Cold-induced alterations on proximate composition and fatty acid profiles of several tissues in gilthead sea bream (Sparus aurata)

Aquaculture 249 (2005) 477 – 486 www.elsevier.com/locate/aqua-online

Cold-induced alterations on proximate composition and fatty acid profiles of several tissues in gilthead sea bream (Sparus aurata) A. Ibarza,T, J. Blascoa, M. Beltra´na, M.A. Gallardoa, J. Sa´ncheza, R. Salab, J. Ferna´ndez-Borra`sa a

Departament de Fisiologia (Centre de Refere`ncia de Recerca i Desenvolupament en Aqu¨icultura, Generalitat de Catalunya), Facultat de Biologia, Universitat de Barcelona, Avd. Diagonal 645, E-08027 Barcelona, Spain b Unitat de Nutricio´ Animal, Departament de Cie`ncia Animal i dels Aliments, Facultat de Veterina`ria, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Barcelona, Spain Received 16 September 2004; received in revised form 18 February 2005; accepted 19 February 2005

Abstract Cultured gilthead sea bream (Sparus aurata L.) are exposed to a multifactorial disease termed bwinter syndromeQ, and low temperatures are the recurrent inducing factor. The effects of low temperatures on tissue composition and fatty acid profiles of polar and non-polar lipid fractions of sea bream were studied in two conditions: a gradual temperature drop (GD: from 18 8C to 8 8C at a rate of 1 8Cd day 1) or two sharp drops (SD: from 18 8C to 12 8C and then from 12 8C to 8 8C completed within a day, with a 1 week interval). No significant differences were detected between GD and SD in any of the variables studied. Animals stopped feeding when the temperature fell below 13 8C, and the resulting fast provoked body weight losses of 12% (GD) and 10% (SD) at the end of the experiment (15th day), and decreased non-polar lipids content in muscle. Unsaturation of polar fatty acids in muscle (SD) and in gills (SD and GD) rose, demonstrating a homeoviscous acclimation of membranes to cold. In contrast, unsaturation of liver polar fatty acids did not rise, but liver showed a great increase in total lipid content on day 15 (+ 35% SD, and + 43% GD), caused by the increase in non-polar lipids, especially n-3 fatty acids. The high deposition of these essential fatty acids, mainly 22:6n-3, in fasting sea bream implied mobilization from extra-hepatic stores and caused dramatic modifications in the physical properties of the liver (larger, more friable and yellowish), often associated with winter disease. Moreover, plasma aspartate aminotransferase (ASAT) levels rose three-fold in the two conditions. No fish succumbed to winter disease, but the incipient appearance of symptoms was evident. The relationships between the changes in muscle, gill, and, especially liver, at low temperature and the clinical signs of dwinter diseaseT are discussed. D 2005 Elsevier B.V. All rights reserved. Keywords: Liver lipid; Fatty acids; Thermal rate; Unsaturation; Plasma ASAT; Winter disease

T Corresponding author. Tel.: +34 934021557; fax: +34 934110358. E-mail address: [email protected] (A. Ibarz). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.02.056

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1. Introduction In recent years, intensive gilthead sea bream farming has spread extensively along Mediterranean coasts. A pathological condition known as dwinter syndromeT or dwinter diseaseT appears among farmed fish during the cold season (Bovo et al., 1995; Doimi, 1996; Tort et al., 1998a). Many causes have been linked to this syndrome, including temperature, nutritional imbalance, immunodepression, infectious agents (Tort et al., 1998a; Sarusic, 1999; Gallardo et al., 2003); temperature being the most important. Winter disease causes chronic mortality during the coldest months and acute mortality episodes when temperatures rise to 14–15 8C in spring (Tort et al., 1998a; Sarusic, 1999). Its incidence varies from year to year and from place to place, but always produces important economic losses. Feeding reduces as temperatures fall (Tort et al., 1998a), stopping altogether below 13 8C (Sa´nchez et al., 1999; Ibarz et al., 2003; Sala-Rabanal et al., 2003). During such long periods of fasting imposed by low temperatures, nutritional deficiencies can occur. Combined with the stress associated with intensive management practices, this can induce a state of immunodepression in the animal (Tort et al., 1998a,b). With all these factors at play, fish become very sensitive to infection by opportunistic bacteria. Dome´nech et al., (1997, 1999) has observed the presence of Pseudomonas anguilliseptica on moribund or dead gilthead sea bream. Symptoms include necrosis of white muscle fibres, pale and friable liver, fatty degeneration of hepatocytes, distended digestive tract, and exocrine pancreas atrophy (Tort et al., 1998a; Galeotti et al., 1999; Contessi et al., 2000; Gallardo et al., 2003). Like other poikilotherms, fish respond to low temperatures by reducing their metabolism. However, some mechanisms that compensate for the effects of cold may become active: e.g. changes in cell protein content produce a compensatory enzymatic activity in green sunfish (Shaklee et al., 1977); and the hypertrophy of heart and liver aids cold acclimation in catfish (Kent et al., 1988). One of the most general reactions to environmental temperature decreases is a rise in the degree of unsaturation of body lipids (Hazel and Prosser, 1974) to compensate for membrane fluidity and permeability (Bell et al., 1986). This mechanism, known as dhomeoviscous adaptationT, has

been reviewed in fish by Hazel (1984). Dey et al. (1993) showed that the effects of temperature on lipid metabolism in liver phospholipids were identical for fish from different habitats. In gilthead sea bream, studies on fatty acids have concentrated on the importance of PUFA in dietary aspects, growth, larvae survival, and tissue composition (Koven et al., 1992; Ibeas et al., 1994, 1996, 1997, 2000; Robaina et al., 1995; Caballero et al., 1999). Aside from our own studies, we have found no information on the effects of cold on sea bream tissues and on lipid alterations. From a previous work (Gallardo et al., 2003), fish with symptoms of dwinter diseaseT showed greater lipid content in the liver and higher plasma ASAT activity than asymptomatic animals that were sampled 3 weeks earlier. In the present study we evaluate the short-term effects of lowering water temperature on liver and muscle composition in juvenile gilthead sea bream and compare them with the effects of winter disease. Specifically, we investigate the effects on lipid content and on fatty acid profiles of lipid fractions, correlating them with an adaptative cold response. As an indicator of liver injury, ASAT plasma activity was also analysed.

2. Materials and methods 2.1. Animals and experimental conditions The experiment was performed at the dCentre d’Aqu¨iculturaT (CA-IRTA, Sant Carles de la Ra`pita, Tarragona, Spain). Gilthead sea bream, obtained from a local fish farm, were transferred to 600 L cylindroconical tanks and acclimated over 15 days at 18 8C and an ambient 9.5:14.5 light:dark (November) photoperiod. The tanks, located indoors and supplied with aerated seawater (3.8%), were connected to a semiclosed system with solid and biological filters, ozone protein skimmer and were partially sterilised with UV. Water quality was controlled throughout the experimental period, maintaining nitrate, nitrite and ammonia concentrations, pH and salinity at initial values. Temperature changes were controlled manually by adjusting the cooling units (EA-2000, CUBIJEI, Bologna, Italy). Two groups of 25 sea bream, initial body weight 98.8 F 3.5 g (mean F S.E.M.), were used to study the

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effects of temperature drops at two different rates. A) Gradual drop (GD): water temperature was lowered from 18 8C to 8 8C, at a rate of approximately 1 8Cd day 1, and then maintained at 8 8C until day 16, the last day of the experiment. B) Two sharp drops of water temperature (SD): from 18 8C to 12 8C, and from 12 8C to 8 8C both completed within a day. After the first drop (day 1), water temperature was maintained at 12 8C for 1 week until a second drop (day 8) to 8 8C. Water temperature was then kept at 8 8C till the end of the experiment (day 16). Food rations equivalent to between 0.75% and 0.40% of body weight (decreasing with temperature over the experiment) were administered daily, but below 13 8C fish refused to feed. Food composition was: 47% protein, 21% lipids, 11% ash, 1.5% fibre and 19.8 MJd kg 1 digestible energy (ProAqua Nutricio´n, Duen˜as, Spain). The fatty acid composition of the extruded diet was analysed using the same procedure as fish samples. In percentage of total lipid, the fatty acid profile was: 14:0, 6.4%; 16:0, 19.4%; 18:0, 2.8%; 16:1(n-7), 6.7%; 18:1(n-9), 19.4%;

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18:2(n-6), 4.6%; 20:1(n-9), 11.6%; 20:5(n-3), 6.4%; 22:1(n-9), 14.3%; 22:6(n-3), 8.5%. It should be noted that over the course of the experiment, only one moribund fish was seen (group GD, day 14). This fish showed erratic movements at the water surface and was immediately sacrificed and analysed, its values treated independently. 2.2. Sampling procedure and analysis Analyses were made on 10 fish from each group at the end of the experimental period (day 16), and results compared with values from 5 fish from each group selected previous to the temperature changes (initial values). Body weights were individually determined and blood samples (approximately 1 mL) were taken from the caudal vessels using EDTA-Li as an anticoagulant. Plasmas were obtained with a centrifuge (5 min, 13 000  g at 4 8C) and kept at 80 8C until analysis. Animals were killed by severing their spinal cord and dissected. Liver weights were recorded and tissue samples of liver, gills, and an

Table 1 Effects of temperature decrease on body weight, ASAT activity, and liver and muscle composition Initial

SD

GD

98.83 F 3.55 a 1.48 F 0.11 1.51 F 0.11 a 13.53 F 0.84 a

86.57 F 3.03 b 1.74 F 0.15 1.88 F 0.14 b 37.43 F 8.49 b

88.80 F 3.10 b 1.73 F 0.08 2.12 F 0.11 b 34.76 F 3.95 b

Liver Water (mgd 100 mg 1 fw) Protein (mgd 100 mg 1 fw) Lipid (mgd 100 mg 1 fw) mg protein total liver mg lipid total liver mg non-polar lipids(2) mg polar lipids(2) mg lipidd mg 1 protein

64.7 F 0.8 12.0 F 0.2 10.8 F 0.9 179.8 F 11.8 155.8 F 8.7 a 105.0 F 5.6 a 50.8 F 4.0 0.89 F 0.06 a

66.7 F 1.0 11.4 F 0.2 13.8 F 1.0 198.9 F 14.9 209.9 F 23.2 b 171.8 F 26.0 b 38.6 F 2.9 1.20 F 0.10 b

65.5 F 0.9 11.3 F 0.4 13.7 F 1.1 202.0 F 8.9 222.6 F 22.5 b 182.6 F 9.4 b 45.6 F 6.0 1.23 F 0.14 b

White muscle Water (mgd 100 mg 1 fw) Protein (mgd 100 mg 1 fw) Lipid (mgd 100 mg 1 fw) Ag lipidd mg 1 protein Ag non-polard mg 1 protein(2) Ag polard mg 1 protein(2)

73.7 F 0.5 21.0 F 0.2 5.0 F 0.5 237.6 F 3.8 202.7 F 4.1 35.0 F 4.8

74.8 F 0.3 21.2 F 0.2 4.1 F 0.2 200.5 F 1.3 152.6 F 3.2 47.9 F 2.5

74.8 F 0.3 21.6 F 0.2 4.0 F 0.3 191.1 F 6.2 152.3 F 8.0 38.8 F 2.0

Body weight (g) Liver weight (g) HSI(1) ASAT (mU ASATd ml

1

plasma)

a a a a

a,b b b b

b b b a

Values are means F S.E.M. of fresh weight (fw). (1) Hepatosomatic index = 100 d [liver weight d body weight 1 ]. Number of fish was 10 (except (2) where N = 5). SD—sharp drop group. GD—gradual drop group. Group differences ( p b 0.05) are indicated with different letters.

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epaxial sample of white muscle for each fish were immediately frozen in liquid nitrogen and stored in the laboratory at 80 8C until analysis. Aspartate aminotransferase (ASAT, EC.2.6.1.1) activity in plasma was determined as described in Gallardo et al. (2003). The samples of liver, gill, and muscle tissue intended for proximate composition analyses were frozen in liquid nitrogen, powdered, and then divided into three subsamples for water, protein, and lipid analyses. Tissue water content was determined gravimetrically by drying one of these subsamples at 105 8C for 24 h. Protein content was estimated from elemental nitrogen, burning subsamples in an elemental analysis-gas chromatograph (EA 1108 CHNS-O, Carlo Erba Instruments, Milano, Italy). To convert elemental nitrogen to protein percentage a correction factor of 6.25 (g proteind g 1 nitrogen) was used. Total lipid content was measured gravimetrically after two extractions with chloroform/methanol (2:1, v/v) and purification according to the procedure used by Folch et al. (1957). Lipid extracts were dried on a

Rotavap (Bu¨chi rotavapor 4-114, Switzerland) and weighed. Crude lipid extracts were separated into polar and non-polar fractions by means of silica SepPak cartridges (Waters, Milford, MA) using chloroform, a mixture of chloroform/methanol (49:1, v/v) and methanol as solvent systems (Juaneda and Rockelin, 1985). All solvents contained 0.01% butylated hydroxytoluene (BHT) as antioxidant. Fatty acid methyl esters of each lipid fraction were prepared by acid-catalyzed transmethylation at 50 8C for 16 h (Christie, 1982) and were extracted and purified as described by Tocher and Harvie (1988). Methyl esters were analysed in a gas chromatograph (Carlo Erba GC 8000 Top) connected to a mass spectrometer (Fisons MD-800). The capillary column used was an HP-5MS 30 m  0.25 mm  0.25 Am using helium as a carrier gas and a biphasic thermal gradient from 80 8C to 300 8C. Individual fatty acid methyl esters were identified by comparison with known standards and quantified using a data processor (Masslab, Thermoquest). All data regarding fatty acids are presented as percentages of total fatty acid mass.

Table 2 Fatty acid composition of polar and non-polar lipids in white muscle Polar

Non-polar

Initial Fraction (%) 16:0 18:0 Total saturated 18:1 (n-9) 24:1 (n-9) 22:1 (n-9) 16:1 (n-7) Total n-9(+n7) 18:3 (n-3) 20:5 (n-3) 22:6 (n-3) 22:5 (n-3) n-3 HUFA Total n-3 18:2 (n-6) 20:4 (n-6) Total n-6 Ratios Unsat/sat EPA/DHA n-9/n-3 HUFA

14.66 F 1.87 20.17 F 0.15 8.15 F 0.22 29.72 F 0.38 12.59 F 0.22 0.65 F 0.03 0.38 F 0.13 1.68 F 0.07 15.29 F 0.33 2.54 F 0.06 9.61F0.16 25.99 F 0.31 2.70 F 0.07 38.23 F 0.23 40.77 F 0.25 9.85 F 0.33 2.06 F 0.04 11.93 F 0.31

SD a

a

a a a a

2.37 F 0.04 a 0.37 F 0.01 0.40 F 0.01 a

24.90 F 1.29 19.73 F 0.40 7.79 F 0.15 28.66 F 0.41 11.78 F 0.43 0.52 F 0.04 0.37 F 0.03 1.67 F 0.10 14.33 F 0.48 2.29 F 0.06 10.22 F 0.28 26.92 F 0.28 2.84 F 0.75 39.98 F 0.47 42.27 F 0.44 10.23 F 0.19 2.08 F 0.06 12.38 F 0.21

GD b

b

b a,b b b

2.53 F 0.03 b 0.38 F 0.01 0.36 F 0.02 b

20.43 F 1.59 20.30 F 0.37 8.15 F 0.17 29.58 F 0.27 11.83 F 0.21 0.45 F 0.02 0.31 F 0.03 1.42 F 0.03 14.02 F 0.18 2.17 F 0.07 9.53 F 0.33 28.18 F 0.62 2.52 F 0.15 40.22 F 0.43 42.40 F 0.38 9.48 F 0.39 2.10 F 0.03 11.63 F 0.39

b

b

b b b b

2.38 F 0.03 a 0.34 F 0.02 0.35 F 0.01 b

Initial

SD

85.34 F 1.87 a 17.94 F 0.60 3.75 F 0.06 27.26 F 0.65 22.64 F 1.33 0.78 F 0.04 a 4.11 F1.04 7.40 F 0.37 34.94 F 1.13 a 3.07 F 0.06 a 4.58 F 0.10 a 8.11 F 0.10 a 1.83 F 0.07 14.52 F 0.26 a 17.59 F 0.31 a 14.30 F 0.39 0.30 F 0.01 a 14.72 F 0.41

76.10 F 1.29 18.20 F 0.15 3.63 F 0.12 26.44 F 0.16 24.30 F 0.17 0.61 F 0.04 4.83 F 0.19 7.74 F 0.13 37.47 F 0.17 2.80 F 0.06 4.21 F 0.03 7.22 F 0.15 1.70 F 0.05 13.13 F 0.20 15.93 F 0.23 14.59 F 0.34 0.26 F 0.01 14.90 F 0.30

2.61 F 0.08 0.57 F 0.01 2.41 F 0.10 a

GD b

b

b b b b b b b

2.72 F 0.02 0.59 F 0.01 2.86 F 0.05 b

79.57 F 1.59 b 18.03 F 0.13 3.78 F 0.06 26.81 F 0.15 24.01 F 0.40 0.69 F 0.01 a,b 4.92 F 0.29 7.60 F 0.09 37.23 F 0.21 b 2.94 F 0.03 a,b 4.15 F 0.15 b 7.19 F 0.11 b 1.70 F 0.05 13.04 F 0.06 b 15.98 F 0.06 b 13.99 F 0.30 0.26 F 0.01 b 14.24 F 0.30

2.66 F 0.20 0.58 F 0.03 2.85 F 0.03 b

Values are means F S.E.M. of percentages of total fatty acid mass. Group differences ( p b 0.05) are indicated with different letters.

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2.3. Statistical analysis Results are presented as mean F S.E.M. Data were subjected to a one-way ANOVA, and where appropriate any differences among the groups (Initial, SD, and GD) were determined by a Tukey’s test ( p b 0.05).

3. Results Fish refused to feed when water temperature was below 13 8C, independent of the rate of temperature change which resulted in either a 16 or 12 day-long fast for SD and GD, respectively. Changes observed in total body weight, liver weight and hepatosomatic index are shown in Table 1. Body weight decreased significantly ( p b 0.05) compared to initial values (12% in SD and 10% in GD), though there were no significant differences in final weight between the two groups. Contrary to the body weight loss, liver size increased, although the change was not significant, perhaps due to individual

481

variability. Nevertheless, the hepatosomatic index reflects the combined effects of body weight loss and liver size increase, rising significantly ( p b 0.05) for both groups. Livers also changed in appearance when temperature decreased, becoming yellowish or whitish and more friable. Visual inspection of animals revealed that all fish that underwent cold temperatures had distended, liquid-filled digestive tracts with fibromucous material inside and reduced levels of perivisceral fat. Plasma ASAT activity of sea bream at low temperatures was threefold greater than in fish at 18 8C (Table 1), indicating some degree of tissue injury. In line with this, one should note that the previously mentioned moribund fish showed considerably higher levels (245 mU/mL) of plasma ASAT. Proximate compositions of liver and muscle are also shown in Table 1. In liver, water and protein percentages did not vary significantly from initial values for either group, but lipid content increased although the increase was not significant. Combined with the overall higher HSI at 8 8C, this translates as a

Table 3 Fatty acid composition of polar and non-polar lipids in gills Polar

Non-polar

Initial

SD

GD

Initial

SD

GD

Fraction (%) 16:0 18:0 Total saturated 18:1 (n-9) 24:1 (n-9) 16:1 (n-7) Total n-9(+n7) 18:3 (n-3) 20:5 (n-3) 22:6 (n-3) 22:5 (n-3) n-3 HUFA Total n-3 18:2 (n-6) 20:4 (n-6) Total n-6

37.74 F 3.18 22.34 F 0.40 11.11 F 0.28 a 36.78 F 0.44 a 12.74 F 0.29 a 1.11 F 0.07 2.50 F 0.07 16.43 F 0.34 a 2.84 F 0.03 6.34 F 0.12 23.88 F 0.37 a 1.59 F 0.05 a 31.81 F 0.51 a 34.65 F 0.48 a 7.01 F 0.12 a 3.69 F 0.14 10.74 F 0.11

50.56 F 6.89 21.74 F 0.31 10.11 F 0.13 b 34.05 F 0.38 b 10.91 F 0.11 b 0.99 F 0.04 2.29 F 0.30 14.32 F 0.15 b 2.83 F 0.06 7.06 F 0.23 25.73 F 0.26 b 1.98 F 0.06 b 34.79 F 0.30 b 37.67 F 0.25 b 8.03 F 0.25 b 3.71 F 0.10 11.78 F 0.25

41.11 F 4.16 22.96 F 0.36 10.63 F 0.40 35.93 F 0.72 12.03 F 0.16 1.11 F 0.07 2.44 F 0.08 15.77 F 0.35 3.01 F 0.07 6.94 F 0.23 26.68 F 0.55 1.72 F 0.03 34.41 F 0.31 37.40 F 0.32 8.50 F 0.26 3.68 F 0.41 10.37 F 0.83

62.26 F 3.18 17.16 F 0.23 3.73 F 0.14 25.68 F 0.93 23.64 F 0.40 0.84 F 0.06 7.72 F 0.21 34.92 F 0.62 3.08 F 0.02 4.90 F 0.06 9.94 F 0.25 1.87 F 0.23 16.71 F 0.30 19.79 F 0.28 13.27 F 0.41 0.49 F 0.03 13.87 F 0.38

49.44 F 6.89 17.47 F 0.18 3.98 F 0.09 a,b 26.81 F 0.28 23.27 F 0.36 0.90 F 0.06 7.87 F 0.09 33.83 F 0.31 a,b 3.07 F 0.08 4.90 F 0.11 11.11 F 0.26 b 1.95 F 0.05 a,b 17.32 F 0.43 20.39 F 0.42 13.60 F 0.22 0.63 F 0.05 a,b 14.35 F 0.23

58.89 F 4.16 16.92 F 0.09 4.24 F 0.07 b 26.95 F 0.66 23.49 F 0.67 0.97 F 0.05 7.81 F 0.43 33.14 F 0.36 b 3.19 F 0.05 5.06 F 0.13 11.50 F 0.39 b 2.20 F 0.27 b 17.66 F 0.71 20.85 F 0.73 13.37 F 0.08 0.71 F 0.07 b 13.94 F 0.26

Ratios Unsat/sat EPA/DHA n-9/n-3 HUFA

1.72 F 0.03 a 0.27 F 0.00 0.52 F 0.02 a

1.91 F 0.03 b 0.27 F 0.01 0.41 F 0.01 b

2.73 F 0.04 0.44 F 0.01 b 1.96 F 0.06

2.72 F 0.09 0.44 F 0.01 b 1.99 F 0.04

a,b a,b a

a

b c b b b

1.83 F 0.05 b 0.27 F 0.01 0.48 F 0.03 a

a

a

a a

a

2.88 F 0.12 0.49 F 0.01 a 2.09 F 0.06

Values are means F S.E.M. of percentages of total fatty acid mass. Group differences ( p b 0.05) are indicated with different letters. 22:1 (n-9) not detected.

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marked rise in the absolute amount of hepatic lipids in both groups, principally attributable to increases in non-polar lipids ( p b 0.05). Amounts of polar lipids did not change significantly. In muscle, lipid content and lipid/protein ratio decreased, though this was only significant in the GD condition. In this tissue, amounts of non-polar lipids significantly decreased (25% in both groups), with corresponding minor increases of protein and water percentages. 3.1. Tissue fatty acid patterns Fatty acid distributions for muscle, gill, and liver tissue are summarised in Tables 2–4, respectively, with polar and non-polar fractions presented separately. Polar fatty acid profiles at the beginning of the experiment (18 8C) were similar for the three tissues, with the following acids dominating: docosahexaenoic acid (DHA; 22:6 n-3) N palmitic acid (16:0) N oleic acid (18:1 n-9) N linoleic acid (18:2 n6) N eicosapentaenoic acid (EPA; 20:5 n-3). More-

over, n-3 highly unsaturated fatty acids (n-3 HUFA) accounted for roughly half of the total polar fraction in liver tissue. Within the non-polar fraction, the most abundant fatty acids in muscle and gill tissue were: oleic acid (18:1 n-9) N palmitic acid (16:0) N linoleic acid (18:2 n-6) N DHA (22:6 n-3). In liver, the nonpolar fatty acid profile was different, with a higher content (30%) of linoleic acid (18:2) and lower percentages of oleic acid (18:1 n-9) and saturated fatty acids (data shown in Table 4). These patterns explain the high unsaturation/saturation ratios observed for non-polar fatty acids (2.61, 2.88, and 4.22 in muscle, gill, and liver, respectively). At 8 8C, absolute non-polar lipid content in muscle decreased. All n-3 fatty acids were affected. EPA and DHA quantities significantly decreased, whilst n-9 fatty acids increased (Table 2). Correspondingly, the polar lipid fraction rose, with n-3 fatty acids, particularly DHA, again being the most affected, this time rising. As a consequence, the n-9/n-3 HUFA ratio decreased in both groups, and the unsaturation/ saturation ratio increased in the SD group ( p b 0.05).

Table 4 Fatty acid composition of polar and non-polar lipids in liver Polar

Non-polar

Initial Fraction (%) 16:0 18:0 Saturated 18:1 (n-9) 24:1 (n-9) 22:1 (n-9) 16:1 (n-7) Total n-9(+n7) 20:5 (n-3) 22:6 (n-3) n-3 HUFA Total n-3 18:2 (n-6) 20:4 (n-6) Total n-6 Ratios Unsat/sat EPA/DHA n-9/n-3 HUFA

32.53 F 1.23 21.06 F 0.42 8.95 F 0.41 31.10 F 0.18 6.49 F 0.37 0.64 F 0.04 0.45 F 0.05 1.27 F 0.05 8.49 F 0.11 9.57 F 0.50 44.12 F 0.53 51.78 F 1.67 51.78 F 1.67 5.64 F 0.42 1.48 F 0.33 7.12 F 0.19

a a a a a a a a a a

2.23 F 0.02 a 0.21 F 0.02 a 0.13 F 0.03 a

SD

GD

Initial

SD

GD

19.45 F 2.46 b 32.72 F 1.77 b 6.00 F 0.06 b 39.86 F 1.81 b 6.75 F 0.44 0.40 F 0.09 b n.d. 1.03 F 0.07 b 8.22 F 0.37 13.85 F 0.93 b 30.97 F 2.02 b 44.82 F 2.42 b 44.82 F 2.42 b 5.50 F 0.44 1.60 F 0.33 7.10 F 0.44

18.66 F 0.85 b 31.15 F 2.06 b 6.58 F 0.55 b 38.83 F 1.79 b 6.97 F 0.27 0.35 F 0.06 b n.d. 0.94 F 0.24 b 8.38 F 0.32 14.31 F 0.90 b 31.51 F1.70 b 45.82 F 2.29 b 45.82 F 2.29 b 5.34 F 0.18 1.64 F 0.43 6.98 F 0.27

67.47 F 1.23 a 14.46 F 1.09 2.83 F 0.17 a 19.24 F 0.68 19.03 F 1.76 0.43 F 0.07 2.30 F 0.34 6.28 F 0.62 28.04 F 1.47 a 4.70 F 0.21 a 13.40 F 1.12 a 18.1 F1.31 a 18.10 F 1.31 a 30.73 F 0.62 4.70 F 0.21 a 35.43 F 0.80

80.55 F 2.46 b 14.25 F 0.48 2.00 F 0.19 b 19.69 F 0.72 17.28 F 1.01 0.39 F 0.04 1.87 F 0.42 6.11 F 0.37 25.66 F 0.93 a,b 3.84 F 0.19 b 19.80 F 0.86 b 23.63 F 0.68 b 23.63 F 0.68 b 30.40 F 0.78 3.80 F 0.11 b 34.15 F 0.85

81.44 F 0.85 13.72 F 0.71 1.76 F 0.14 18.56 F 1.05 15.92 F 0.78 0.39 F 0.06 2.51 F 0.16 6.16 F 0.30 24.97 F 1.04 3.65 F 0.05 17.52 F 0.59 21.17 F 0.57 21.17 F 0.57 30.20 F 1.10 3.72 F 0.21 34.05 F 1.05

1.53 F 0.13 b 0.46 F 0.04 b 0.19 F 0.02 a,b

1.60 F 0.13 b 0.46 F 0.03 b 0.19 F 0.01 b

4.22 F 0.19 0.36 F 0.02 a 1.59 F 0.17 a

4.12 F 0.21 0.20 F 0.02 b 1.09 F 0.06 b

b b

b b b c c b

4.45 F 0.27 0.21 F 0.01 b 1.19 F 0.08 b

Values are means F S.E.M. of percentages of total fatty acid mass. Group differences ( p b 0.05) are indicated with different letters. 18:3 (n-3) and 22:5 (n-3) not detected.

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Lipid contents of gill tissue, in mg per 100 mg w.w., were 4.08 F 0.34 (Initial group), 3.53 F 0.12 (SD group), and 4.06 F 0.41 (GD group). No significant differences were observed among groups in total lipid, and non-polar and polar fractions, although fatty acids profiles were modified. Thus, after a decrease in temperature, DHA and estearic acid (18:0) in the nonpolar fraction significantly increased whereas the n-9 family decreased in the GD condition. In the polar fraction, the most relevant change was an increase in the unsaturation/saturation ratio ( p b 0.05) in both 8 8C-exposed groups, caused by a rise in n-3 HUFA and a decrease in saturated fatty acids. In both groups, the short-term effect on liver tissue of lower temperatures was a rise in the non-polar lipid fraction, at the expense of the polar fraction (Table 4). All fatty acids increased in absolute amounts, but not all by the same proportion. In the non-polar fraction, DHA percentage increased by 50%, but estearic acid (18:0), EPA, arachidonic acid (20:4n-6) and n-9 fatty acid percentages significantly decreased. Compared with the initial samples at 18 8C, the unsaturation/ saturation ratio did not change, while EPA/DHA and n-9/n-3 HUFA ratios lowered in both groups. In the polar fraction of liver lipids, HUFA decreased significantly, DHA again being the most affected fatty acid. This decrease produced a significant change in the unsaturation/saturation ratio in both groups, as well as a significant increase in the EPA/ DHA and n-9/n-3 HUFA ratios.

4. Discussion Cold induced a reduction of food intake in farmed gilthead sea bream (Tort et al., 1998a; Sarusic, 1999), and, as already observed, at temperatures below 13 8C sea bream stop feeding altogether (Ibarz et al., 2003). This cold-induced fasting explains the significant decreases of body weight, and also the observed loss of perivisceral fat and non-polar lipids in muscle tissue. It is well known that in physiological states demanding a higher consumption of fuel reserves, such as fasting or stress, stored triacylglicerols are catabolized, glycerol used in gluconeogenesis (Moon, 1988), and fatty acid used as an energy source (Gurr and Harwoord, 1991). The relationship between stress and the use of non-polar lipids was well established

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by Sheridan (1988) in coho salmon parr by cortisol implants. Rotllant et al. (2000) showed that temperature drops induced an acute increase of plasma cortisol in sea bream. The mobilisation and use of lipids from perivisceral fat and muscle observed in our study therefore seems to be a direct response to thermal stress as well as the associated fasting, though these effects were independent of the rate of temperature decrease. The energy needs of fish diminish in cold conditions, and we have shown that the Q10 of the metabolic rate of sea bream was near 2 when water temperature decreased from 18 8C to 8 8C (Ibarz et al., 2003). Thus, fasting effects would not be very severe at low temperatures. Another general response to cold challenge is homeoviscous adaptation. To maintain the appropriate fluidity of biological membranes, increased unsaturation of fatty acids of phospholipids occurs (Hazel and Prosser, 1974). The initial fatty acid profile has an important role in this cold adaptation and a high dietary intake of HUFA enhances cold tolerance in a number of species (Viola et al., 1988; Craig et al., 1995; Kelly and Kohler, 1999). It is worth noting that, owing to their diet, the initial levels of unsaturated fatty acids in our sea bream were higher than those described in Ibeas et al. (1997, 2000). There are no studies on adaptation in sea bream and the present results are a first approximation to the problem. Small increases in gill and muscle polar n-3 HUFA, with a significant increment in EPA and, especially, DHA levels were observed, matching data from other species as tilapia and carp (Viola et al., 1988), red drum (Craig et al., 1995), or striped bass (Kelly and Kohler, 1999). In liver tissue, the cold did not induce any mobilization of fat stores or homeoviscous adaptation of membranes, though we show an accumulation of lipids. Such increases in liver lipid depots have been reported as a seasonal response in golden ide (Segner and Braunbeck, 1990), as an adaptative mechanism to the cold in Antarctic fishes (Giardina et al., 1998) and as a preparation for the winter months (Kaushik, 1997). The rapid deposition in the current study (over 8 or 10 days), however, accompanied as it was by cold-induced fasting, must have relied on an imbalance between liver uptake from extrahepatic tissues and the liver output of lipids. Deposition of lipids in the liver was so notable that it caused an increment in

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organ mass, reductions in the percentages of other principal components, and alterations of colour and compactability. These changes of appearance and the distension of digestive tracts were in concordance with alterations observed in fish affected by bwinter diseaseQ (Gallardo et al., 2003). Moreover, although plasma ASAT activity was not so high as in symptomatic fish, it was three-fold higher after the cold treatment, therefore beyond the normal range possibly indicating the onset of illness. Supporting this idea, the liver of the sole moribund fish in this study was found to be similar to those of symptomatic fish. Further, its HSI was abnormally high (3.9%) and plasma ASAT levels were well beyond the normal range (245 mU/mL). Our group’s early studies on bwinter diseaseQ indicate that almost all symptomatic fish showed lipid infiltration in liver tissue (Tort et al., 1998a; Gallardo et al., 2003), and Penrith et al. (1994), Spisni et al. (1998), and Caballero et al. (1999) have shown that in marine fish liver steatosis provoked tissue injury. So, we hypothesise that acute cold effects on liver may be caused by a malfunction of the organ. We have shown that low temperatures induce in sea bream a hypoglycaemic state with significant decreases in different plasma protein fractions, particularly albumin, a1globulins and fibrinogen (Sala-Rabanal et al., 2003). Moreover, lower plasmatic levels in protein fractions were also present in bwinter diseaseQ symptomatic fish described by Gallardo et al. (2003). Some of these physiological variables have been strongly related with liver function. So, liver alteration in sea bream at low temperatures can be related to exacerbated lipid accumulation, which, should environmental conditions remain unfavourable, would lead to a pathological condition resulting in bwinter diseaseQ. In summary, gilthead sea bream submitted to shortterm cold challenge stress by lowering water temperature from 18 8C to 8 8C at two different rates ceased feeding below 13 8C, resulting in a decline in body weight. Results provide evidence of significant effects on lipid depots: the non-polar fraction of muscle lipids decreased, while liver mass increased through the accumulation of non-polar lipids. Cold acclimation increased the unsaturation of polar lipids in gill and muscle tissue, but not in liver. Final tissue composition and fatty acid profiles did not vary with rate of temperature drop.

Acknowledgements Special thanks are due to Dr. D. Furones for providing the necessary facilities and R. Carbo´ and M.A. Soubrier at the IRTA-Centre Aqu¨icultura for their technical assistance. This study was supported by grant CICYT-MAR97-0408-C02-0 from the Spanish government, and A. Ibarz and M. Beltra´n received fellowships from the Ministerio de Educacio´n y Cultura and the Generalitat de Catalunya, respectively.

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