Ulfert Focken

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.

Effect of different lipid extraction methods on δ13C of lipid and lipid-free fractions of fish and different fish feeds

For many ecological applications of stable carbon isotope techniques, it is necessary to separate the lipid and lipid-free fractions. The effect of different lipid extraction methods on the isotope signature of the remaining lipid-free matter as well as the lipid fraction was tested. A hot extraction form of the Soxhlet method using petrol-ether was compared with two liquid-liquid extraction methods for lipid determination described by Bligh and Dyer and Smedes. Solid samples of fish and different natural food items were subjected to extraction and the carbon isotope ratios in lipid and lipid-free matter determined by IRMS. All methods were suitable for lipid extraction from all samples analysed here and did not cause biologically relevant differences (>1) in carbon isotopic ratios, except the Bligh and Dyer extraction method using chloroform which caused systematic errors for δ 13C when applied to diatoms.

Stable Isotopes in Animal Ecology: The Effect of Ration Size on the Trophic Shift of C and N Isotopes Between Feed and Carcass

Abstract The feeding ecology of living or extinct animal species is now frequently studied by analysis of stable isotope ratios in small quantities of carcass or remains such as bones or teeth. Although there are numerous papers on these applications in natural systems, the theoretical and experimental basis of this method is weak. In order to evaluate the effect of different feeding levels on the carbon and nitrogen trophic shift, an experiment was carried out in which fish (Nile tilapia, Oreochromis niloticus, initial weight 40.8g) were fed for 4 weeks at three levels from slightly above maintenance to almost satiation. For each treatment, three fish were reared individually. The isotopic ratios of carbon and nitrogen in feeds and fish carcasses were determined and in the case of carbon this was done separately for lipids and lipid-free matter. The trophic shift was calculated at each feeding level from the δ13C and δ15N ratios of feed and fish. There was a significant trend towards higher values for the trophic shift at higher feeding rates in all fractions analysed. Although further research is required, it can be concluded that the effect of feeding level cannot be ignored when the diet of an animal has to be calculated from the ratios of isotopes in its body.

Evaluation of an unconventional legume seed, Sesbania aculeata, as a dietary protein source for common carp, Cyprinus carpio L.

Abstract A 7-week feeding trial was conducted in a recirculation system at 27±0.2°C to evaluate the nutritive value of Sesbania aculeata seed meal as a possible protein source in the diet of common carp. Sesbania seed meal was included in the diets at various levels (12%, 24%, 36% and 48% for diets 2, 3, 4 and 5, respectively, which correspond to 10%, 20%, 30% and 40% of dietary crude protein) and the response of fish fed these diets was compared to fish fed a fish meal based control diet (40% crude protein). All the diets were made iso-nitrogenous and iso-energetic. Each treatment had two replicates, eight fish per replicate, with mean initial mass of 5.25±0.03 g. Fish were fed six times of their maintenance requirement (3.2×body mass (g)×kg0.8) ration daily. On the basis of the observed growth rate, feed conversion ratio, protein efficiency ratio, apparent net protein utilisation and energy retention, diet 1 (control) and diet 2 (containing 12% Sesbania meal) were similar and significantly (P 12%) of Sesbania meal had significantly higher whole body moisture, lower lipid and gross energy content. The lower growth performance of fish fed diets containing higher levels of Sesbania meal is thought to be due to the presence of various antinutrients such as tannins and saponins in Sesbania seeds. The results of this study indicated that inclusion of up to 12% of untreated Sesbania seed meal (10% of the dietary protein) in the diet did not affect the growth performance and nutrient utilisation in common carp.

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.

Contribution of natural food and supplemental feed to the gut content of Penaeus monodon Fabricius in a semi-intensive pond system in the Philippines

Abstract Juvenile Penaeus monodon were stocked in grow-out ponds and fed a compound diet at high rates for 19 weeks under semi-intensive conditions. At three stages of the rearing period (weeks 6, 11 and 16), the gut content of the shrimp was analysed microscopically at every hour of the day. Additionally, possible sources of natural food (lablab, lumut, zoobenthos, etc.) were analysed microscopically and where possible for proximate composition. At week 6, the gut content consisted of 28.9% supplemental feed, 42.3% plant materials (other than from the pelleted diet), 1.8% crustacean parts and 27.0% diverse detrital matter. For week 11, the percentages (same order) were 47.5%, 21.1%, 22.8%, 8.6% and for week 16, 21.7%, 34.3%, 31.7% and 12.9%, respectively. Food preference did not change with time of the day. At week 6, most feeding activity occurred at night, later, feeding activity shifted to day-time. Reduction of the maximum gut content at dissolved oxygen levels below 4 mg l −1 at night indicated a cessation of feeding in which case shrimp fed during the day-time, when dissolved oxygen levels were higher.

Effect of soaking and soaking followed by autoclaving of Sesbania seeds on growth and feed utilisation in common carp, Cyprinus carpio L.

An 8-week feeding trial was conducted in a recirculation system at 27±0.2°C to observe the effect of soaking and soaking+autoclaving of Sesbania aculeata seed meal on growth and feed utilisation in common carp. Sesbania seeds were treated by 24-h water soaking and 24-h soaking followed by autoclaving at 121°C at 15 psi (1.05 kp cm−2) for 30 min. Six iso-nitrogenous and iso-energetic diets were formulated. Diet 1 (control) was prepared with fish meal as the major source of protein. Untreated Sesbania seed meal was included in diet 2 at 20% level of dietary protein. Each of soaked and soaked+autoclaved Sesbania seed meal was included at two dietary levels of 23 (diets 3 and 5) and 35% (diets 4 and 6) which corresponds to 20% and 30% of total dietary protein, respectively. Each treatment had two replicates, eight fish per replicate, with mean initial body weight of 6.2±0.02 g. Fish were fed six times of their maintenance requirement (3.2 g×bodyweight (kg)−0.8d−1) ration daily. Fish fed the control diet showed the best (P<0.05) growth performance and feed utilisation. The growth performance of fish fed diet 3 containing 23% soaked seed meal was significantly higher than that of fish fed diets 2 and 5 containing similar amounts of untreated and soaked+autoclaved seeds, respectively. Diets 4 and 6 (containing 35% soaked and soaked+autoclaved seed, respectively) resulted in significantly lower fish growth and feed utilisation compared to rest of the diets. Fish fed diets 2, 4, 5 and 6 had significantly lower muscle and plasma cholesterol levels compared to the control and diet 3. The results of the study indicated that use of soaked and soaked+autoclaved seed significantly improved the growth performance and feed utilisation of common carp compared to that of untreated seeds but not to a level of performance obtained with a fish meal based control diet. The reduced growth of fish in diets with Sesbania seeds compared to the control might be related to the non-starch polysaccharide and the low protein solubility of Sesbania seeds.

Microcystins in natural blooms and laboratory cultured Microcystis aeruginosa from Laguna de Bay, Philippines.

Laguna de Bay, the largest freshwater lake in the Philippines, experiences periodic blooms of the cyanobacteria Microcystis aeruginosa. Blooms of these cyanobacteria in 1996, 1998 and 1999 were sampled. HPLC and MALDI-TOF mass spectrometry were used to analyze for microcystins. A total of 16 structural variants of the toxin were isolated from the samples with microcystin LR (MC-LR) as the most abundant variant in the samples from 1996 and 1999 making up 77 to 85% of the total, respectively. MC-RR was the dominant variant in the 1998 bloom making up 38%. The samples from 1996 had the highest total toxin concentration (4049 microg g(-1)) followed by those from 1998 (1577 microg g(-1)) and 1999 (649 microg g(-1)). A strain of M. aeruginosa previously isolated from the lake was also cultured in the laboratory under different nitrogen concentrations (1, 3 and 6 mg L(-1)) and elevated phosphorus concentration (0.5 mg L(-1)) to determine the influence of these factors on toxin production. A total of 9 different structural variants of microcystin were isolated from the laboratory cultures with MC-LR consisting more than 75% of the total in all treatments. No significant differences in the total toxin concentration as well as the % distribution of the different variants among treatments were observed. However, the strain of M. aeruginosa cultured in the laboratory had from 3 to 20 times higher total microcystin than those harvested from the lake.

A review of the fish feeding model MAXIMS

A review of the ICLARM fish feeding model MAXIMS is presented, including a literature review of previous publications using the model. This model calculates feeding period, ingestion and evacuation rates and daily ration from sets of 24-h stomach content field data without the need for laboratory studies to determine any of these parameters. The best fit to the data is determined by means of a lowest sum of squared residuals (SSR) approach. The model has four main routines, giving the option of either one (Model 1) or two (Model 2) feeding periods and ingestion rates that are either constant (Models 1.1 and 2.1) or inversely dependent on the level of stomach fullness (Models 1.2 and 2.2). When both the constant and inversely dependent model are fitted to the same data set, nearly the same fit and a slightly lower SSR value is obtained using the latter model; the mathematical reasons for and statistical significance of this phenomenon are discussed. The authors attempt to give biological guidelines on the choice of model for different fish species depending on feeding type and environmental circumstances, as well as a mathematical way to test the goodness of fit of the Models 1.1/2.1 curves against that of the Models 1.2/2.2 curves so that these biological consideration may gain statistical support. An improved version using the software package SAS for Windows is also presented, calculating confidence limits for the various parameters so that curves for different data sets may be compared against each other.