2. Materials and methods
2.1. Animals and husbandry Ž
. Rainbow trout, Onchorhynchus mykiss 23.22 4.81 g; 124.7 6.35 mm , were
2
Ž .
randomly distributed into eight 600-l, 1-m surface area fibreglass tanks n s 40rtank . Tanks were supplied with aerated ground water at a flow rate of 3 l min
y1
. Water temperature and oxygen levels were monitored continuously throughout using Oxygaurd
w
and M q S datalogging systems. Over the period of study, water temperature ranged between 6.08C and 11.28C. Oxygen concentrations at the water outlet were maintained
above 7.0 mg O l
y1
. Photoperiod was controlled to provide a 12:12 h dark–light cycle.
2
Prior to trial start, animals were acclimated to experimental conditions for 10 days and Ž
. individually identified using passive integrated transponders Fish Eagle, UK . Fish were
fed according to commercial feeding tables 6 daysrweek, using a band feeder system Ž
. BioMar, Brande, Denmark .
2.2. Diet manufacture Ž .
Ž
y1
. Four diets were produced, consisting of A feed containing high 2083 mg kg
, Ž .
Ž
y1
. Ž
B low 208.3 mg kg concentrations of lipase Grindamyle Exel 16, Danisco
. Ž . Ž
. Ž
y1
. Cultor, Brabrand, Denmark , C heat-treated inactivated lipase 2083 mg kg
, and Ž .
Ž .
D the basal control diet. Heat treatment of lipase diet C was by autoclave for 30 min at 1208C followed by oven heating at 1208C for 1 h. Feeds were formulated in
accordance to commercial standards for high-energy diets, to contain 25 lipid. A non-extruded 3-mm pellet containing 8.29 lipid was used for the preparation of feeds.
Ž .
These pellets were extruded using an experimental extruder BioMar and coated with Ž
. heated 30–358C fish oil under vacuum. For lipase-supplemented diets, a fungal lipase
complex, originating from Thermomyces, but produced by fermentation in Aspergillus Ž
. oryzae, was employed. The lipase was in powder form and comprised protein lipase
and a starch carrier. The appropriate amount of enzyme for each feed type was mixed in fish oil by gentle stirring over 10 min and then added to feeds as described above.
Dietary proximate composition and FFA profile is presented in Table 1.
2.3. Analytical procedures 2.3.1. Growth performance
Individual weight and length measurements were taken for all animals at monthly intervals. Prior to handling, animals were fasted for 2 days. Animals were anaesthetised
Ž .
0.0002 benzocaine; Sigma during manipulations. FCR was calculated using the formula:
amount of feed ingested g
Ž .
FCR s .
weight increase g
Ž .
Table 1 Ž
Compositional analysis and fatty acid content of the basal diet employed during the present study all values .
are presented as percentage of diet Component
Protein 45.180.22
Lipid 25.350.28
Moisture 6.550.06
Ash 7.010.02
C 8.0 0.1
C 14.0 6.5
C 15.0 0.5
C 16.0 14.5
C 16.1 5.5
C 17.0 0.3
C 18.0 2.2
C 18.1 11.7
C 18.2 2.6
C 18.3 1.7
C 20.0 0.2
C 20.1 9.7
C 20.2 4.6
C 20.3 0.2
C 20.5 8.5
C 22.0 0.1
C 22.1 13.8
C 22.5 0.7
C 22.6 10.2
C 23.0 0.4
Others -C 18.3 2.5
Others C 18.3 3.5
Ž .
Ž .
Condition factor CF and weight and length specific growth rates SGRrday were Ž
. Ž
. computed as described in McLean et al.
1997 and Bassompierre et al.
1998 , Ž
respectively. Somatic indices were recorded for liver, heart and gut n s 35 per
. Ž
. treatment , using the methods described in Christensen and McLean 1998 , while fillet
yield and carcass percentage were evaluated according to the methods in Ostenfeld et al. Ž
. 1995 .
2.3.2. Fatty acid analysis Fatty acid composition of feeds and fillets were determined using extracted oil
Ž .
samples using a modified Bligh and Dyer 1959
method. Oil samples were Ž
. Ž
. esterifiedrinterestified with methanol anhydrous by means of an acid catalyst HCl .
The resultant methylesters were extracted with MTBE and determined by means of Ž
. gas–liquid chromatography Perkin-Elmer 8320 Capillary Gas Chromatograph . The
carrier gas used was helium, and the column used was a Cabowax 20. The response factors for each component relative to C-18 fatty acid methyl ester was calculated from
Ž .
standard 102 Danisco and the calculated area prescribed as 100.
2.3.3. Proximate composition Compositional analyses were undertaken on fillets from eight animals per treatment;
n s 4 per duplicate tank per treatment. Dry matter and moisture fractions were deter- mined by heating homogenized samples at 1058C for 24 h. The homogenized sample’s
inflammable components were removed by incineration at 5508C for 22–24 h for ash Ž
. determination. Calculation of protein
Kjeldahl-N was accomplished according to
Ž .
AOAC 1984 . Lipid presence was quantified using the method described by Bligh and Ž
. Dyer 1959 .
2.3.4. Quantification of lipase actiÕity Lipase activity, following addition to fish oil and feed was quantified indirectly by
following the appearance of free fatty acids. Feed samples were examined at 0, 60, 120 and 240 min post-lipase addition whereas for oil, control and high lipase-treated samples
Ž were examined at 0 and 240 min post-enzyme addition. Enzymatic reversibility i.e., the
. possibility of fatty acids reverting back to triglycerides was evaluated 24 h after enzyme
addition. The lipase reaction was stopped with the addition of 37 HCl. Titrations were Ž
. performed after lipid extraction Bligh and Dyer, 1959 . Lipase activity was examined
for all experimental feed types. 2.4. Experimental model and tests
Each treatment was undertaken in duplicate with tanks being randomly assigned. Since each tank was given one treatment, tanks were nested within treatments. The
experiment was performed as a two-factor nested design, where the factor treatment was considered as a fixed factor and fish tanks as a random factor. The two-factor nested
design model applied to the ANOVA was:
yijk s m q Ai q T A j i q ´ ij k ,
Ž . Ž . Ž .
Ž . where A was the treatment, T A the tank effect with fixed treatment, and ´ the
residual. By performing an analysis of variance upon recorded data, the effects of the Ž .
treatment and tank were estimated as a probability value P . Data from the evaluation of lipase activity were examined through a two-factor model:
yij s m q Ai q Tj q AT ij q ´ ij.
Ž .
Factors were treatment and time. An analysis of variance was performed upon recorded data. The effects of the treatment and time were estimated as a probability
Ž . value P . The data from investigation of FFA in the fillet were investigated using a
one-factor model: yij s m q Ai q ´ ij
Ž .
with the factor being a combination of treatment and time i.e., A end . An analysis of variance was performed upon recorded data. The effects of the combined treatmentrtime
Ž . factor were estimated as a probability value
P . All statistical analyses employed Ž
. SigmaStat software packages Jandel Scientific and results are presented as 95
Ž .
confidence limits Walpole and Myers, 1993 .
3. Results
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