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Towards a semi-automated metabolic profile analysis of fish blood plasma by 1H NMR

ABSTRACT:

Aquafeed is one of the main costs in aquaculture and major efforts are being made to find sustainable ingredients that could improve production sustainability and efficiency. Our research focused on the inclusion of glycerol in the feed of European seabass (Dicentrarchus labrax), as it is a cheap carbon substrate expected to enter metabo- lism through the glycolytic pathway. The Nuclear Magnetic Resonance (NMR) spectroscopy provides accurate and high-throughput metabolic profiles, allowing quantification and evaluation of the effects of experimental diets in the context of fish nutrition. The aim of the present study was to assess the applicability of the automated on-line profiling routine Bayesil to fish blood plasma and compare with the manual targeted metabolite profiling approach. After a 60-day feeding trial (0%, 2.5%, 5.0% glycerol supplemented diets), fish were sacrificed at 6 h and 24 h after last meal and blood was sampled. Filtered plasma samples were analysed by NMR, followed by two metabolite-profiling approaches: i) an automated spectral profiling provided by the Bayesil platform, and ii) a manual metabolite profiling. Spectra were analysed with the automated Bayesil routine, identifying and quantifying 51 metabolites, in a reproducible, fast and user-independent method. On the other hand, the manual approach only managed to quantify 20 metabolites. However, under supraphysiological concentrations of glycerol, namely in the 5.0% diet, the Bayesil algorithm was not able to properly integrate the glycerol and glycine signals. The Bland-Altman and PLS-DA analysis revealed differences; nonetheless, with the exception of acetic acid, creatinine, formate, glutamine and threonine, all other metabolites presented a low bias and/or a strong correlation coefficient. Overall, it is expected a reasonable tradeoff between the accuracy in metabolite quantification and the increase of variables.The automated Bayesil database may be applied as a useful tool for systematic monitoring of fish plasma profile in aquaculture research and industrial quality control analysis, to generate robust variance-based multivariate statistical models. To our knowledge, this is the first time a NMR-based high-throughput automated routine is evaluated in aquaculture research.

INSTRUMENT(S): Nuclear Magnetic Resonance (NMR)

SUBMITTER: Mariana Palma

PROVIDER: MTBLS1145 | MetaboLights | 2022-02-14

REPOSITORIES: MetaboLights

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Project description:Background: The decreasing availability of fishmeal as a protein source in aquaculture diets will require aquaculture to develop an econmoical and sustainable protien replacement. Plant proteins are readily available and are being tested as a promising alternative to replace a substantial portion of fishmeal which currently provides most of the protein content in aquaculture diets. The types of plant protein feasible for incorporation into aquaculture diets will likely contain various anti-nutritional compounds, carbohydrates, fiber, and a different amino acid profile than what is found in fishmeal. Substantial genetic variation was previously observed for growth on plant based dits in rainbow trout Hence, it will be beneficial to identify metabolic and physiologic pathways related to enhanced plant protein utilization which will aid in identifying genes that contribute to this genetic variation. Results: Microarray analysis of liver samples from two families of rainbow trout that differed in their growth responses when compared between individuals grown on a fish meal or plant protein based diet. Differential expression relating to dietary utilization between the two families found significant changes in expression of 33 ESTs. Eight of the differntially expressed ESTs had identified mammalian homologs that had been previously researched with identified cellular interactions and functions. Conclusions: Utilizing pathway analysis software to analyze sequences annotated with known mammalian genes were were ablet o map gene pathway and process interactions. From this information we were able to infer that the metabolic changes associated with utilization of plant protein versus fishmeal were associated with differential reaulation of genes related to cell oxidative stress, proliferation, growth, and survival. Furthermore, we inferred from the changes we observed in immune response genes expression that ingestion of this plant based diet upregulated the expression of genes involved in immunoregulatroy processes. Genotyped samples linked to families that had 4 or more members sampled on each diet were identified and ranked according to average family weight on each diet. Size rankings corresponded to large (>750 g), medium (600-640 g), and small (<500 g). From these identified groups 2 families were chosen for microarray expression analysis that demonstrated individuals with growth differences between the two diets. Data for the two families used for analysis and slide arrangements are listed in table 3. The families compared for growth differences between diets used for this study only differed by one growth range, large fish on fish meal compared to medium fish on plant-based diet and medium sized fish on fishmeal diet compared to small fish on plant-based diet. This strategy to use size separation within family was an attempt to identify gene changes more specific to diet utilization than specific for growth differences.

Project description:INTRODUCTION: Feed optimization is a key step to the environmental and economic sustainability of aquaculture, especially for carnivorous species. Plant-derived ingredients can contribute to reduce costs and nitrogenous effluents while sparing wild fish stocks. However, the metabolic use of carbohydrates from vegetable sources by carnivorous fish is still not completely understood.OBJECTIVES: We aimed to study the effects of diets with carbohydrates of different digestibilities, gelatinized starch (DS) and raw starch (RS), in the muscle metabolome of European seabass (Dicentrarchus labrax).METHODS: We followed an NMR-metabolomics approach, using two sample preparation procedures, the intact muscle (HRMAS) and the aqueous muscle extracts (1H NMR), to compare the variations in muscle metabolome between the two diets.RESULTS: In muscle, multivariate analysis revealed similar metabolome shifts for DS and RS diets, when compared with the control diet. HRMAS of intact muscle, which included both hydrophobic and hydrophilic metabolites, showed increased lipid in DS-fed fish by univariate analysis. Regardless of the nature of the starch, increased glycine and phenylalanine, and decreased proline were observed when compared to the Ctr diet. Combined univariate analysis of intact muscle and aqueous extracts indicated specific diet related changes in lipid and amino acid metabolism, consistent with increased dietary carbohydrate supplementation.CONCLUSIONS: Due to differential sample processing, outputs differ in detail but provide complementary information. After tracing nutritional alterations by profiling fillet components, DS seems to be the most promising alternative to fishmeal-based diets in aquaculture. This approach should be reproducible for other farmed fish species and provide valuable information on nutritional and organoleptic properties of the final product.

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Towards a semi-automated metabolic profile analysis of fish blood plasma by 1H NMR

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