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Aquaculture, 73 (1988) 207-216 Elsevier Science Publishers B.V., Amsterdam

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Effect of Frequency of Feeding on Nitrogen and Energy Balance in Rainbow Trout under Maintenance Conditions SADASIVAM

J. KAUSHIK’

and EMIDIO F. GOMES’

‘Laboratoire de Nutrition des Poissons, INRA, 64310 Saint Pde-sur-Niuelle (France) (To whom reprint requests should be addressed) “Znstituto de Ciencias Biomkdicas “Abel Salazar”, Universidade do Porto (Portugal) (Accepted 4 December

1987)

ABSTRACT Kaushik, S.J. and Gomes, E.F., 1988. Effect of frequency of feeding on nitrogen and energy balance in rainbow trout under maintenance conditions. Aquaculture,73: 207-216. Three groups of rainbow trout were fed at maintenance level (daily, or once every 2 or 4 days) for a period of 4 weeks. Changes in the individual body weights and in body composition at the end of the trial were recorded. Patterns of nitrogen excretion and of oxygen consumption rates after a meal were followed in these groups, based on which some characteristics of protein utilization for energy purposes under routine and under postprandial conditions were calculated. Trout fed a ration to cover the maintenance needs once every 2 days showed significant conservation of body protein compared to other groups.

INTRODUCTION

The protein requirements for growth of salmonids and the physiological bases of such needs have been subjected to some analysis in the past few years (Huisman, 1976; Kaushik et al., 1981; Cowey and Luquet, 1983; Cho and Kaushik, 1985). The dependence of carnivorous fish on a high protein diet derives not only from a highly developed capacity to metabolize protein (Smith et al., 1978) but also from a limited ability to catabolize carbohydrates (Cowey and Luquet, 1983). It is now well established that, despite the consequent high participation of protein in energy metabolism of fish, protein requirements for maintenance are much lower in fish than in mammals. The maintenance needs for protein of rainbow trout have been estimated by direct methods after carcass analysis (Nose, 1961) or by dose-response curves extrapolated to zero growth (Kaushik et al., 1981; Kaushik and Luquet, 1983 ). According to these last authors, the maintenance needs of rainbow trout would

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amount to 1.8 2 0.3 gprotein/kg

body weight per day for fish weighing between

40 and 200 g. The effects of feeding frequency and of fasting (Andrews and Page, 1975; Grayton and Beamish, 1977; Luquet et al., 1981), of protein supply (Steffens, 1981; Fauconneau and Luquet, 1984) and of the level and type of dietary protein (Cho et al., 1976) have been well studied in salmonids under growing conditions. In contrast, very little is known of the nutrient utilization in fish under maintenance conditions. Besides, there are phases in the cycle of fish production where the maintenance of fish weight rather than growth is sought (e.g. adaptation to the market. needs) and where the economy of labour involved in feed distribution might become important. The present work is intended to study the effect of feeding a maintenance ration at different frequencies on nitrogen and energy balance in rainbow trout. MATERIAL AND METHODS

Fish, feeding, growth and digestibility Three groups of ten rainbow trout (Salmo gairdneri R. ) each (mean body weight 147.7 +- 16.5 g) were maintained in tanks of 60 1 capacity, with a water flow of 2 l/min, in a recirculated, thermoregulated ( 18 ? 1 ‘C ) system. They were fed a commercial diet (see proximate composition in Table 1) at a level estimated to cover their maintenance needs of about 2 g protein/kg per day (Kaushik and Luquet, 1983), at three different feeding frequencies: 0.5% of live weight every day (batch 1 ), 1% of live weight every 2 days (batch 2) and 2% every 4 days (batch 3 ). Fish were individually marked with alcyan blue using a dermojet. Individual fish weights were recorded at the beginning, at weekly intervals and at the end of the trial which lasted 28 days. A sample of ten other fish was drawn for carcass composition at the start of the experiment and weighed individually. They were eviscerated and weights of liver and of viscera were noted. At the TABLE 1 Composition of the diet Dry matter ( % ) Protein (NX6.25) (%) Energy (kJ/g DM) Cr,O, (% DM)

86.7 50.9 21.0 1.2

Digestible protein (% DM) Digestible energy (kJ/g DM)

42.5 17

Digestibility coefficients ( % ) dry matter protein energy

71.60 84.50 80.37

209

end of the trial, the same procedure was followed for every fish of the three treatments. Samples were kept frozen until analysed. Digestibility coefficients were determined by the indirect method using 1% Cr,03 in the diet. Faeces were collected using the automatic faecal collector described by Choubert et al. ( 1982 ). Metabolic studies

On the fourth day of the trial, after food distribution to the three treatment groups of fish, continuous monitoring of ammonia nitrogen excretion was carried out in each tank, during 3 consecutive 24-h cycles, following the procedures outlined by Kaushik (1980). The oxygen content of the water at the inlet and at the outlet of the tanks was also measured during the 72-h cycle using a portable electrode (Ponselle, France) to determine the oxygen uptake by fish. On the basis of these daily patterns, the pre-feeding, basal levels as well as the increase in metabolic rates due to feeding were calculated both for ammonia excretion and for oxygen uptake. Blood metabolites

On the 28th day, 8 h after a meal, individual blood samples were drawn from the caudal vessel (using heparinized syringes) of six anaesthetized (monophenyl ether, 1 : 2500) fish from each group. The blood samples were centrifuged (3000 rpm, 20 min) , and the plasma was separated and stored frozen before analyses for ammonia and free amino acids. Analytical methods

Proximate analyses of the diet, carcasses and faeces were made following the usual procedures: dry matter after drying in an oven at 104 oC for 24 h; protein (N x 6.25) by the Kjeldahl method after acid digestion; energy in an adiabatic bomb calorimeter (Gallenkamp); fat content of carcasses by chloroform : methanol extraction (Folch). Chromic oxide in the diet and faeces was measured using a semiautomatic procedure involving perchloric acid digestion (Bolin et al., 1952) in a block digestor (Technicon Inc., BD-40) followed by the automatic determination of bichromate ( Autoanalyser, Technicon ) in the digesta, using diphenylcarbazide (Mathieson, 1970). Ammonia concentrations in water were measured according to Le Corre and Treguer (1976) and the instantaneous rates calculated as explained by Kaushik ( 1980). Plasma ammonia and total free amino acids (as glycine equivalents of amino groups using trinitrobenzene sulphonate) were analysed in an autoanalyser (Technicon) using slightly modified methodologies of Assous et al. ( 1960) and Palmers and Peters (1965), respectively.

Statistical analyses were made following methods outlined by Snedecor and Cochran (1956). RESULTS

Growth and digestibility At the end of the 4 weeks of the trial, the individual average weights of the fishes in the three treatments were not significantly different from their initial weights. The hepatosomatic indices (1.1%) and dressed weights (88%) also did not vary much (Table 2). As regards body composition, at the end of 4 weeks, fish of the group fed once every 2 days (batch 2) had a lower fat and a higher protein content than those in the other two groups. The apparent digestibility coefficients of protein and energy of the diet used were respectively 84.5 and 80.4%. Nitrogen excretion Fig. 1 shows the patterns of ammonia-N excretion by fish from each of the batches during a 72-h cycle. In all groups, there was a postprandial rise in ammonia excretion immediately after a meal; the peak rates as well as the time of appearance of such peak values were affected by the amount of feed intake. The ammonia excretion rates reached the peak values 8, 10 and 13 h after a meal in batch 1, 2 and 3 respectively. Cumulative daily values are reported in Table 3. After the first day, the fish fed 2% every 4 days (batch 3) reacted to fasting with a gradual reduction in their rate of ammonia excretion, reaching a lower basal level (120 mg N/kg BW per day) than those of the other two TABLE2 Growth

and body composition

of fish fed maintenance Initial

Batch

(g)

-

Final average weight (g ) Weight variation (%/day) Body composition Dressed weight (% ) Hepatosomatic index

-

Initial average weight

Dry matter Protein (N~6.25) Energy (kJ/g DM) Lipids (% DM)

(% DM)

rations

88.24+ 1.1 l.lOkO.2 25.65 69.48 23.27 19.80

at different

1

frequencies

(0.5%/day)

Batch 2 (1%/2 days)

Batch 3 (2%/4 days)

153.0 + 18.0 152.0 + 14.7 -0.08+ 0.15

141.4 k 16.5 137.7 * 18.7 -0.11 k 0.29

148.8 + 15.7 153.7 + 16.6 0.15+ 0.18

90.66+ 1.10* 24.23 70.14 23.59 20.10

1.1 0.2

88.45k 1.10% 22.89 74.76 22.49 16.74

1.2 0.2

87.51? 1.4 l.OOf 0.1 24.15 70.08 22.87 18.55

211

60

hours

Fig. 1. Postprandial patterns of ammonia nitrogen excretion in trout fed maintenance rations at three different feeding frequencies. Arrows indicate meals. I to III: batches 1 to 3.

groups (170 and 150 mg N/kg per day in batch 1 and 2 respectively). The percentage of ammonia nitrogen excretion, relative to nitrogen intake, was not very different between the three groups. Energy utilization As can be seen in Table 3, the routine oxygen uptake was slightly lower in batch 1, fed 0.5% daily, and higher in batch 2, fed 1% every 2 days. On the basis of data on the postprandial rise in oxygen uptake, the specific dynamic action (SDA) was calculated and the values are also reported in Table 3 as a proportion of the digestible energy (DE) intake. The SDA as a percent of DE intake

212 TABLE 3 Nitrogen excretion and oxygen uptake vaiues in trout fed maintenance frequencies

Ammonia excretion Routine rate (mg N/kg BW per day) Total excretion/meal (mg N/kg BW) Rise due to feeding (mg N/kg BW) N excreted/N intake (%) Oxygen uptake Routine rate (g Oz/kg BW per day) Total uptake/meal (g O,/kg BW) Rise due to feeding (g O,/kg BW) SDA as % DE”

rations at different feeding

Batch 1 (0.5%/day)

Batch 2 (1%/2 days)

Batch 3 (2%/4 days)

168 321 153 42

148 624 328 46

120 1160 680 41

3.4 5.2 1.8 25.3

4.5 12.7 3.2 24.7

3.8 19.8 4.6 16.3

“SDA, specific dynamic action; DE, digestible energy intake. Energy expenditure for SDA was calculated from values on the rise in oxygen uptake due to feeding, using oxycaloric equivalents of 13.4 kJ/g oxygen.

TABLE 4 Basal and postprandial energy expenditure and the protein to energy ratios in trout under maintenance conditions fed at different frequencies

Energy expenditure basal (kJ/kg per day) SDA (kJ/kg per day) Protein/energy ratios (mg N/kJ) basal postprandial carcass analysis Relative contribution of protein catabolism to energy release (% )”

Batch 1 (0.5%/day)

Batch 2 (I%/2 days)

Batch 3 (2%/4 days)

45.6 24.4

61.0 24.12

51.4 15.5

3.7 6.3 5.3 42.8

2.4 6.7 1.4 34.0

2.4 10.9 3.3 40.6

*Calculated from ammonia quotient (A&) values (mole to mole ratio of N excreted/oxygen uptake) per meal, assuming that an AQ of 0.33 would correspond to total aerobic protein catabolism (Kutty, 1978).

was lower in those fish fed every 4 days (16%) than in the other two groups (25%). In Table 4 are given the energy expenditures calculated from the oxygen uptake data partitioned into basal and postprandial values. Also given are the

213

values for the relative contribution of protein to this energy expenditure (protein : energy ratios as mg NfkJ) under basal and post-feeding conditions. Basal energy expenditure was lowest in fish fed daily (46 kJ/kg BW per day) than in the other two groups (61 and 51 kJ/kg BW per day in groups 2 and 3 respectively ). The contribution of proteins to this basal energy expenditure was higher in group 1 (3.7 mg N/kJ) than in the other two groups (2.4 mg N/kJ). In the postprandial state, however, this participation of protein in energy metabolism was nearly twice as high in fish fed every 4 days (11 mg N/kJ) as in the other groups (6.5 mg N/kJ). Further, on the basis of changes in body composition, the relative loss of body proteins to that of energy loss in the three groups is also reported in Table 4. This contribution of protein to satisfy the energy requirements, estimated by carcass analysis, was lower in batch 2 than in the others. Plasma

metabolites

The ammonia and free amino acids levels in the plasma 8 h after a meal are reported in Table 5. Plasma ammonia levels of fish in the batch fed OA%/day were lower than those of the other groups, Free amino acid levels were, on the contrary, higher in this group than in the other two groups. DISCUSSION

The maintenance requirement is defined as the level of dietary intake at which the animal neither loses nor gains weight, or in other words, when the relative rate of growth equals zero. From this definition, an attempt can be made to estimate the protein level required for maintenance. These estimations in rainbow trout provide values of 1.25 g/kg per day (Nose, 1971) and 1.74 g/kg per day (Kaushik and Luquet, 1983), estimated by zero nitrogen retention and zero growth, respectively. Kaushik et al. ( 1981) tried to estimate maintenance protein needs of rainbow trout by feeding them protein and nonprotein energy components separately. From the present work, it can be calTABLE Ammonia

5 and free amino acid levels in plasma of trout fed different rations (values at 8 h after a

meal) Batch

Ammonia (mg N&N/l)

Free amino acids (mM equiv. glycine)

1

5.80 2 1.10

5.26 k 0.12

2 3

7.78 io.91 7.00 k 0.64

3.96 + 0.56 4.04 i 0.55

214

culated that 2.6 g digestible protein/kg BW per day would be required to maintain zero nitrogen balance in rainbow trout carcasses. Temperature and protein quality affect protein needs for maintenance (Birkett, 1969; Nose, 1971). Besides, as pointed out by Luquet and Kaushik (1979), usually the endogenous body protein losses in fish estimated by carcass analysis yield higher values than the ones obtained through other means, like the measurement of endogenous nitrogen excretion, which do not take into account all body protein losses. The higher value observed here might also reflect the maintenance needs of trout fed a complete diet as against results obtained under separate-feeding conditions (Kaushik et al., 1981). Recently, Dabrowski et al. (1987) showed that considerable variations in the utilization of protein and energy can occur even within a diurnal cycle, depending on the nutritional quality of diets fed to sturgeon, Acipencer baeri. It seems also that protein synthetic processes might have a cyclical pattern (Goolish and Adelman, 1983). The data on the variations in the patterns of ammonia excretion (peak rates and the time to reach SUC_~ rates in relation to nitrogen intake) are very much in accordance to our own earlier observations (Kaushik, 1980). The differences, although small, in plasma ammonia and free amino acids levels suggest that fish adapted to a fasting rhythm utilize more rapidly the dietary input of greater amounts of amino acids than those fed daily. The lower levels of free amino acids in these two groups 8 h after a meal cannot be attributed to slower absorption rates of dietary amino acids but to slower rates of deamination, as shown by higher plasma ammonia levels and by the later occurrences of maximal rates of ammonia excretion. The difference in the time of appearance of peak levels of circulating nitrogenous metabolites is known to be influenced by the previous nutritional history (Kaushik, 1979) and hence care should be taken as regards the choice of time interval after a meal for blood sampling used for the assessment of the nutritional status of fish on the basis of such biochemical criteria. Grayton and Beamish (1977) stated that the responses to infrequent feeding, such as hyperlipogenesis, decreased metabolic rate and increased growth efficiency, are likely generated in fish only when long intervals between meals are imposed. The effects of feed frequency and of periodic fasting in rainbow trout were studied by Luquet et al. (1981) who found that the responses, in terms of growth performance and body composition, to fasting were different from those obtained by a reduction in feed frequency. Both Jobling (1980) in plaice (Pleuronectes platessa) and Jezierska et al. (1982) in rainbow trout studied the long-term effects of starvation on body lipid mobilization and showed that perivisceral and to a lesser extent muscular lipid reserves were depleted. It is of considerable interest to note that, for fish fed under maintenance conditions, the decrease in lipid content was variable depending on the frequency of food supply. Calculations based on the ammonia quotient (AQ =mole to mole ratio of ammonia excreted to oxygen consumed) values

215

(Table 4) show that in fish fed every 2 days, the contribution of protein to energy expenditure is in fact reduced in comparison to the two other groups. It is evidence that some interaction between feeding level and the frequency of feeding occurs in terms of energy requirements for maintenance and in terms of protein contribution to such needs. Overall postprandial protein : energy ratios show that this also holds true in trout fed maintenance rations. In terms of SDA, those fish fed once every 4 days seem to have the lowest SDA per unit DE intake. Despite higher basal metabolic rates, fish fed 1% every 2 days showed a better conservation of body protein, with a lower protein contribution to maintenance energy needs, also evidenced by the higher lipid mobilization observed in this group. Under maintenance ration levels, it therefore seems appropriate to feed rainbow trout once every 2 days for better conservation of body proteins.

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