Stable isotope analyses are often used to calculate relative contributions of multiple food sources in an animal's diet. One prerequisite for a precise calculation is the determination of the diet-tissue fractionation factor. Isotopic ratios in animals are not only affected by the composition of the diet, but also by the amount of food consumed. Previous findings regarding the latter point are controversial. As stable isotope analyses have often been used to investigate aquatic food webs, an experiment with carp (Cyprinus carpioL.) was carried out to test the influence of the feeding level and individual metabolic rate on delta(13)C and delta(15)N values of the whole body. After an initial phase, 49 carp were assigned randomly to four groups and fed the same diet at different levels for 8 weeks. For 15 fish, the energy budget was determined by indirect calorimetry. Feed and individual fish were analysed for their proximate composition, gross energy content and delta(13)C and delta(15)N values. delta(13)C and delta(15)N values differed significantly at different feeding levels. While delta(13)C values of the lipids and delta(15)N values decreased with increasing feeding rate, delta(13)C values of the lipid-free matter showed a non-linear pattern. Data obtained from fish held in the respirometric system revealed a relationship between delta(13)C values and the percentage retention of metabolizable energy. Our results show that reconstructing the diets of fish from the isotopic ratios when the feeding level and individual metabolic rates are unknown would introduce an error into the data used for back-calculation of up to 1 per thousand for both delta(13)C and delta(15)N values and may have substantial effects on the results of calculated diets. As other workers have pointed out, the development and application of stable isotopes to nutritional ecology studies is a field in its infancy and gives rise to erroneous, misleading results without nutritional, physiological and ecological knowledge.

Many scientists use naturally occurring stable isotopes to reconstruct the diets of animals. However, isotopic ratios may be affected not only by the composition of the diet but also by the amount of food consumed. Thus, an experiment using tilapia (Oreochromis niloticus) was carried out to test the influence of feeding level on delta13C and delta15N of fish given a semi-synthetic wheat gluten/wheat starch based diet. In addition, the effect of diet quality was tested by comparing tilapia given this feed with tilapia fed a fish meal/wheat meal based diet. Forty-four tilapia were reared individually. After a prefeeding phase, fish were randomly assigned to five groups, four on the semi-synthetic diet at different feeding levels and one group on the fish meal/wheat meal based diet fed at the equivalent of the highest level of the semi-synthetic diet. The experiment lasted eight weeks. Proximate composition, gross energy content and delta13C and delta15N values were determined in feed and fish, for delta13C separately in the lipids and the lipid-free matter. Delta13C in the lipids and the lipid-free matter and delta15N of tilapia fed the semi-synthetic diet decreased significantly with increasing feeding rate. The absolute values of the trophic shift in fish fed the semi-synthetic wheat based diet were significantly higher than in fish fed the fish meal/wheat meal based diet. The different delta13C and delta15N values in tilapia fed the same diet at different feeding levels and the influence of feed quality on the trophic shift add to the uncertainty involved in the use of stable isotopes in ecological research.

This study provides data for the effect of dietary non-essential amino acid composition on the delta(13)C values of individual amino acids in rainbow trout (Oncorhynchus mykiss) using liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS). In this experiment, trout were reared either on a control diet or on three experimental diets, differing in the composition of non-essential/conditionally essential amino acids, for a period of 6 weeks. The control diet was a commercial trout starter feed with fish meal as the main protein source. The experimental diets contained no protein, only synthetic amino acids. Diet 1 resembled the composition of fish meal in both essential and non-essential amino acids, Diet 2 had all essential amino acids, but cysteine, glycine, proline and tyrosine were replaced by the corresponding amounts of their precursors, and in Diet 3 all non-essential amino acids were replaced by glutamate. LC/IRMS was used for the determination of delta(13)C values of individual amino acids from diets and tissues without derivatization. Diet affected the delta(13)C of individual amino acids in fish. For fish on Diets 1-3 amino acid delta(13)C values showed a similar trend: phenylalanine showed very little change from diet to body tissue. Arginine, lysine, tyrosine and proline showed strong depletion from diet to body tissue and glycine, alanine, aspartate and serine all showed variable but strong enrichment in (13)C. Improvements are necessary before all amino acid delta(13)C values can be determined; however, this study demonstrates that measuring amino acid isotopic signatures by LC/IRMS is a promising new technique for nutritional physiologists.

Although stable isotope ratios in animals have often been used as indicators of the trophic level and for the back-calculation of diets, few experiments have been done under standardized laboratory conditions to investigate factors influencing delta(15)N and delta(13)C values. An experiment using Nile tilapia [ Oreochromis niloticus (L.)] was therefore carried out to test the effect of different dietary protein contents (35.4, 42.3, and 50.9%) on delta(15)N and delta(13)C values of the whole tilapia. The fish were fed the isoenergetic and isolipidic semi-synthetic diets at a relatively low level. delta(15)N and delta(13)C values of the lipid-free body did not differ between the fish fed the diets with different protein contents, but the trophic shift for N and C isotopes decreased with increasing protein accretion in the individual fish, for N from 6.5 per thousand to 4 per thousand and for C in the lipid-free body from 4 per thousand to 2.5 per thousand. This is the first study showing the strong influence of the individual protein balance to the degree to which the isotopic signature of dietary protein was modified in tissue protein of fish. The extrapolation of the trophic level or the reconstruction of the diet of an animal from stable isotope ratios without knowledge of the individual physiological condition and the feeding rate may lead to erroneous results.

The influence of different feeding levels below and slightly above maintenance on whole body d 13 C and d 15 N values of Nile tilapia Oreochromis niloticus was examined. The energy budget of each fish was determined by indirect calorimetry. The d 13 C values of the lipid-free material of Nile tilapia fed below and slightly above maintenance level did not differ between the feeding groups, but the d 13 C values in the lipids and the d 15 N values of the lipid-free material showed small but significant differences. Those fish with a negative lipid retention had significantly higher d 13 C values in the lipid fraction compared to fish that synthesized fatty acids. There was a significant negative correlation between the amount of energy metabolized by the fish and both the d 13 C values in the lipids and the d 15 N values of the lipid-free material. Fasting and feeding below the maintenance level may influence the isotopic composition of animals and should therefore be considered in ecological and nutritional studies.