An aliquot (1 mL) of the chloroform phase was transferred <str<strong>on</strong>g>in</str<strong>on</strong>g>to a test tube thenevaporated to dryness <strong>on</strong> a water bath (ca. 40°C) us<str<strong>on</strong>g>in</str<strong>on</strong>g>g nitrogen gas. Samples were dissolved <str<strong>on</strong>g>in</str<strong>on</strong>g>to4 or 1 mL of water-saturated butanol (feeds <str<strong>on</strong>g>and</str<strong>on</strong>g> muscles, respectively). The soluti<strong>on</strong> was filtered(0.45 µm; M<str<strong>on</strong>g>in</str<strong>on</strong>g>isart SRP15, Sartorius), <str<strong>on</strong>g>in</str<strong>on</strong>g>to the sample vials, sealed <str<strong>on</strong>g>and</str<strong>on</strong>g> storage at-80 °C untilanalysis. All carotenoid extracti<strong>on</strong>s were c<strong>on</strong>ducted under dim light to avoid sample degradati<strong>on</strong>by photo-oxidati<strong>on</strong>.Carotenoids extracts were analysed accord<str<strong>on</strong>g>in</str<strong>on</strong>g>g to Abdel-Aal et al. (2007) by liquidchromatography (1100 series liquid chromatographer, Aligent, Mississauga, ON). <str<strong>on</strong>g>Pigment</str<strong>on</strong>g>s wereseparated us<str<strong>on</strong>g>in</str<strong>on</strong>g>g a short C30 column (YMC Carotenoid, Waters, Mississauga, ON, Canada), setup at 35°C. Compositi<strong>on</strong> of the eluted mobile phase was (A) methanol/methyl tert-butilether/nanopure water soluti<strong>on</strong> (81:15:4, v/v/v) <str<strong>on</strong>g>and</str<strong>on</strong>g> (B) methyl tert-butyl ether/methanol (90:10,v/v) soluti<strong>on</strong>, programed to run as: 0-9 m<str<strong>on</strong>g>in</str<strong>on</strong>g>, 100 - 75% A; 9 - 14 m<str<strong>on</strong>g>in</str<strong>on</strong>g>, 75 - 20% A; 14 - 15 m<str<strong>on</strong>g>in</str<strong>on</strong>g>,25 - 0% A; 15 - 18 m<str<strong>on</strong>g>in</str<strong>on</strong>g>, hold at 0% A; 18 -20 m<str<strong>on</strong>g>in</str<strong>on</strong>g> hold at 100% A. Detecti<strong>on</strong> <str<strong>on</strong>g>and</str<strong>on</strong>g> measurementof different pigments were c<strong>on</strong>ducted at 450 nm ( for all-trans-lute<str<strong>on</strong>g>in</str<strong>on</strong>g>, all-trans zeaxanth<str<strong>on</strong>g>in</str<strong>on</strong>g>, alltrans-β-cryptoxanth<str<strong>on</strong>g>in</str<strong>on</strong>g><str<strong>on</strong>g>and</str<strong>on</strong>g> all-trans-β-carotene) <str<strong>on</strong>g>and</str<strong>on</strong>g> 478 nm (for all-trans-astaxanth<str<strong>on</strong>g>in</str<strong>on</strong>g>),respectively.Identificati<strong>on</strong> <str<strong>on</strong>g>and</str<strong>on</strong>g> quantificati<strong>on</strong> of carotenoids was accomplished us<str<strong>on</strong>g>in</str<strong>on</strong>g>g pure authenticcarotenoids st<str<strong>on</strong>g>and</str<strong>on</strong>g>ards (all-trans-astaxanth<str<strong>on</strong>g>in</str<strong>on</strong>g>, all-trans-lute<str<strong>on</strong>g>in</str<strong>on</strong>g>, all-trans-β-carotene, all-transzeaxanth<str<strong>on</strong>g>in</str<strong>on</strong>g>, all-trans-β-cryptoxanth<str<strong>on</strong>g>in</str<strong>on</strong>g>) (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada). Thel<str<strong>on</strong>g>in</str<strong>on</strong>g>earity of the resp<strong>on</strong>se for each pigment (resp<strong>on</strong>se area v/s <str<strong>on</strong>g>in</str<strong>on</strong>g>jected amount) was evaluated <str<strong>on</strong>g>and</str<strong>on</strong>g>the coefficient of determ<str<strong>on</strong>g>in</str<strong>on</strong>g>ati<strong>on</strong> (R 2 ) for all-trans-astaxanth<str<strong>on</strong>g>in</str<strong>on</strong>g>, all-trans-lute<str<strong>on</strong>g>in</str<strong>on</strong>g>, all-transzeaxanth<str<strong>on</strong>g>in</str<strong>on</strong>g>, all-trans-β-cryptoxanth<str<strong>on</strong>g>in</str<strong>on</strong>g>, <str<strong>on</strong>g>and</str<strong>on</strong>g> all-trans-β-carotene were 0.9997, 0.9996, 0.9996,0.9994 <str<strong>on</strong>g>and</str<strong>on</strong>g> 0.9999, respectively.43
3.2.4 Statistical analysisData from the Plackett-Burman design were analyzed us<str<strong>on</strong>g>in</str<strong>on</strong>g>g a multiple regressi<strong>on</strong> analysisperformed by the Analyst applicati<strong>on</strong> of the SAS/STAT software (SAS <str<strong>on</strong>g>in</str<strong>on</strong>g>stitute, Cary, NC,USA). The design for the Box-Behnken design was generated <str<strong>on</strong>g>and</str<strong>on</strong>g> analyzed us<str<strong>on</strong>g>in</str<strong>on</strong>g>g the ADXapplicati<strong>on</strong> of the SAS/STAT software (SAS <str<strong>on</strong>g>in</str<strong>on</strong>g>stitute, Cary, NC, USA).Data from the fish muscle pigmentati<strong>on</strong> trial were analysed as a complete r<str<strong>on</strong>g>and</str<strong>on</strong>g>om designus<str<strong>on</strong>g>in</str<strong>on</strong>g>g the general l<str<strong>on</strong>g>in</str<strong>on</strong>g>ear model of the SAS/STAT software (SAS <str<strong>on</strong>g>in</str<strong>on</strong>g>stitute, Cary, NC, USA). Themeans of dependent variables were compared us<str<strong>on</strong>g>in</str<strong>on</strong>g>g Tukey’s h<strong>on</strong>estly significant difference(HSD) test; significance was c<strong>on</strong>sidered when p0.05) effects under theexperimental c<strong>on</strong>diti<strong>on</strong>s (Table 3.6).44
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Pigment Re
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Table 4. 3 - Carcass chemical proxi
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Table 4. 4 - Retain</strong
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Table 4. 5 - Pigment</stron
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Table 4. 6 - Colour attributes <str
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FiguresFigure 4. 1 - Growth curves
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Figure 4. 3 - Muscle colour attribu
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the effects of dietary reduced-caro
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astaxanthin deposi
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After steeping, ke
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0.9994 and 0.9999,
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under acidic and a
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oxidation within t
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colour difference usin</str
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TablesTable 5. 1 - Analyzed proxima
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Effects of
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Effects of
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Effects of
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Effects of
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Data are mean (n=2).a Steepwater pH
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CHAPTER - 6 GENERAL DISCUSSIONCurre
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significant reduction in</s
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Reduction of pigme
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though wheat gluten meal conta<stro
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BibliographyAas, G.H., Storebakken,
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Bjerkeng, B., Hatlen, B., Wathne, E
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Choubert, G., Cravedi, J., Laurenti
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Fauconneau, B., Andre, S., Chmaitil
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Johnston, I., Li, X., Vieira, V., N
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Li, M.H., Oberle, D.F., Lucas, P.M.
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Peter, C.M., Han, Y., Bolin
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Skonberg, D.I., Hardy, R.W., Barrow
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Ytrestoyl, T., Struksnæs, G., Kopp