The aim of the present study was to investigate the effects of dietary supplementation with either methionine, cysteine or MCT on growth, digestibility, nitrogen and energy
retention, levels of plasma amino acids and activity of pancreatic enzymes in Atlantic salmon under pair-feeding and conditions of limited protein supply.
2. Materials and methods
2.1. Fish and facilities The trial was conducted at the research station of AKVAFORSK, Sunndalsøra,
Ž .
Norway. Atlantic salmon Salmo salar L. of the Sunndalsøra breed, weighing on
average 180 g, were distributed in 12 fibreglass tanks containing seawater. Each tank contained an initial biomass of approximately 12 kg fish. Water surface in the tanks was
1 m
2
, water depth 58 cm, and the water supply was 19 lrmin. Oxygen level was 80 of saturation. At trial termination, water temperature had risen to 118C from an initial 88C.
Total sum of degrees was 632 and the duration of the trial was 65 days. 2.2. Diets and feeding regimes
The experiment involved four diets, each fed to fish in three tanks under a pair-feeding regime. Tables 1 and 2 show feed composition and results of chemical
analysis. All diets were based on the same basal mash containing low temperature dried Ž
. Ž
. LT fish meal as the main protein source. Yttrium oxide Y O , 100 mgrkg, was used
2 3
Ž .
as an external indicator for digestibility determinations see below . To optimise
conditions for detection of improvements in protein utilisation, the protein level was low Ž
. ca. 14.5 g protein per MJ digestible energy and the diets were formulated to be
isonitrogenous. Wheat bran was used as a filler to be able to reach the intended protein-to-energy ratio without risking leakage of fat from the diets. The amino acids
were added on an equal nitrogen basis to the diet mash before extrusion. The diet Ž
. supplemented with alanine ALA-diet served as the reference diet.
Feed was provided 24 h a day using automatic feeders delivering feed every 15 min. Feed delivery and waste were recorded each day. The tank of fish with the lowest feed
intake determined the feeding level in the remaining tanks. Feed was delivered in excess during the first week of feeding to find the limiting group. Thereafter, this group
determined the amount of feed distributed to the other tanks the next day. Fish were fed the same amount of feed according to biomass in each tank. Feed intake was calculated
as the difference between distributed feed and waste feed collected.
2.3. Records and sampling All fish in each tank were bulk-weighed and counted at the start and end of the
experiment. At the start of the trial, three groups of 10 fish from the same population as the experimental fish were randomly sampled, and at the end, groups of 10 fish were
sampled from each tank for recording of body length and of weight, and for analysis of
Table 1 Formulations and chemical composition of the test diets
ALA-diet MET-diet
CYS-diet MCT-diet
y 1
Ingredients g kg Wheat
120 119
119 120
Wheat bran 262
262 262
262
a
Fish meal 354
353 354
354 Ž
. Fish oil Sandeel
245 245
245 146
MCT oil 100
Yttrium oxide 0.1
0.1 0.1
0.1
b
Vitamin and mineral premixes 15
15 15
15
c
Pigment 0.050
0.050 0.050
0.050
d
L
-Alanine 3.7
3.7
d
L
-Methionine 6.2
e
L
-Cysteine 5.0
Chemical composition as analysed
y1
Ž .
Water g kg 87
66 66
66
y1
Ž . Ž
. Protein g kg
nitrogen=6.25 297
298 303
310
y1
Ž .
Starch g kg 140
151 144
145
y1
Ž .
Ash g kg 52
55 53
53
y1
Ž .
Fat g kg 285
289 293
291
y1
Ž .
Total ny3 fatty acids of fat g kg 246
246 246
167
y1
Ž .
Total ny6 fatty acids of fat g kg 48
48 48
39
y1
Ž .
Total monounsaturated fatty acids of fat g kg 416
416 416
296
y1
Ž .
Total saturated fatty acids of fat g kg 225
225 225
470
y1
Ž .
Gross energy MJ kg 22.5
23.0 23.3
22.7
y1 f
Ž .
DPrDE g MJ 14.6
14.3 14.3
15.0
a
Norse LT 94, Nordsildmel, Norway.
b
Prioritary to BioMar, Brande, Denmark.
c
Ž .
Carophyll Pink 8 astaxanthin formulation Hofmann-LaRoche, Basel, Switzerland .
d
Sigma, St. Louis, MO, USA.
e
Fluka Chemie, Buchs, Switzerland.
f
DPrDE s gram digestible protein MJ
y1
of digestible energy. Gross energy for the nutrients and results of digestibility analysis in the present experiments were used in the calculation.
whole body composition. At the end, an additional 15 fish were sampled from each tank. Blood samples were drawn from five fish and centrifuged. Plasma samples were frozen
in liquid nitrogen and stored below y208C. Thereafter, the fish were killed by a blow to Ž
. the head, and the complete gastrointestinal GI tracts of the 15 fish were dissected,
wrapped in aluminium foil, frozen in liquid nitrogen and stored below y208C. Body measurements were taken. Livers from 10 of the 15 fish were weighed and sampled.
In a half-thawed state, the GI tracts were divided into the following four regions: Ž
stomach ST, J-shaped region with muscular closures in both ends, the cardiac and the .
Ž pyloric sphincters , pyloric region PR, the proximal region of the intestine, starting at
the distal side of the pyloric sphincter and ending at the location of the distal-most .
Ž caeca , mid-intestine MI, the region distal to the pyloric region and proximal to the
. Ž
increase in intestinal diameter and distal intestine DI, from the distal end of the .
mid-intestine to the anus . When divided into two equal-sized pieces, the mid- and distal
Table 2 Ž
1
. Amino acid composition of diets g kgy
Amino acid ALA-diet
MET-diet CYS-diet
MCT-diet Alanine
21.0 17.3
18.2 20.8
Arginine 18.3
18.6 18.3
18.0 Aspartic acid
27.6 27.9
28.4 27.2
Glutamic acid 47.0
47.4 48.2
46.6 Glycine
16.4 16.7
16.3 16.3
Cysteinercystine 4.2
4.3 8.9
4.0 Histidine
7.1 7.1
7.2 7.0
Isoleucine 13.8
13.6 14.1
13.3 Leucine
24.1 23.8
24.1 23.8
Lysine 22.5
22.3 23.3
22.4 Methionine
9.7 14.4
10.6 9.7
Phenylalanine 12.9
13.1 13.1
12.8 Proline
14.7 15.0
15.1 15.2
Serine 15.3
15.1 15.3
14.8 Threonine
14.3 14.0
14.2 13.7
Tyrosine 10.5
10.0 10.1
9.8 Valine
16.6 16.6
16.6 16.3
Sum 296.0
297.2 302.0
291.7
Ž .
Ž .
intestines were termed MI1 proximal half
and MI2 distal half , DI1 and DI2,
respectively. The intestines were opened and the luminal contents separated from the intestinal wall and pH measured directly in the chyme before it was collected in test
tubes. After sampling of fish for registration and analysis, the remaining fish were anaes-
Ž .
thetised and faeces stripped according to Austreng 1978 for determination of apparent digestibility of dietary protein, fat, starch, and energy and of apparent amino acid
absorbability.
2.4. Analyses 2.4.1. Feed, chyme, faeces and fish
Ž .
Nitrogen, fat gravimetrically and ash analysis were carried out as described by the Ž
. Association of Official Chemists 1990 . Fat in feed, chyme and faeces was extracted
with dichloromethane and homogenates of fish with ethylacetate. Protein content was Ž .
estimated as nitrogen N = 6.25. Feed dry matter was determined by oven drying at 1058C until the weight was stable. Energy content in feed and faeces was analysed by
Ž .
bomb calorimetry Association of Official Chemists, 1990 . Faeces and chyme were pooled within each tank and intestinal section and subjected to freeze-drying before
analysis. Dry matter was obtained from weight reduction during freeze-drying. Starch was determined as glucose after hydrolysis with a heat stable glucosidase under boiling
Ž .
conditions employing hexokinase, liberating reduced nicotinamide dinucleotide NADH , Ž
. detected spectrophotometrically Boehringer Mannheim, kit. no. 737 160 . Determina-
tion of amino acids was carried out according to the methods described by Spackman et
Ž .
Ž .
al. 1958 and Moore 1963 . Samples of approximately 50 mg were hydrolysed with 5 ml 6 M HCl containing 1 mgrml phenol and 5000 nmol norleucine as internal standard
for 24 h at 110 8C in thoroughly evacuated, sealed glass tubes. Following hydrolysis, 0.5 ml of each hydrolysate was evaporated with a rotatory evaporator in in vacuo and the
residues were taken up in the 2 ml pH 2.2 buffer. Aliquots of 50 ml were analysed with a BIOTRONIK LC 5001 amino acid analyser using the extended physiological system
with lithium citrate buffers and ninhydrin detection. Data collection was done with a two-channel Shimadzu C-R2AX integrator. Methionine and half-cysteine were deter-
mined as methionine sulfone and cysteic acid, respectively, in separate samples hydrol- ysed following oxidation with performic acid. The cysteine results therefore represent
the sum of cysteine and cystine in the sample. The oxidised samples were analysed using a LKAB Alpha-Plus amino acid analyser using the standard protein hydrolysate
system with sodium citrate buffers. The results were normalised on the basis of the sample weight and the recovery of the added internal standard norleucine. The amino
acid analysis was carried out at the Swedish Agricultural University, Uppsala, Sweden. Fatty acid composition of the feeds was determined by use of gas chromatography as
Ž .
previously described by Røsjø et al. 1994 . Yttrium was detected with an ICP
Ž . Ž
spectrometer model 1100, Termo JarrellrAsh, Franklin, MA, USA Refstie et al.,
. Ž
. 1997 . Enzyme activities were determined in 10 wrv water extracts of the freeze-
Ž .
dried samples. Total proteolytic activity was assayed according to Holm et al. 1988 with casein as substrate. Casein hydrolysis was used to express the total activity of the
intraluminal proteinases and peptidases along the intestinal tract. Lipolytic activity was Ž
. assayed according to Gjellesvik et al.
1992 using 4-nitrophenyl myristate as a
substrate. All enzyme activities are expressed per milligram of yttrium. As the diets contained the same concentration of yttrium, the given enzyme activities indicated
activities per unit of food eaten.
2.5. Blood Ž
. Free amino acids and taurine were determined in deproteinised trichloroacetic acid
Ž .
samples of plasma after prederivatisation with phenyl isothiocyanate PITC using a Ž
w
. Ž .
Waters HPLC amino acid analyser system Pico Tag Cohen et al., 1990 at the
Institute of Nutrition, Directorate of Fisheries, Bergen, Norway. 2.6. Calculations
The following calculations were performed: Specific growth rate SGR s
lnW y lnW rd = 100
Ž .
Ž .
Ž .
2 1
where W s weight at end, W s weight at start, d s number of days in the period.
2 1
Ž ŽŽ
Apparent digestibility or absorbability of a compound s 1 y compound in fae- . Ž
... cesryttrium in faeces r compound in dietryttrium in diet
= 100. In the present
study nitrogen absorbability was taken as an estimate of protein digestibility.
Ž .
Ž . Ž
Feed conÕersion ratio FCR s feed eaten r final biomass q biomass of dead fish y .
initial biomass Ž
. Ž
Ž . Ž Ž
..
3
. Condition factor CF s fish weight g r Fish length cm
= 100
Ž .
Ž .
Hepatosomatic index HSI s liver weightrbody weight = 100 Ž
. Retention of nitrogen s nitrogen increase in fishrnitrogen intake = 100
Ž .
Retention of energy s energy increase in fishrenergy intake = 100. 2.7. Statistics
Statistical evaluation of differences between diets was conducted using analysis of Ž
. Ž .
variance general linear model SAS Institute, 1989 . Multiple comparisons of growth,
specific growth rate, feed conversion, plasma amino acids, body composition, nutrient retention and digestibility of protein, fat, starch and energy, of amino acids absorbabil-
ity, and of enzyme activities between diets were conducted using the Duncan multiple range test, with tank mean as observation. For lipolytic and proteolytic activity,
comparisons were also carried out within each intestinal region separately. A signifi- cance level of P F 0.05 was chosen for all tests. As the variance of the amino acid
absorbabilities within each intestinal segment were fairly similar, a comparison of the
Ž absorbabilities was carried out on the basis of 95 confidence range least significant
. difference , estimated as 4 = SEM.
3. Results
