Fish Physiology and Biochemistry vol. 14 no. 2 pp 153-164 (1995) Kugler Publications, Amsterdam/New York

Effect of feeding on the tissue free amino acid concentrations in rainbow trout (Oncorhynchus mykiss Walbaum) C.G. Carter1 ,2, Z-Y. He l , D.F. Houlihanl, I.D. McCarthyl and I. Davidson 3 IDepartment of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen, AB9 2TN, U.K.; 2 Department of Aquaculture, University of Tasmania, P.O. Box 1214, Launceston, Tasmania 7250, Australia; 3 Department of Molecular and Cell Biology, University of Aberdeen, Marischal College, Broad Street, Aberdeen, AB9 AS, U.K. Accepted: October 19, 1994 Keywords: rainbow trout, free amino acid pools, stomach, caeca, liver, white muscle, amino acid flux Abstract This study investigates whether tissue free amino acid (FAA) pools in rainbow trout, Oncorhynchus mykiss (Walbaum), are altered following feeding and the relationships between the amount of food consumed and the FAA pool size. Trout were starved for 7 days to provide baseline data and then refed on day 8. Individual food intake was measured by radiography and the consumption of amino acids (AA) calculated from dietary protein consumption. Total FAA concentrations in the stomach, liver and white muscle were little changed at various times after the meal and this pattern was repeated for the majority of individual FAA. Overall, the most notable change was a reduction in essential FAA concentrations (principally in valine, leucine and isoleucine) in the white muscle following feeding. However, in the caeca total FAA, total essential FAA and a number of individual FAA were significantly elevated at 4, 9 and 15h following feeding. There were few significant correlations between dietary amino acid consumption and total tissue FAA and essential FAA concentration in the stomach, caecum and white muscle; correlations were stronger in the liver. In order to explain the relative constancy of total FAA concentrations in the tissues following food intake (which represents over 100% of the total FAA pool) a model is presented that quantifies the AA flux through the free pools and considers the role of protein turnover in regulating FAA pool size.

Introduction Rates of absorption of dietary amino acids (AA), AA oxidation and protein turnover are likely to be the most important mechanisms controlling tissue free amino acid (FAA) concentrations in fish (Dabrowski 1982; Millward and Rivers 1988; Houlihan et al. 1993, 1995). Ogata et al. (1985) measured the total essential FAA concentration in the whole body of the European eel, Anguilla anguilla L., and noted that it was positively correlated with dietary protein content. Significant correlations have also been reported between essential AA concentration in the diet and plasma of rainbow trout,

Oncorhynchus mykiss (Nose 1972; Yamada et al. 1981; Walton and Wilson 1986; Murai et al. 1987); common carp, Cyprinus carpio(Plakas et al. 1980; Dabrowski 1982; Ogata 1986) and channel catfish, Ictaluruspunctatus (Wilson et al. 1985). Although there were no data on the individual food consumption rates in these studies, Dabrowski (1982) suggested that individual variation in blood and plasma FAA concentrations were largely due to differences in the amount of food ingested. In contrast, other studies have reported that the FAA concentrations change little following feeding (Walton and Wilson 1986; Lyndon et al. 1993); FAA concentrations in the white muscle and stomach were relative-

154 Table 1. The AA composition of the diet (g per 100 g determined AA) Amino acid

Abreviation

Essential AA Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine

Arg His Ile Leu Lys Met Phe Thr Val

Non-essential AA Alanine Glycine Proline Serine Tyrosine Asparagine + Aspartate Glutamine + Glutamate

Ala Gly Pro Ser Tyr Asn + Asp Gin + Glu

g 100g-l AA

4.21 1.41 2.62 6.95 3.75 1.10 2.97 3.90 4.32

7.17 9.20 5.54 5.02 1.56 7.66 12.65

Tryptophan and cysteine were not measured after acid hydrolysis.

ly unchanged by a single meal of known weight eaten by individual Atlantic cod, Gadus morhua (Lyndon et al. 1993). It has subsequently been proposed by Houlihan et al. (1993) that increased rates of protein synthesis could account for the observed stability in the tissue FAA pools in the face of destabilisation from the dietary AA influx. The aim of the present paper was to measure tissue FAA concentrations in rainbow trout at various times following feeding. Individual rates of food consumption were measured using radiography (McCarthy et al. 1993). FAA concentrations in the stomach, caeca, liver and white muscle of rainbow trout were investigated in relation to AA consumption. A model of AA flux and protein turnover was constructed to test the hypothesis that rates of protein synthesis could account for the constancy of tissue FAA concentrations in rainbow trout.

Material and methods Husbandry and feeding Twenty rainbow trout with a mean ( SEM) weight of 264.8 + 38.4 g were held outdoors in a tank containing 350 1 of aerated water and supplied with fresh water at a rate of 30 1 h- 1. Fish were individually marked with alcian blue dots using a Panjet (Hart and Pitcher 1969), and were fed a commercial diet (North Eastern Farmers, Aberdeen, U.K.) once a day for 14 days at 20 mg of moist diet per gram initial body weight per day. The water temperature ranged between 10 and 12°C during the experimental period. After 7 days without food a group of 4 fish was removed, killed (see below) and used as the prefeeding zero time group. The remaining 16 fish were fed normally with a batch of commercial diet labelled with ballotini glass beads (McCarthy et al. 1993). Individual rates of food consumption were measured by radiography 2h after feeding (radiography details as in Carter et al. 1992). The AA consumption (mol g-1 wet weight) was calculated from the food (protein) consumed by each fish and the AA profile of the diet (Table 1). Protein was converted to AA equivalents assuming 9 mmol of FAA per gram of protein and taking into account the loss of water due to peptide bond formation (Ronnestad et al. 1992). The protein content of the diet was 50.6°70 dry material. The fish were returned to the tank and groups of 4 fish removed at 4, 9, 15 and 24h after feeding. Each fish was individually netted, removed and then killed by a blow to the head and transection of the spinal cord. Samples (500 mg) of stomach, caecal, liver and white muscle tissue were quickly dissected out on ice and frozen in liquid nitrogen. Samples were stored at -70°C until analysis.

Sample preparationand amino acid analysis To determine the tissue FAA concentrations, weighed tissue samples (approximately 200 mg) were treated exactly as described by Lyndon et al. (1993) and with 50 M norleucine in 0.1 M HCI as

155 Table 2. Total free AA (AA) and total free essential AA (EAA) concentrations (mol g- ) in stomach, caecum, liver and white muscle tissue before (Oh) and at various times after feeding. Comparison was made between the Oh group and each refed group (mean + SEM: n = 4, except liver at 9h when n = 3: *, p < 0.05; ***, p < 0.001) Hours after feeding

Stomach AA EAA Caecum AA EAA Liver AA EAA White msucle AA EAA

Oh

4h

9h

15h

24h

32.58 + 3.88 2.98 ± 0.27

35.00 + 2.93 4.62 + 1.65

34.63 + 1.26 3.03 ± 0.39

31.04 ± 2.01 3.34 ± 0.34

37.19 + 3.27 2.96 ± 0.33

40.05 + 2.45 5.19 ± 0.31

49.15 + 2.89* 8.76 ± 0.95*

51.00 + 3.97* 9.69 ± 0.24***

46.35 ± 3.74 8.57 + 0.93*

46.44 + 3.93 8.15 + 1.45

33.83 ± 1.19 5.06 + 0.61

40.65 ± 1.76* 5.32 ± 0.46

37.05 ± 0.45 5.37 ± 0.55

40.39 ± 1.19* 6.44 ± 0.55

36.49 + 1.77 5.13 ± 0.70

31.56 + 1.92 17.05 + 0.93

31.27 ± 1.03 13.97 + 0.48*

34.58 + 2.67 14.24 + 0.37*

34.02 ± 2.14 14.08 + 0.84*

32.40 ± 2.77 11.84 + 2.33

an internal standard. FAA concentration in the tissues were expressed as Ixmol per gram of fresh tissue. Weighed samples of food (approximately 20 mg) were hydrolysed under vacuum in 5 ml of 6M HCI for 18h at 110°C and 10 1lsub-samples were added to 10 il of the norleucine solution before dilution to 500 tl with distilled water. The AA composition of the food was expressed as grams of AA per 100 g of determined AA (Table 1). Arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine have been defined as essential AA for fish (Wilson 1989) and the remaining AA are termed non-essential AA. The non-essential AA not included in Table 1 were phosphoserine (Psr), hydroxyproline (Hyp), taurine (Tau) and ornithine (Orn).

Statistics Means + SEM are used throughout. Statistical significance was accepted at probabilities of 5% or less. Data were subjected to analysis of variance (ANOVA) and group means were compared by a Bonferroni's multiple comparison between the Oh group and each refed group. Pearson correlation coefficients were calculated to examine relationships between AA consumption and tissue FAA concentration. Statistical analysis was performed

by Statistix 4.0 (Analytical Software, St. Paul, Minnesota, U.S.A.).

Results Total FAA concentrations The total FAA and total essential FAA concentrations in the tissues of the unfed group were compared with those in the four refed groups (Table 2). In the caeca, total FAA and total essential FAA concentrations were both significantly higher at 4 and 9h after feeding and peaked at 9h. FAA concentrations in the stomach, liver and white muscle tissues were less affected by feeding. There were no significant differences in total FAA concentration in the stomach and white muscle or in the concentration of total essential FAA in the liver. In the white muscle there was a significant decrease in total essential FAA concentration at 4, 9 and 15h after the meal compared with the unfed (Oh) group. There were no significant differences in the total essential FAA concentrations in the stomach and liver. The total FAA and essential FAA concentrations in the tissues of the unfed and 24h groups were not significantly different from each other in any of the tissues.

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Effect of feeding on the tissue free amino acid concentrations in rainbow trout (Oncorhynchus mykiss Walbaum).

This study investigates whether tissue free amino acid (FAA) pools in rainbow trout,Oncorhynchus mykiss (Walbaum), are altered following feeding and t...
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