Tissue and fixative dependent shifts of delta13C and delta15N in preserved ecological material

Carbon and nitrogen stable isotope analyses are routinely used to investigate aquatic food webs, and have potential application in retrospective investigations using archived materials. However, such analyses assume that storage does not alter isotopic signatures of materials preserved, or that changes in isotopic composition during storage are predictable. Here we examine preservation shifts on cod (Gadus morhua) muscle, roe and liver tissue over 21 months following preservation in 80% ethanol, in 4% formaldehyde, and by freezing. Preservation shifts were not consistent among tissues. High protein tissues exhibited greater delta(15)N shifts than low protein tissues in 4% formaldehyde, while greater delta(13)C shifts occurred in relatively higher fat tissues when preserved in alcohol. Freezing did not change isotopic signatures. Responses of delta(15)N and delta(13)C are explained by differences in the preservative's isotopic signature and the reaction properties and biochemical composition of the tissues preserved. The results clarify some of the processes that lead to isotopic change during preservation.     

Serial analysis of stable nitrogen and carbon isotopes in hair: monitoring starvation and recovery phases of patients suffering from anorexia nervosa

Stable nitrogen and carbon isotopic ratios of hair strands of six patients suffering from anorexia nervosa were measured to monitor a dietary change from near starvation to recovery. This paper presents the results of a first-time study of nitrogen and carbon balance of the patients prior to and after admittance to a hospital and therapy. Sequential analysis of the isotopic ratios of hair strands of all patients could be related to the respective body mass index (BMI) of each patient. Our hypothesis concerning the diachronic change in delta15N and delta13C during therapy was met: The delta15N values were inversely related to the BMI, indicating a slow-down in catabolism of bodily protein due to the process of gluconeogenesis during the starvation phase. In contrast, the delta13C values and BMI were in phase: an increase in BMI resulted in an increase in the delta13C values. This rise in delta13C ratios is best interpreted by an increased supply of protein in the diet. Furthermore, delta15N and delta13C were inversely related. We conclude that hair, which is easily and non-traumatically sampled, is an adequate monitor that reflects dietary change and nitrogen balance within days. This isotopic method may also be applied in forensic studies with regard to cases of deprivation, and starvation, and may be a method for investigating starvation in historic populations.     

A comparison of muscle- and scale-derived delta13C and delta15N across three life-history stages of Atlantic salmon, Salmo salar

Stable isotope signatures were obtained from paired scale and muscle tissue samples from smolt, post-smolt and one-sea-winter adult Atlantic salmon (Salmo salar). Post-smolt and adult scales were separated into central and outer (marine) portions with analyses carried out on the marine growth section of both life-history stages and the central portion for the adult scales. Muscle and scale delta(13)C and delta(15)N signatures were assessed (1) to determine whether a linear relationship exists between tissue types, (2) to determine if a constant offset exists between tissue signatures across all life-history stages, and (3) to evaluate whether underplating imparts a significant bias to life-history scale segments that would preclude their use in retrospective analyses of any ontogenetic dietary changes between life-history stages. Significant correlations were found to exist between muscle and scale stable isotope signatures obtained from smolts (delta(13)C and delta(15)N) and adults (delta(15)N). Both the muscle and the scale signatures captured the dietary shift associated with the transition from freshwater to the marine environment. Post-smolt and adult scales were depleted relative to muscle tissue, which may be attributed to isotopic differences in amino acid composition between muscle and scale tissues. The results suggest that scales may better represent dietary carbon sources because they are not influenced by lipid dynamics. The scale, however, appears less responsive to short-term shifts in diet relative to muscle and, therefore, must be used only to infer seasonally integrated dietary patterns for slow-growing life-history stages. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Nitrogen isotopic composition in hair protein is different in liver cirrhotic patients

The stable-isotopic composition of nitrogen (delta15N) or carbon (delta13C) of body tissues depends on the isotopic composition of food sources and on shifts due to isotopic fractionation during metabolism. As little is known about the effects of pathophysiological conditions we measured delta15N and delta13C values in hair and hair amino acids of patients with cirrhosis (n = 21) and compared the results with those of healthy subjects (n = 100) randomly selected from the 1987-1988 VERA German nutrition survey population. Cirrhosis was reflected in lower hair 15N abundances (6.7 vs. 9.9 per thousand delta15N; P < 0.001) whereas hair 13C abundances did not differ from healthy subjects (-19.4 vs. -19.6 per thousand 13C). Distinct patterns of delta15N and delta13C values were measured in hair amino acids. The delta15N values of phenylalanine were significantly higher in cirrhotics (P < 0.001). With the exception of isoleucine, threonine, and proline all other measured amino acids showed lower delta15N values than healthy subjects (P < 0.001). Lower hair delta15N values were associated with cirrhotic liver disease which suggests that under this condition the altered liver amino acid metabolism affects the nitrogen isotopic composition of the amino acids used for hair protein synthesis. It remains to be determined in controlled studies whether the altered nitrogen isotopic composition directly reflects the pathophysiological condition or is related to differences in dietary protein intake from plant or animal food sources.     

Effects of temperature on isotopic enrichment in Daphnia magna: implications for aquatic food-web studies

Laboratory experiments were conducted with Daphnia magna and Hyalella sp. grown on a single food source of known isotopic composition at a range of temperatures spanning the physiological optima for each species. Daphnia raised at 26.5 degrees C were enriched in delta(13)C and delta(15)N by 3.1 and 2.8 per thousand, respectively, relative to diet. Daphnia raised at 12.8 degrees C were enriched 1.7 and 5.0 per thousand in delta(13)C and delta(15)N, respectively. Results imply a significant negative relationship between the delta(13)C and delta(15)N of primary consumers when a temperature gradient exists. Similar responses were observed for Hyalella. Results indicate a general increase in delta(13)C enrichment and decrease in delta(15)N enrichment as temperature rises. Deviations from the commonly applied isotopic enrichment values used in aquatic ecology were attributed to changes in temperature-mediated physiological rates. Field data from a variety of sources also showed a general trend toward delta(13)C enrichment with increasing temperature in marine and lacustrine zooplankton. Multivariate regression models demonstrated that, in oligotrophic and mesotrophic lakes, zooplankton delta(13)C was related to lake-specific POM delta(13)C, lake surface temperature and latitude. Temperature-dependent isotopic separation (enrichment) between predator and prey should be taken into consideration when interpreting the significance of isotopic differences within and among aquatic organisms and ecosystems, and when assigning organisms to food-web positions on the basis of observed isotope values.     

Effects of preservation methods on stable isotope signatures in bird tissues

Increasing use is being made of stable isotopes as indicators of habitat use and trophic ecology of animals. Preservation of tissues can alter stable isotope signatures. We investigated the effects of addition of ethanol and NaCl solution (hereafter 'salt'), and of freezing and drying, on carbon and nitrogen isotopic values in blood of the spectacled petrel Procellaria conspicillata, and compared these with those from simultaneously growing feathers. The mean delta(13)C values of blood preserved in ethanol was significantly higher, and of blood preserved in salt was significantly lower than that of dried or frozen samples. delta(13)C values in ethanol showed high variation according to brand and batch and could account for the differences found in delta(13)C ratios in ethanol-preserved blood samples. Mean delta(13)C and delta(15)N values in growing feathers were higher than in blood, suggesting tissue-specific fractionation. We conclude that different methods of preserving tissues such as blood may bias stable isotope values, and urge researchers to consider this issue. Air drying is proposed as a practical and unbiased method for blood preservation in field situations where freezing is not a practical option, and a mathematical approach is suggested to permit comparison between studies using different preservation methods or tissues.     

Nitrogen balance and delta15N: why you're not what you eat during pregnancy

Carbon (13C/12C) and nitrogen (15N/14N) stable isotope ratios were longitudinally measured in human hair that reflected the period from pre-conception to delivery in 10 pregnant women. There was no significant change in the delta13C results, but all subjects showed a decrease in delta15N values (-0.3 to -1.1 per thousand) during gestation. The mechanisms causing this decrease in hair delta15N have not been fully elucidated. However, since the delta15N values of dietary nitrogen and urea nitrogen are significantly lower compared to maternal tissues, it is hypothesized that the increased utilization of dietary and urea nitrogen for tissue synthesis during pregnancy resulted in a reduction of the steady state diet to a body trophic level effect by approximately 0.5-1 per thousand. An inverse correlation (R2 = 0.67) between hair delta15N and weight gain was also found, suggesting that positive nitrogen balance results in a reduction of delta15N values independent of diet. These results indicate that delta15N measurements have the ability to monitor not only dietary inputs, but also the nitrogen balance of an organism. A potential application of this technique is the detection of fertility patterns in modern and ancient species that have tissues that linearly record stable isotope ratios through time.     

Stable isotope ratio analysis as a tool to discriminate between rainbow trout (O. mykiss) fed diets based on plant or fish-meal proteins

The use of stable isotope ratio analysis (SIRA) as a rapid analytical tool to characterize and discriminate farmed fish on the basis of the feedstuffs included in the diet formulation is discussed. Two isoproteic (44.8%) and isolipidic (19.6%) extruded diets were formulated: a fish-meal-based diet (FM diet), containing fish meal as the sole protein source; a plant-protein-based diet (PP diet), where pea protein concentrate and wheat gluten meal replaced 80% of fish meal protein. The diets were fed to eight groups of rainbow trout (initial body weight: 106.6g) for 103 days in two daily meals under controlled rearing conditions. Growth performance (final body weight: 318.5 g; specific growth rate: 1.06%) and feed-to-gain ratio (0.79) were not affected by the dietary treatment. The differences in isotopic values of the two diets were clearly reflected in the different carbon and nitrogen isotopic values in rainbow trout fillets. The delta(13)C and delta(15)N values of muscle of farmed rainbow trout showed differences between farmed fish fed a fish-protein-based diet (-20.47 +/- 0.34 and 12.38 +/- 0.57 for delta(13)C and delta(15)N, respectively) and those fed a plant-protein-based diet (-23.96 +/- 0.38 and 7.15 +/- 0.51 for delta(13)C and delta(15)N, respectively). The results suggest that SIRA provides a robust and verifiable analytical tool to discriminate between fish fed on a plant or a fish protein diet.     

The effect of acidification on the determination of organic carbon, total nitrogen and their stable isotopic composition in algae and marine sediment

We investigated the effects of sample acidification on the stable carbon and nitrogen isotopic composition (delta13C and delta15N), as well as the organic carbon (OC) and total nitrogen (TN) composition, of an algal culture and a marine sediment. Replicate measurements of untreated and acid-treated samples were made using 1 M, 2 M and 6 M HCl, 6% H2SO3 and 1 M H3PO4. For all treatments the precision of the analysis for the acid-treated sample was equal to or less than that in the non-acidified sample. For the algae, analysis of variance (ANOVA) indicated no significant differences in the mean OC and TN concentration, or delta13C and delta15N composition, between any acid treatment and non-acidified samples. For the sediment sample a comparison could only be made between the different acid treatments because the untreated contained significant amounts ( approximately 30%) of carbonate carbon. ANOVA indicated that the mean OC determined in sediment samples after the 1 M HCl treatment and the mean delta13C values after the 6% H2SO3 and 1 M H3PO4 treatments were significantly different (p < 0.013 and < .05, respectively) from all other treatments. Mass balance calculations indicate that in some instances delta13C values were biased due to a contribution from unreacted carbonate carbon. There were no significant differences in the mean TN between any acid-treated and non-acidified samples. The mean delta15N values after 6 M HCl, 6% H2SO3 and 1 M H3PO4 treatments were significantly different from the untreated sediment sample (p < 0.044). Based on the significant bias observed for the delta15N and delta13C values, a weak (1-2 M) HCl solution is confirmed as the most appropriate acid for the removal of inorganic carbon from natural materials requiring elemental and isotopic analysis.     

Stable isotopes to discriminate lambs fed herbage or concentrate both obtained from C(3) plants

This study was aimed at determining whether isotopic ratio mass spectrometry (IRMS) enables us to discriminate between lambs fed herbage or concentrate, both obtained from C(3) plants, and those fed a concentrate obtained from C(4) plants. Thirty-four Comisana male lambs (age 45 days) were assigned to three feeding treatments. Fourteen lambs were fed vetch (Vicia sativa) ad libitum. Another fourteen lambs received a barley-based concentrate. The remaining six lambs were fed a maize-based concentrate. After 60 days of experimental treatment the animals were slaughtered and the wool, perirenal fat and muscle longissimus dorsi were sampled. The delta(13)C and delta(15)N values of the muscle, wool and feed were measured by continuous flow elemental analysis (CF-EA)-IRMS. The delta(13)C of the fat was determined likewise. The isotopic composition of the tissues reflected that of the three diets. For the lambs which were fed herbage the muscle delta(13)C values were higher (P < 0.0005) and delta(15)N values were lower (P < 0.0005) than those of the lambs receiving concentrates. The delta(15)N and delta(13)C values in the muscle and delta(13)C values in the adipose tissue allowed perfect discrimination between the lambs fed the three different diets. The regression between the delta(13)C values measured in muscle and in wool of lambs was linear (R(2) = 0.99; P < 0.0005). This result shows that delta(13)C measured in the wool can predict muscle delta(13)C distribution, suggesting that wool is a valuable matrix for meat authentication.     

Identification of energy consumption and nutritional stress by isotopic and elemental analysis of urine in bonobos (Pan paniscus)

A mounting body of evidence suggests that changes in energetic conditions like prolonged starvation can be monitored using stable isotope ratios of tissues such as bone, muscle, hair, and blood. However, it is unclear if urinary stable isotope ratios reflect a variation in energetic condition, especially if these changes in energetic condition are accompanied by shifts in dietary composition. In a feeding experiment conducted on captive bonobos (Pan paniscus), we monitored urinary δ(13)C, δ(15)N, total C (carbon), total N (nitrogen), and C/N ratios and compared these results with glucocorticoid levels under gradually changing energy availability and dietary composition. Measurements of daily collected urine samples over a period of 31 days showed that while shifts in urinary isotope signatures of δ(13)C and δ(15)N as well as total C were best explained by changes in energy consumption, urinary total N excretion as well as the C/N ratios matched the variation in dietary composition. Furthermore, when correcting for fluctuations in dietary composition, the isotope signatures of δ(13)C and δ(15)N as well as total C correlated with urinary glucocorticoid levels; however, the urinary total N and the C/N ratio did not. These results indicate for the first time that it is possible to non-invasively explore specific longitudinal records on animal energetic conditions and dietary compositions with urinary stable isotope ratios and elemental compositions, and this research provides a strong foundation for investigating how ecological factors and social dynamics affect feeding habits in wild animal populations such as primates.     

Two new organic reference materials for delta13C and delta15N measurements and a new value for the delta13C of NBS 22 oil

Analytical grade L-glutamic acid is chemically stable and has a C/N mole ratio of 5, which is close to that of many of natural biological materials, such as blood and animal tissue. Two L-glutamic acid reference materials with substantially different 13C and 15N abundances have been prepared for use as organic reference materials for C and N isotopic measurements. USGS40 is analytical grade L-glutamic acid and has a delta13C value of -26.24 per thousand relative to VPDB and a delta15N value of -4.52 per thousand relative to N2 in air. USGS41 was prepared by dissolving analytical grade L-glutamic acid with L-glutamic acid enriched in 13C and 15N. USGS41 has a delta13C value of +37.76 per thousand and a delta15N value of +47.57 per thousand. The delta13C and delta15N values of both materials were measured against the international reference materials NBS 19 calcium carbonate (delta13C=+1.95 per thousand ), L-SVEC lithium carbonate (delta13C=-46.48 per thousand ), IAEA-N-1 ammonium sulfate (delta15N=0.43 per thousand ), and USGS32 potassium nitrate (delta15N=180 per thousand ) by on-line combustion continuous-flow and off-line dual-inlet isotope-ratio mass spectrometry. Both USGS40 and USGS41 are isotopically homogeneous; reproducibility of delta13C is better than 0.13 per thousand, and that of delta15N is better than 0.13 per thousand in 100-microg amounts. These two isotopic reference materials can be used for (i) calibrating local laboratory reference materials, and (ii) quantifying drift with time, mass-dependent fractionations, and isotope-ratio-scale contraction in the isotopic analysis of various biological materials. Isotopic results presented in this paper yield a delta13C value for NBS 22 oil of -29.91 per thousand, in contrast to the commonly accepted value of -29.78 per thousand for which off-line blank corrections probably have not been quantified satisfactorily.     

Seabird satellite tracking validates the use of latitudinal isoscapes to depict predators' foraging areas in the Southern Ocean

Stable isotopes are increasingly being used to trace wildlife movements. A fundamental prerequisite of animal isotopic tracking is a good knowledge of spatial isotopic variations in the environment. Few accessible reference maps of the isotopic landscape ("isoscapes") are available for marine predators. Here, we validate for the first time an isotopic gradient for higher trophic levels by using a unique combination of a large number of satellite-tracks and subsequent blood plasma isotopic signatures from a wide-ranging oceanic predator. The plasma δ(13)C and δ(15)N values of wandering albatrosses (n = 45) were highly and positively correlated to the Southern Ocean latitudes at which the satellite-tracked individuals foraged. The well-defined latitudinal baseline carbon isoscapes in the Southern Ocean is thus reflected in the tissue of consumers, but with a positive shift due to the cumulative effect of a slight (13)C-enrichment at each trophic level. The data allowed us to estimate the carbon isotopic position of the main oceanic fronts in the area, and thus to delineate robust isoscapes of the main foraging zones for top predators. The plasma δ(13)C and δ(15)N values were positively and linearly correlated, thus suggesting that latitudinal isoscapes also occur for δ(15)N at the base of the food web in oceanic waters of the Southern Ocean. The combination of device deployments with sampling of relevant tissues for isotopic analysis appears to be a powerful tool for investigating consumers' isoscapes at various spatio-temporal scales.     

Fractionation and metabolic turnover of carbon and nitrogen stable isotopes in black fly larvae

Diet-tissue fractionation factors and metabolic turnover rates of delta15N and delta13C were assessed in laboratory-reared black fly (Simulium vittatum IS-7) larvae fed isotopically distinct diets. Five treatments consisted of using food with different delta15N signatures throughout the experiments (19-26 days), a sixth shifted from a low to high delta15N signature diet (uptake) on day 14, and the last shifted from a high to low delta15N signature diet (elimination) on day 14. In the larvae, diet-tissue fractionation factors for delta13C, which were in steady state with food, ranged from -0.61 to 2.0, with a median of 1.87. The delta15N diet-tissue fractionation factors were mostly negative, ranging from +2.85 to -24.96 per thousand, with a single positive value from the elimination treatment in which larval delta15N did not achieve steady state with the food. Diet-tissue fractionation factors also had a significant negative relationship (r2 = 0.98) with delta15N values in the food suggesting that nitrogen diet-tissue fractionation factors are 15N concentration-dependent. The delta15N of shed head capsules and feces were enriched in 15N and could be mechanisms for elimination of 15N by the larvae. For delta15N, metabolic turnover values based on the Hesslein model were highly consistent (0.40 to 0.43 delta15N*day(-1)) between uptake and elimination phases and across experiments and were an order of magnitude greater than growth rates. The rapid turnover of nitrogen in black fly larvae, which was orders of magnitude greater than measured in vertebrates, makes them an excellent indicator of short-term changes in nitrogen inputs to aquatic systems.     

Reconstruction of the isotopic history of animal diets by hair segmental analysis

Carbon and nitrogen isotope signatures (delta(13)C and delta(15)N) of animal tissues provide information about the diet and, hence, the environment in which the animals are living. Hair is particularly useful as it provides a stable archive of temporal (e.g. seasonal) fluctuations in diet isotope composition. It can be sampled easily and with minimal disturbance from living subjects. However, derivation of the temporal record along the hair length may be subject to errors and uncertainties. This study investigates (and suggests means to minimize) several sources of error, including (a) incomplete sampling, (b) sampling during the quiescent (telogen) phase, (c) non-representative sub-sampling, (d) ignorance of hair growth rate, i.e. time-position relationship of isotope signatures, and (e) non-optimal compromise between analytical/procedural precision and effort/cost. Cattle tail switch hair was collected from animals of different breed, sex and age. Hair was washed, sectioned, and 5- or 10-mm-long sections were analyzed for C and N isotope composition. Signatures along paired hairs were similar (r(2) approximately 0.8) and distances between isotopic minima and maxima nearly identical, indicating that a single hair constituted a representative sample and (except for telogen hair) hair growth rate was the same for paired hairs. However, cutting hair, instead of plucking, caused a variable loss of recently grown hair and information. Telogen hair was identified and data loss due to cutting error reduced when more than one hair from the same animal and sampling region was compared to spot and delimit common and missing regions. Similarly, comparison of isotopic profiles from hair collected at different times identified the segment produced during the respective interval and allowed calculation of average hair growth rate, which varied between animals (0.69-1.06 mm d(-1)). Analysis of alternate 10-mm-long sections for two hairs per animal provided a good compromise between precision/resolution and effort. The method should be applicable to other mammalian species including man.     

Identifying migratory Salmo trutta using carbon and nitrogen stable isotope ratios

Many Salmo trutta populations consist of non-anadromous (freshwater-resident) brown trout and anadromous (sea-run migratory) sea trout. Although adult brown trout and sea trout can usually be identified using differences in size and body colouration, it is not possible to easily identify eggs/alevins as the progeny of brown trout or sea trout. In this study we show that delta(13)C and delta(15)N, measured using a continuous flow isotope ratio mass spectrometer (CF-IRMS), can accurately identify fish eggs as the progeny of freshwater-resident (delta(13)C(egg) = -25.7 +/- 1.9 per thousand,delta(15)N(egg) = 9.2 +/- 1.8 per thousand) or migratory (delta(13)C(egg) = -19.9 +/- 1.1 per thousand, delta(15)N(egg) = 14. 3 +/- 1.5 per thousand) adult female Salmo trutta. Case studies show that stable isotope analysis is a more reliable technique for distinguishing anadromous adult fish than differentiation using morphological characteristics. For example, stable isotope analysis of brown trout from Loch Eck, Scotland, revealed that some individuals possessed delta(13)C and delta(15)N signatures indicative of marine feeding despite visual identification as freshwater-resident fish. It is most likely that these fish are misidentified sea trout although it possible that these fish may be brown trout that have adopted an estuarine feeding strategy to avoid interspecific competition for food within Loch Eck with salmon, powan and Arctic charr. Most stable isotope studies of fish ecology use terminal tissue sampling to provide sufficient biological material for isotopic analysis; however, our study suggests that adipose fin tissue could provide a comparable measure of delta(13)C and delta(15)N. Such a strategy would be invaluable when studying the trophic ecology or migration patterns of fish of high conservation value.     

Stable carbon and nitrogen isotope analysis of avian uric acid

We report results obtained using a new technique developed to measure the stable-isotope composition of uric acid isolated from bird excreta (guano). Results from a diet-switch feeding trial using zebra finches suggest that the delta(13)C of uric acid in the guano equilibrates with the diet of the bird within 3 days of a change in diet, while the equilibration time for delta(15)N may be longer. The average carbon isotope discrimination between uric acid and food before the diet switch was +0.34 +/- 1 per thousand (1sigma) while after the diet switch this increased slightly to +0.83 +/- 0.7 per thousand (1sigma). Nitrogen isotope discrimination was +1.3 +/- 0.3 per thousand (1sigma) and +0.3 +/- 0.3 per thousand (1sigma) before and after the diet switch; however, it is possible that the nitrogen isotope values did not fully equilibrate with diet switch over the course of the experiment. Analyses of other chemical fractions of the guano (organic residue after uric acid extraction and non-uric acid organics solubilised during extraction) suggest a total range of up to 3 per thousand for both delta(13)C and delta(15)N values in individual components of a single bulk guano sample. The analysis of natural samples from a range of terrestrial and marine species demonstrates that the technique yields isotopic compositions consistent with the known diets of the birds. The results from natural samples further demonstrate that multiple samples from the same species collected from the same location yield similar results, while different species from the same location exhibit a range of isotopic compositions indicative of different dietary preferences. Given that many samples of guano can be rapidly collected without any requirement to capture specimens for invasive sampling, the stable-isotope analysis of uric acid offers a new, simple and potentially powerful tool for studying avian ecology and metabolism.     

Determination of underivatized amino acid delta(13)C 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.     

Alteration of the carbon and nitrogen stable isotope composition of beef by substitution of grass silage with maize silage

This study investigated the effect of substituting grass silage (C3 photosynthetic plant product) with maize silage (C4 photosynthetic plant product) on the natural abundance carbon (delta13C) and nitrogen (delta15N) stable isotope composition of bovine muscle tissue. Forty-five continental crossbred heifers were assigned to one of three diets consisting of 3 kg of a barley-based concentrate plus grass silage, maize silage or an equal mixture (dry matter basis) of grass silage and maize silage, fed ad libitum, for 167 days. Substitution resulted in less negative delta13C values (P<0.001) in lipid-free muscle and in lipid, and also a lower delta15N (P<0.001) in lipid-free muscle. Feeding of maize silage was clearly reflected in the delta13C of muscle, with each 10% difference in the dietary C4 carbon intake resulting in a 0.9 to 1.0 per thousand shift of delta13C in lipid-free muscle and a 1.0 to 1.2 per thousand in lipid. Minimum detectable mean differences (95% confidence, power 0.80, n=15) in this experiment were about 0.5 per thousand and 1.0 per thousand for delta13C of lipid-free muscle and lipid, respectively, and about 0.5 per thousand for delta15N of lipid-free muscle. The power analysis presented here is useful for estimating minimum isotopic differences that can be detected between any two groups of beef samples with a given number of replicates. It is concluded that carbon stable isotope ratio analysis of meat can be used to quantify C3/C4 dietary constituents in beef production.     

Should we use one‐, or multi‐compartment models to describe 13C incorporation into animal tissues?

Understanding rates of isotopic incorporation and discrimination factors between tissues and diet is an important focus of ecologists seeking to use stable isotopes to track temporal changes in diet. We used a diet-shift experiment to measure differences among tissues in (13)C incorporation rates in house sparrows (Passer domesticus). We predicted faster incorporation rates in splanchnic than in structural tissues. We also assessed whether isotopic incorporation data were better supported by the one-compartment models most commonly used by ecologists or by multi-compartment models. We found large differences in the residence time of (13)C among tissues and, as predicted, splanchnic tissues had faster rates of isotopic incorporation and thus shorter retention times than structural tissues. We found that one-compartment models supported isotopic incorporation data better in breath, excreta, red blood cells, bone collagen, and claw tissues. However, data in plasma, intestine, liver, pectoralis muscle, gizzard, and intestine tissues supported two-compartment models. More importantly, the inferences that we derived from the two types of models differed. Two-compartment models estimated longer (13)C residence times, and smaller tissue to diet differences in isotopic composition, than one-compartment models. Our study highlights the importance of considering both one- and multi-compartment models when interpreting laboratory and field isotopic incorporation studies. It also emphasizes the opportunities that measuring several tissues with contrasting isotopic residence times offer to elucidate animal diets at different time scales. Copyright (c) 2008 John Wiley & Sons, Ltd.