Effect of buffer pH on methane production and fermentation characteristics of three forages tested in vitro.

BACKGROUND: Low rumen pH is proposed to be a major mechanism for low methane (CH4) emissions from sheep fed forage rape. However, it is difficult to separate this from other in vivo factors, such as rumen passage rate. The objective of this study was to determine the effect of pH alone on CH4 production in vitro using different pH buffers. Ryegrass, white clover and forage rape were incubated in vitro using three different incubation buffers with starting pH values of 5.5, 6.2 and 6.8. RESULTS: Decreasing pH reduced overall in vitro CH4 emission relative to fermented hexoses (CH4/FHex) by up to 54% and overall fermentation by 40%. pH also changed fermentation profiles where the acetate + butyrate to propionate + valerate ratio decreased when pH decreased. Within the three forages, forage rape led to the lowest CH4/FHex, but only in pH 5.5 and 6.2 buffer, and this was enhanced when the pH fell below 6. CONCLUSION: Reducing pH in vitro decreased CH4 production and overall fermentation across all forages. The lower pH reached by forage rape compared to ryegrass and white clover appears to drive the lower CH4 production relative to the extent of fermentation from forage rape compared to the other forages. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Tailored Nanoparticles With the Potential to Reduce Ruminant Methane Emissions.

Agricultural methane produced by archaea in the forestomach of ruminants is a key contributor to rising levels of greenhouse gases leading to climate change. Functionalized biological polyhydroxybutyrate (PHB) nanoparticles offer a new concept for the reduction of enteric methane emissions by inhibiting rumen methanogens. Nanoparticles were functionalized in vivo with an archaeal virus lytic enzyme, PeiR, active against a range of rumen Methanobrevibacter species. The impact of functionalized nanoparticles against rumen methanogens was demonstrated in pure cultures, in rumen batch and continuous flow rumen models yielding methane reduction of up to 15% over 11 days in the most complex system. We further present evidence of biological nanoparticle fermentation in a rumen environment. Elevated levels of short-chain fatty acids essential to ruminant nutrition were recorded, giving rise to a promising new strategy combining methane mitigation with a possible increase in animal productivity.

Effect of Divergent Feeding Regimes During Early Life on the Rumen Microbiota in Calves.

The objective of this study was to determine whether divergent feeding regimes during the first 41 weeks of the life of a calf are associated with long-term changes in the rumen microbiota and the associated fermentation end-products. Twenty-four calves (9 ± 5 days of age) were arranged in a 2 × 2 factorial design with two divergent treatments across three dietary phases. In phase 1 (P01), calves were offered a low-milk volume/concentrate starter diet with early weaning (CO) or high-milk volume/pasture diet and late weaning (FO). In phase 2 (P02), calves from both groups were randomly allocated to either high-quality (HQ) or low-quality (LQ) pasture grazing groups. In phase 3 (P03), calves were randomly allocated to one of two grazing groups and offered the same pasture-only diet. During each dietary phase, methane (CH4) and hydrogen (H2) emissions and dry matter intake (DMI) were measured in respiration chambers, and rumen samples for the evaluation of microbiota and short-chain fatty acid (SCFA) characterizations were collected. In P01, CO calves had a higher solid feed intake but a lower CH4 yield (yCH4) and acetate:propionate ratio (A:P) compared with FO calves. The ruminal bacterial community had lower proportions of cellulolytic bacteria in CO than FO calves. The archaeal community was dominated by Methanobrevibacter boviskoreani in CO calves and by Mbb. gottschalkii in FO calves. These differences, however, did not persist into P02. Calves offered HQ pastures had greater DMI and lower A:P ratio than calves offered LQ pastures, but yCH4 was similar between groups. The cellulolytic bacteria had lower proportions in HQ than LQ calves. In all groups, the archaeal community was dominated by Mbb. gottschalkii. No treatment interactions were observed in P02. In P03, all calves had similar DMI, CH4 and H2 emissions, SCFA proportions, and microbial compositions, and no interactions with previous treatments were observed. These results indicate that the rumen microbiota and associated fermentation end-products are driven by the diet consumed at the time of sampling and that previous dietary interventions do not lead to a detectable long-term microbial imprint or changes in rumen function.

Effect of Methane Inhibitors on Ruminal Microbiota During Early Life and Its Relationship With Ruminal Metabolism and Growth in Calves.

The present study aimed to determine whether dietary supplementation with methanogen inhibitors during early life may lead to an imprint on the rumen microbial community and change the rumen function and performance of calves to 49-weeks of rearing. Twenty-four 4-day-old Friesian x Jersey cross calves were randomly assigned into a control and a treatment group. Treated calves were fed a combination of chloroform (CF) and 9,10-anthraquinone (AQ) in the solid diets during the first 12 weeks of rearing. Afterward, calves were grouped by treatments until week 14, and then managed as a single group on pasture. Solid diets and water were offered ad libitum. Methane measurements, and sample collections for rumen metabolite and microbial community composition were carried out at the end of weeks 2, 4, 6, 8, 10, 14, 24 and 49. Animal growth and dry matter intake (DMI) were regularly monitored over the duration of the experiment. Methane emissions decreased up to 90% whilst hydrogen emissions increased in treated compared to control calves, but only for up to 2 weeks after treatment cessation. The near complete methane inhibition did not affect calves' DMI and growth. The acetate:propionate ratio decreased in treated compared to control calves during the first 14 weeks but was similar at weeks 24 and 49. The proportions of Methanobrevibacter and Methanosphaera decreased in treated compared to control calves during the first 14 weeks; however, at week 24 and 49 the archaea community was similar between groups. Bacterial proportions at the phylum level and the abundant bacterial genera were similar between treatment groups. In summary, methane inhibition increased hydrogen emissions, altered the methanogen community and changed the rumen metabolite profile without major effects on the bacterial community composition. This indicated that the main response of the bacterial community was not a change in composition but rather a change in metabolic pathways. Furthermore, once methane inhibition ceased the methanogen community, rumen metabolites and hydrogen emissions became similar between treatment groups, indicating that perhaps using the treatments tested in this study, it is not possible to imprint a low methane microbiota into the rumen in the solid feed of pre-weaned calves.

Condensed Tannins in White Clover (Trifolium repens) Foliar Tissues Expressing the Transcription Factor TaMYB14-1 Bind to Forage Protein and Reduce Ammonia and Methane Emissions in vitro.

Grazing ruminants contribute to global climate change through enteric methane and nitrous oxide emissions. However, animal consumption of the plant polyphenolics, proanthocyanidins, or condensed tannins (CTs) can decrease both methane emissions and urine nitrogen levels, leading to reduced nitrous oxide emissions, and concomitantly increase animal health and production. CTs are largely absent in the foliage of important temperate pasture legumes, such as white clover (Trifolium repens), but found in flowers and seed coats. Attempts at enhancing levels of CT expression in white clover leaves by mutagenesis and breeding have not been successful. However, the transformation of white clover with the TaMYB14-1 transcription factor from Trifolium arvense has resulted in the production of CTs in leaves up to 1.2% of dry matter (DM). In this study, two generations of breeding elevated foliar CTs to >2% of DM. The CTs consisted predominantly of prodelphinidins (PD, 75-93%) and procyanidins (PC, 17-25%) and had a mean degree of polymerization (mDP) of approximately 10 flavan-3-ol subunits. In vitro studies showed that foliar CTs were bound to bovine serum albumin and white clover proteins at pH 6.5 and were released at pH 2.-2.5. Using rumen in vitro assays, white clover leaves containing soluble CTs of 1.6-2.4% of DM significantly reduced methane production by 19% (p ≤0.01) and ammonia production by 60% (p ≤ 0.01) relative to non-transformed wild type (WT) controls after 6 h of incubation. These results provide valuable information for further studies using CT expressing white clover leaves for bloat prevention and reduced greenhouse gas emissions in vivo.

Assessment of toxicity and persistence of Yersinia entomophaga and its Yen-Tc associated toxin.

BACKGROUND: The insect-pathogenic bacterium Yersinia entomophaga MH96 is currently under development as a microbial pesticide active against various pasture and crop pests such as the diamondback moth Plutella xylostella and the cotton bollworm Helicoverpa armigeria. To enable nonrestricted field trials of Y. entomophaga MH96, information on the persistence and nontarget effects of the bacterium and its Yen-Tc proteinaceous toxin are required. RESULTS: The Y. entomophaga Yen-Tc associated toxin was found to have limited persistence on foliage and is inactivated by UV light. The Yen-Tc was rapidly degraded in ovine or bovine rumen fluid or the intestinal fluid of H. armigera. In H. armigera an intestinal protein of >50 kDa was found to cleave the Yen-Tc bond. Assessment of Y. entomophaga persistence on foliage and in soil found that after 42 days the bacterium could not be detected in soil at 20% soil moisture content but persisted for 72 days at 30-40% soil moisture. Nontarget effects of Y. entomophaga towards earthworms found that the bacterium afforded no adverse effects on worm growth or behavior. A summary of historic Yen-Tc and Y. entomophaga persistence and toxicity data is presented. CONCLUSION: The bacterium Y. entomophaga and its Yen-Tc associated toxin have limited persistence in the environment, with the Yen-Tc being susceptible to UV inactivation and proteolytic degradation, and the bacterium persisting longer in soil of a high moisture content. © 2020 Society of Chemical Industry.

Naturally Produced Lovastatin Modifies the Histology and Proteome Profile of Goat Skeletal Muscle.

This study was conducted to examine the effects of different levels of lovastatin on the histological and sarcoplasmic proteome profile of goat skeletal muscle. A total of 20 intact male Saanen goats were randomly assigned in equal numbers to four groups and fed a total mixed ration containing 50% rice straw, 22.8% concentrates and 27.2% of various proportions of untreated or treated palm kernel cake (PKC) to achieve the target daily intake levels of 0 (Control), 2 (Low), 4 (Medium) or 6 (High) mg lovastatin/kg BW. A histological examination discovered that the longissimus thoracis et lumborum muscle of animals from the Medium and High treatment groups showed abnormalities in terms of necrosis, degeneration, interstitial space and vacuolization. Our preliminary label-free proteomics analysis demonstrates that lovastatin supplementation induced complex modifications to the protein expression patterns of the skeletal muscle of the goat which were associated with the metabolism of carbohydrate and creatine, cell growth and development processes and other metabolic processes. The changes in these biochemical processes indicate perturbations in energy metabolism, which could play a major role in the development of myopathy. In conclusion, the present study suggests that supplementation of naturally produced lovastatin above 4 mg/kg BW could adversely affecting the health and wellbeing of treated animals.

Effects of naturally-produced lovastatin on feed digestibility, rumen fermentation, microbiota and methane emissions in goats over a 12-week treatment period.

Twenty male Saanen goats were randomly assigned to four levels of lovastatin supplementation and used to determine the optimal dosage and sustainability of naturally produced lovastatin from fermentation of palm kernel cake (PKC) with Aspergillus terreus on enteric methane (CH4) mitigation. The effects on ruminal microbiota, rumen fermentation, feed digestibility and health of animal were determined over three measuring periods (4-, 8- and 12-weeks) and the accumulation of lovastatin in tissues was determined at the end of the experiment. The diets contained 50% rice straw, 22.8% concentrates and 27.2% of various proportions of untreated or treated PKC to achieve the target daily intake level of 0 (Control), 2, 4 or 6 mg lovastatin/kg body weight (BW). Enteric CH4 emissions per dry matter intake (DMI), decreased significantly (P<0.05) and equivalent to 11% and 20.4%, respectively, for the 2 and 4 mg/kg BW groups as compared to the Control. No further decrease in CH4 emission thereafter with higher lovastatin supplementation. Lovastatin had no effect on feed digestibility and minor effect on rumen microbiota, and specifically did not reduce the populations of total methanogens and Methanobacteriales (responsible for CH4 production). Similarly, lovastatin had little effect on rumen fermentation characteristics except that the proportion of propionate increased, which led to a decreasing trend (P<0.08) in acetic: propionate ratio with increasing dosage of lovastatin. This suggests a shift in rumen fermentation pathway to favor propionate production which serves as H+ sink, partly explaining the observed CH4 reduction. No adverse physiological effects were noted in the animals except that treated PKC (containing lovastatin) was less palatable at the highest inclusion level. Lovastatin residues were detected in tissues of goats fed 6 mg lovastatin/kg BW at between 0.01 to 0.03 µg/g, which are very low.

Prediction of enteric methane production, yield, and intensity in dairy cattle using an intercontinental database.

Enteric methane (CH4 ) production from cattle contributes to global greenhouse gas emissions. Measurement of enteric CH4 is complex, expensive, and impractical at large scales; therefore, models are commonly used to predict CH4 production. However, building robust prediction models requires extensive data from animals under different management systems worldwide. The objectives of this study were to (1) collate a global database of enteric CH4 production from individual lactating dairy cattle; (2) determine the availability of key variables for predicting enteric CH4 production (g/day per cow), yield [g/kg dry matter intake (DMI)], and intensity (g/kg energy corrected milk) and their respective relationships; (3) develop intercontinental and regional models and cross-validate their performance; and (4) assess the trade-off between availability of on-farm inputs and CH4 prediction accuracy. The intercontinental database covered Europe (EU), the United States (US), and Australia (AU). A sequential approach was taken by incrementally adding key variables to develop models with increasing complexity. Methane emissions were predicted by fitting linear mixed models. Within model categories, an intercontinental model with the most available independent variables performed best with root mean square prediction error (RMSPE) as a percentage of mean observed value of 16.6%, 14.7%, and 19.8% for intercontinental, EU, and United States regions, respectively. Less complex models requiring only DMI had predictive ability comparable to complex models. Enteric CH4 production, yield, and intensity prediction models developed on an intercontinental basis had similar performance across regions, however, intercepts and slopes were different with implications for prediction. Revised CH4 emission conversion factors for specific regions are required to improve CH4 production estimates in national inventories. In conclusion, information on DMI is required for good prediction, and other factors such as dietary neutral detergent fiber (NDF) concentration, improve the prediction. For enteric CH4 yield and intensity prediction, information on milk yield and composition is required for better estimation.

Does Dietary Mitigation of Enteric Methane Production Affect Rumen Function and Animal Productivity in Dairy Cows?

It has been suggested that the rumen microbiome and rumen function might be disrupted if methane production in the rumen is decreased. Furthermore concerns have been voiced that geography and management might influence the underlying microbial population and hence the response of the rumen to mitigation strategies. Here we report the effect of the dietary additives: linseed oil and nitrate on methane emissions, rumen fermentation, and the rumen microbiome in two experiments from New Zealand (Dairy 1) and the UK (Dairy 2). Dairy 1 was a randomized block design with 18 multiparous lactating cows. Dairy 2 was a complete replicated 3 x 3 Latin Square using 6 rumen cannulated, lactating dairy cows. Treatments consisted of a control total mixed ration (TMR), supplementation with linseed oil (4% of feed DM) and supplementation with nitrate (2% of feed DM) in both experiments. Methane emissions were measured in open circuit respiration chambers and rumen samples were analyzed for rumen fermentation parameters and microbial population structure using qPCR and next generation sequencing (NGS). Supplementation with nitrate, but not linseed oil, decreased methane yield (g/kg DMI; P<0.02) and increased hydrogen (P<0.03) emissions in both experiments. Furthermore, the effect of nitrate on gaseous emissions was accompanied by an increased rumen acetate to propionate ratio and consistent changes in the rumen microbial populations including a decreased abundance of the main genus Prevotella and a decrease in archaeal mcrA (log10 copies/g rumen DM content). These results demonstrate that methane emissions can be significantly decreased with nitrate supplementation with only minor, but consistent, effects on the rumen microbial population and its function, with no evidence that the response to dietary additives differed due to geography and different underlying microbial populations.

Dietary fish oil supplements modify ruminal biohydrogenation, alter the flow of fatty acids at the omasum, and induce changes in the ruminal Butyrivibrio population in lactating cows.

Four lactating cows fitted with ruminal cannulae and fed a grass silage-based diet were used in a 4 × 4 Latin square with 28-d periods to investigate the effects of incremental dietary fish oil (FO) supplementation (0, 75, 150, or 300 g/d) on the flow of fatty acids at the omasum and populations of rumen bacteria capable of biohydrogenation. FO decreased silage intake and ruminal volatile fatty acid concentrations and promoted an increase in molar butyrate and propionate proportions at the expense of acetate. Extensive ruminal biohydrogenation of 20:5(n-3) and 22:6(n-3) resulted in corresponding increases in numerous 20- and 22-carbon unsaturated fatty acids at the omasum. Omasal flow of several 20-, 21-, and 22-carbon all-cis (n-3) PUFA exceeded the intake from FO. Supplements of FO also induced a dose-dependent decrease in 18:0 and increased trans 18:1 and trans 18:2 flow at the omasum. Trans-11 was the major 18:1 intermediate in digesta, while FO induced quadratic increases in trans-10 18:1 flow, reaching a maximum of 300 g/d. FO had no substantial influence on omasal flow of CLA. Results suggest that one or more fatty acids in FO inhibit the reduction of trans-18:1 and trans-18:2 intermediates by ruminal microorganisms. qPCR based on 16S rRNA genes in omasal digesta indicated that key Butyrivibrio spp. declined linearly in response to FO. Dose-dependent increases in ruminal outflow of biohydrogenation intermediates containing one or more trans double bonds in response to FO has major implications for host metabolism and the nutritional quality of ruminant foods.

Strategies to reduce methane emissions from farmed ruminants grazing on pasture.

Methane emissions from livestock are a significant contributor to greenhouse gas emissions and have become a focus of research activities, especially in countries where agriculture is a major economic sector. Understanding the complexity of the rumen microbiota, including methane-producing Archaea, is in its infancy. There are currently no robust, reproducible and economically viable methods for reducing methane emissions from ruminants grazing on pasture and novel innovative strategies to diminish methane output from livestock are required. In this review, current approaches towards mitigation of methane in pastoral farming are summarised. Research strategies based on vaccination, enzyme inhibitors, phage, homoacetogens, defaunation, feed supplements, and animal selection are reviewed. Many approaches are currently being investigated, and it is likely that more than one strategy will be required to enable pastoral farming to lower its emissions of methane significantly. Different strategies may be suitable for different farming practices and systems.

Forage type and fish oil cause shifts in rumen bacterial diversity.

Despite evidence supporting improved incorporation of beneficial polyunsaturated fatty acids (PUFA) into ruminant products, such as meat and milk, following red clover and fish oil (FO) inclusion in the ruminant diet, little is known regarding the concomitant bacterial diversity. We evaluated the effects of feeding grass vs. red clover silage with incremental FO inclusion on known lipolytic, biohydrogenating, cellulolytic and proteolytic rumen bacterial communities of steers. Following 14 days of dietary adaptation, liquid-associated (LAB) and solid-associated (SAB) bacterial communities were harvested, DNA extracted and bacterial denaturing gradient gel electrophoresis (DGGE) and specific-bacterial quantitative PCR (QPCR) were undertaken. DGGE-derived dendrograms showed that diet caused the greatest change in LAB and SAB bacterial diversity, with FO inclusion at the 2% and 3% dry matter intake also causing some changes. QPCR revealed that diet resulted in changes in the DNA concentration of Anaerovibrio lipolytica, the Butyrivibrio proteoclasticus group, Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. FO inclusion caused changes in A. lipolytica, F. succinogenes and R. flavefaciens DNA concentration only. In the B. proteoclasticus group, which are the only known bacteria with the capacity to biohydrogenate PUFA to 18:0, DNA concentration did not correlate to 18:0 flow to the duodenum, however, suggesting that other bacteria may play a role in biohydrogenation. A greater understanding of microbial changes that accompany beneficial dietary changes will lead to novel strategies to improve ruminant product quality.

Rumen protozoa are rich in polyunsaturated fatty acids due to the ingestion of chloroplasts.

Within this study, we investigated whether the polyunsaturated fatty acids (PUFA)-rich nature of rumen protozoa is a consequence of ingestion of PUFA-rich chloroplasts. Four Hereford x Friesian steers were offered hay [low 18:3 (n-3) and low chlorophyll concentration] followed by freshly cut perennial ryegrass [high 18:3 (n-3) and high chlorophyll concentration] for 16 days. On the 14th and 16th days, rumen protozoa as well as attached and planktonic bacteria were fractionated 1 h before (-1 h), 2 and 6 h postfeeding, and their fatty acid concentrations determined. Protozoa fractionated from fresh grass-fed steers were richer (P<0.05) in PUFA, except conjugated linoleic acid, for all time points compared with those from hay-fed steers. Protozoal density was higher (P<0.05) for grass compared with hay. Entodinomorphid abundance was 3.4 times higher on fresh grass (P<0.01) compared with hay. Confocal microscopy and transmission electron microscopy confirmed that Epidinium spp. were commonly saturated with intracellular cytoplasmic chloroplasts. These data suggest that engulfment of chloroplasts is a major contributor to the high 18:3 (n-3) concentration of protozoa.

Fish oil increases the duodenal flow of long chain polyunsaturated fatty acids and trans-11 18:1 and decreases 18:0 in steers via changes in the rumen bacterial community.

Ruminant fat is rich in SFA, partly due to the biohydrogenation of dietary PUFA to SFA in the rumen. This process can be inhibited by the dietary inclusion of fish oil. The only bacteria isolated from the rumen capable of converting PUFA to SFA are closely related to Clostridium proteoclasticum. The aim of this study was to investigate if a correlation could be found in vivo between dietary fish oil inclusions and the composition of the ruminal bacterial community and specifically of C. proteoclasticum. Six Hereford x Friesian steers, prepared with ruminal and duodenal cannulae, received grass silage plus 1 of 3 concentrates resulting in total dietary fish oil contents of 0, 1, or 3% of dry matter. A dual flow marker technique was employed to estimate the relative flow of fatty acids. Steers fed the 3% fish oil diet had 100% increases in trans 18:1 flow, whereas 18:0 flow declined to 39% of steers fed the control diet. 16S ribosomal RNA-based denaturing gradient gel electrophoresis profiles obtained from ruminal digesta showed major changes in the bacterial community within steers fed the 3% fish oil diet. Quantitative PCR indicated only a weak relation between numbers of C. proteoclasticum and 18:0 flow between treatments and in individual steers (P < 0.05, but the percentage variance accounted for only 22.8) and did not provide unambiguous evidence that numbers of C. proteoclasticum in the rumen dictate the ratios of SFA:PUFA available for absorption by the animal. Understanding which microbes biohydrogenate PUFA in the rumen is key to developing novel strategies to improve the quality of ruminant products.

The impact of saponins or saponin-containing plant materials on ruminant production--a review.

Saponins are steroid or triterpene glycoside compounds found in a variety of plants. Some saponin-containing plants, mainly legumes, have been used as animal feed, but others are toxic. Several studies on the effect of saponins on ruminant production have also been reported. Some in vitro and in vivo experiments that demonstrate the beneficial effects of saponin such as defaunation of the rumen and manipulation of the end products of fermentation are described. Defaunation is the selective removal of protozoa from the rumen microbial ecosystem by a cell membrane cholesterol-saponin interaction, which causes cell rupture. Because protozoa in the rumen cause protein turnover by predating on bacteria, defaunation increases the nitrogen utilization of the ruminant and may lead to an increase in growth, milk, or wool production. The growth-promoting effect was evident in the high roughage diet suggesting that the application of saponins or saponin-containing plant materials may be beneficial for the subsistence farmers in developing countries. Saponins are deglycosylated by rumen microbes. Some sapogenins have been detected in the digestive tract of ruminants; however, the direct action of these compounds on the host animal is still unclear. No information on the effects of saponin on ruminant reproduction is available. There is an urgent need for a systematic evaluation of the most active structural components of the saponins, and their interaction with the microbial community, the host animal, and the diet. Along with these studies, the direct effects of saponins or their microbial degradation products on the host must be examined in order to get the full understanding of the metabolism and beneficial effects of saponins on animals.

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.