Julia Gaye-Siessegger

Feeding level and diet quality influence trophic shift of C and N isotopes in Nile tilapia (Oreochromis niloticus (L.)).

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 δ 13C and δ 15N 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 δ 13C and δ 15N values were determined in feed and fish, for δ 13C separately in the lipids and the lipid-free matter. δ 13C in the lipids and the lipid-free matter and δ 15N 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 δ 13C and δ 15N 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.

Individual protein balance strongly influences δ15N and δ13C values in Nile tilapia, Oreochromis niloticus

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 δ15N and δ13C 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 δ15N and δ13C values of the whole tilapia. The fish were fed the isoenergetic and isolipidic semi-synthetic diets at a relatively low level. δ15N and δ13C 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‰ to 4‰ and for C in the lipid-free body from 4‰ to 2.5‰. 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.

Determination of underivatized amino acid δ13C by liquid chromatography/isotope ratio mass spectrometry for nutritional studies: the effect of dietary non‐essential amino acid profile on the isotopic signature of individual amino acids in fish

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.

Dietary lipid content influences the activity of lipogenic enzymes in the liver and on whole body δ13c values of Nile tilapia, Oreochromis niloticus (L.)

The use of stable isotope techniques for the reconstruction of diets has increased over the last decade. However, isotopic ratios in an animal are not only affected by the composition of the feed, but also by the amount of feed consumed. An uncertainty of up to 1 ‰ for both δ13C and δ15N values has been observed when the feeding level is unknown. This may have substantial effects on the results of back-calculation. As the feeding level of animals is unknown in nature, an additional indicator for their nutritional status is needed. High feeding levels and a consequent surfeit of dietary energy lead to the synthesis of lipids. In order to test whether the level of lipogenesis could be used as an indicator, Nile tilapia (Oreochromis niloticus) were fed four isonitrogenous and isoenergetic wheat-based semi-synthetic diets with different lipid contents (2.0 %, 4.5 %, 9.5 % and 13.3 %) for eight weeks. Body composition, gross energy content and δ13C values in the lipids and the lipid-free material were determined in diets and fish bodies. The livers of three fish per feeding group were assayed for the activity of two lipogenic enzymes, ATP-citrate lyase and malic enzyme. There was a strong negative correlation between δ13C values in the lipids of the individual fish and the apparent lipid conversion. The activities of lipogenic enzymes decreased with rising lipid content in the diet. The δ13C values in the lipids decreased significantly with increasing specific activity for both enzymes. In this experiment where lipogenesis was influenced by the composition of the diet, it was possible to determine the exact value for the trophic shift in relation to the enzyme activities. Further experiments to investigate the use of enzyme activities in situations where the feeding level of an animal is unknown are recommended. Revised version of a paper presented at the 26th Annual Meeting of the German Association for Stable Isotope Research (GASIR) October, 6 to 8, 2003, Cologne, Germany

Dietary back-calculation using stable isotopes: can activities of enzymes involved in amino acid metabolism be used to improve estimates of trophic shifts in fish?

The aim of this study was (1) to assess the effects of dietary protein content and feeding level on trophic shifts of C and N isotopes (Δ δ13Ctissue−diet and Δ δ15Ntissue−diet) and (2) to test whether the measurement of the activities of two enzymes involved in the metabolism of amino acids could improve the accuracy of estimation of the trophic shifts of C and N isotopes. For this, 36 Nile tilapia (Oreochromis niloticus) were kept under controlled conditions for 8 weeks and fed at three different levels (2, 4 and 8 g kg−0.8 d−1) with three diets differing in their protein content only (20, 29 and 39 %). For each fish, food to fish body trophic shifts of C and N isotopes were measured as well as the hepatic activities of aspartate aminotransferase (ASAT) and glutamate dehydrogenase (GDH). The feeding level affected the activities of ASAT and GDH as well as the trophic shifts of C and N isotopes significantly but the dietary protein content had no significant effect except on the specific activity of ASAT. Fish fed at the lowest level had significantly higher trophic shifts of C and N isotopes than fish fed at higher levels. The trophic shifts were significantly lower in fish with a high protein utilisation. Values of the ‘goodness-of-fit’ for linear regressions between enzyme activities and trophic shifts were low. Thus, activities of ASAT and GDH are not suitable for predicting estimates of trophic shifts in situations where the amount of food consumed or the dietary protein content is not known. In further studies, activities of enzymes involved in the metabolism of amino acids combined with measurements of the activities of other enzymes should be used to try and improve the accuracy of estimates of trophic shifts.

Improving estimates of trophic shift (Δδtrophic) for diet reconstruction studies using enzyme activities

For diet reconstruction studies using stable isotopes, accurate estimates of trophic shift (Δδtrophic) are necessary to get reliable results. Several factors have been identified which affect the trophic shift. The goal of the present experiment was to test whether measurements of the activities of enzymes could improve the accuracy of estimation of trophic shift in fish. Forty-eight Nile tilapia (Oreochromis niloticus) were fed under controlled conditions with two diets differing in their protein content (21 and 41%) each at four different levels (4, 8, 12 and 16gkg(-0.8)d(-1)). At the end of the feeding experiment, proximate composition, whole body δ(13)C and δ(15)N as well as the activities of enzymes involved in anabolism and catabolism were measured. Step-wise regression specified contributing variables for Δδ(15)N (malic enzyme, aspartate aminotransferase and protein content) and Δδ(13)Clipid-free material (aspartate aminotransferase and protein content). Explained variation by using the significant main effects was about 70% for Δδ(15)N and Δδ(13)Clipid-free material, respectively. The results of the present study indicate that enzyme activities are suitable indicators to improve estimates of trophic shift.