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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

Nitrogen-free extract (NFE)

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.

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