Conclusion and future workFish density and water quality both influenced the number of spots present in different amounts in Tris- and in CHAPS-urea extracts. Some of them seemed to be caused by proteolysis or by active growth (and protein synthesis), as indicated by the presence of anti-myosin heavy chain immunoreactive fragments in the CHAPS-urea extracts.

Future works will comprise the sequencing of selected discriminatory spots to map the effect of the farming conditions on the muscle protein expression, which in turn will reflect the fish welfare and affect the quality of the flesh.

Acknowledgment. The financial support of the Norwegian Research Council (Project 154 137/130) is gratefully acknowledged

References[1] Martinez, I., Slizyte, R. & Dauksas, E. (2006) High resolution two-dimensional electrophoresis as a tool to differentiate wild from farmed cod (Gadus morhua) and to assess the protein composition of klipfish. Food Chem., in pres.[2] Molloy, M. P., Herbert, B. R., Walsh, B. J., Tyler, M. I., Traini, M., Sanchez, J. C., Hochstrasser, D. F., Williams, K. L. & Gooley, A. A. (1998) Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis, 19, 837-844.[3] Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4354.[4] Martinez, I. & Pettersen, G.W. (1992) Temperature-induced precocious transitions of myosin heavy chain isoforms in the white muscle of the Arctic charr (Salvelinus alpinus). BAM, 2: 89-95.

Effect of water quality on the expression of proteins in white skeletal muscle of cod (Gadus morhua L.) examined by high resolution two-dimensional electrophoresis.

A preliminary study

Martinez, I.*1, Dauksas, E.1, Slizyte, R.1, Damsgård, B.2 and Toften, H. 21 SINTEF Fisheries and Aquaculture, N-7465 Trondheim, Norway; 2, Fiskeriforskning, PO Box 6122, N-9291 Tromsø, Norway

*E-mail: iciar.martinez@sintef.noIntroductionProtein extracts from the white skeletal muscle of wild and commercially farmed cod analyzed by two-dimensional electrophoresis (2DE) differed in some spots present only in extracts from cultivated cod. The spots were attributed to increased protein degradation that might have been caused by stressful breeding conditions [1]. One known and relevant stressful factor during cultivation, whose effect is relevant to map, is the water quality: while low quality has a negative effect on fish welfare, it is more convenient for the farmer and environmentally friendly to reduce as much as possible the use of water. Since cultivated fish is intended for human consumption, it is of interest to map the effect of the water quality not only on the fish survival andwelfare but also on the protein composition of the edible part, namely the skeletal muscle. The aim of this work was to establish the conditions for 2DE analysis and identify spots with the potential to serve as indicators of the effect of fish density and water quality on the protein expression in the white skeletal muscle of the cod.

Material and methodsIndividually labelled cod of 70-200 g were cultivated during 12 weeks under low fish density and good water quality (group A), high fish density and good water quality (group B) and high density and poor water quality (group C). The fish were killed by standard methods to avoid stress and transported in ice to the laboratory. The white skeletal dorsal muscle was extracted the next day as described by [2] using the ReadyPrep Sequential Extraction Kit of Bio-Rad. IEF, was performed as described by [1] on 13 cm Immobiline DryStrips pH 4-7 (AmershamBiosciences). 200 or 100 μg of protein from Tris and CHAPS-urea extracts respectively were loaded. IEF was performed in the Protean IEF System at 15oC for: 30 min at 250 V and 1 h at 4,000 V; then the voltage was increased to 9,000 until 70,000 Vh were reached and finally decreased to 500 V until the run was stopped. The second dimension (SDS-PAGE) was performed in 12.5% SDS-PAGE, after equilibration of the strips in 6 M urea, 2 % SDS, 50 mM Tris, pH 8.8, 30 % glycerol and 1% DTT for 10 min followed by another 10 min using the same buffer but with 4.5% iodoacetamide instead of DTT. All samples were analyzed individually and as pools. To detect myosin heavy chain and its fragments, the proteins of the CHAPS-urea extracswere separated by 12.5% SDS-PAGE, transferred to nitrocellulose [3] and probed with rabbit serum anti-myosin heavy chain [4].

Results

SINTEF Fisheries and Aquaculture

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Figure 2.- Immunoreactivities of myosin heavy chain fragments (after 12.5% SDS-PAGE) in the CHAPS-urea extracts from white skeletal muscle of cod of groups A, B, and C, as indicated. Each lane is one individual fish. The arrowhead indicates the intact myosin heavy chain (200 kDa). All extracts contained myosin heavy chain fragments.

Figure 1.- 2DE analyses (Immobiline DryStrips, pH 3-10; 12.5% SDS-PAGE) of farmed cod. Each gels is a pool several fish (n), as indicated in the figure. A, actin, T, tropomyosin, M, myosin heavy chain; 1,2 and 3 are the fast myosin light chains type 1, 2 and 3 respectively.

Numerous spots - areas enclosed in squares - were identified present in different amounts depending on the farming conditions and therefore with the potential to serve as markers for the effect of the water quality and fish density on the muscle protein expression.

Tris extracts CHAPS-Urea extracts

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Low fishdensityHigh water quality

Group B:High fishdensityHigh water quality

Group C:High fishdensityLow water quality

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