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Author's personal copy Aquaculture 279 (2008) 11–17

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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Assessment of “discreet” vertebral abnormalities, bone mineralization and bone compactness in farmed rainbow trout M.-H. Deschamps a, A. Kacem b, R. Ventura a, G. Courty a, P. Haffray c, F.J. Meunier d, J.-Y. Sire a,⁎ a

Équipe «Évolution et développement du squelette», UMR 7138, Université Pierre & Marie Curie — Paris 6, 7 quai St-Bernard, 75251, Paris, France Département de Biotechnologie Marine et Aquaculture, Institut Supérieur de Biotechnologie de Monastir, 5000, Monastir, Tunisie SYSAAF, Campus de Beaulieu, 35042, Rennes, France d Équipe “Biodiversité et Dynamique des Communautés aquatiques”, UMR 5178, Muséum national d’histoire naturelle, rue Cuvier, 75005 Paris, France b c

A R T I C L E

I N F O

Article history: Received 5 July 2007 Received in revised form 29 January 2008 Accepted 24 March 2008 Keywords: Oncorhynchus mykiss Vertebral abnormalities Bone mineralization Bone compactness Aquaculture

A B S T R A C T The present study aimed to evaluate the importance of “discreet” vertebral abnormalities in normally-shaped rainbow trout in relation to vertebral bone condition in French fish farms. A total of 373 trout (262 ± 2 mm in total length) from 20 fish farms sampled were studied. The fish were radiographed and the axial skeleton examined for vertebral abnormalities. Vertebrae from the middle axial region (V32-38) were selected to evaluate vertebral bone condition. Bone mineralization (BM, %) was estimated by the ratio of ash and dry weight. Bone compactness (BC,%) was measured using Bone Profiler 3.23 images software on digitized radiographs of transverse sections (125 ± 10 μm). Statistical analyses were performed to test the relationships between the occurrence of vertebral abnormalities, and BM and BC. The occurrence of affected trout ranged from 0 to 55% depending on the farm. Trout displayed vertebrae with low BM (b 54.6%) and low BC (b 28.1%) in 40% and 55% of the farm, respectively. No relationships were observed between bone condition parameters (BM, BC) and the occurrence of vertebral abnormalities. These results could be explained by a wide and variable plastic response of bone characters (i.e., vertebral abnormalities, BM and BC) to the various rearing conditions in the fish farms sampled. © 2008 Elsevier B.V. All rights reserved.

1. Introduction In France, rainbow trout is, by far, the most common reared fish, with an annual production exceeding 35 000 tons (FEAP, 2006). The economical importance of this species is associated to constant efforts aiming to optimize performance, yield and product quality. In the past decades, major progresses were obtained in selecting various genetic strains for growth and processing yields (Haffray et al., 2004) and in improving rearing conditions. In parallel to these gains in growth rate and muscle quality, the occurrence of skeletal abnormalities was shown to be a general problem for all world wide salmonids industry (it exceeds more than 35% in some fish farms) compared to the 2–3% observed in wild salmonids (Gill and Fisk, 1966; Poynton, 1987). In reared trout, skeletal anomalies have been related to a large number of either genetical and/or physiological disorders principally related to environmental factors, which could act at various life periods. Triploidization type of treatment (Sadler et al., 2001), inadequate light intensity or temperature (Fjelldal et al., 2004; Sfakianakis et al., 2004), water current intensity (Kihara et al., 2002; Sfakianakis et al., 2006), non-inflation of the swimbladder (Chatain, 1994), pathologic

⁎ Corresponding author. Tel.: +1 44 27 35 72; fax: +1 44 27 35 72. E-mail addresses: [email protected], [email protected] (J.-Y. Sire). 0044-8486/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2008.03.036

events (Madsen et al., 2001) and nutritional imbalance (for a review, see Cahu et al., 2003) can lead to vertebral abnormalities such as fused and/or compressed vertebrae. These skeletal anomalies represent a real problem for producers as they affect yield of production (survival and reduction of market value in affecting trout morphology). Also, as vertebral abnormalities may arise late in production (Witten et al., 2006), they increase sorting costs. Furthermore, during filet production, some waste can occur due to “visually undetectable” abnormalities (called here “discreet” abnormalities, i.e., not prominent or not readily noticeable), as knives strip against cartilaginous callus and/or abnormal vertebrae (Koumoundouros et al., 1997, 2001; Boglione et al., 2001; Witten et al., 2006). For these reasons, there is a real need to find reliable indicators either to prevent the development of such vertebral abnormalities or to limit their importance. Development and growth of a healthy vertebral skeleton takes time and relies on two important constraints. First, the vertebral skeleton plays a biomechanical role as it ensures muscle anchoring, flexibility and elasticity during propulsion (Webb, 1975). Second, the vertebrae are the main locus of mineral storage and they contribute to calcium and phosphorus homeostasis regulation (Carragher and Sumpter, 1991; Meunier and François, 1992; Persson et al., 1994; Skonberg et al., 1997). A recent study assessing vertebral histo-morphometric abnormalities in farmed rainbow trout revealed extended resorption of the

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Table 1 Assessment of vertebral bone condition in the 373 rainbow trout sampled in 20 French fish farms Total

Lots 1

Number of observed individuals Affected individuals (n) Affected individuals (%) Number of total abnormala vertebrae Individual average of abnormala vertebrae in all trout Individual average of abnormala vertebrae in affected trout BM (%) in normal trout BM (%) in affected trout BC (%) in normal trout BC (%) in affected trout

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

373

20

2 20

20

20

19

20

20

20

20

20

20

16

20

20

20

9

9

20

20

20

82 22 403

10 50 78

3 15 10

6 30 14

1 5 2

4 21 21

4 20 26

11 55 53

8 40 57

1 5 2

3 15 11

6 30 24

1 6 3

6 15 7

5 25 18

4 20 17

0 0 0

4 44 16

5 5 8

3 5 14

3 15 22

1.1 (3.0)

1.3 (1.7)

2.7 (2.9)

2.9 (5.1)

0.6 (1.5)

1.2 (2.0)

0.2 (0.7)

0.4 (0.9)

0.9 (1.8)

0.9 (2.6)

0 0

1.6 (2.1)

0.4 (1.4)

0.7 (1.5)

1.1 (3.5)

7.1 2.0 (5.8) –

3.7 (2.1)

4.0 (1.1)

3.0 –

2.3 (0.7)

3.6 (2.1)

4.3 – (4.5) –

4.0 (0.8)

4.0 (2.8)

4.7 (3.0)

7.3 (8.6)

52.6 51.7 52.6 51.9 56.5 (1.0) (1.8) (2.1) (3.0) (4.2) 51.5 51.9 51.5 50.5 57.7 – (1.6) (1.4) – (7.4) 32.0 25.8 28.8 27.4 28.0 (5.7) (4.4) (4.6) (4.5) (4.4) 33.0 24.2 30.3 33.0 29.1 – (5.0) (5.9) – (6.1)

54.9 (3.7) 56.2 (3.3) 32.8 (5.6) 34.6 (6.0)

54.8 (1.8) 55.2 (1.1) 28.7 (7.1) 30.8 (3.5)

57.8 55.8 58.0 (1.3) (1.3) (2.4) 57.4 54.9 57.8 (1.0) (4.6) (1.4) 24.9 33.8 28.8 (3.9) (6.4) (5.0) 25.2 38.9 26.4 (5.4) (1.3) (2.5)

58.6 (0.9) 58.3 (0.7) 25.7 (4.5) 25.3 (6.3)

1.1 (2.7)

3.9 0.5 (4.7) (1.5)

0.7 (1.2)

0.1 (0.4)

4.9 (3.6)

7.8 (4.1)

3.3 (3.0)

2.3 2.0 (0.9) –

5.3 (4.7)

6.5 (5.9)

4.8 (2.3)

54.6 56.1 (3.4) (1.8) 54.5 56.3 (3.0) (2.6) 28.1 26.0 (5.7) (5.4) 28.3 24.4 (5.6) (3.7)

51.4 (5.7) 50.8 (7.7) 24.0 (4.3) 21.9 (10.2)

56.3 (1.5) 56.8 (0.9) 26.6 (6.8) 27.8 (6.2)

54.4 (1.5) 55.0 (0.5) 26.7 (3.1) 25.7 (5.4)

55.4 (1.2) 54.5 (2.3) 25.0 (3.3) 28.0 (2.2)

52.4 51.0 (2.0) (1.0) 52.3 51.7 (1.5) (0.8) 32.5 26.8 (4.7) (3.6) 29.5 29.5 (5.5) (5.2)

54.8 (1.2) 54.5 – 28.0 (4.3) 32.5 –

0.1 (0.5)

57.1 (0.9) – – 24.9 (5.0) – –

The values above the individual average of abnormal vertebrae and below the mean bone mineralization (BM) and bone compactness (BC) are indicated in bold (mean values calculated from all individuals). a Abnormal vertebrae include incompletely and completely fused vertebrae and malformed vertebrae; Standard deviations between brackets.

vertebral body, as a result of osteoclast activity (Kacem et al., 2004). This phenomenon leads to a significant reduction of bone compactness and was thought to be related to a dysfunction in phosphorus and calcium metabolism induced by increasing growth speed in unfavorable rearing conditions. However, the study by Kacem et al. (2004) was limited to a few specimens from a single fish farm, and the question remained whether vertebral bone resorption was widely spread in fish farms. Indeed, it was tempting to propose the hypothesis of a relationship between extended vertebral bone resorption and vertebral abnormalities observed in farmed rainbow trout. Bone mineralization and bone compactness are useful parameters for estimating “bone condition”, as they result from bone “reshaping” (remodeling) resulting from physiological demands and/or biomechanical constraints (Francillon-Vieillot et al., 1990; Fleming, 1996; Persson et al., 1994, Persson, 1997; Vielma and Lall, 1998; Yamada et al., 2001). An inadequate bone condition could predispose individuals to vertebral abnormalities. For instance, low or over-mineralized vertebrae were reported as vertebral abnormalities (Helland et al., 2005, 2006; Kranenbarg et al., 2005a,b). To date, only a few studies have attempted to link bone condition and vertebral abnormalities in salmonids (Fjelldall et al., 2006; Gil Martens et al., 2006; Witten et al., 2006). In the present study, we i) evaluate the importance of discreet vertebral abnormalities in normally-shaped rainbow trout sampled in several French fish farms, and ii) quantify the variability of two vertebral bone condition parameters (bone mineralization and bone compactness). Our objective was to check whether a high number of discreet vertebral abnormalities could be correlated to variations of the vertebral bone condition in reared trout.

2.2. Typology of vertebral abnormalities Trout were radiographed using a Faxitron@Cabinet X-Ray systems model 43 855C, adjusted to 90 kV and 160 ms. X-rays were scanned and digitized using Digital Linear X-Ray Scanner EZ 320 set at 60 cm of the source and iX-Pect for EZ 320 X-Ray acquisition software. Each radiograph was enlarged and the axial skeleton examined for vertebral abnormalities. Two types of abnormalities were distinguished: vertebrae that were either asymmetrical, compressed, hypoor hyper-mineralized were considered malformed vertebrae; vertebrae that were either juxtaposed, lacking intervertebral space but still exhibiting a distinguishable body and one pair of neural/hemal arches (incomplete vertebral fusion), or possessed a single vertebral body showing several pairs of neural/hemal arches (complete vertebral fusion), were considered fused vertebrae (Fig. 1).

2. Materials and methods 2.1. Sampling A total of 373 rainbow trout, Oncorhynchus mykiss, were sampled in 20 French fish farms (9–20 individuals per lot, Table 1). Trout of similar total length (market size: 262 ± 2 mm TL) were randomly sampled. Particular attention was paid to retain normally-shaped specimens only, i.e. showing no external deformities. Lots were randomly assigned to respect fish farm confidentiality.

Fig. 1. Typology of vertebral abnormalities used in the present study from enlarged radiographs of the rainbow trout axial skeleton. Top: normal vertebrae showing regularly-spaced bodies and their single pairs of arches. Bottom: vertebrae showing abnormalities (asymmetries, compressions, hypo- or hyper-mineralization, complete and incomplete fusions) and the types (malformations and fusions) in which they were categorized.

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Fig. 2. Transverse sections (inversed radiographs) of vertebrae showing slight (on the left, bone compactness (BC) = 38%) and important (on the right, BC = 27%) bone resorptions. Measurements (bone area, total vertebra area and notochord area) were obtained using Bone profiler software 3.23 designed for 2D bone tissue analyses (Girondot and Laurin, 2003). BC values (BC, %) were calculated as indicated in the figure.

Fig. 3. Percentage of affected trout (mean ± S.D.) in function to increasing number of total abnormal vertebrae (i.e., including incompletely and completely fused vertebrae and malformed vertebrae) in the 20 fish farms sampled.

2.3. Measurements of bone quality parameters

varies from a cross-section to another and does not contain mineralized tissues. Vertebral BC (%) was calculated as [bone area / (total vertebra area − notochord area)] × 100 (Fig. 2).

Soft tissues were removed from spines and bones. In each trout, seven adjacent vertebrae from the middle region of the axial skeleton (V32 to 38) were selected, as they possess the largest bone volume (Kacem et al., 1998). The vertebrae were dehydrated in a graded series of ethanol, and lipids were removed in acetone, then in trichloroethylene. Four vertebrae (V32-33, V37-38) were pooled, dried for 72 h at 37 °C and weighed (Wdry) at the nearest mg. They were incinerated for 8 h at 800 °C, the ashes weighed (Wash) at the nearest mg, and vertebral bone mineralization (BM, %) calculated: (Wash / Wdry) × 100. Three adjacent vertebrae (V34-36) were embedded in resin (98% stratyl, 2% Luperox catalysor) and sectioned into 125 ± 10 μm transverse sections using a Leitz 1600 Saw Microtome. The single section through the mid-region of the vertebrae, in which the notochord canal is the narrowest and the bone tissue area the largest, was retained for bone compactness (BC) analysis. The sections were radiographed using a CGR Sigma 2060 generator, adjusted to 8 kV and 6 mA, on a Kodak Industrex film Ready Pack set at 30 cm of the source. The enlarged radiographs (35×) were digitized using an Olympus Camedia digital camera mounted on an Olympus SZX12 binocular microscope. The pictures were transformed into binary images with Adobe Photoshop 7.0 software, using a threshold, which took into consideration the bone present in the entire section thickness (Fig. 2). Bone area, total vertebra area and notochord area were obtained from binary images using Bone Profiler 3.23 software designed for bone tissue analysis (Girondot and Laurin, 2003). When calculating vertebral BC, the notochord area was always subtracted, as this central region of the vertebral section

Table 2 Mean vertebral bone condition parameters (bone mineralization, BM; bone compactness, BC) in reared rainbow trout affected or not by abnormal vertebrae (i.e., incompletely or completely fused vertebrae and malformed vertebrae) Total

Number of individuals BM (%) BC (%)

373 54.6 (3.3) 28. 1 (5.2)

Number of abnormal vertebrae 0

1–5

6–10

1–20

291 54.2 (3.5) 28.2 (5.2)

54 54.3 (3.3) 28.6 (5.2)

20 54.4 (2.9) 27.8 (6.6)

8 55.0 (3.3) 28.9 (5.3)

2.4. Statistics BM and BC are percentages; therefore, prior to statistical analyses, an arcsine transformation was performed to ensure normality, using the formula: p′ = arcsin√p × 100 (Zar, 1999). However, to allow comparisons, BM and BC are expressed in their initial values (mean ± standard deviation (%); Tables 1 and 2) rather than in p′ values. Bone quality parameters p′(BM) and p′(BC) in lots were statistically evaluated using one-way analysis of variance (ANOVA). Simple regression analyses were also performed to put in relation the bone conditions parameters (BM and BC) and the number of abnormal vertebrae. Analyses were realized taking all trout together, and normal and affected trout separately. All statistics were performed using JMP™ Statistical Software version 5.1 with a significance level (α) of 5%. 3. Results 3.1. Vertebral abnormalities Out of the 373 trout analyzed in this study, 82 (22%) possessed at least one discreet abnormal vertebra (Table 1; Fig. 3). The occurrence of affected fish ranged from 0 to 55% depending on the fish farm, and the number of abnormal vertebrae ranged from 0 to 20 (Fig. 3). The individual average of abnormal vertebrae was comprised between 0 and 3.9 ± 4.7 per lot, with a mean overall value of 1.1 ± 2.7 (Table 1). When looking to affected trout only, the individual average of abnormal vertebrae ranged from 2.0 ± 0.0 to 7.8 ± 4.1 per lot, with a mean overall value of 4.9 ± 3.6 (Table 1). Only a few trout showed more than 8 abnormal vertebrae (Fig. 3). Lots with the highest number of affected individuals were not always those having the highest value in the individual average of abnormal vertebrae, and conversely (Table 1). For instance, lot 1 and lot 7 had 50 and 55% of affected individuals, while individual average of abnormal vertebrae of affected trout was of 7.8 and 4.8, respectively. Fused vertebrae represented 77% of the total vertebral abnormalities. Vertebral abnormalities were not randomly distributed along the axial skeleton (Fig. 4). Using the morpho-functional division suggested by Ramzu and Meunier (1999) and Meunier and Ramzu (2006), vertebral abnormalities were mostly found in the post-cranial region

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Fig. 4. Schematic drawing of rainbow trout skeleton showing the vertebral regionalization defined by e.g., Ramzu and Meunier (1999) and Meunier and Ramzu (2006) and frequency (%) of discreet vertebral abnormalities observed along the axial skeleton. Our results (black histograms) are compared to those obtained by Kacem et al. (2004) from a single fish farm (grey histograms).

(i.e., the first truncal vertebrae) and in the ural regions (i.e., the last caudal vertebrae), and only a few abnormal vertebrae were found in the middle region of the axial skeleton (Fig. 4). 3.2. Bone condition parameters Taking all trout together (N = 373), the two bone condition parameters, BM (p b 0.0001) and BC (p b 0.0001), varied significantly among the 20 fish farms sampled and, sometimes, large differences were observed between lots (Table 1). These trends were not affected when analyzing the bone condition parameters in normal and affected trout separately (Table 1). Indeed, the mean BM and mean BC were not significantly different in trout having a larger number of abnormal vertebrae compared to less affected individuals (Table 2). Therefore, in the following, we present the results obtained in all trout taken together. Vertebral BM varied between 35.8 and 63.9%, with a mean value of 54.6% and a coefficient of variation of 6.0%. When considering lots, the mean BM ranged from 51.3 ± 1.2% to 58.6 ± 0.8%. If one considers the mean BM (54.6%) as a possible “normal value” for vertebral bone, trout had a vertebral BM below this level in 40% of the fish farms sampled (Table 1: lots 2, 7, 8, 9, 10, 11, 12 and 16). Vertebral BC ranged from 15.6 to 46.0% when considering all individuals, with a mean value of 28.1% and a coefficient of variation of 18.6%. Among lots, the mean BC varied from 23.6 ± 4.3% to 34.3 ± 5.9%. If one considers the mean BC (28.1%) of all individuals as a possible “normal value” for the vertebral bone in reared trout, in 55% of the fish farms sampled trout had a vertebral BC below this level (Table 2: lots 1, 2, 3, 5, 6, 8, 10, 12, 16, 17 and 20). 3.3. Vertebral abnormalities and bone condition parameters Plotting BM against BC for all trout taken together (N = 373) or for normal and affected trout separately did not reveal any significant relationship between these two parameters. No significant correlation

was found between BM and BC, and either the individual average of abnormal vertebrae or the occurrence of affected individuals within lots (Tables 1 and 2). Interestingly, however, lots having affected trout with a high number of abnormal vertebrae (i.e., above the mean of all affected individuals, e.g., ≥5) had also a low vertebral BC, suggesting that a decrease of BC could favor the appearance of discreet vertebral abnormalities. 4. Discussion To our knowledge, this is the first study assessing “discreet” vertebral abnormalities and vertebral bone condition of reared trout from a large sampling (20 fish farms, 373 individuals). 4.1. Occurrence of vertebral abnormalities As reported in other studies (Loy et al., 2000; Sfakianakis et al., 2006), ‘abnormal vertebrae’ is a too vague description. Indeed, vertebral abnormalities can range from a few over-mineralized but normally-shaped vertebrae, to severe malformations and fusions involving a high number of vertebrae. In addition, given the number of phenotypes observed in market-size trout, abnormalities are probably the result of different factors that could have play a role at various periods of ontogeny. Therefore, the current terminology must be improved and we need more accurate and standardized definitions in the near future to describe all types of vertebral abnormalities in reared fish. This is particularly important to discriminate among “discreetly”-expressed bone abnormalities as some can be related to inappropriate conditions during development periods and other to factors acting during growth (Sfakianakis et al., 2006; Witten et al., 2006). Producers generally discard malformed trout during growth to minimize feeding cost. As a consequence, assessment of vertebral abnormalities cannot be based on a few specimens displaying anatomical divergences from the normal type. In addition, in individuals showing

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obvious external deformation, bone quality parameters can be severely affected, precluding good comparisons (Kacem et al., 2004; Helland et al., 2005, 2006). For these reasons, the vertebral abnormalities considered in our study were only discreet abnormalities [i.e., which are not yet revealed by external morphological changes and, therefore, do not present any problem for market-size trout. However, discreet vertebral abnormalities can lead to some troubles when filleting large individuals or could result in external deformities if they are aggravated during further growth (Witten et al. (2006)]. This is why the occurrence of severe vertebral abnormalities was probably underestimated when neglecting externally-deformed trout. It is, therefore, somewhat surprising that not only the occurrence of discreet vertebral abnormalities was high (22%), compared to the 2–3% observed in wild salmonids (Gill and Fisk, 1966; Poynton, 1987), but also these abnormalities were widely spread among the 20 fish farms studied. We found only a single fish farm with no affected trout while the number of abnormal specimens reached 55% in some lots. High percentages of deformed individuals were reported in several reared species such as, e. g., 15–50% in Sparus aurata, the gilthead sea bream (Balbelona et al., 1993; Koumoundouros et al., 1997), 34–90% in Dicentrarchus labrax, the European sea bass (Barahona-Fernandes, 1982; Boglione et al., 1994), 100 to 500 times the values observed for wild specimens in Plecoglossus sp., the ayu (Komada, 1980), 1.5 to 25.2% in Salmo salar, the Atlantic salmon (Gjerde et al., 2004; Kause et al., 2005). In this latter species, the estimation of vertebral malformations based on external criteria revealed to be largely underestimated: 1.5% specimens showed external deformities while 7% of specimens were affected by discreet vertebral abnormalities (Fjelldal et al., 2005). This means that a high percentage of individuals housing discreet vertebral abnormalities, which potentially could lead to external deformities (Witten et al., 2006), do exist in these species. The number of affected individuals cannot be used for fish farm comparison, as the individual average of abnormal vertebrae varied greatly among lots and within lots. However, in some fish farms such a high percentage of trout with abnormal vertebrae could represent a serious problem if one considers that this phenomenon reflects inappropriate environmental conditions and could lead to deformed trout in subsequent growth. Nevertheless, the high variability in the number of abnormal trout within each fish farm, although specimens being from a same origin is interesting. Indeed, this strongly suggests different susceptibilities of individuals to environmental conditions. We wonder whether selecting individuals that do not develop abnormal vertebrae (while congeners in the same lot are affected) would not be an efficient approach toward a reduction of the prevalence of vertebral abnormalities in fish farms. The occurrence of discreet vertebral abnormalities reported in our study was well beneath the percentage reported in rainbow trout by Kacem et al. (2004) (see Fig. 4). The difference could be due to the material analyzed in both studies; a few specimens from a single fish farm and the analysis of trout displaying external malformations only in the latter, versus 373 trout from 20 farms and discreet abnormalities analyzed in our study. We found that a high percentage of discreet vertebral abnormalities only were preferentially encountered in post-cranial (i.e., first truncal vertebrae) and ural regions (i.e., last caudal vertebrae) of the vertebral column. Kacem et al. (2004) found also a high number of vertebral abnormalities in median regions of deformed trout. They suggested that the vertebrae located in the median region develop severe abnormalities because they sustain strong muscles inherent to the subcanrangiform swimming of trout (Meunier and Ramzu, 2006). Our study reveals that this previous finding should not be generalized to all fish farms of rainbow trout, and points to the necessity of comparing trout from several fish farms. Kacem et al. (2004) reported a phenomenon occurring in a particular fish farm, and we can also suspect that the high number of abnormal vertebrae in the median regions is more related to the selection of

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deformed specimens rather than representative of the general condition in fish farms. 4.2. Vertebral bone condition parameters In 40% and 55% of the fish farms studied, trout displayed vertebrae with low BM (b54.6%) and low BC (b28.1%) values, respectively. In these fish farms, the mean BM value was below the 57.3–60.2% reported by Casadevall et al. (1990) for rainbow trout (but only 3 specimens examined) and above the mean value (52.7%) reported for a single farm by Kacem et al. (2004). The mean BC values were far below the values (37–40%) reported by Kacem et al. (2004). This could suggest that important structural changes related to an extended resorption of the vertebral body, as reported first by Kacem et al. (2004), are a generalized phenomenon in reared trout. However, the structural changes occurring during trout vertebra growth cannot be accurately described using the mean BC value only. Indeed, vertebrae showing weak resorption but narrow trabeculae can have BC values similar to that of vertebrae with extended resorption but thick trabeculae. Therefore, changes in the vertebra structure resulting from bone remodeling (bone resorption and bone deposition) cannot be accurately described. In addition, the measures of BC could vary depending on the operator and the software used. This is why we have improved the characterization of these extended vertebral resorptions using a new model (Deschamps et al., unpublished data). In reared fish, vertebral bone condition assessed here using BM and BC is related to calcium and, mostly to phosphorus metabolism, as these ions are directly involved in the development and maintenance of skeleton mineralization through the formation of hydroxyapatite crystals (for a review, see Francillon-Vieillot et al., 1990). Bone remodeling involves bone matrix formation (assessed using BC) and mineralization (assessed using BM) followed by a resorption process which occurs mostly through osteoclast activity (Sire et al., 1990), but demineralization can also be mediated by osteocytes in teleosts possessing cellular bone, as salmonids and eels (i.e., a periosteocytic osteolysis; Lopez, 1973) and/or be diffuse (i.e., the so-called halastasy; Lopez, 1973). Taken together these processes allow calcium and phosphorus mobilization from the skeleton to fulfill various demands, principally coming from physiological processes: e.g., osmo-regulation, muscular activity, and reproduction (Fleming, 1996; Kacem et al., 1998, 2000, Kacem and Meunier, 2000; Witten and Hall, 2003). Mineral imbalance induced by insufficient ion uptake from the food or disadvantageous environmental conditions disturbing animal physiology leads generally to a low vertebral bone condition (i.e., poorly mineralized; review in Sugiura et al., 2004), although hypermineralized vertebrae were also reported (Helland et al., 2005, 2006; Kranenbarg et al., 2005a,b). In salmonids, during intensive growth dietary uptake and absorption of minerals from the surrounding water may fail to fulfill body requirements long before detrimental effects are detected on skeletal growth (Lall, 2002; Helland et al., 2005; Kaushik, 2005). Indeed, mineral intake allowing optimal bone mineralization is generally higher than that required for growth. This means that in several fish farms studied here the rearing conditions were not sufficient to sustain an optimal vertebral bone condition in rainbow trout. From now on, our study brings a clear idea of the range of variation of two vertebral bone parameters in reared rainbow trout, enabling comparisons in the next future. 4.3. Relations between vertebral abnormalities and bone quality Our preliminary observations of vertebral bone quality in reared trout (data not shown) had raised the question of a possible relationship between BM and BC. In brown and rainbow trout, Paxton et al. (2006) observed that the first post-cranial vertebrae were more mineralized than vertebrae located in the trunk region. As the structure of the former is different from that of the latter, the authors

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suggested that such an increase of BM (resulting probably from increased mechanical stress) could compensate the decrease in “stiffness” observed in vertebral geometry (decrease in BC?). These previous findings lead us to the following hypothesis: BM increase could compensate for BC decrease as a possible mean to maintain the resistance of the vertebrae to mechanical constraints generated by muscle forces but resulting eventually in a more fragile vertebral bone, which could predispose individuals to vertebral abnormalities. However, our results do not support such hypothesis of compensation between the two parameters, at least in the vertebral region (V32-38) analyzed in our study. Moreover, trout showing discreet vertebral abnormalities did not show a particular variation of the vertebral bone condition parameters (either low or high values). This could be explained by a wide and variable plastic response of BM and BC to rearing conditions. In fact, numerous factors have been already evocated as possibly favoring the appearance of vertebral abnormalities and/or acting on bone condition parameters in fish farms: genetics (McKay and Gjerbe, 1986; Gjerde et al., 2004; Kause et al., 2005; Takle et al., 2005; Tiira et al., 2006; Villeneuve et al., 2006) and ploidy (Sadler et al., 2001), inadequate light or temperature (Jensen et al., 1989; Poxton, 1991; Baeverfjord et al., 1998, 1999; Gabillard et al., 2003; Fjelldal et al., 2004; Sfakianakis et al., 2004; Finn, 2007), water acidity (Helland et al., 2005; Gil Martens et al., 2006), water flow intensity (Kihara et al., 2002; Sfakianakis et al., 2006), non-inflation of the swimbladder (Chatain, 1994), pathologic events (Madsen et al., 2001) and nutrition imbalance (review in Cahu et al., 2003). Several handling factors (e.g., transfer of eggs and/or fry from one farm to another, crowding, netting, draining, noise, etc.; review in Schreck et al., 2001) can also have detrimental effects on axial skeleton mineralization and can favor the appearance of vertebral abnormalities. Mechanical injuries and also stress can disturb mineral balance in salmonids, via regulatory hormones (cortisol and stanniocalcin; Pierson et al., 2004) that control bone resorption (Björnsson et al., 1987; Flik and Perry, 1989) and gill function (Gomez et al., 1997; Sbaihi, 2001; Pierson et al., 2004; Jentoft et al., 2005). Unfortunately, in the absence of experimental data, we are not able to identify factors responsible for the changes in the vertebral bone condition and, eventually, resulting in the appearance of discreet abnormalities. Indeed, a number of factors can lead to similar symptoms, they have frequently synergistic effects, and they could act during a wide span of the developmental period (Koumoundouros et al., 2002). Further work is needed to study the relationship between the rearing conditions and bone condition in reared trout. Improving rearing conditions in general should be considered not only in term of bone quality but also with regard to the increasing society demands concerning animal welfare (FSBI, 2002; Chandroo et al., 2004). 5. Conclusion Despite the current improvement of rearing conditions of trout in fish farms, vertebral abnormalities are still a matter of concern for producers, and especially discreet abnormalities, which cannot be detected during production by external examination only. Assessment of vertebral bone condition in fish farms turns out to be a precious mean to improve our knowledge on fish bone physiology. At this time, no relationship was found between the two bone condition parameters (BM and BC) and the occurrence of discreet vertebral abnormalities. This highlights the necessity to perform well-defined experimental studies aiming to understand the influence of the numerous rearing conditions on bone condition and vertebral anomalies. Notwithstanding, our study brings a clear idea on the range of variation of vertebral bone condition parameters and of discreet vertebral abnormalities in reared rainbow trout, enabling comparisons in the future.

Acknowledgements We are grateful to the producers for sending us trout and for their efforts in collecting data of rearing conditions. We would like to thank P. LeGal (Salmodis, Rungis, France) for receipt and keeping sampled trout for us, and C. Deshayes and M. Levadoux (CIPA), and P. Girard (independent veterinary) for providing trout samples from some farms, P. Rault (SYSAAF) for fruitful discussions. M. Hautecoeur (Muséum national d'Histoire naturelle) provided her expertise for whole fish radiographs. This research was funded by Office national interprofessionnel des produits de la mer et de l'aquaculture (OFIMER, contract No. 050/04/C), the European Union (IFOP/DPMA, contract No. 2005/010) and the Comité Interprofessionnel des Produits de l'Aquaculture (CIPA). References Baeverfjord, G., Lein, I., Aasgaard, T., Rye, M., Storset, A., 1998. High temperature during egg incubation may induce malformations in Atlantic salmon (Salmo salar). In: Publication, E.A.S.S. (Ed.), Aquaculture and Water, pp. 24–25. Baeverfjord, G., Lein, I., Aasgaard, T., Rye, M., Storset, A., Siikavuopio, S.I., 1999. Vertebral deformations induced by high temperatures during embryogenesis in Atlantic salmon. In: Publication, E.A.S. (Ed.), Towards Predictable Quality, pp. 6–7. Balbelona, M.C., Morinigo, M.A., Andrades, J.A., Santamaria, J.A., Becerra, J., Borrero, J.J., 1993. Microbiological study of gilthead sea bream (S. aurata L.) affected by lordosis (a skeletal deformity). Bull. Eur. Assoc. Fish Pathol. 13, 33. Barahona-Fernandez, M.H., 1982. Body deformation in hatchery reared European sea bass Dicentrarchus labrax (L.). Types, prevalence and effect on fish survival. J. Fish Biol. 21, 239–249. Björnsson, B.T., Yamauchi, M., Nishioka, R.S., Deftos, L.J., Bern, H.A., 1987. Effects of hypophysectomy and subsequent hormonal replacement therapy on hormonal and osmoregulatory status of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 68, 421–430. Boglione, C., Gagliardi, F., Scardi, M., Finoia, M.G., Cadaudella, S., 1994. Anatomical aspects for seed quality assessment in sea bass (D. labrax): hatchery and wild populations. In: Kestemont, P., Sevila, F., Williot, P. (Eds.), Measures for Sucess. Cemagref-Edition. Boglione, C., Gagliardi, F., Scardi, M., Cataudella, S., 2001. Skeletal descriptors and quality assessment in larvae and post-larval of wild-caught and hatchery-reared gilthead sea bream (Sparus aurata L. 1758). Aquaculture 192, 1–22. Cahu, C.L., Zambonito Infante, J.L., Takeuchi, T., 2003. Nutritional components affecting skeletal development in fish larvae. Aquaculture 227, 254–258. Carragher, J.F., Sumpter, J.P., 1991. The mobilization of calcium from calcified tissues of rainbow trout (Oncorhynchus mykiss) induced to synthesize vitellogenin. Comp. Biochem. Physiol. 99, 169–172. Casadevall, M., Casinos, A., Viladiu, C., Ontanon, M., 1990. Scaling of skeletal mass and mineral content in teleosts. Zool. Anz. 225, 144–150. Chandroo, K.P., Duncan, I.J.H., Moccia, R.D., 2004. Can fish suffer?: perspectives on sentence, pain, fear and stress. Appl. Anim. Behav. Sci. 86, 225–250. Chatain, B., 1994. Abnormal swimbladder development and lordosis in sea bass (Dicentrarchus labrax) and sea bream (Sparus auratus). Aquaculture, 119, 371–379. FEAP, 2006. European Production. . Available on ‘Federation of European Aquaculture Producers’ website: http://www.aquamedia.org/production/default_en.asp. Finn, R.N., 2007. The physiology and toxicology of salmonid eggs and larvae in relation to water quality criteria. Aquat. Toxicol. 81, 337–354. Fjelldal, G., Grotmol, S., Kryvi, H., Gjerdet, N.R., Taranger, G.L., Hansen, T., Porter, M.J.R., Totland, G.K., 2004. Pinealectomy induces malformation of the spine and reduces the mechanical strength of the vertebrae in Atlantic salmon, Salmo salar. J. Pineal Res. 36, 132–139. Fjelldal, P.G., Nordgarden, U., Berg, A., Grotmol, S., Totland, G.K., Wargelius, A., Hansen, T., 2005. Vertebrae of the trunk and tail display different growth rates in response to photoperiod in Atlantic salmon, Salmo salar L., post-smolts. Aquaculture 250, 516–524. Fjelldal, P.G., Lock, E.-J., Grotmol, S., Totland, G.K., Nordgarden, U., Flik, G., Hansen, T., 2006. Impact of smolt production strategy on vertebral growth and mineralization during smoltification and the early seawater phase in Atlantic salmon (Salmo salar, L.). Aquaculture 261, 715–728. Fleming, I.A., 1996. Reproductive strategies of Atlantic salmon: ecology and evolution. Rev. Fish Biol. Fish. 6, 379–416. Flik, G., Perry, S.F., 1989. Cortisol stimulates whole body calcium uptake and the branchial calcium pump in freshwater rainbow trout. J. Endocrinol. 120, 75–82. Francillon-Vieillot, H., de Buffrénil, V., Castanet, J., Géraudie, J., Meunier, F.J., Sire, J.-Y., Zylberberg, L., de Ricqlès, A., 1990. Microstructure and mineralization of vertebrate skeletal tissues. In: Carter, J.G. (Ed.), Skeletal Biomineralization: Patterns, Processes and Evolutionary trends. Van Nostrand Reinhold, New York, pp. 471–530. FSBI, 2002. Fish Welfare. Briefing 2. Fisheries Society of the British Isles, Cambridge, pp. 1–25. Gabillard, J.C., Weil, C., Rescan, P.Y., Navarro, I., Gutiérrez, J., Le Bail, P.Y., 2003. Environmental temperature increases plasma GH levels independently of nutritional status in rainbow trout (Oncorhynchus mykiss). Gen. Comp. Endocrinol. 133, 17–26.

Author's personal copy M.-H. Deschamps et al. / Aquaculture 279 (2008) 11–17 Gjerde, B., Pante, M.J.R., Baeverfjord, G., 2004. Genetic variation for vertebral deformity in Atlantic salmon (Salmo salar). Aquaculture 244, 77–87. Gil Martens, L., Witten, P.E., Fivelstad, S., Huysseune, A., Sævareid, B., Vikeså, V., Obach, A., 2006. Impact of high water carbon dioxide levels on Atlantic salmon smolts (Salmo salar L.): effects on fish performance, vertebrae composition and structure. Aquaculture 261, 80–88. Gill, C.D., Fisk, D.M., 1966. Vertebral abnormalities in Sockeye, Pink, and Chum Salmon. Trans. Am. Fish. Soc. 95, 177–182. Girondot, M., Laurin, M., 2003. Bone Profiler: a tool to quantify, model and statistically compare bone section compactness profile. J. Vertebr. Paleontol. 23, 458–461. Gomez, J.M., Boujard, T., Boeuf, G., Solari, A., Le Bail, P.Y., 1997. Individual diurnal plasma profiles of thyroid hormones in rainbow trout (Oncorhynchus mykiss) in relation to cortisol, growth hormone and growth rate. Gen. Comp. Endocrinol. 107, 74–83. Haffray, P., Pincent, C., Rault, P., Coudurier, B., 2004. Domestication et amélioration génétique des cheptels piscicoles français dans le cadre du SYSAAF. INRA Prod. Anim. 17, 243–252. Helland, S., Refstie, S., Espmark, A., Hjelde, K., Baeverfjord, G., 2005. Mineral balance and bone formation in fast-growing Atlantic salmon parr (Salmo salar) in response to dissolved metabolic carbon dioxide and restricted dietary phosphorus supply. Aquaculture 250, 364–376. Helland, S., Denstadli, V., Witten, P.E., Hjelde, K., Storebakken, T., Skrede, A., Åsgård, T., Baeverfjord, G., 2006. Hyper dense vertebrae and mineral content in Atlantic salmon (Salmo salar L.) fed diets with graded levels of phytic acid. Aquaculture 261, 603–614. Jensen, A.J., Johnsen, B.O., Saksgard, L., 1989. Temperature requirements in Atlantic salmon (Salmo salar), brown trout (Salmo trutta), and artic char (Salvelinus alpinus) from hatching to initial feeding compared with geographic distribution. Can. J. Fish. Aquat. Sci. 46, 786–789. Jentoft, S., Aastveit, A.H., Torjesen, P.A., Andersen, O., 2005. Effects of stress on growth, cortisol and glucose levels in non-domesticated Eurasian perch (Perca fluviatilis) and domesticated rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol., A 141, 353–358. Kacem, A., Meunier, F.J., Baglinière, J.L., 1998. A quantitative study of morphological and histological changes in the skeleton of Salmo salar during its anadromous migration. J. Fish Biol. 53, 1096–1109. Kacem, A., Gustafsson, J.-A., Meunier, F.J., 2000. Demineralization of the skeleton in Atlantic salmon Salmo salar L. during spawning migration. Comp. Biochem. Physiol., A 125, 479–484. Kacem, A., Meunier, F.J., 2000. Mise en évidence de l'ostéolyse périostéocytaire vertébrale chez le saumon atlantique Salmo salar (Salmonidae, Teleostei) au cours de sa migration anadrome. Cybium 24, 105–112. Kacem, A., Meunier, F.J., Aubin, J., Haffray, P., 2004. Caractérisation histo-morphologique des malformations du squelette vertébral chez la truite arc-en-ciel (Oncorhynchus mykiss) après différents traitements de triploïdisation. Cybium 28, 15–23. Kause, A., Ritola, O., Paananen, T., Wahlroos, H., Mäntysaari, E., 2005. Genetic trends in growth, sexual maturity and skeletal malformations, and rate of inbreeding in a breeding programme for rainbow trout (Oncorhynchus mykiss). Aquaculture 247, 177–187. Kaushik, S.-J., 2005. Besoins et apports en phosphore chez les poissons. INRA Prod. Anim. 18, 203–208. Kihara, M., Ogata, S., Noriaki, K., Kubota, I., Yamaguchi, R., 2002. Lordosis induction in juvenile red sea bream, Pagrus major, by high swimming activity. Aquaculture 212, 149–158. Komada, N., 1980. Incidence in gross malformations and vertebral abnormalities of natural and hatchery Plecoglossus altivelis. Copeia 1, 29–35. Koumoundouros, G., Gagliardi, F., Divanach, P., Boglione, C., Cataudella, S., Kentouri, M., 1997. Normal and abnormal osteological development of caudal fin in Sarus aurata L. fry. Aquaculture 149, 215–226. Koumoundouros, G., Divanach, P., Kentouri, M., 2001. The effect of rearing conditions on the development of saddleback syndrome and caudal fin deformities in Dentex dentex (L.). Aquaculture 200, 285–304. Koumoundouros, G., Maingot, E., Divanach, P., Kentouri, M., 2002. Kyphosis in reared sea bass (Dicentrarchus labrax L.): ontogeny and effects on mortality. Aquaculture 209, 49–58. Kranenbarg, S., van Cleynenbreugel, T., Schipper, H., van Leeuwen, J.L., 2005a. Adaptative bone formation in acellular vertebrae of sea bass (Dicentrarchus labrax L.). J. Exp. Biol. 208, 3493–3502. Kranenbarg, S., Waarsing, J.H., Muller, M., Weinans, H., van Leeuwen, J.L., 2005b. Lordotic vertebrae in sea bass (Dicentrarchus labrax L.) are adapted to increased loads. J. Biomech. 38, 1239–1246. Lall, S.P., 2002. The minerals, In: Halver, J.E., Hardy, R.W. (Eds.), Fish nutrition, third edition. Elesevier, San Diego, pp. 259–308. Lopez, E., 1973. Étude morphologique et physiologique de l'os cellulaire des poissons téléostéens. Mem. Mus. Natl. Hist. Nat. 80, 1–90. Loy, A., Boglione, C., Gagliardi, F., Ferrucci, L., Cataudella, S., 2000. Geometric morphometrics and internal anatomy in sea bass shape analysis (Dicentrarchus labrax L., Moronidae). Aquaculture 186, 33–44. Madsen, L., Arnbjerg, J., Dalsgaard, I., 2001. Radiological examination of the spinal column in farmed rainbow trout Oncorhynchus mykiss (Walbaum): experiments with Flavobacterium psychrophilum and oxytetracycline. Aquac. Res. 32, 235–241.

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McKay, L.R., Gjerbe, B., 1986. Genetic variation for spinal deformity in Atlantic salmon, Salmo salar. Aquaculture 52, 263–272. Meunier, F.J., François, Y., 1992. La croissance du squelette chez les téléostéens. Ann. Biol. 31, 169–219. Meunier, F.J., Ramzu, M.-Y., 2006. La régionalisation morphofonctionnelle de l'axe vertébral chez les téléostéens en relation avec le mode de nage. C. R. Palevol. 5, 499–507. Paxton, H., Bonser, R.H.C., Winwood, K., 2006. Regional variation in the microhardness and mineralization of vertebrae from brown and rainbow trout. J. Fish Biol. 68, 481–487. Persson, P., Sundell, K., Björnsson, B.T., 1994. Estradiol-17b-induced calcium uptake and resorption in juvenile rainbow trout Oncorhynchus mykiss. Fish Physiol. Biochem. 13, 379–386. Persson, P., 1997. Calcium Regulation During Sexual Maturation of Female Salmonids: Estradiol-17b and Calcified Tissues, Department of Zoophysiology. Göteborg University, Göteborg. Pierson, P.M., Lamers, A., Flik, G., Mayer-Gostan, N., 2004. The stress axis, stanniocalcin, and ion balance in rainbow trout. Gen. Comp. Endocrinol. 137, 263–271. Poxton, M.G., 1991. Incubation of salmon eggs and rearing of alevins: natural temperature fluctuations and their influence on hatchery requirements. Aquac. Eng. 10, 31–53. Poynton, S.L., 1987. Vertebral column abnormalities in brown trout, Salmo trutta L. J. Fish. Dis. 10, 53–57. Ramzu, M., Meunier, F.J., 1999. Descripteurs morphologiques de la zonation de la colonne vertébrale chez la truite arc-en-ciel Oncorhynchus mykiss (Walbaum, 1792) (Teleostei, Salmoniforme). Ann. Sci. Nat. 3, 87–97. Sadler, J., Pankhurst, P.M., King, H.R., 2001. High prevalence of skeletal deformity and reduced gill surface area in triploid Atlantic salmon (Salmo salar L.). Aquaculture 198, 369–386. Sbaihi, M., 2001. Interaction des stéroïdes sexuels et du cortisol dans le contrôle de la reproduction et du métabolisme calcique chez un téléostéen migrateur, l'anguille (Anguilla anguilla L.), Département de physiologie de la reproduction. Université Pierre et Marie Curie, Paris, p. 214. Schreck, C.B., Contreras-Sanchez, W., Fitzpatrick, M.S., 2001. Effects of stress on fish reproduction, gamete quality, and progeny. Aquaculture 197, 3–24. Sfakianakis, D.G., Koumoundouros, G., Divanach, P., Kentouri, M., 2004. Osteological development of the vertebral column and of the fins in Pagellus erythrinus (L. 1758). Temperature effect on the developmental plasticity and morpho-anatomical abnormalities. Aquaculture 232, 407–424. Sfakianakis, D.G., Georgakopoulou, E., Papadakis, I.E., Divanach, P., Kentouri, M., Koumoundouros, G., 2006. Environmental determinants of haemal lordosis in European sea bass, Dicentrarchus labrax (Linnaeus, 1758). Aquaculture 254, 54–64. Sire, J.-Y., Huysseune, A., Meunier, F.J., 1990. Osteoclasts in teleost fish: light- and electron-microscopical observations. Cell Tissues Res. 260, 85–94. Skonberg, D.I., Yogev, L., Hardy, R.W., Dong, F.M., 1997. Metabolic response to dietary phosphorus intake in rainbow trout (Oncorhynchus mykiss). Aquaculture 157, 11–24. Sugiura, S.H., Ferraris, R.P., 2004. Dietary phosphorus-responsive genes in the intestine, pyloric ceca, and kidney in rainbow trout. Am. J. Physiol., Regul. Integr. Comp. Physiol. 287, R541–R550. Takle, H., Baeverfjord, G., Lunde, M., Kolstad, K., Adersen, O., 2005. The effect of heat and cold exposure on HSP70 expression and development of deformities during embryogenesis of Atlantic salmon (Salmo salar). Aquaculture 249, 515–524. Tiira, K., Piironen, J., Primmer, C.R., 2006. Evidence for reduced genetic variation in severely deformed juvenile salmonids. Can. J. Fish. Aquat. Sci. 63, 2700–2707. Vielma, J., Lall, S.P., 1998. Phosphorus utilization by Atlantic salmon (Salmo salar) reared in freshwater is not influenced by higher dietary calcium intake. Aquaculture 160, 117–128. Villeneuve, L.A.N., Gisbert, E., Moriceau, J., Cahu, C.L., Zambonito Infante, J.L., 2006. Intake of high levels of vitamin A and polyunsaturated fatty acid during different developmental periods modifies the expression of morphogenesis genes in European sea bass (Dicentrarchus labrax). Brit. J. Nutr. 95, 677–687. Webb, P.W., 1975. Hydrodynamics and energetics of fish propulsion. Bull. Fish. Res. Board Can. 190, 1–159. Witten, P.E., Hall, B.K., 2003. Seasonal changes in the lower jaw skeleton in male Atlantic salmon (Salmo salar L.): remodeling and regression of the kype after spawning. J. Anat. 203, 435–450. Witten, P.E., Obach, A., Huysseune, A., Baeverfjord, G., 2006. Vertebrae fusion in Atlantic salmon (Salmo salar): development, aggravation and pathways of containment. Aquaculture 258, 164–172. Yamada, Y., Okamura, A., Tanaka, S., Utoh, T., Horie, N., Mikawa, N., 2001. The roles of bone and muscle as phosphorus reservoirs during the sexual maturation of female Japanese eels, Anguilla japonica Temminck and Schlegel (Anguilliformes). Fish Physiol. Biochem. 24, 327–334. Zar, J.H., 1999. Biostatistical Analysis, 4th edition. Simon & Schuster/A Viacom Company, Upper Saddle River. 663 p.