Fish and experimental system
Juvenile rainbow trout (O. mykiss) weighing 88.0-109.3 g were
grown at the research facilities of the Technical University of Denmark at the
North Sea Research Centre in Hirtshals.
Three consecutive experiments were carried out to investigate the postprandial
changes in plasma amino acid concentrations, each one with a different daily
feed ratio. For each one of the experiments, a group of 32 fish were
transferred to a flow through system consisting of eight 230 L aquaria, which
were divided into four chambers, each one with a 57L volume, were they were
individually housed. Fish were allocated randomly to one of 8 groups (n=4),
corresponding to the sampling times after feeding. During an acclimation period
of one week, fish were fed 0.5% body weight per day of a commercial diet (see
section 2). After the acclimation period, fish were reweighed and the six day
experimental period started, when fish were fed a ratio of either 0.5%, 0.9% or
1.5% body weight per day in each one of the experiments. Each group of four
fish was hand fed daily over a period of 20 min, and all the uneaten food was
recorded.
Diet
An experimental diet used for (XXXXXX
experiment look for citation) the three experiments, was formulated and
prepared by Biomar Ltd, Denmark (Table 1)
Table 1 Diet formulation and gross chemical composition of the experimental
diet.
Diet composition | % |
Fish meal SA 68
Superprime | 58.65 |
Wheat | 23.56 |
Fish Oil Std 18 | 18.63 |
Premix DK 3 | 0.30 |
Ethoxiquin 66% dry | 0.02 |
Barox Becp dry | 0.03 |
| |
Gross chemical composition | % |
Crude protein | 42.90 |
Crude fat | 24.01 |
Moisture | 6.00 |
Ash | 10.53 |
Phosphorus | 1.37 |
16.34 |
Sampling regime and growth
At the end of each experiment, fish were euthanized
using an overdose of Aqui-S (company). Sampling was carried out at 0, 2, 4, 6,
8, 12 and 24h after the last feeding. Fish were weighed and dissected through a
mid-sagittal incision and the heart exposed. Blood was collected using
heparinised syringes by direct puncture to the ventricle. Blood was centrifuged
at 1,500 g for 5 mins at 4oC and plasma stored at -80oC.
Following blood collection, the liver was excised, the gall bladder removed and
then weighed to obtain the hepatosomatic index (HSI) value, which was
calculated as:
HSI= [liver weight (g) / body weight
(g)]*100
Using the weights obtained at the start and
end of the experimental period (7 d), specific growth rate was calculated with the
following equation using data from individual fish:
SGR= Ln (W(ti)/W(t0))/(ti-t0)
x 100
Where W is
the individual weight of the fish, and t0 and ti
represent the first and the last day of the experimental period,
respectively. The feed conversion ratio
(FCR) was calculated using the equation:
FCR= feed
consumed/biomass gained.
Amino acid analyses
Acid hydrolysis of feed
Feed was hydrolysed as previously described
by Larsen et al with slight modifications. Feed was finely ground using a
mortar and pestle, and approximately 9 mg of feed were weighed and resuspended in a 5 mL vacuum hydrolysis
tube using 1.6 mL of 0.2% phenol in 6 N HCl. Samples were heated at 110oC in a
Reacti-Therm Heating module under vacuum conditions for 24h. After hydrolysis,
an internal standard (60 µL of 200 µM nor-leucine) was added, samples were transferred to an 8 mL glass
tube and HCl was removed by vacuum centrifugation at 100oC for 2h
(CentriVap, Labconco). Samples were then
resuspended in 2 mL 60% acetonitrile v/v and stored at 4oC until
analysis.
Plasma processing
Plasma samples were deproteinised by
thoroughly mixing 20 µL plasma with 50 µL 100% acetonitrile, followed by centrifugation at 5,000 g for 10 min
at room temperature. Supernatant was transferred into 0.2 µm filtering units (VWR…), and
centrifuged once more as above.
HPLC
Feed samples were diluted 1:3 v/v in 60%
acetonitrile before derivatization. Plasma and feed samples were derivatised
using AccQ-Fluor Reagent Kit (Waters) following manufacturer’s
instructions. Samples were then diluted
1:4 v/v in acetate buffer and amino acid content was measured in a Flexar FX-10
HPLC system (PerkinElmer Inc., Waltham, Mass, USA), using buffer gradients of
0.1 M sodium acetate and 60% acetonitrile. Two different gradients had to be prepared
to differentiate every amino acid peak in the chromatograms; pH of the sodium
acetate buffer was adjusted at 5.0 and 5.2 for each gradient. A standard 18 amino
acid mixture (Sigma-Aldrich), supplemented with hydroxiproline, glutamine,
asparagine and nor-leucine was analysed under the same conditions as the
samples and used to quantify the amino acid content. Tryptophan could not be
identified in the feed samples, as this aminoacid is destroyed by oxidation
under acidic conditions. The Chromera Flexar v3.2.0. 4847 software (PerkinElmer
Inc.) was used for analysis of amino acid peaks and quantification. Amino acid
concentrations in plasma are expressed as nmol/ml, and in feed as g/100g feed.
Data analyses
One way analyses of variance were performed
in growth data and HIS, two-way analyses of variance, with time and feeding
ratio as factors, were conducted on the plasma amino acid data. All analyses
were carried out in GraphPad Prism 5 (…).
Results
Amino acid composition of diet.
Amino acid (AA) | Abbreviation | g per 100 g of feed |
Essential AAs | | |
Arginine | Arg | |
Histidine | His | 1,10±0,03 |
Isoleucine | Ile | |
Leucine | Leu | |
Lysine | Lys | |
Methionine | Met | |
Phenylalanine | Phe | |
Threonine | Thr | |
Valine | Val | |
Tryptophan | Trp | |
Sum of essential AAs | | |
| | |
Non-essential AAs | | |
Aspartate (+ asparagine) | Asp (+ Asn) | 4,441±0,07 |
Alanine | Ala | |
Cysteine | Cys | |
Glutamate (+ glutamine) | Glu (+ Gln) | 3,01±0,06 |
Glycine | Gly | |
Hydroxy-proline | Hpro | 0,34±0,00 |
Proline | Pro | |
Serine | Ser | 1,63±0,02 |
Taurine | Tau | |
Tyrosine | Tyr | |
Sum of non-essential AAs | | |
| | |
Growth parameters
Amino acid profiles in plasma over time.
Add amino acid composition?