Simple Summary Zebrafish have emerged as invaluable model organisms in biological research, offering a cost-effective alternative to rodents. However, the absence of standardized feeding protocols and nutritional requirements poses a significant challenge, potentially compromising the validity of experimental outcomes, particularly in preclinical studies. Our systematic review analyzes a plethora of studies focused on zebrafish feed intake, feeding regimes, and diet compositions. We uncover substantial variability in dietary parameters, notably in crude protein and lipid content. Despite this diversity, energy levels remain quite consistent across zebrafish diets. By using these insights, we propose a novel feeding protocol for zebrafish of different developmental stages, based on a formulated feed optimized for protein and energy content. This protocol might represent the first step towards standardizing zebrafish feeding practices, thereby enhancing experimental reproducibility and data reliability. Abstract Zebrafish are one of the most used animal models in biological research and a cost-effective alternative to rodents. Despite this, nutritional requirements and standardized feeding protocols have not yet been established for this species. This is important to avoid nutritional effects on experimental outcomes, and especially when zebrafish models are used in preclinical studies, as many diseases have nutritional confounding factors. A key aspect of zebrafish nutrition is related to feed intake, the amount of feed ingested by each fish daily. With the goal of standardizing feeding protocols among the zebrafish community, this paper systematically reviews the available data from 73 studies on zebrafish feed intake, feeding regimes (levels), and diet composition. Great variability was observed regarding diet composition, especially regarding crude protein (mean 44.98 ± 9.87%) and lipid content (9.91 ± 5.40%). Interestingly, the gross energy levels of the zebrafish diets were similar across the reviewed studies (20.39 ± 2.10 kilojoules/g of feed). In most of the reviewed papers, fish received a predetermined quantity of feed (feed supplied). The authors fed the fish according to the voluntary intake and then calculated feed intake (FI) in only 17 papers. From a quantitative point of view, FI was higher than when a fixed quantity (pre-defined) of feed was supplied. Also, the literature showed that many biotic and abiotic factors may affect zebrafish FI. Finally, based on the FI data gathered from the literature, a new feeding protocol is proposed. In summary, a daily feeding rate of 9–10% of body weight is proposed for larvae, whereas these values are equal to 6–8% for juveniles and 5% for adults when a dry feed with a proper protein and energy content is used.

Zebrafish Feed Intake: A Systematic Review for Standardizing Feeding Management in Laboratory Conditions

Manipulating feeding rate and protein quality may improve growth and feeding efficiency of cultured species. However, whether feeding rate, protein quality, or their interaction has a greater effect on growth and feeding efficiency response variables is unknown. To determine whether feeding rate and protein quality individually or interactively affect growth and feeding efficiency, juvenile Zebrafish (Danio rerio) were either offered nutritionally similar diet consisting of either menhaden fishmeal protein or a 100% replacement of fishmeal with soybean meal-based protein restrictively or to satiation. Total length, weight, feed intake, and feed conversion ratio (FCR) were measured throughout the duration of the study. Protein quality and feeding rate individually and interactively affected feed intake and FCR: Zebrafish offered feed to satiation had higher growth and FCR than those fed restrictively, and Zebrafish fed soybean meal-based diet showed lower growth and higher FCR and feed intake compared to those fed fishmeal-based diet, although magnitude of response depended on feeding rate. These findings likely indicate lower digestibility of soybean meal or the presence of antinutritional factors in soybean meal that led to impaired nutrient absorption of fish offered soybean meal-based diet. Differences in measured response variables between protein qualities and feeding rates highlight the importance of determining interactive effects in nutritional studies.

Feeding Rate and Protein Quality Differentially Affect Growth and Feeding Efficiency Response Variables of Zebrafish (Danio rerio).

Filamentous fungi have potential as an alternative protein source for use in aquafeeds due to their high protein content and bioactive components. Paecilomyces variotii (PEKILO® mycoprotein) is a β-glucan- and nucleotide-rich filamentous fungi with a crude protein (CP) content of 60–70%. A 9-week feeding trial was conducted to evaluate the effects of P. variotii in diets for Atlantic salmon (initial average body weight of 24 g fish−1) reared in freshwater. Four experimental diets were formulated. Diet 1 was a control diet based on fish meal, soy protein concentrate, and wheat gluten meal. Diets 2, 3, and 4 were formulated so that P. variotii replaced 5, 10, and 20% of the CP content of the diets, respectively. Results showed that dietary inclusion of P. variotii was associated with changes in physical pellet quality, including significant linear (p < 0.0001) and/or quadratic (p < 0.01) decreases in pellet length, width, expansion, and durability. Conversely, significant linear (p < 0.0001) and quadratic (p < 0.025) increases in water activity, bulk density, and water stability index were associated with increasing replacement of the CP content of the diets with P. variotii. There were no significant differences in weight gain, growth rate or feed intake among fish fed the experimental diets (p > 0.05), but a significant linear improvement (p < 0.025) in feed conversion ratio (FCR) with increasing dietary inclusion of P. variotii was observed. The apparent digestibility coefficients for CP and gross energy of the experimental diets decreased linearly (p < 0.01) with increasing dietary levels of P. variotii. Similar results were observed for the essential and non-essential amino acids. Despite reduced digestibility, linear increases in nitrogen (p < 0.01), energy (p < 0.025), and mineral (p < 0.001) retention efficiencies were associated with increasing dietary levels of P. variotii. In the distal intestine, dietary inclusion of P. variotii resulted in significant (p < 0.05) upregulation of several cytokines (ifng, il10, and tgfb), effector molecules (inos, arg1, sod), and transcription factors (irf4), suggesting both activation and control of the inflammatory response in this key mucosa-associated lymphoid tissue. Overall, replacement of 20% of the CP content of the diet with P. variotii reduced digestibility of the experimental diets, but did not affect growth performance and improved FCR and nutrient utilization efficiency of salmon. Thus, P. variotii is a promising ingredient with potential for use in salmonid aquafeeds to improve fish performance and health. 

Paecilomyces variotii (PEKILO®) in novel feeds for Atlantic salmon: Effects on pellet quality, growth performance, gut health, and nutrient digestibility and utilization

Zebrafish (Danio rerio) has ascended as a pivotal model organism in the realm of gut-brain axis research, principally owing to its high-throughput experimental capabilities and evolutionary alignment with mammals. The inherent transparency of zebrafish embryos facilitates unprecedented real-time imaging, affording unparalleled insights into the intricate dynamics of bidirectional communication between the gut and the brain. Noteworthy are the structural and functional parallels shared between the zebrafish and mammalian gut-brain axis components, rendering zebrafish an invaluable model for probing the molecular and cellular intricacies inherent in this critical physiological interaction. Recent investigations in zebrafish have systematically explored the impact of gut microbiota on neurodevelopment, behaviour, and disease susceptibility, underscoring the model's prowess in unravelling the multifaceted influence of microbial communities in shaping gut-brain interactions. Leveraging the genetic manipulability inherent in zebrafish, researchers have embarked on targeted explorations of specific pathways and molecular mechanisms, providing nuanced insights into the fundamental functioning of the gut-brain axis. This comprehensive review synthesizes pivotal findings and methodological advancements derived from zebrafish-based gut-brain axis research, accentuating the model's potential to significantly advance our understanding of this complex interplay. Furthermore, it underscores the translational significance of these insights, offering promising avenues for the identification of therapeutic targets in neuro-gastroenterological disorders and psychiatric conditions intricately linked with gut-brain interactions. 

Zebrafish: A trending model for gut-brain axis investigation

Clostridium butyricum, a new probiotic in recent years, can produce butyric acid and short-chain fatty acids. It has the characteristics of strong acid and alkali resistance, high temperature resistance, and strong resistance to most antibiotics, and has more advantages than other probiotics. However, the action mechanism of C. butyricum on Eriocheir sinensis is still unclear and needs further study. In this study, when C. butyricum was added to the basic diet, the number of living bacteria was 0, 1 × 106 and 1 × 108 CFU/g, respectively. The E. sinensis were randomly divided into three groups: (blank control group, experimental group 1 (1 × 106 CFU/g) and experimental group 2 (1 × 108 CFU/g)). They were fed an experimental diet for 28 days. The effects of C. butyricum on E. sinensis were studied by detecting the differences in non-specific immune indexes, intestinal microflora, and metabolites between serum and hepatopancreas. The results showed that C. butyricum could improve the antioxidant ability of E. sinensis serum and hepatopancreas, protect intestinal tissues, and promote the absorption of nutrients. At the same time, it can enhance the microbial diversity and richness of the E. sinensis gut flora. LC-MS metabolomics was used to detect the metabolism of intestinal flora. It was found that C. butyricum could up-regulate lysophosphatidylcholine in the intestine. Through the KEGG enrichment pathway, it was found that significantly different metabolites were mainly concentrated in six metabolic pathways. The purine metabolism and alanine, aspartate, and glutamate metabolism pathways showed a downward trend, indicating that the addition of C. butyricum to feed could reduce purine metabolism, promote the water-salt balance of the organism's cells, and reduce inflammation. In this study, it was found that the addition of certain concentrations of C. butyricum to feed could improve the antioxidant ability of E. sinensis, improve the intestinal flora environment, and promote the growth of beneficial bacteria in the gut. This can promote the body's metabolism, which is more conducive to its growth. 

Effects of Clostridium butyricum on Intestinal Microflora and Metabolism of Eriocheir sinensis

A reduction in daily caloric or nutrient intake has been observed to promote health benefits in mammals and other vertebrates. Feed Restriction (FR), whereby the overall food intake of the organism is reduced, has been explored as a method to improve metabolic and immune health, as well as to optimize productivity in farming. However, less is known regarding the molecular and physiological consequences of FR. Using the model organism, Danio rerio, we investigated the impact of a short-term (month-long) FR on growth, gut morphology and gene expression. Our data suggest that FR has minimal effects on the average growth rates, but it may affect weight and size heterogeneity in a sex-dependent manner. In the gut, we observed a significant reduction in gut circumference and generally lower mucosal heights, whereas other parameters remained unchanged. Gene Ontology (GO), EuKaryotic Orthologous Groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified numerous metabolic, reproductive, and immune response pathways that were affected by FR. These results broaden our understanding of FR and contribute towards growing knowledge of its effects on vertebrate health.

Feed Restriction Modulates Growth, Gut Morphology and Gene Expression in Zebrafish

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Feed Restriction Modulates Growth, Gut Morphology and Gene Expression in Zebrafish