Violeta Kalemi

Background/Objectives: This study presents a gas chromatography–mass spectrometry (GC–MS) method for the derivatization-free quantification of the three major short-chain fatty acids (SCFAs)—acetate, propionate, and butyrate—in gilthead sea bream (Sparus aurata) fecal samples. SCFAs are key metabolites involved in intestinal physiology and are widely recognized as indicators of gut microbiota activity and host health across animal species. In aquaculture research, SCFA profiling can provide valuable insight into diet–microbiota interactions and gut functional status. Methods: The method was developed and optimized using gut-content samples collected from 105 gilthead sea bream at the end of two independent feeding trials, yielding 42 pooled samples (18 from FT1 and 24 from FT2). An extraction protocol based on acidified aqueous extraction with water followed by liquid–liquid extraction using methyl tert-butyl ether was developed (ExA) and subsequently optimized (ExB) to improve extraction consistency and efficiency. SCFA separation and quantification were performed by GC–MS using external calibration. Method validation included evaluation of linearity, matrix effects, and partition coefficients between water and methyl tert-butyl ether. Results: The optimized extraction method (ExB) demonstrated high analytical performance, with linearity coefficients exceeding 0.992 and matrix effects below 17.4%. Conclusions: The proposed method represents a practical and reliable approach for routine SCFA analysis in fish fecal samples. It provides a useful analytical tool for studies investigating diet–microbiota interactions, gut health, and functional responses to nutritional interventions in aquaculture species.

Aquaculture sustainability requires a reduction in the reliance on marine-derived raw materials such as fish oil in aquafeeds while maintaining fish health and product quality. This study investigated the effects of replacing fish oil with plant oils supplemented with DHA-rich Schizochytrium limacinum biomass on the gut microbiota of European sea bass (Dicentrarchus labrax). S. limacinum SR21—an oleaginous microalga naturally rich in omega-3 fatty acids—was produced through heterotrophic fermentation using crude glycerol, a waste stream from biodiesel production, within a circular economy framework. A 21-week feeding trial was conducted in an indoor recirculating aquaculture system using 280 fish distributed across eight tanks. Four experimental diets were tested: fish oil-based (FO), plant oil-based without microalga (VO + 0), and plant oil-based supplemented with 5% (VO + 5) or 10% (VO + 10) microalgal biomass. Gut microbiota was analyzed in 22 fish per group using 16S rRNA gene sequencing. While alpha and beta diversity analyses of gut microbiota revealed modest structural shifts at phylum and class ranks, genus-rank differences were evident, with Lactobacillus and Clostridium sensu stricto associated with FO and VO + 0 diets, and Pseudomonas and Staphylococcus enriched in microalga-supplemented groups. Functional inference highlighted enhanced bile acid biosynthesis and carbohydrate metabolism in VO + 0, whereas antioxidant-related pathways, including ubiquinone and carotenoid biosynthesis, were stimulated in VO + 5 and VO + 10 groups. These results demonstrate that S. limacinum biomass modulates microbiota functional capacity, potentially contributing to oxidative stress mitigation and host resilience. The findings support microbiota-informed feed formulation strategies to advance sustainable aquaculture.

To reduce dependence on oceanic resources, poultry-based ingredients and fortified feeds have become valid alternatives to fish meal (FM) and fish oil (FO). While their impact on growth performance is well established, effects on host-associated microbiota remain less characterized. This study examines the gut microbiota of European sea bass (Dicentrarchus labrax) following FM and FO replacement with poultry- and additive-based diets, applying machine learning (ML) to evaluate diet and genotype effects. A secondary analysis of microbial profiles from two prior trials employed classification models to determine associations between microbial abundance and categorical groupings, and regression models to assess the predictive power of ingredient variations on microbial abundance. Regressors showed limited predictive capacity, whereas classifiers performed better, particularly when genotype was considered. For poultry-based diets, average accuracy was approximately 0.4 for synergistic effects, 0.6 for diet effects, and 0.8 for genotype effects; for fortified-feed diets, average accuracy was approximately 0.2, 0.4, and 0.5, respectively. Feature selection detected microbial genera encompassing beneficial (Brevundimondas, Clostridium, Idiomarina, Lactobacillus, Marinobacter, Pseudoalteromonas, Salinisphaera), neutral (Enterovibrio, Flavobacterium, Photobacterium), opportunistic (Acinetobacter, Escherichia-Shigella, Streptococcus), and undercharacterized (Acholeplasma, Cutibacterium, Enhydrobacter, Micrococcus, Peptoniphilus, Salegentibacter) taxa. ML techniques thus reveal diet- and genotype-specific signatures, underlining the importance of integrated computational-microbiological pipelines.

Technological advancements in computational power and algorithm design have enabled artificial intelligence to become a transformative force in microbiome research. This paper presents a concise overview of recent applications of this computational paradigm in human and animal health, with a particular emphasis on aquaculture. International projects focused on the intestinal microbiome have allowed human research to consistently dominate in terms of application cases, offering insights into various pathological conditions. In contrast, animal research has leveraged artificial intelligence in microbiome analysis to promote sustainable productivity, addressing environmental and public health concerns linked to livestock husbandry. In aquaculture, on the other hand, artificial intelligence has mainly supported management practices, improving rearing conditions and feeding strategies. When considering microbiome manipulation, however, fish farms have often relied on traditional methods, without harnessing the immense potential of artificial intelligence, whose recent applications include biomonitoring and modeling interactions between microbial communities and environmental factors in farming systems. Given the paradigm shift currently underway in both human health and animal husbandry, we advocate for a transition in the aquaculture industry toward smart farming, whose interconnected infrastructure will allow to fully leverage artificial intelligence to seamlessly integrate both biological measurements and rearing parameters.

This study addressed the urgent need for sustainable protein sources in aquaculture due to the depletion of marine resources and rising costs. Animal protein sources, particularly poultry by-product meal (PBM) and insect exuviae meal, were investigated as viable alternatives to fishmeal (FM). The research study confirmed the successful replacement of FM with a combination of PBM and insect exuviae meal (up to 50%) in the diet of European seabass without compromising growth, feed conversion, gut health, and liver fat content. In particular, growth was robust with both PBM formulations, with the 25% PBM diet showing better results. Histological examinations showed good gut and liver health, contradicting the concerns of previous studies. This paper emphasizes the importance of holistic analyzes that go beyond growth parameters and include histomorphological investigations. The results show that PBM in combination with insect/exuviae meal is well tolerated by seabass, which is consistent with reports in the literature of it mitigating negative effects on gut health. A detailed analysis of the microbiota revealed a decrease in the Firmicutes/Proteobacteria ratio due to an increase in potentially pathogenic bacteria. However, the formulation containing insect exuviae partially counteracted this effect by preserving the beneficial Lactobacillus and promoting the synthesis of short-chain fatty acids (SCFAs), particularly butyrate. Chitin-rich components from insect exuviae were associated with improved gut health, which was supported by the increased production of SCFAs, which are known for their anti-inflammatory properties. This paper concludes that a combination of PBM and insect/exuviae meal can replace up to 50% of FM in the diet of seabass, supporting sustainable aquaculture practices. Despite some changes in the microbiota, the negative effects are mitigated by the addition of insect exuviae, highlighting their potential as a prebiotic to increase fish productivity and contribute to a circular economy in aquaculture.

This study addresses the urgent need for sustainable protein sources in aquaculture due to the depletion of marine resources and rising costs. Animal protein sources, particularly poultry by-product meal (PBM) and insect exuviae meal, are investigated as viable alternatives to fishmeal (FM). Research confirms the successful replacement of FM with a combination of PBM and insect exuviae meal (up to 50%) in the diet of European seabass without compromising growth, feed conversion, gut health and liver fat content. In particular, growth performance was robust with both PBM formulations, with the 25% PBM diet showing better results. Histological examinations showed good gut and liver health, contradicting the concerns of previous studies. The study emphasizes the importance of holistic analyzes that go beyond growth parameters and include histomorphological investigations. The results show that PBM in combination with insect/exuviae meal is well tolerated by seabass, which is consistent with literature reports of mitigating negative effects on gut health. A detailed analysis of the microbiota revealed a decrease in the Firmicutes/Proteobacteria ratio due to an increase in potentially pathogenic bacteria. However, the formulation containing insect exuviae partially counteracted this effect by preserving the beneficial Lactobacillus and promoting the synthesis of short-chain fatty acids (SCFA), particularly butyrate. Chitin-rich components from insect exuviae were associated with improved gut health, which was supported by increased production of SCFA, which are known for their anti-inflammatory properties. The study concludes that a combination of PBM and insect/exuviae meal can replace up to 50% of FM in the diet of seabass, supporting sustainable aquaculture practices. Despite some changes in the microbiota, the negative effects are mitigated by the addition of insect exuviae, highlighting their potential as a prebiotic to increase fish productivity and contribute to a circular economy in aquaculture.