Indirect calorimetry method was used to measure postprandial utilization of energy substrates by atipa, #Hoplosternum littorale$ (#Callichthyidae$, Siluriform). A model was adjusted to the data on respiratory exchanges and nitrogen excretion for this purpose. As for other fish species, protein was actively catabolized after feeding during the 60 hr measurement time. As long as carbohydrates were available, atipa was also able to use these substrates for its energy metabolism. A gradual increase of contribution of lipids was then noticed. Over a 24 hr period following the food intake, a 30 g fish catabolized 90 mg of proteins and 43 mg of carbohydrates. At the same time, the results led to a synthesis of 3.7 mg of fat. An estimation of the increase of the global metabolism associated to food intake was obtained from integration of the exponential terms of the models. (Résumé d'auteur

Boujard, T.

Luquet, Pierre

Medale, F.

Moreau, Yann

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POSTPRANDIAL UTlLlZATlON OF ENERGY 
SUBSTRATES BY A TROPICAL CATFISH, 
HOPLOSTERNUM LITTORALE: 
INDIRECT CALORIMETRY ANALYSIS 
Y. MOREAU,* F. MEDALE,** T. BOUJARD and P. LUQUET 
Laboratoire dHydrobiologie, INRA, BP 709, 97387 Kourou Cedex, Guyane francaise 
*Present address: Centre de Recherches Océanographiques, BP V18, Abidjan 01, Ivory Coast 
Laboratoire de Nutrition des Poissons, INRA, BP 3, 64310 St f é e  sur Nivelle, France ** 
ABSTRACT 
Indirect calorimetry method was used to measure postprandial utilization of energy substrates by atipa, 
Hoplosternum littorale (Callichthyidae, Siluriform). A model was adjusted to the data on respiratory 
exchanges and nitrogen excretion for this purpose. A s  for other fish species, protein was actively ca- 
tabolized after feeding during the 60 hr measurement time. A s  long as carbohydrates were available, 
atipa was also able to use these substrates for its energy metabolism. A gradual increase of contribu- 
tion of lip,ids was then noticed. Over a 24 hr period following the food intake, a 30 g fish catabolized 
90 mg of proteins arid 43 mg of carbohydrates. At the same time, the results led to a synthesis of 
3.7 mg of fat. An estimation of the increase of the global metabolism associated to food intake was 
obtained from integration of the exponential terms of the model. 
In fish, as in other animals, necessary nutrients for growth are provided by food. 
But food also provided fuels required by the energy metabolism. Among the three 
different energy substrates (proteins, lipids, and carbohydrates) which could be 
used by living beings, fish are reported to mainly use proteins even if they can 
burn fat too. 
Referring to their final destiny, the best energy substrates must be those 
which are not concrete parts of growth products. In this way, non-protein en- 
ergy substrate, such as fat *and carbohydrates, should be more adequate. The 
apparent poor retention of non-protein substrates, observed among tropical fish, 
indicates their better utilization for energy needs (Luquet and Moreau, 1989). As 
they disappear, they are not misused by transformation into body fat for example. 
To obtain both qualitative and quantitative evaluations of energy substrates- 
utilization by fed animals, indirect calorimetry is the only available method. This 
method was already applied to several fish species (Gnaiger, 1983; Van Wa- 
versveld et al., 1988; Kaushik et al., 1989; Medale and Kaushik, 1991). It is based 
250 
on respiratory gas exchanges which reflect cellular oxidation of energy-supplying 
substrates, and on nitrogen excretion arising from catabolism of nitrogen com- 
pounds. One of the first trials in this line was made by Kutty (1972) to study the 
metabolic rate associated with swimming activity in Oreochromis mossambicus. 
The attractions of this method in evaluating the standard metabolic rate of fish 
have been underlined by Brafield (1985) and compared to direct calorimetry by 
Van Waversveld et al. (1 989). 
Atipa (Hoplosfernum littorale, Hancock 1828) is an air-breathing siluriform fish 
of the Callichthyidae family living in the marshy areas of northern South America. 
So far, the data on this species mainly dealt with its respiratory system (Carter and 
Beadle, 1931 ; Gee and Graham, 1978), its behaviour (Winemiller, 1987; Gautier 
et al., 1988; Boujard et al., 1990, Boujard and Meunier, 1991), its rearing, and 
its feeding (Luquet et al., 1989; Moreau and Luquet, 1991). In earlier studies, the 
utilization of endogenous substrates was observed following body losses during 
starvation (Luquet et d., 1991) or using the indirect calorimetry method (Moreau 
et al., 1991). The aim of the present work is to identify and quantify the energy 
substrates supplied by food and used by this tropical catfish. 
MATERIALS AND METHODS 
One month before trials, fish reared in our experimental fish farm (Kourou, French 
Guiana) were transferred to the laboratory in a close circuit system. Trials were 
performed on five batches of five fish (31.8 g mean weight). Each batch was 
kept in 200 I tanks and submitted to natural photoperiod. Fish were fed daily 
with commercial food pellets at 3% of their body weight (Aqualim, 45% crude 
protein). The food was given at the end of the night, in accordance with their 
natural rhythm of food intake (Boujard et al., 1990). To be sure of a good food 
consumption, the fish were kept for at least 1 hr in contact with food. They were 
then carefully transferred into a respirometer which allowed the measurement of 
exchanges occurring in water and in air, as previously described by Moreau et 
al. (1991). Ammonia excretion was used to determine total nitrogen excretion 
since urea excretion was found negligible. Oxygen uptake (from air and water), 
carbon dioxide, and ammonia excretion were measured, every 2 hr for 48 hr. The 
postprandial 60 hr period was covered by selecting the time of transfer for each 
fish group (Feeding time + 1 hr, batch 1; .FT + 3 hr, batch 2 and 3; FT + 12 hr, 
batch 4 and 5). 
If calculations are made from a set of uptake or excretion data observed at 
the same time, some discrepancies can occur. As exchanges do not necessarily 
take place at the same time, a models was adjusted to the data to describe the 
general tendency of uptake and excretion during the whole measurement period. 
A delay between O, uptake and CO, excretion might result from an increase in 
fish activity (Wood and Perry, 1985, cit. in Playle et al., 1990) as during bursts- 
through the surface for air breathing or from the air breathing itself, some of the 
oxygen measured as uptake being trapped in air bubbles before their use. 
25 1 
Food intake induces a large income of metabolites, which are gradually ab- 
sorbed by metabolism. An exponential model is often used to describe this kind 
of process involving a steady treatment (or utilization) of products. But, as this for- 
mulation tends towards zero, it does not take into account a routine level reached 
at the end of the process. The model designed to adjust the data was then as 
follows: 
y = bo + b, ë y  
where y is the expected value of oxygen uptake, carbon dioxide, or ammonia 
excretion, and t the time in hours. bo can be assimilated to the routine metabolism 
rate, b, to the income associated to feed intake, and T to the rate of the different 
metabolism processes. 
The analytical methods were the same as those described in Moreau et al. 
(1 991). Energy expenditure and quantity of oxidized substrates were calculated 
according to Van Waversveld et al. (1 988). The SAS statistics software package 
has been used for statistical inference. 
RESULTS AND DISCUSSION 
Postprandial gas exchanges and ammonia excretion are plotted on Fig. 1. The 
postprandial increase of oxygen uptake and ammonia excretion usually described 
(Brett and Groves, 1979; Vellas, 1981) was not observed. This step should be 
missing as the first observations were made in fact 3 hr after feeding, i.e., sooner 
transfer time + 2 hr. All exchanges were only decreasing steadily over the mea- 
suring period to level off at the routine metabolism rate. 
The equations obtained for the different models are reported in Table I .  From 
these three equations, the types and amounts of energy substrates used by fish 
have been calculated (Fig. 2). Just after feeding, proteins are actively catabolized 
and their utilization decreases during the entire period of measurement to reach a 
level of about 50 mg/kg/hr after 58 hr. During the first 14 hr after feeding, a large 
amount of carbohydrates is also catabolized. At the same time, the observed 
RQ values come to a negative value for lipids which could be interpreted as a 
synthesis. Afterwards the lipids begin to be involved in the energy metabolism 
and their contribution increases as the involvement of carbohydrates tends to 
disappear. Fifty-five hours later, the carbohydrates are no longer used and lipids 
contribution represents about 16 mg/kg/hr. The amounts of substrate oxidized 
by a fish during the post-feeding periods can be calculated from the integration 
of the equations obtained for O, uptake and CO, and NH, excretion (Table 2). 
Furthermore, the contribution of each substrate to the total energy expenditure is 
deduced using the heat of combustion given for them (Brafield, 1985). 
Proteins are an important source of energy for atipa, as for other species. 
But this species is also able to burn carbohydrates. As carbohydrates are nearly 
absent from the whole body composition (Luquet et al., 1991), carbohydrates 
utilization must be related to their availability from feed. Increase in the carbo- 
hydrates level of feed is generally associated with a high body fat deposition 
252 
(Steffens, 1989). In this way carbohydrates will not be consumed, as a fuel but 
12 
I 
2 1  
O 10 20 30 40 50 60 70 
12 I I 
o '  I 
O 10 20 30 40 50 60 70 
2.5 
. .  . .  
O '  
O 10 20 30 40 50 60 
A 
Feeding Time (hours) 
Fig. I .  Evolution of oxygen uptake, carbon dioxide and ammonia excretion in H. littorale during 
the hours following food intake. The solid lines represent the adjustment of the data to a model, 
y = bo + ble-Ptz  and the broken lines are the 95% confidence levels of the means. 
Table 1. Values obtained after adjustment of gas exchange and nitrogen (in pmol/kg/hr) to a model, 
y = bo + b1e-l'' (with t in hours). n is the number of observations. Significance is given by the 
probability of finding an F statistic greater to f calculated between mean squares explained by the 
model'and residuals mean squares 
bo bl r n Prob (F > f) 
O2 uptake 2,591 7,114 0.03 1 00 < 0.001 
CO2 excretion 2,454 7,893 0.04 96 < 0.001 
NH3 excretion 524 1,772 0.06 97 < 0.001 
2 6  12 18 24 30 36 42 40 5 4 5 8  
Time (hours after feeding) 
Fig. 2. Utilization of different energy substrates by H. littorale during the hours following food intake. 
Table 2. Utilization of different substrates and'their contributions to the total energy expenditure by 
H. littorale, after a meal. The values are given for 30 g fish. Energy equivalents are calculated from 
the heat of combustion values given by Brafield (1 985): 19.67 kJ/g for proteins to ammonia, 39.5 kJ/g 
for lipids and 17.2 kJ/g for carbohydrates. 
Hours after Substrates Oxidized amount Energy contribution 
feeding (mg) (% of total) 
Proteins 
Carbohydrates 
1-14 Lipids 
57.6 
30 
(6) 
69 
31 
15-54 
Proteins 
Lipids 
Carbohydrates 
84.6 
16.8 
21 
62 
25 
13 
Proteins 
Carbohydrates 
55-60 Lipids 
7.5 
2.4 
609) 
61 
39 
transformed as fat. Based on one meal per day, total amounts of involved carbo- 
hydrates and lipids can be evaluated from integration over 24 hr. For a 30 g fish, 
this will give 43 mg of catabolized carbohydrates and simultaneously 3.7 rng of 
254 
produced fat. If all food was ingested and fish fed twice daily, the values would 
correspond to the post-feeding period of 1-14 hr (Table 2). 
The amount of carbohydrates needed to produce such an amount of fat can 
be estimated from metabolic fatty acid synthesis pathway. Three moles of car- 
bohydrates carbon are necessary to obtain 2 mol of fatty acid’carbon (Stryer, 
1988). Using the global formulas given by Gnaiger (1983) for carbohydrates and 
f,at, 1 g of carbohydrate is required to make 380 mg of lipids. Even if fish were 
becoming more fatty, this could not be a simple transfer from carbohydrates. If 
so, the .amount of deposit would be much higher. Thus, carbohydrates are used 
as energy supply. 
Over a 24 hr period, the amount of catabolized protein is near 90 mg for a 30 g 
fish. This value represents about 22% of the protein supplied in the food, which 
is much less than the value previously reported on rainbow trout (Oncorhynchus 
míkyss) fed at a lower rate (Kaushik et al., 1989), or those given for eel (An- 
guilla anguilla) or catfish (Clarias gariepinus) fed at a close level but continuously 
(Heinsbroek et al., 1989). 
Integration of the model for nitrogen excretion gives an estimation of the 
amount of catabolized proteins. For five days, a 100 g fish would use approx- 
imately 800 mg of proteins. This is close to the values of endogenous losses 
reported for the same species (Luquet et al., 1991). Conversely, estimates for 
lipids are not consistent. This could largely result from differences in the general 
state of the studied fish. For the former fasting experiment, fish were taken directly 
from the rearing pond, whereas those of the present study stayed one month in 
tanks. Nevertheless, the great similarity between the estimates of protein utiliza- 
tion seems to indicate that this is quite reliable. 
Studies on fasted fish show that the nature and amount of catabolited 
substrates were not constant over the fasting period (Luquet et al., 1991). So, 
the metabolism of starving fish changes as fasting time increases. Therefore, 
metabolism of starved fish could not be strictly taken as an estimate of standard or 
routine metabolism. Thus, specific dynamic action, or more precisely metabolism 
rate associated with feeding (including rise of activity), cannot be calculated by 
the difference between fed and unfed fish. In the model used for the present 
study, the exponential term corresponds to the overall process of ingested food, 
whereas the constant term could be assumed as the routine metabolism of fed 
fish. In fact these values are much higher than those found in fish fasted for 
10 days, as routine metabolism of fish had certainly changed during the fasting 
period. Moreover, the increase in metabolism resulting from food intake can be 
evaluated by the integration of the exponential term of the adjusted models. This 
leads to an energy expenditure of 2.97 kJ for a 30 g fish provided by utilization 
of 77.8 mg of proteins (52% of supplied energy), 27.6 mg of lipids (37%), and 
19.6 mg of carbohydrates (1 lYo). At the same time, 80% of the energy expenditure 
for routine metabolism (0.034 J/hs) came from protein catabolism. 
In summary, these results confirm the main role of protein as energy-yielding 
substrate for fed Hoplosternum littorale, as for starved ones. In this regard, atipa 
does not seem to be mainly different from other species (Cho and Kaushik, 1990). 
255 
Besides, the effective contribution of carbohydrates to energy metabolism indi- 
cates that the metabolism of carbohydrate is active in this species. 
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Reprint 
Journal 
of 
Aquaculture 
in the 
Tropics 


Postprandial utilization of energy susbtrates by a tropical catfish, Holosternum littorale : indirect calorimetry analysis

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