The aim of this study was to evaluate in marketable size turbot the effects of dietary fat levels on nutrient utilisation, tissue composition, fat deposition, lipogenesis, sensory
and physical quality of flesh.
2. Material and methods
2.1. Experimental diets Ž
Four isonitrogenous digestible protein: 59, calculated from apparent digestibility Ž
. .
coefficients ADC of protein diets were formulated to contain 10, 15, 20 and Ž
. 25 of crude fat, using gradient increases in fish oil. Yttrium oxide 0.1 was added as
an indicator for digestibility measurements. Diets were manufactured industrially by Ž
. Nutreco Aquaculture Research Centre, Stavanger, Norway , using a twin-screw ex-
truder, in the form of 9-mm-diameter pellets. Ingredient and chemical composition of the diets are reported in Table 1.
Table 1 Ingredients and proximate composition of the experimental diets
Experimental diets 10
15 20
25 Ingredients
Ž .
Fish meal Norseamink 50.0
50.0 50.0
50.0 Soluble fish protein concentrate
8.0 8.0
8.0 8.0
Whole wheat 22.1
15.3 8.3
1.5 Wheat gluten
13.1 14.7
16.4 18.0
Fish oil 3.8
9.0 14.3
19.5
a
Mineral mix 1.0
1.0 1.0
1.0
a
Vitamin mix 1.0
1.0 1.0
1.0 Ž
. Binder Edelbind
0.9 0.9
0.9 0.9
Yttrium oxide 0.1
0.1 0.1
0.1 Proximate composition
Ž .
Dry matter DM , 89.8
90.1 91.0
93.6 Crude protein, DM
61.3 62.2
62.5 61.3
Crude fat, DM 11.3
16.2 21.4
27.7
b
Phospholipids, DM 2.7
3.0 3.2
3.8 Ash, DM
8.5 8.2
8.1 8.3
Gross energy, kJrgDM 22.1
23.2 24.1
24.6
c
Ž .
Digestible protein DP , DM 58.7
59.9 59.9
59.1 Ž
. Digestible energy DE , kJrgDM
19.0 20.9
21.6 22.3
DPrDE ratio, mgrkJ 30.8
28.6 27.7
26.5
a
Ž .
Ž .
Proprietary mixtures Nutreco, ARC providing levels meeting requirements as proposed by NRC 1993 .
b
Levels of phospholipids were calculated from lipids of Folch analyses.
c
Ž .
DP and DE values are based on apparent digestibility coefficients ADC of protein and energy reported in Table 2.
2.2. Digestibility measurements ADC of the experimental diets were determined with triplicate groups of fish. Fifteen
Ž .
turbot body weight: 815 8 g; mean S.D. were allotted to cylindroconical tanks, each of which was equipped with a flat-bottomed large-mesh basket and was supplied
Ž .
with fresh seawater temperature maintained at 17 18C; mean S.D. in a flow-through system. Fish were fed to satiety once a day and faecal samples were collected using a
Ž .
faeces settling column similar to the one developed by Cho et al. 1982 . For each treatment, faecal samples were collected once a day each morning over 2 consecutive
Ž .
weeks and were centrifuged 3000 = g at 68C for 20 min and kept frozen at y208C. After freeze-drying, the faeces were analysed for yttrium, crude protein, crude fat and
gross energy. The ADC of the experimental diets was calculated as follows:
Tracer in Diet Nutrient or Energy in Faeces
ADC s 100 y 100 =
Ž .
ž
Tracer in Faeces Nutrient or Energy in Diet
2.3. Growth study Ž
The growth trials were conducted in the experimental facilities of IFREMER Centre .
de Brest at a constant seawater temperature of 17 0.58C with a salinity of 35‰ in a flow through system for a period of 12 weeks. Twenty-two turbot, Psetta maxima,
Ž .
Ž initial body weight: 657 6 g; mean S.D. were randomly allotted to each tank, 1
3 y1
. m
tank with a water flow rate of 18 l min . A 12r12 h lightrdark cycle was
adopted. The different diets were randomly allotted and triplicate groups were fed by Ž
. hand once a day to visual satiety visual observation of first feed refusal over a 90-min
period and feed intake was recorded. Each group was weighed every 3 weeks to follow growth and feed utilisation.
2.4. Samplings At the beginning of the growth study, six fish from an initial pool of fish were
sampled and stored at y208C for analyses of whole body composition. Six fish from the same initial pool were also withdrawn to weigh the liver and the digestive tract for
Ž .
Ž .
calculating hepatosomatic index HSI and viscerosomatic index VSI . At the end of the trial, the same protocol of slaughter was followed for each tank.
Ž Five fish per tank were withdrawn for comparative carcass analyses water, ash, protein,
. Ž
. fat and energy and nutrient retention calculation. Blood samples 1 ml were withdrawn
Ž from the best replicate using heparinised syringes. After centrifugation 1200 = g, 10
. min, 58C , plasma was separated and stored frozen at y208C as separate aliquots for
analyses of triglyceride and total cholesterol concentrations. Six livers and viscera were withdrawn from fish from the same replicate for analyses of lipid and hepatic enzyme
Ž .
activities G6PD, acetyl Coenzyme-A carboxylase, AcoAC; and ME . Enzyme activities
were measured in fresh liver samples. For quality analyses, fish were stunned, bled to death in ice cold water before dissection. After slaughter, gutting was done immediately
and filleting was over within 30 min. Eight fish per tank were withdrawn to weigh the liver and the digestive tract for calculating HSI and VSI and for the following analyses:
Ž .
three fish fillets with skin for sensory analyses, three other fish for instrumental Ž
. analyses of texture fillets within skin and, finally, two fish were withdrawn for lipid
Ž .
analyses in muscle dorsal and ventral fillets and skins. Samples of muscle and organs for lipid analyses were frozen in liquid nitrogen and stored at y808C.
2.5. Analytical methods The fish for whole body composition were ground frozen and a representative part
was freeze-dried before analysis. Proximate composition analyses of diets, faeces and chemical composition of whole body and tissues were conducted following standard
Ž .
Ž laboratory procedures AOAC, 1984 : dry matter after desiccation in an oven 1058C for
. Ž
. Ž
24 h , ash incineration at 5508C for 12 h , crude protein N = 6.25 using a Dumas .
Ž nitrogen analyser, Fisons Instrument , crude fat dichloromethane extraction by Soxhlet
. Ž
. method and gross energy IKA Adiabatic Calorimeter C4000 A . Plasma total choles-
Ž terol and triglyceride concentrations were determined using commercial kits Boehringer
. no. 1442341, Mannheim, Germany and bioMerieux no. PAP150, France, respectively .
´
Analysis of yttrium in the diets and faecal matter was done in predigested samples as Ž
. detailed in Mambrini et al. 1999 .
In liver, viscera, muscle and skin samples, total lipid was determined according to Ž
. Folch et al. 1957 , chloroform being replaced by dichloromethane. The separation of
neutral lipids and phospholipids were realised according to the procedure described by Ž
. Juaneda and Rocquelin 1985 . The total lipid extracts were fractionated on silica
Ž .
cartridges Sep-Pack, Waters , neutral lipids were eluted by chloroform and phospho- lipids by methanol.
For assays of hepatic lipogenic enzyme activities, liver samples were homogenised in Ž
three volumes of ice-cold buffer 0.02 M Tris–HCl, 0.25 M sucrose, 2 mM EDTA, 0.1 M sodium fluoride, 0.5 mM phenyl methyl sulphonyl fluoride, 0.01 M b-mercapto-
. ethanol, pH 7.4 and the homogenates were centrifuged at 20,000 = g at 48C for 20 min.
Soluble protein content of liver homogenates was determined by the method of Bradford Ž
. 1976 using bovine serum albumin as the standard. Selected lipogenic enzyme activities
Ž .
were assayed on supernatant fractions: G6PD EC 1.1.1.49 according to Bautista et al. Ž
. Ž
. Ž
. Ž
. 1988 , ME EC 1.1.1.40 according to Ochoa 1955 and ACoAC EC 6.4.1.2 as per
Ž .
Ž .
Holland et al. 1984 . Enzyme activity units IU defined as mmol substrate converted to Ž
. product per min at assay temperature 308C , were expressed per mg hepatic soluble
Ž .
protein and per g liver tissue wet weight . 2.6. Flesh quality eÕaluation
Ž .
Sensory profiling Stone et al., 1974; ISO 11035 was performed on cooked fillets by Ž
. an internal sensory panel IFREMER, Nantes consisting of 20 individuals selected for
their interest, availability and sensorial capacities of memorising stimuli or discriminat- ing intensities. All were volunteers and received regular training sessions to develop
their sensory performances and knowledge of marine products. Sessions were conducted in an air-conditioned room designed for sensory analysis divided into ten individual
Ž .
boxes with standardized light T s 65008K and equipped with a computerised system Ž
. Fizz, Biosystemes, Dijon . These conditions were conducive to concentration and
`
Ž avoided communication between assessors and disturbance by external factors AFNOR
. V-09-105, 1995 . The samples with skin were cooked for 2.5 min in a microwave oven
Ž .
Panasonic NN8550, 850 W in closed bowl before analyses. Products were assigned three-digit numbers, randomised and served simultaneously. The samples were evaluated
Ž .
using a continuous scale presented on a computer screen from 0 low intensity to 10 Ž
. high intensity for the following attributes: intensity, ApotatoesB, fat fish, marine-iodine
and milky for odour; white colour and fatness for visual aspect; intensity, fatty fish, marine, sweet, acid flavour and bitter aftertaste for flavour; and hardness, elasticity,
Ž .
moisture, fat, exudation and sticky for texture in mouth see Table 7 . The odour attributes were assessed immediately after opening the bowl, while flavour and texture
in mouth were evaluated after cutting the samples. The data were immediately trans- ferred by the network to a central computer for statistical processing.
Instrumental colour and texture analysis were performed 7 days after slaughter, on Ž
fillets kept in ice in sealed vacuum packing and were done on raw and cooked fillets 60 .
Ž .
min at 708C in vacuum packing . For texture, a Kramer shear press 64 = 64 mm was Ž
used with compression up to breaking using a universal testing machine INSTRON, .
model 4501 with a load cell of 5 kN and during this test, a constant speed of 1 mmrs into the fillets was adopted. Samples were withdrawn from the caudal part of fish and
measured 64 mm long and fillet size in width. The weight of each Kramer sample was Ž
. recorded to avoid possible size differences Szczesniack et al., 1970 . Instrumental
colour analysis of raw or cooked fillets was made on the internal part of the fillet with a Ž
. chromameter Micro-Color, Dr. Lange using light source D65 and 108 observer angle.
Five measurements per fish were made directly on the fillets behind the head to the tail. Data were expressed as L
a b
system, representing lightness, redness and yellow- Ž
. ness, respectively, recommended by CIE 1976 . The pH of fillets was measured using a
pH meter Metrohm on internal part of fillet. The instrumental analyses followed Ž
. procedures described by Laroche et al. 1996 .
2.7. Statistical analysis All data except sensory analysis were subjected to one-way analysis of variance to
test the effects of experimental diets. In cases where significant differences occurred Ž
. significance level s 0.05 , the means were compared using Newman–Keuls test. The
Ž .
statistical tests ANOVA and regression were performed using the STAT-ITCF soft- Ž
. ware ITCF, 1988 . The results of sensory analyses were subjected to two-way analysis
of variance to test the effects of experimental diets and assessors. In cases where significant differences occurred, the means were compared using Duncan test with the
FIZZ, Biosystemes software.
`
3. Results
