Effects of drought-stressed temperate forage legumes on the degradation and the rumen microbial community in vitro.

According to climate change scenarios, central Europe may expect extending drought periods during summer. Lower water availability may influence the ruminal digestion of individual forage legume species differently. To test this hypothesis, Lotus corniculatus L. (var. Bull), Medicago lupulina L. (var. Ekola), Medicago falcata L. (wild seeds) and Trifolium repens L. (var. Rivendel) were each grown in parallel lots of control and drought-stressed monocultures. Rainout shelters (installed in May 2011 on a regrowth after first cut until harvest in mid of June) withheld rainfall of 40 mm in the drought stress treatment. Samples of dried (60°C) and milled (5 mm screen) forage legumes were incubated in a simulation experiment using Rusitec to assess drought effects on parameters for microbial metabolism. Degradability of dry matter and organic matter as well as methane production decreased in incubations with drought-stressed compared to control variants of legume species. Degradability of crude protein, neutral detergent fibre, acid detergent fibre and residual organic matter including non-fibre carbohydrates and lipids were affected by interactions between drought stress and species. Significant interactions were also found for ammonia concentrations, molar SCFA proportions and the microbial communities. It is concluded that drought stress for growing forage legumes influences their ruminal degradation and fermentation as well as the ruminal microbial communities of Bacteria and Archaea differently in a legume species-dependent manner.

Egg production dynamics and fecundity of Heterakis gallinarum residing in different caecal environments of chickens induced by fibre-rich diets.

In modern husbandry systems, feed rations for laying hens may contain high levels of dietary fibre, which may only be fermented intestinally. Depending on fermentation properties, the type of dietary fibre may influence the intra-caecal environment constituting the predilection site of Heterakis gallinarum. We investigated egg production dynamics and fecundity of H. gallinarum residing in different caecal environments induced through different types of dietary fibre. Growing layers were fed a standard (CON) or an insoluble- (I-) or soluble- (S-) non-starch polysaccharides-(NSP) supplemented diet for the first 11 weeks (wk) of life in a twice-replicated experiment. At 3 wk of age, the birds were infected with 200 embryonated eggs of H. gallinarum. Starting from 3 wk post-infection (p.i.), individual daily total excreta were collected. The number of eggs per gram of faeces (EPG) was determined (N = 2240), and the number of eggs per day (EPD) were estimated. The birds were necropsied 8 wk p.i. and the worm burdens were quantified. The nematode began to lay eggs as early as 23 d.p.i. and thereafter laid on average 436 eggs/d. I-NSP- and S-SNP-supplemented diets expedited the onset of patency by approximately 5 days, and increased total egg excretion by 110% and 185%, respectively, due to higher worm counts. The latter diet (S-SNP) additionally increased total egg excretion by 94% due to enhanced fecundity. Longitudinally collected data showed that fecundity of the nematode (EPD/female) was relatively stable starting from the second week of the patent phase. Results suggest that NSP-induced enlargement of caeca favoured establishment of the nematode while effects of an intensified microbial fermentation through S-NSP were crucial for an enhancement in the worm fecundity. Based on the long-term data obtained from daily collections of total excreta in experimentally H. gallinarum-infected chickens, it is concluded that a diet enriched with pea bran as source of insoluble NSP increased worm egg production merely through elevated worm counts, whereas a diet supplemented with chicory root meal as source of soluble NSP elevated worm egg production through both increased worm counts, and more profoundly through enhanced fecundity.

Segmental diversity of electrogenic glucose transport characteristics in the small intestines of weaned pigs.

It has been shown in several species that the intestinal Na(+)-dependent glucose co-transporter 1 (SGLT1) is more abundant in the jejunum than in ileum. In contrast, the efficiency of intestinal glucose uptake rates in suckling piglets or weaned pigs is not clearly fitting with this segmental distribution. The aim of this study was to evaluate SGLT1 mediated glucose absorption in the jejunum and ileum of growing pigs (Sus scrofa) in more detail. In Ussing chambers, basal short-circuit currents were significantly more positive in the jejunum. It could be demonstrated that the electrogenic ileal glucose transport was significantly more pronounced in different breeds and occurred at 5 mmol∙L(-1) glucose 7 times faster in the ileum, although slightly higher jejunal expression of glycosylated SGLT1 was detected by Western blotting. This expression pattern was connected to significantly lower phlorizin sensitivity in the jejunum. As the more efficient ileal glucose absorption was also observable with glucose uptake studies into isolated brush-border membrane vesicles without differences in abundance and activity of the Na(+)/K(+)-ATPase in both segments, we conclude that the segmental differences in porcine glucose transport characteristics may be based on direct or indirect modulations of SGLT1 activity.

Ruminal fermentation characteristics and microbial nitrogen assimilation in sheep fed differently composed grass silages.

The investigation aimed at examining if the composition of grassland silage affects the microbial nitrogen assimilation in the rumen of sheep. The silages were made of vegetative summer re-growths consisting of 48% grasses, 28% legumes and 24% other forbs (GCF) or of pure grass (G). Silage GCF contained more intermediately degradable non-structural and less slowly degradable carbohydrates, more crude protein (CP), a narrower ratio between slow and very slow degradable nitrogen (N), and exhibited higher in situ degradability of organic matter and CP than Silage G. Four adult wethers equipped with rumen fistulae were used in a two factorial trial. Feed was offered either as silage alone or as a mixture of silage and barley (60:40). Microbial N was estimated using continuous intraruminal 15N infusion and measurement of 15N-enrichment in microbes isolated from rumen liquor samples. With the exception of trends for ruminal butyrate concentrations, no interactions were detected between silage and barley feeding. Sheep receiving Silage GCF exhibited larger diurnal fluctuations of ammonia, and produced more microbial N (p < 0.05) than sheep on Silage G. Feeding the silages with barley decreased ruminal pH and elevated the concentrations of butyrate (p < 0.05). The 15N incorporation into microbial N was reduced by barley feeding (p < 0.05) along with a trend to accelerated rumen fluid turnover, resulting in similar microbial N yields as found in sheep receiving silage without barley. It is concluded that the larger and better balanced amounts of intermediately degradable carbohydrate- and N-containing fractions favoured the ruminal microbial protein synthesis in sheep consuming Silage GCF instead of Silage G.

Nitrogen excretion of adult sheep fed silages made of a mixed sward or of pure unfertilised grass alone and in combination with barley.

Four adult rumen-fistulated wether sheep were fed silages combined with barley. The silages consisted of 48% grasses, 28% legumes and 24% other forbs (GCF) or of pure grass (G). The swards received no mineral fertiliser. The dry matter (DM) and fibre contents were lower in GCF than in G. Crude protein content of DM in GCF and G were 145 g/kg and 102 g/kg respectively. DM content as ash, lipids and non-fibrous carbohydrates were rather similar in both silages. About 40g DM were offered per kg BW0.75 and day either as silage alone or as a mixture of silage and barley (60:40). Faecal N excretion was greater with GCF than with G. The proportion of faecal bacterial and endogenous debris N reached 75 and 73% when GCF or G was fed, respectively. Undigested dietary N represented about 20%, and water soluble N accounted for 5-6% of faecal N. GCF caused more urinary N than G. Barley reduced urinary N excretion when supplemented to GCF. No dietary influence on urinary non-urea nitrogenous compounds was shown. GCF caused higher urinary urea N excretion than G and barley reduced this fraction when replacing part of GCF. Based on the urinary urea N proportions, it is concluded that N intake exceeded N requirement for any of the four diets fed. Dietary supplementation of ruminally fermentable carbohydrates can reduce urinary N excretion and this improves the efficiency of utilisation of N in N-unfertilised biodiverse grassland/ruminant farming systems.

Precision, repeatability and representative ability of faecal egg counts in Heterakis gallinarum infected chickens.

This study investigated whether a precise and repeatable quantification of Heterakis gallinarum egg excretion, which considerably reflects the actual worm burdens, can be achieved based on collection of the daily total amount of faeces from chickens. Three-week-old birds (N=64) were infected with 200 embryonated eggs of H. gallinarum, and placed into individual cages 3 wk after infection for 5 wk to collect daily faeces (N=2240). The total daily faeces was mixed and a randomly taken sample per bird was analyzed to estimate the numbers of eggs per gram of faeces (EPG) and total number of eggs excreted within 24h (EPD). A total of 235 daily faecal collections were randomly selected and further examined to determine between and within sample variations of EPG counts as a measure of precision. For this, two random faecal samples were taken from the daily produced faeces by a bird, and the EPG was determined for each of the samples (EPG1 and EPG2). The second faecal sample was analyzed once more to determine a parallel EPG2 count (EPG2a) of the suspended sample. Precision of an EPG count was defined as its relative closeness to the average of two EPG counts using a relative asymmetry index (Index(EPG)). At an age of 11 wk, i.e. 8 wk p.i. the birds were slaughtered and their worm burdens were determined. There were no significant differences between EPG1 and EPG2 (P=0.764) nor between EPG2 and EPG2a (P=0.700), suggesting that the differences between or within the samples were not different from zero. Correlations between EPG counts, as between and within sample coherences, were r=0.85 and r=0.86, respectively. Precision of EPG counts, as measured by Index(EPG), was not influenced by consistency (P=0.870) and total amount of faeces (P=0.088). However, concentration of eggs in faeces (mean EPG) had a significant effect on the precision of the EPG counts (P<0.001). Similar results were also observed for the within sample precision (Index(EPG2)). A segmented regression analysis indicated an abrupt change in the precision of EPG counts as the response to changing egg concentration in the examined faecal samples. The precision of analyses remarkably heightened up to a breakpoint with an EPG count of ≤ 617. A similar breakpoint was also determined for within sample precision (EPG2 ≤ 621). Moderate repeatabilities (R=0.49) for EPG and EPD were estimated in the first week of egg excretion, whereas the estimates were higher (R=0.67-0.84) in the following weeks. Correlations between number of female worms with daily measured EPG and EPD increased to an almost constant level (r ≥ 0.70; P<0.05) in a few days after the nematode excreted eggs and predominantly remained so for the rest of the sampling period. It is concluded that mixing daily total faeces provides samples with random homogenous distribution of H. gallinarum eggs. Precision of the EPG counts increases as the egg concentration in faecal sample increases. Egg excretion of H. gallinarum, quantified either as EPG or EPD, is highly repeatable and closely correlated with the actual worm burden of birds starting as early as in 5 th wk of infection.

Pathobiology of Heterakis gallinarum mono-infection and co-infection with Histomonas meleagridis in layer chickens.

Little is known about the induction and modulation of gut-associated immune reactions after nematode infection in the chicken. The objective of the present study was to compare the pathogenesis, induction of immune reactions and electrophysiological changes of the gut after mono-infection with Heterakis gallinarum and after dual infection with H. gallinarum and Histomonas meleagridis in layer chickens. In two experiments 3-week-old chickens were inoculated with embryonated H. gallinarum eggs, which were positive for H. meleagridis. While birds of the first experiment were left untreated, those of the second experiment were treated with dimetridazol to prevent H. meleagridis co-infection. Mild to moderate histological lesions and local immune reactions with a significant increase in CD4(+), CD8α(+), TCRαß(+) and TCRδγ(+) cells in the lamina propria and induction of the T-helper type 2 (Th2) cytokine interleukin-13 dominated the H. gallinarum immune response at 2 weeks post infection. Co-infection with H. gallinarum and H. meleagridis induced an increase in mRNA expression of the T-helper type 1 (Th1) cytokine interferon-γ, a decrease in splenic CD4(+) cells and severe destruction of the caecal mucosa in association with strong T-cell infiltration in the caecal lamina propria. There was no obvious effect on the chloride secretion of the caecal epithelium, which was investigated once the mucosa had almost recovered from the infection, in either experiment. These results suggest that the local T-cell reactions to nematode infections in chickens may be comparable with mammals and may be shifted from a Th2-dominated to a Th1-dominated response when accompanied by a protozoan infection.

Pantothenic acid supplementation to support rumen microbes?

Based on repeatedly reported extensive pantothenic acid disappearance in the rumen, the present study is aimed at examining if pantothenic acid is used for a more efficient ruminal fermentation and microbial growth in an artificial rumen (Rusitec). Three substrates differing in roughage/concentrate ratio were incubated with and without the addition of Ca-D-pantothenate. Pantothenic acid was extensively degraded without notably influencing fermentation, microbial protein synthesis and the status of other B-vitamins such as riboflavin, vitamin B6 and niacin. Therefore, pantothenic acid supplementation cannot be expected to contribute to microbial benefit for the ruminant animal.

Immunopathogenesis of Ascaridia galli infection in layer chicken.

Gastro-intestinal nematode infections in mammals are associated with local T lymphocyte infiltrations, Th2 cytokine induction, and alterations in epithelial cell secretion and absorption. This study demonstrates that Ascaridia (A.) galli infection in chicken also elicits local gut-associated immune reactions and changes in the intestinal electrogenic nutrient transport. In A. galli-infected birds we observed infiltrations of different T cell populations in the intestinal lamina propria and accumulation of CD4+ lymphocytes in the epithelium. The Th2 cytokines IL-4 and IL-13 dominated the intestinal immune reactions following A. galli infection. A. galli-specific systemic IgY antibodies were detected after two weeks post infection, and did only poorly correlate with detected worm numbers. Electrogenic transport of alanin and glucose was impaired in A. galli-infected chicken. Our data provide circumstantial evidence that local immune responses and electro-physiological intestinal functions may be connected and contribute to the elimination of worm infection.

Effects of dietary non-starch polysaccharides on establishment and fecundity of Heterakis gallinarum in grower layers.

It was hypothesized that the establishment and fecundity of Histomonas meleagridis free Heterakis gallinarum may be affected by dietary non-starch polysaccharides (NSPs). One-day-old female layer chicks (N=670) were fed ad libitum for 11wk one of the following diets in a three-times repeated experiment: basal diet (CON), basal diet plus pea bran rich in insoluble NSP (I-NSP), basal diet plus chicory root meal as a source of inulin rich soluble NSP (S-NSP). At the end of wk three, each feeding group was subdivided into an uninfected and an infected group of birds each being inoculated with a placebo or with 200 H. meleagridis free eggs of H. gallinarum. The birds were slaughtered 8wk post infection and their worm burdens, the nematode egg excretion, caeca sizes and weights as well as intracaecal pH and volatile fatty acid (VFA) concentrations were determined. The NSP supplemented diets and also infection led to reduced body weights (BWs) of birds and impaired the feed conversion rate (P<0.001). The NSP supplemented diets increased average length of caecum (P<0.001) with S-NSP exerting a stronger effect than I-NSP (P<0.05). Full caeca weight was increased by S-NSP (P<0.001). Feeding S-NSP lowered intracaecal pH and molar proportion of acetate and increased that of butyrate compared to CON and I-NSP (P<0.001). Caecal pool of VFA was increased with S-NSP (P<0.001). The NSP-diets elevated incidence of infection (P<0.01), average number of larvae (P<0.009) and total worm burden (P<0.001) compared to CON. The daily amount of faeces increased in NSP-fed birds (P<0.001). Number of eggs per gram of faeces (EPG), number of eggs excreted per worm population of a bird within 24h (EPD) and female worm fecundity (EPD/female worm) were elevated after feeding S-NSP (P≤0.002), whereas I-NSP led to lower EPG/female worm (P<0.05). The EPD increased in the sequence of CON

Non-starch polysaccharides alter interactions between Heterakis gallinarum and Histomonas meleagridis.

Nutrition of the host animal may not only influence interactions between the host and its parasites, but also relations between different parasites species residing on the same host. We investigated effects of insoluble and soluble non-starch polysaccharides (NSP) on establishment and development of Heterakis gallinarum in chicken being treated or left untreated against Histomonas meleagridis. Six groups of one-day-old birds were allocated to three diets, two on each diet. The birds were fed ad libitum either a basal diet (CON), or CON+insoluble NSP (I-NSP) or CON+soluble NSP (S-NSP) until an age of 11wk. At an age of 19d, one of each diet groups was prophylactically treated for 9d with dimetridazole (0.05%, w/v) via drinking water against histomonas. The remaining three groups were left un-treated. Two days after starting dimetridazole treatment (at 3wk), each of the 6 groups was divided into two sub-groups. One dimetridazole treated and one dimetridazole un-treated groups of birds on each diet (6 groups) were infected with 200 embryonated eggs of H. gallinarum that were previously harvested from histomonas-carrying H. gallinarum infected chickens. The remaining 6 groups of uninfected birds, either treated or left un-treated against H. meleagridis, served as controls. Worm burdens of infected birds were determined 8wk p.i. Treatment against H. meleagridis significantly increased incidence of H. gallinarum infection and average worm length in all infected groups independent of the diet consumed (p<0.001). An interaction between effects of diet and dimetridazole treatment on worm burden (p<0.001) indicated that the S-NSP diet resulted in lowest worm burden in dimetridazole un-treated birds, whereas it caused the highest worm burden in the treated birds (p<0.05). Furthermore, the treatment resulted in higher worm burdens when compared to un-treated birds on the corresponding diets (p<0.05). Infection with H. gallinarum impaired body weight (BW) of the chicks (p<0.05) and H. meleagridis aggravated this effect (p<0.05). Dimetridazole treated and un-treated uninfected birds developed similar BW (p>0.05). Both NSP supplemented diets resulted in lower (p<0.05) BW when compared with the CON diet, S-NSP being inferior to I-NSP (p<0.05). It is concluded that H. meleagridis harms the definitive host as well as H. gallinarum. Both insoluble and soluble NSP supplemented diets favor H. gallinarum infection while S-NSP additionally intensifies histomonas infection, which then impairs establishment and development of H. gallinarum.

Effect of extra dietary lysine in Ascaridia galli-infected grower layers.

The hypothesis examined in this study was that extra dietary lysine (Lys) above the recommended standard exerts beneficial effects on the defence reactions, growth performance and nutrient utilization in Ascaridia galli-infected chickens. Therefore, 1-day-old female Lohmann Selected Leghorn chickens were at first fed standard Lys diet (8.5 g Lys/kg DM). At 4 weeks of age, the birds were allocated to groups 1 and 3, both being continued on standard Lys diet, whereas birds in groups 2 and 4 were switched to extra Lys diet (10.5 g Lys/kg DM). Birds in groups 3 and 4 were additionally infected at this age (4 weeks) with 250 embryonated eggs of A. galli. All the birds were slaughtered 7-week-post-infection (p.i.) at an age of 11 weeks. Infected birds on standard Lys diet consumed more feed and reached a similar level of Lys intake as birds on extra Lys diet 7-week-p.i. Utilization of feed, crude protein, and Lys for body weight (BW) gain was adversely affected in infected birds on standard Lys diet (P<0.001). Extra Lys diet reduced the incidence of infection (P<0.05), but worm counts, worm length, faecal egg counts and female worm fecundity were not different between the infected groups (P>0.05). Extra Lys diet led to higher final BW in uninfected and infected birds (P<0.05). It is concluded that extra Lys above the normal recommended standard supports the defence reactions and compensates the negative effect of infection on growth performance and nutrient utilization in A. galli-infected birds.

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 (Delta delta(13)C(tissue-diet) and Delta delta(15)N(tissue-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.

Studies on the biotin flow at the duodenum of dairy cows fed diets with different concentrate levels and types of forages.

Biotin is involved in many vital metabolic pathways and must be provided for an efficient fermentation in the rumen, as well as for the intermediary metabolism of the host animal. Factors influencing ruminal biotin metabolism and output are widely unknown at present. Therefore, dairy cows fitted with permanent cannulas in the dorsal rumen and in the proximal duodenum were fed differently composed diets, and the biotin flow at the proximal duodenum was measured. The diets (on DM basis) consisted of 8.9 kg grass hay (Diet 1), 8.9 kg corn silage plus 2.0 kg concentrate (Diet 2), or 7.3 and 7.4 kg grass silage plus 10.0kg concentrate (Diets 3 and 4). The concentrate in Diets 3 and 4 contained 87% wheat and corn grain, respectively. The cows were pre-fed the rations for 21 days. Thereafter duodenal digesta was sampled every two h for 5 days. Cr2O3 served as a flow marker and the microbial proportion of total nitrogen at the duodenum was estimated by near infrared spectroscopy (NIRS). The duodenal flow of biotin was not related to biotin intake, but to the amount of fermented organic matter (FOM) and the amount of microbial protein (Biotin [mg/d] = 0.518 kg FOM - 0.300; r=0.85 and biotin [mg/d] = 0.012 x g microbial protein + 1.478; r = 0.84), irrespective of the composition of the diet fed. Mean daily biotin flow was 0.48 +/- 0.11 mg/kg FOM without any systematic effect of diet composition. The ruminal biotin balance, calculated as the difference between biotin flow at the duodenum and biotin intake, was positive (1.4 - 2.0 mg/d) in cows fed the mixed roughage/concentrate diets and negative (-0.71 mg/d) when the pure hay diet was fed.

Dietary lipid content influences the activity of lipogenic enzymes in the liver and on whole body delta13C 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 per thousand for both delta13C and delta15N 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 delta13C 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 delta13C 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 delta13C 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.

Individual protein balance strongly influences delta15N and delta13C 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 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.

Feeding level and individual metabolic rate affect delta 13C and delta 15N values in carp: implications for food web studies.

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.