 Jaen, Spain

b Department of Biochemistry and Molecular Biology, Centre for Biological Sciences, Uni�ersity of Granada,A�enida Fuentenue�a s/n, E18001 Granada, Spain

c Department of Animal Biology and Ecology, Centre for Biological Sciences, Uni�ersity of Granada, A�enida Fuentenue�a s/n,E18001 Granada, Spain

Received 10 October 2000

Abstract

We have studied the growth rate, nucleic-acid concentration, protein-accumulation rate (KG), and several otherparameters relating to protein turnover, such as the protein-synthesis (KS), and protein-degradation rates (KD),protein-synthesis capacity (CS), protein-synthesis efficiency (KRNA), protein-synthesis rate per DNA unit (KDNA) andprotein-retention efficiency (PRE), in the white muscle of rainbow trout during development. Both growth rate andrelative food intake decreased significantly with age and weight, as did the food-efficiency ratio (FER) andprotein-efficiency ratio (PER). Although absolute RNA and DNA contents increased with age, their relativeconcentrations decreased. The RNA/DNA ratio increased sharply from 14 to 28 weeks but afterwards decreasedtowards initial values. Hypertrophy increased rapidly to the 28-week stage but thenceforth increased much moreslowly. Hyperplasia, on the other hand, continued to increase linearly, resulting in a significant four- to fivefoldpredominance in this type of growth at the end of the 96-week experimental period. KG decreased significantly withage, as did KS, and CS, whereas at the 14-week stage, KD was significantly lower than at other ages. KRNA increaseduntil 28 weeks. KDNA increased significantly in juvenile fish compared to both fingerlings and adults, where it showedsimilar lower values. PRE remained high at all ages. © 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Fish; Hyperplasia; Hypertrophy; Protein-degradation rate; Protein-growth rate; Protein-synthesis rate

www.elsevier.com/locate/ijbcb

Abbre�iations: AGL, absolute protein-accumulation rate; AS, absolute protein-synthesis rate; CS, protein-synthesis capacity; KGL,protein-accumulation rate; KS, protein-synthesis rate; KD, protein-degradation rate; KRNA, protein-synthesis efficiency; KDNA,protein-synthesis rate/DNA unit; PRE, protein-retention efficiency.

� Publication c191 from ‘Drugs, Environmental Toxics and Cell Metabolism Research Group’, Department of Biochemistry andMolecular Biology, Centre of Biological Sciences, University of Granada, Granada, Spain.

* Corresponding author. Tel./fax: +34-958-243-089.E-mail address: jlcara@ugr.es (J.A. Lupianez).

1357-2725/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.

PII: S1357-2725(01)00088-7

J. Peragon et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 1227–12381228

1. Introduction

There are two tissues of paramount importanceto the physiological and organic functions of thebody, the liver and muscle tissue, both of whichbehave in intrinsically different ways. The role ofthe liver is first and foremost a homeostatic one,redistributing its chemical proceeds to the rest ofthe body tissues. The muscle, on the other hand,serves a mainly mechanical function, but becauseit accounts for the largest part of the animal, thecharacteristics of its growth and development di-rectly influence the growth of the animal as awhole. Muscle tissue therefore tends to react moreslowly in metabolic terms than does the liver; it iswell known in rats, for example, that total proteinturnover takes around five times longer in muscletissue than it does in the liver [1], due basically tothe fact that metabolic turnover is much higher inthose cells and tissues which, like the liver andintestinal mucosa, need to adapt rapidly and con-tinuously to changes in the chemical compositionof different exogenous nutritional elements.

In fish, the white muscle is the largest tissue andthe main site of protein accumulation and theamino acids deriving from its protein turnoveraccount for some 30% of the body’s total proteinsynthesis [2]. Although its fractional rate ofprotein synthesis is the lowest of all the tissues, itis the one which most efficiently accumulates al-ready synthesised protein. A clear relationshipbetween the rate of protein synthesis and growthhas been described in the cod (Gadus morhua)when fed different quantities of the same diet [2].The specific growth rate of white muscle is verysimilar to that of the organism as a whole and theevolution of muscle growth determines the growthand size of the fish [3].

White-muscle growth involves an increase inboth the length and diameter of pre-existing mus-cle fibres, known as hypertrophy, and the genera-tion of new smaller fibres, known as hyperplasia(also referred to by some authors as cellular re-cruitment or fibre recruitment). Post-embryonicmuscle growth in rats is characterised by an earlyinhibition of hyperplasia, muscle growth there-after resulting from an increase in fibre diameter[4]. In the rainbow trout, however, post-embry-

onic muscle growth is believed to be the productof variable contributions of both hyperplasia andhypertrophy. According to Weatherley and Gill[5], for example, experimentally induced somaticgrowth differences in trout are strictly related toproportional adjustments of recruitment and hy-pertrophy of the fibres, so that a proper distribu-tion of fibre diameter is maintained for any givensize of fish. Hypertrophy of muscle cells predomi-nates during rapid-growth stages, particularlyduring the early phases of development, althoughin larger fish the recruitment of new fibres is ofgreater importance [6,7].

RNA concentration and the RNA/DNA ratiohave usually been related to the growth rate ofdifferent tissues in immature fish during somestages of their development [8–12], but to ourknowledge no studies have been devoted toanalysing the behaviour of protein turnover andits relationship to the nucleic-acid content of themuscle during fish growth from fingerling toadulthood. We have completed our work with astudy of the evolution of protein-turnover ratesand their relationship with nucleic-acid concentra-tions throughout the development of rainbow-trout white muscle from fingerling to adult stages.Protein growth rate and its relationship with mus-cle hypertrophy and hyperplasia has also beeninvestigated.

Our initial hypothesis, which we have beenworking on in several recent studies, is thatprotein-turnover rates in fish tissue determine andcontrol the rate and design of tissue-growth dur-ing development and thus, if we can discover thenature of this relationship and how proteinturnover rates are controlled we can equally wellcontrol tissue and whole-body growth rate. In aprevious study carried out into protein-turnoverrates in the liver of rainbow trout during develop-ment [13] our experimental results partially confi-rmed this hypothesis.

The results of this new study reveal the basicprinciples controlling protein accumulation in thewhite muscle during trout development and itsrelationship with growth rate. These results add toour knowledge about how protein turnover ratesregulate growth rate in different fish tissues, eachone having a specific metabolic design. Once we

J. Peragon et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 1227–1238 1229

understand the principal characteristics governingprotein turnover in the white muscle, and thus thegrowth of the fish in general, it should be possibleto choose the ideal moment to intervene artifi-cially in the growth process by modifying some ofthese parameters and thus achieve a faster growthrate at a lower cost to the fish farmer.

2. Materials and methods

2.1. Chemicals

L-[2,6-3H]phenylalanine (2.22 TBq/mmol) wassupplied by Amersham (UK). L-Tyrosine decar-boxylase from Streptococcus faecalis, L-phenylala-nine, bovine-serum albumin, �-phenylethylamine,leucylalanine and pyridoxal 5-phosphate camefrom Sigma (USA). All other chemical com-pounds were bought from Fluka (Switzerland)and were of analytical grade.

2.2. Fish and maintenance

Rainbow trout (Oncorhynchus mykiss) were ob-tained from a local fish farm (Riofrıo, Granada,Spain). The different development stages observedin this study were characterised according to theage/weight of the fish. The fingerling stage was agroup of fifty 14-week-old fish with an initialmean weight of 5.01�0.05 g. The juvenile stagesincluded three groups of 50 fish each, 24, 28 and40 weeks old, with initial mean weights of37.58�1.46, 42.27�0.68, and 140.10�1.10 g.The adult stage was represented by a group oftwenty-five 96-week-old fish with a mean weightof 402.93�2.71 g.

We bought prime specimens of trout of eachexperimental age group from the same fish farm.We then kept each separate group in 350-l fibre-glass tanks in fresh, continuously aerated water(1.5 l kg fish−1 min−1) at 15.0�0.1 °C and acontrolled 12:12-h photoperiod. Before the experi-mental period the trout were kept under fish farmconditions and fed on a standard diet with differ-ent pellet sizes according to the age/size of thefish. During the experiment each group of fish wasfed twice daily for 25 days on a commercial trout

diet containing: 45.2% protein, 11.3% lipid, 8%digestible carbohydrate and 14.7 MJ kg−1. Thesize of the pellet used during our experimentalperiod was 1.5 mm for 14-week-old fish, 3 mm for24 and 28 weeks and 5 mm for fish of 40 and 96weeks. During the experimental period, we usedthe same batch of diet, stored at 4 °C.

The nutrient and energy contents were analysedby standard methods [14]. The fish were fed byhand to satiation and the precise quantity of foodsupplied at each feeding was recorded in order tocalculate feed efficiency (FER) and protein-effi-ciency ratio (PER). Daily food intake and weeklyweight gains were recorded throughout the experi-ment. The relative daily intake was calculated bydividing the total daily diet intake by the meanbody weight of the fish, as obtained from thegrowth curve.

Each fish was killed by a sharp blow to thehead and immediately put onto ice, where it wascleaned of everything superfluous to the muscle,except for the bones. The entire muscle wasweighed, including the bones, and a section wasthen removed from beneath the skin above andon either side of the spine just in front of thedorsal fin, discarding any superficial red muscle.The white muscle thus obtained was freezeclamped and stored in liquid nitrogen for protein-turnover assays.

2.3. Growth cur�es

Growth curves were drawn for whole-body andwhite-muscle weights and protein-accumulationrate (KG). All the fish were weighed at the begin-ning of the experimental period, after 2 weeks andthen again after 25 days to obtain the data for thegrowth curves. This information was supple-mented every week with the weights of six fishfrom each group (four from the 96-week-oldgroup), which were sampled at random to mea-sure their whole-body weight, white-muscleweight and protein concentration. At the end ofthe experimental time all the remaining fish wereused to provide the final data for the growth,muscle-protein and muscle-weight curves.

J. Peragon et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 1227–12381230

2.4. Growth rates

Specific growth rate (Gr) was calculated as thepercentage of weight increase per day using thefollowing equation [15]:

Gr (% day−1)=100 (loge W2− loge W1)/(t2− t1)

where W1 and W2 are the body weight at times t1

and t2.The white-muscle protein-accumulation rate

(KG) was calculated as the percentage of white-muscle-protein increase per day, using theequation:

KG (% day−1)=100 (loge P2− loge P1)/(t2− t1)

where P1 and P2 are the total muscle-proteincontents at time t1 and t2 [16]. Protein concentra-tion was determined according to Ref. [17].

2.5. Protein-turno�er rates

The fractional protein-synthesis rate was deter-mined by the method described in Ref. [18] andmodified in Refs. [19,20]. The caudal-vein injec-tion contained a solution of 135 mM L-phenyl-alanine containing L-[2,6-3H]phenylalanine (Am-ersham) at 100 �Ci ml−1 with a specific radioac-tivity of 1640 dpm nmol−1. The dose was 50 �Ci100 g body weight−1 at a volume per dose of 0.5ml 100 g body weight−1, accepting a blood vol-ume of 4–10% [21], thus representing 5–12.5% ofthe total blood volume.

Four fish were killed 2 min after injection andseven after 45 min. They were immediately put onice and sections of white muscle were removedand freeze-clamped in liquid nitrogen, as de-scribed in Section 2.2. The tissues were ho-mogenised (1:10 w/v) with cold 0.2 M HClO4 andseparated into two equal fractions, one for deter-mining the protein-synthesis rate and the other forquantifying DNA and RNA.

After centrifuging one of the fractions for 15min at 2800×g (rav=8.94 cm) at 4 °C, saturatedtripotassium citrate was added to the acid-solublefraction to pH 6 and later the precipitate ofKClO4 was eliminated by centrifuging at 2800×gfor 15 min. The insoluble protein fraction waswashed twice with 96% ethanol and once with

ether, and the pellet was then hydrolysed in 6 MHCl for 24 h at 110 °C. HCl was removed byevaporation and the amino acids were resus-pended in saturated sodium citrate.

The final concentrations of phenylalanine andL-[2,6-3H]phenylalanine incorporated into the sol-uble supernatant (SA) and insoluble protein (SB)fractions were calculated after the conversion ofphenylalanine to �-phenylethylamine (�-PEA).The �-PEA concentrations were determined byspectrofluorescence using a standard curve basedon an analysis of 0–15 nmol of �-PEA [22]. Tocalculate the values for KS the mean value of thefour fish killed after 2 min was subtracted fromthe values for each individual fish killed 45 minafter injection. These seven individual values werethen used to arrive at a final overall mean valuewith its corresponding standard mean error(S.E.M.). The specific radioactivity of protein-bound Phe increased linearly over 45 min [19].

The results are expressed as a function of SA

and SB (dpm nmol−1). The fractional protein-syn-thesis rate, KS (% protein synthesis per day) wascalculated as:

KS= [(SBt2−SBt1)/SA(t2− t1)] · [1440/(t2− t1)]×100

where SBt1 and SBt2 are the protein-bound specificradioactivity at 2 and 45 min after injection andSA(t2− t1) is the average free pool of specific ra-dioactivity over the period (t2− t1) and 1440 is thenumber of minutes in a day [18].

The absolute protein-synthesis rate (AS) wascalculated as the product of KS multiplied by thetotal protein content of the tissue and expressedas mg of protein synthesised per day. The frac-tional protein-degradation rate (KD) was taken tobe the difference between the protein-synthesis(KS) and protein-accumulation (KG) rates, calcu-lated for a period of 43 min and expressed as apercentage per day [23]. Protein-retention effi-ciency (PRE) was defined as the ratio betweenprotein retained as growth and total protein syn-thesised and was calculated as (KG/KS)×100 [24].Both, the values of KD and PRE for each age wereexpressed in relation to the values obtained for14-week-old fish, these being the smallest fish forwhich we could obtain reliable values.

J. Peragon et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 1227–1238 1231

2.6. Determination of DNA and RNAconcentrations in the white muscle

DNA and RNA were separated, purified andquantified from the second fraction of muscleprecipitated with 0.2 M HClO4 by the methodrecommended in Ref. [25]. The pellet was washedtwice with 0.2 M HClO4 to remove low-molecu-lar-weight contaminants (nucleotides, coenzymes,inorganic phosphate and phosphorus). The RNAand DNA fractions were separated by digestion inan alkaline medium (0.3 M KOH) at 37 °C for 1h followed by acidification in 1.2 M HClO4.

The supernatant was diluted up to 0.1 MHClO4 and the RNA concentration was deter-mined by measuring the absorbance at 260 and232 nm, according to the equation proposed inRef. [26]. The pellet was dissolved in 0.1 M KOHand the DNA concentration was estimated by theIndol test [27] using herring sperm as DNA stan-dard. The concentrations of protein, RNA andDNA in individual fish were determined and ex-pressed as mg g tissue−1. The protein/DNA andRNA/DNA ratios were calculated together withthe total-protein, RNA and DNA contents.

The values of the protein-synthesis rate are to agreat extent proportional to RNA concentrationsand thus protein-synthesis capacity (CS) can alsobe defined as a ratio of RNA/protein and ex-pressed as mg g−1 [28]. Protein-synthesis effi-ciency (KRNA) is defined as the amount (g) ofprotein synthesised per day and RNA unit (g) andis calculated as [(KS/CS)×10] [28]. Protein-synthe-sis rate/DNA unit (KDNA) is defined as theamount (g) of protein synthesised per day andDNA unit (g) and is calculated as [(KS/100)×(protein/DNA)] [28].

2.7. Statistical analysis

The results are expressed as means�standardmean error (S.E.M.). To compare the groups offish we used ANOVA followed by Duncan’s mul-tiple range test. The differences between meanswere also analysed with Student’s unpaired t-test.Differences were considered to be significant froma value of P�0.05. Regression analysis was ap-plied to the results. The linear and logarithmic

regressions were determined by a least-squareslinear-regression analysis.

3. Results

Food intake, growth rate and food-conversionand protein-conversion efficiencies during devel-opment are shown in Table 1. Both growth rateand food intake (mg g−1) decreased significantlywith age. The food-efficiency ratio (FER) andprotein-efficiency ratio (PER) also decreased sig-nificantly from the fingerling to adult stages.Growth rate decreased by 67% from the 7- to420-g groups (Table 1). This decrease in growthrate was related to whole weight according to theequation Gr=5.6074W −0.2880 (r= −0.98, P�0.01, n=30). The KG of white muscle decreasedconcomitantly with age in the same way as didwhole-body growth rate; this relationship is ad-justed to a curve defined by the expression KG=A×W b (Fig. 1).

The relationship between white-muscle weightand protein content, RNA, DNA and the protein/DNA ratio are shown in Table 2 and Fig. 1.Protein concentration was not related to eitherage or weight (Table 2) but when tissue proteincontent was expressed per mg of DNA it wasfound that this parameter increased sufficientlyduring development to indicate an increase inmuscle hypertrophy with age. The results concern-ing muscle nucleic-acid content at all five stages ofdevelopment are shown in Table 2. AbsoluteRNA and DNA contents increased with develop-ment. Nevertheless, mg DNA g tissue−1 de-creased significantly in fish of more than 14weeks, but remained approximately constantthereafter. After the age of 14 weeks the mgRNA/mg DNA ratio underwent a sharp increaseto reach its highest values in the 24- to-28-weekfish, tending thereafter to decrease to initialvalues.

While KG decreased in relation to whole-bodyand muscle weight, accompanied by a generaldecrease in DNA and RNA concentrations intissue, there was a concomitant increase in boththe number and size of DNA units. These in-creases were different in nature, however, the

J. Peragon et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 1227–12381232

increase in cell numbers being linear whilst that ofcell size was hyperbolic. The increase in DNA-unit size (hypertrophy) occurred in two phases: arapid phase in fish of 14–28 weeks, followed by aslower one in older fish. At the same time thenumber of DNA-units (hyperplasia) continued toincrease linearly so that after 96 weeks this fac-tor’s contribution to cell growth was between fourand five times that of hypertrophy. That is to saythat white muscle-cell growth in fish more than 28weeks old (65 g) was due rather to hyperplasiathan to hypertrophy (Table 2 and Fig. 1).

The results for white-muscle protein-turnoverrates at all five development stages studied are setout in Table 3. The fractional protein-synthesisrate (KS) decreased considerably with age and wassignificantly lower in mature fish. The absoluteprotein-synthesis rate (AS), however, was lower inyounger fish and at its highest in adults. In addi-tion, protein synthesis capacity (CS: mg RNA gprotein−1) showed a continuous and significant

decrease with increasing age and correlated sig-nificantly with KG and KS.

Protein-synthesis efficiency (KRNA, g proteinsynthesised/day×g RNA), an index of ribosomalcapacity for protein synthesis, tended to increasewith age up to the 28-week group. The protein-synthesis rate per DNA unit (KDNA) increasedsignificantly in juvenile fish (24, 28, 40 weeks)compared to fingerling (14 weeks) and adult (96weeks) fish. The KDNA values in fingerling andadult fish were considerably alike. The trajectoriesof KS, CS, mg RNA g muscle−1 and KDNA areshown in Fig. 2.

The behaviour of KD and of the protein-reten-tion-efficiency (PRE) values during developmentare shown in Fig. 3. KD increased significantlyuntil 28 weeks but thenceforth remained constant.The PRE values on the other hand were highregardless of age, although they decreased signifi-cantly until 28 weeks and showed a slight increasein the adult stage.

Table 1Body and white-muscle growth, food intake, food-conversion and protein-conversion efficiencies from the fingerling to adult stagesof rainbow trout

P-value dfF-ratioAge (weeks)a

14 24 28 40 96

Body weights (g) and growth rate140.10�1.105.01�0.05 402.93�2.71 7029.6 �0.0001 22437.58�1.46 42.27�0.68Initial196.50�5.4210.78�0.22 439.65�15.02 1100.3 �0.0001 �7850.14�0.56 85.94�2.33Final

421.29�1.11168.50�0.70Mean 64.10�0.2243.46�0.157.38�0.041.51�0.07c 1.30�0.02d 0.97�0.04e 38.9 �0.0001 111Gr 2.94�0.03a 1.70�0.02b

White-muscle weights (g)7982.5281.12�7.7589.73�5.0028.03�0.31 �0.000124.95�1.252.74�0.003Initial 224

50.63�2.28 137.46�5.00 285.08�9.02 2506.8 �0.0001 �786.00�0.13Final 31.38�0.91

Relati�e daily ingestion (mg g fish−1) and nutritional indices25.90�0.30a 16.20�0.10b 18.20�0.12b 17.70�0.30bDiet 15.00�0.10c 16.2 �0.0001 124

10.80.53�0.01d0.79�0.01c0.84�0.01b1.04�0.01a1.07�0.03a 124FER �0.0052.31�0.02a 2.19�0.01b 1.81�0.01c 1.79�0.02cPER 1.20�0.01d 9.8 �0.005 124

The experiment began with 50 specimens for groups of 14-, 24-, 28- and 40-week-old fish, and 25 specimens for the 96-week-oldgroup. The results are expressed as the mean�S.E.M. and have been compared between groups. The experimental time was in allcases 25 days. The terms ‘initial’, ‘final’ and ‘mean’ indicate the average initial, final and mean weights of all experimental trout. Themean value was used to refer to the different groups (fingerling: 7 g; juvenile: 45, 65 and 170 g; adult: 420 g; represented, respectively,by 14-, 24-, 28-, 40- and 96-week-old fish. P�0.05 or less was considered to be statistically significant. Data in a row followed bydifferent superscript letters are statistically different. df, degrees of freedom; PER, weight gain (g) protein intake (g)−1. FER, weightgain (g) diet intake (g)−1.

a Age at the beginning of the experiments.

J. Peragon et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 1227–1238 1233

Fig. 1. Relationship between fractional white-muscle protein-accumulation rate (panel a), total protein and total RNAcontent (panel b), protein/DNA ratio and total DNA content(panel c) and white-muscle weight (WM, g) during the develop-ment of rainbow trout (Oncorhynchus mykiss). The regressionequations for the different curves are: KG (% day−1)=3.78×WM (g)−0.30 (n=36, r= −0.96, P�0.001), total protein(g)=0.14×WM (g)+0.38 (n=36, r=0.99, P�0.001), totalRNA (mg)=4.82×WM (g)0.63 (n=40, r=0.97, P�0.001),protein/DNA (g mg−1)=0.28×WM (g)0.26, (n=30, r=0.85,P�0.001), total DNA (mg)=0.12×WM (g)+2.63 (n=40,r=0.92, P�0.001).

tion (r= −0.95, P=0.02) was found between ageand both food-conversion and protein-conversionefficiency, possibly as a result of decreasing re-quirements. Other authors have reported a similardecrease in relative food intake and conversionindices versus age in fish [10,31].

The nature of liver growth can be fairly easilyinterpreted in terms of increases in cell numbersor cell size [13] but in white-muscle tissue, wherethe possibility of polynucleate cells arises, changesin total DNA and the protein/DNA ratio cannotbe so easily explained in the same terms. Never-theless, when the DNA unit is considered to bethe cytoplasmic volume controlled by a singlenucleus the terms hypertrophy and hyperplasia canbe used to describe changes in the size and num-ber of muscle DNA units [32]. Thus, an increasein the size of DNA units (hypertrophy) occurredin two distinct phases: a rapid one in fish of 14weeks (7 g) to 28 weeks (65 g) followed by aslower one from 40 weeks (170 g) to 96 weeks(420 g). Whilst the rate of hypertrophy sloweddown considerably after 28 weeks, the number ofcell nuclei, indicating hyperplasia, continued toincrease steadily. This difference in growth be-haviour agrees with the findings of Kiessling et al.[7], who observed hypertrophy during rapidgrowth in trout whilst slow-growth stages wereassociated with hyperplasia.

Furthermore, Houlihan et al. [33], when com-paring cardiac, red and white muscle of trout,found that a correlation existed between oxidativepotential and KS. Thus, the proportion of musclevolume occupied by mitochondria may well fallwith growth [34], resulting in a greater proportionof the muscle being occupied by contractileproteins, which have lower turnover rates thanmitochondrial proteins [32]. In the growing rat anincrease in total body protein has been associatedwith an increase in DNA-unit size and a slightdecrease in RNA [32], just as we have observed inthis study with rainbow trout.

White-muscle RNA and DNA concentrationsdecreased concomitantly with the age of the fish.RNA concentrations have been related to growthrate; for example, Bastrop et al. [10], using thesame methods as ours, found a positive correla-tion between growth rate and muscle RNA con-

4. Discussion

Body growth rate (Gr), expressed as a percent-age of body-weight increase per day, fell with age.Previous studies have shown that both parametersare related in such a way that decreasing growthrate is related to body weight (W) according tothe equation Gr= ln A×W b or Gr=a×W b

[29,30]. We found negative allometry between KG

and weight for both the whole-body and white-muscle, with a similar behaviour to that proposedin Ref. [30] when the fish were fed at maximumrates. A decrease in growth rate with age wasassociated with a lower use of food for growthpurposes. In fact, a significant negative correla-

J. Peragon et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 1227–12381234

tent. Similarly, Ferguson [8] found that RNA andDNA concentrations in the white muscle of im-mature rainbow trout were significantly higherthan in that of mature fish. In addition, Mathers

et al. [35] have also provided evidence to showthat the concentration of RNA in juvenile fish ishigher than in mature ones, showing that anincrease in body weight in the larvae of tilapia

Table 2Protein and nucleic-acid contents of trout white muscle at different development stages

P-value dfF-ratioAge (weeks)a

14 96402824

White-muscle protein concentrationsmg g tissue−1 �0.051.19142.41�7.02a �49167.82�6.53a139.03 146.98�7.77a133.42

�13.97a �15.11a

0.68�0.02a 4.31�0.11b 7.49�0.33c 20.54�0.46d 40.18�13.38e 369.8 �0.01 �49g protein total0.36�0.03a 0.83�0.05b 0.98�0.06b 0.88�0.12b 1.19�0.11c 14.43 �0.005g protein mg �49

DNA−1

White-muscle RNA and DNA concentrations (mg)2.24�0.18a 1.42�0.20b 0.47�0.02d1.65�0.04bmg RNA g 67.55 �0.0051.03�0.07c �49

tissue−1

47.56�2.35b 72.72�3.18c 126.09�2.81d 131.18�5.67d 26.21mg RNA total �0.00511.58�0.77a �49�49mg DNA g 0.16�0.01b0.39�0.04a 0.15�0.01b 0.19�0.05b 0.12�0.02b 31.25 �0.005

tissue−1

mg DNA total 5.10�0.22b1.92�0.14a 7.65�0.06c 23.07�2.95d 34.21�4.09e 587.87 �0.001 �49�49�0.00540.463.92�0.84c5.42�0.39amg RNA mg 9.47�0.90b10.31�0.50b5.74�0.28a

DNA−1

Results are expressed as the mean�S.E.M. of at least seven individual values and have been compared between groups. Theexperimental time was in all cases 25 days. The different groups (fingerling: 7 g; juvenile: 45, 65 and 170 g; adult: 420 g) wererepresented, respectively, by 14-, 24-, 28-, 40- and 96-week-old fish. P�0.05 or less was considered to be statistically significant. Datain a row followed by different superscript letters are statistically different. df, degrees of freedom.

a Age at the beginning of the experiments.

Table 3White-muscle protein-synthesis rates of rainbow trout at different development stages

Age (weeks)a dfF-ratio P-value

24 28 4014 96

1.25�0.06aKS 1.23�0.07a1.48�0.20a 1.12�0.06a 0.47�0.06b 8.63 �0.005 �34�34�0.0001161.90281.26�35.90e285.51�15.29dAS 92.13�5.24c53.87�2.59b10.06�1.36a

12.37�0.85b 9.66�0.85c 6.14�0.33dCS 3.27�0.14e16.11�0.16a 80.94 �0.0001 �34�346.06 �0.0051.75�0.07b 1.82�0.24b 1.42�0.21bKRNA 0.92�0.13a 1.01�0.13a

10.42�0.48b 12.05�0.99b 9.89�0.68b 5.58�0.55c5.28�0.66a 38.92KDNA �0.0001 �34

Results are mean�S.E.M. of at least seven values and have been compared between groups. The experimental time was in all cases25 days. The different groups (fingerling: 7 g; juvenile: 45, 65 and 170 g; adult: 420 g; were represented, respectively, by 14-, 24-,28-, 40- and 96-week-old fish. P�0.05 or less was considered to be statistically significant. Data in a row followed by differentsuperscript letters are statistically different. df, degrees of freedom. KS, protein-synthesis rate is expressed as % day−1; AS, absoluteprotein-synthesis rate [(KS/100)×[protein]Total] is expressed as mg of white-muscle protein (fish×day)−1; CS, protein-synthesiscapacity (mg RNA g protein−1); KRNA, protein-synthesis efficiency [(KS/CS)×10, g protein synthesised/day×g RNA]; KDNA,protein-synthesis rate/DNA unit [(KS/100)×(Protein: DNA), g protein synthesised/day×g DNA].

a Age at the beginning of the experiments.

J. Peragon et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 1227–1238 1235

Fig. 2. Relationship between fractional white-muscle protein-synthesis rate (KS, panel a), RNA concentrations (mg gmuscle−1 and mg g protein−1, CS, panel b) and protein-syn-thesis rate: DNA unit (KDNA, panel c) and white-muscleweight (WM, g) during the development of rainbow trout(Oncorhynchus mykiss). The relationships are defined by theequations: KS (% day−1)=2.31×WM (g)−0.22 (n=40, r= −0.67, P�0.001), mg RNA g muscle−1=5.21×WM (g)−0.39

(n=40, r= −0.94, P�0.001), CS (mg RNA g protein−1)=35.43×WM (g)−0.39 (n=40, r= −0.94, P�0.001).

it was 80% less in 96-week-old fish compared tofingerlings. The decrease in KG in white musclecan be put down to a significant reduction (68%)in KS concomitant with a 250% increase in KD.

Another important difference in the behaviourof protein turnover between the two tissues is tobe seen in the evolution of protein-synthesis effi-ciency (KRNA). In trout liver KRNA diminishedsteadily until 40 weeks, at which point it was atabout 36% of its original value in the fingerlings,and then it increased again somewhat in 96-week-old fish [13]. In white muscle, on the other hand,KRNA increased significantly until in 40-week-oldfish it was 100% more than its original value, andthen it diminished slightly until 96 weeks. Similardifferences in behaviour occurred in both tissueswith regard to the fractional protein-synthesis rateper DNA unit (KDNA).

In addition, we also found a continuous de-crease in both KS and CS associated with a de-crease in growth rate (KG) during thedevelopment of our trout. As the decrease in CS

was more continuous and pronounced than thatof KRNA it might be deduced that the main factorresponsible for the decrease in KS is the fall in

Fig. 3. Evolution of white-muscle protein-degradation rate(KD) and protein-retention efficiency (PRE) values during thedevelopment of rainbow trout (Oncorhynchus mykiss). Thevalues of KD and PRE for trout of 24, 28, 40 and 96 weeks areexpressed as percentages using as 100% the values obtained in14-week-old trout. Superscript letters above the bars indicatestatistical differences (P�0.05).

(Oreochromis mossambicus) is inversely related tothe RNA/protein ratio (CS), KS and KG.

A simple analysis of the parameters that char-acterise protein turnover in trout liver and whitemuscle, two tissues with different organic func-tions, reveals several different trends in their be-haviour during the fish’s development. As we haveshown in a previous paper [13], fractional growthrate in the liver (KG) undergoes no significantchange throughout the period of developmentstudied, due to significant and similar decreases ofaround 70% both in the fractional protein-synthe-sis rate (KS) and protein-degradation rate (KD).White muscle, on the other hand, behaves in acompletely different way in that KG diminishedsignificantly during development to the extent that

J. Peragon et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 1227–12381236

RNA concentrations. A decrease in KS andRNA concentration with age has also been de-scribed in mammals [32]. Several authors [36,37]have furthermore observed a two- to threefolddecrease in KS in the muscle of the growing ratas a result of a progressive decrease in the ca-pacity for muscle protein synthesis with age. Inthe same way, a gradual reduction in KS con-comitant with a slowing in the growth rate hasalso been observed in the rainbow trout [33,38].Nevertheless, whilst other authors have reportedthat the relationship between KS and body-weight is allometric, we found that the relation-ship between KS and muscle weight appears tobe isometric [2,33].

The values of KRNA increased progressivelyfrom 14 to 28 weeks and stayed fairly highthereafter. KDNA behaved differently, however,increasing progressively with age to reach itshighest values at 28 weeks and decreasing there-after. KDNA increased until the DNA-unitsreached maximum size, the subsequent increasein the number of nuclei being responsible forthe decreasing KDNA values observed in olderfish.

From the differences observed in the be-haviour of the protein turnover rate in the liverand white muscle of the trout, together with thevalues for hypertrophy, hyperplasia and specificgrowth rate (Gr), it is possible to conclude withconsiderable certainty not only that the growthrate of the whole fish follows the same patternas that of the white muscle but also to estimatethe most opportune moment to manipulate theturnover rates in such a way as to improvegrowth, with the considerable commercial ad-vantages which this implies. In conclusion,throughout the development of rainbow trout,the growth rate of the whole fish clearly andsignificantly diminishes concomitantly with a sig-nificant decrease in protein-accumulation andprotein-synthesis rates. This trend may in alllikelihood reflect a significant fall in protein-syn-thesis capacity from the fingerling to adultstages, which is in turn related to the transcrip-tion rates. An important result of these pro-cesses is that during early development stagesthe whole-body and muscle growth of fish de-pends to a much greater extent upon hypertro-

phy than hyperplasia, whilst in older fish theopposite is true, with the effects of hyperplasiafar outweighing those of hypertrophy.

As far as the practical application of our re-sults is concerned, the highest protein-turnoverrates and protein-retention efficiency are to befound in trout during the early stages of theirdevelopment, at the fingerling and juvenilestages, when it would be easiest to influence ar-tificially either the protein-synthesis or degrada-tion rates with the aim of obtaining highergrowth yields. To this end we are currently en-gaged in studies with natural specific inhibitorsof protein degradation in an attempt to directthe balance of protein turnover towards a highersynthesis rate, thus encouraging tissue growth.

Acknowledgements

This work was supported by a grant from theComision Interministerial de Ciencia y Tecnolo-gıa (CICYT), Ministerio de Educacion y Cien-cia, Spain, and a research grants (Ns CVI 157 yRNM 156) from the Plan Andaluz de Investiga-cion (PAI), Junta de Andalucıa, Spain. The au-thors thank their colleague A.L. Tate for hercomments and suggestions.

References

[1] P. Siekevitz, The turnover of protein and the usage ofinformation, J. Theor. Biol. 37 (1972) 321–334.

[2] D.F. Houlihan, S.J. Hall, C. Gray, B.S. Noble, Growthrates and protein turnover in Atlantic cod (Gadusmorhua), Can. J. Fish. Aquat. Sci. 43 (1988) 951–964.

[3] A.H. Weatherley, H.S. Gill, The role of muscle in deter-mining growth and size in teleost fish, Experientia 45(1989) 875–878.

[4] G. Goldspink, Growth of muscle, in: D.F. Goldspink(Ed.), Development and Specialization of Skeletal Mus-cle, Cambridge University Press, London, 1980, pp. 19–35.

[5] A.H. Weatherley, A.H. Gill, Growth dynamics of whitemyotomal muscle fibres in the bluntose minnow,(Pimephales notatus) Rafinesque, and comparison withrainbow trout, (Salmo gairdneri ) Richardson, J. FishBiol. 25 (1984) 13–24.

J. Peragon et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 1227–1238 1237

[6] A.H. Weatherley, Approaches to understanding fishgrowth, Trans. Am. Fish. Soc. 116 (1990) 662–672.

[7] A. Kiessling, T. Storebakken, T. Asgard, K.H. Kiessling,Changes in the structure and function of the epaxialmuscle of rainbow trout (Oncorhynchus mykiss) in rela-tion to ration and age. I. Growth dynamics, Aquaculture93 (1991) 335–356.

[8] M.M. Ferguson, Enzyme heterozygosity and growth inrainbow trout: genetic and physiological explanations,Heredity 66 (1992) 115–122.

[9] K. Jurss, T. Bittorf, The relationship between biochemicalliver status and growth in immature rainbow trout (Salmogairdneri Richardson) I. Effects of feeding and salinity,Zool. Jb. Physiol. 94 (1990) 474–485.

[10] R. Bastrop, K. Jurss, R. Wacke, Biochemical parametersas a measure of food availability and growth in immaturerainbow trout (Oncorhynchus mykiss), Comp. Biochem.Physiol. 102A (1992) 151–161.

[11] J. Peragon, J.B. Barroso, L. Garcıa-Salguero, F. Aranda,M. de la Higuera, J.A. Lupianez, Selective changes in theprotein-turnover rates and nature of growth induced introut liver by long-term starvation followed by re-feeding,Mol. Cell. Biochem. 201 (1999) 1–10.

[12] J. Peragon, J.B. Barroso, L. Garcıa-Salguero, M. de laHiguera, J.A. Lupianez, Dietary alterations in protein,carbohydrates and fat increase liver protein-turnover rateand decrease overall growth rate in the rainbow trout,Mol. Cell. Biochem. 209 (2000) 97–104.

[13] J. Peragon, J.B. Barroso, M. de la Higuera, J.A. Lupi-anez, Relationship between growth and protein turnoverrates and nucleic acids in the liver of rainbow trout(Oncorhynchus mykiss) during development, Can. J. Fish.Aquat. Sci. 55 (1998) 649–657.

[14] AOAC (Association of Official Analytical Chemists), in:S. Williams (Ed.), Official Methods of Analysis of theAssociation of Official Analytical Chemists, 14th edition,AOAC, Washington, DC, 1984, pp. 152–160.

[15] W.E. Ricker, Growth rates and models, in: W.S. Hoar,D.J. Randall, J.R. Brett (Eds.), Fish Physiology, vol. 8,Academic Press, New York, 1979, pp. 677–743.

[16] R.J. Wootton, The Ecology of Teleost Fishes, Chapmanand Hall, London, 1990.

[17] O.H. Lowry, N.J. Rosebrough, J.L. Farr, R.J. Randall,Protein measurement with the Folin phenol reagent, J.Biol. Chem. 193 (1951) 265–275.

[18] P.J. Garlick, M.A. McNurlan, V.R. Preedy, A rapid andconvenient technique for measuring the rate of proteinsynthesis in tissues by injection of [3H]phenylalanine,Biochem. J. 192 (1980) 719–723.

[19] J. Peragon, F. Ortega-Garcıa, J.B. Barroso, M. de laHiguera, J.A. Lupianez, Alterations in the fractionalprotein turnover rates in rainbow-trout liver and whitemuscle caused by an aminoacid-based diet and changes inthe feeding frequency, Toxicol. Environ. Chem. 36 (1992)217–224.

[20] J. Peragon, J.B. Barroso, L. Garcıa-Salguero, M. de laHiguera, J.A. Lupianez, Dietary protein effects on growth

and fractional protein synthesis and degradation rates inliver and white muscle of rainbow trout (Oncorhynchusmykiss), Aquaculture 124 (1994) 35–46.

[21] D.R. Jones, D.J. Randall, The respiratory and circulatorysystems during exercises, in: J.C. Rankin, F.B. Jensen(Eds.), Fish Physiology, vol. 7, Chapman and Hall, Lon-don, 1978, pp. 45–71.

[22] O. Suzuki, K. Yagi, A fluorometric assay for �-phenylethylamine in rat brain, Anal. Biochem. 75 (1976)192–200.

[23] D.J. Millward, P.J. Garlick, J.C. Stewart, D.O. Nnamyel-ngo, J.C. Waterlow, Skeletal-muscle growth and proteinturnover, Biochem. J. 150 (1975) 235–243.

[24] A.R. Foster, D.F. Houlihan, C. Gray, F. Medale, B.Fauconneau, S.J. Kaushik, P.Y. Le Bail, The effects ofovine growth hormone on protein turnover in rainbowtrout, Gen. Comp. Endocrinol. 82 (1991) 111–120.

[25] H.N. Munro, A. Fleck, The determination of nucleicacids, in: G. Glick (Ed.), Methods of Biochemical Analy-sis, vol. 14, Wiley-Interscience, New York, 1966, pp.113–176.

[26] A.J. Ashford, V.M. Pain, Effect of diabetes on the ratesof synthesis and degradation of ribosomes in rat muscleand liver in vivo, J. Biol. Chem. 261 (1986) 4059–4065.

[27] G. Ceriotti, A microchemical determination of deoxyri-bonucleic acid, J. Biol. Chem. 198 (1952) 297–301.

[28] P.H. Sugden, S.J. Fuller, Regulation of protein turnoverin skeletal and cardiac muscle, Biochem. J. 273 (1991)21–37.

[29] J.R. Brett, Environmental factors and growth, in: W.S.Hoar, D.J. Randall, J.R. Brett (Eds.), Fish Physiology,vol. 8, Academic Press, New York, 1979, pp. 599–677.

[30] M. Jobling, Growth studies with fish — overcoming theproblems of size variation, J. Fish Biol. 22 (1983) 153–157.

[31] I.D. McCarthy, C.G. Carter, D.F. Houlihan, Correlatingfood intake and specific growth rate of rainbow trout ofdifferent sizes, Aquaculture 100 (1992) 326–334.

[32] J.C. Waterlow, P.J. Garlick, D.J. Millward, Proteinturnover in mammalian tissues and in the whole body,J.C. Waterlow (Ed.) Elsevier North-Holland, Amsterdam,1978.

[33] D.F. Houlihan, D.N. McMillan, P. Laurent, Growthrates, protein synthesis and protein degradation rates inrainbow trout: effects of body size, Physiol. Zool. 59(1986) 482–493.

[34] O. Mathieu, R. Krauer, H. Hoppeler, P. Gehr, S.L.Lindstedt, R.M.N. Alexander, C.R. Taylor, E.W. Weibel,Design of the mammalian respiratory system. VII. Scalingmitochondrial volume in skeletal muscle to body mass,Respir. Physiol. 44 (1981) 113–128.

[35] E.M. Mathers, D.F. Houlihan, I.D. McCarthy, L.J. Bur-ren, Rates of growth and protein synthesis correlated withnucleic acid content in fry of rainbow trout,Oncorhynchus mykiss : effects of age and temperature, J.Fish Biol. 43 (1993) 245–263.

J. Peragon et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 1227–12381238

[36] S.E.M. Lewis, F.J. Kelly, D.I. Goldspink, Pre- and post-natal growth and protein turnover in smooth muscleheart and slow and fast-twitch skeletal muscles of the rathindlimb, Differentiation 25 (1984) 168–175.

[37] T.A. Davis, M.L. Fiorotto, H.V. Nguyen, P.J. Reeds,

Protein turnover in skeletal muscle of suckling rats, Am.J. Physiol. 257 (1989) R1141–R1146.

[38] K.R. Dabrowski, Ontogenetic aspects of nutritional re-quirements in fish, Comp. Biochem. Physiol. 85A (1986)639–655.