A Bacterial-Sourced Protein Diet Induces Beneficial Shifts in the Gut Microbiome of the Zebrafish, Danio rerio.

Background: Bacterial-sourced single-cell proteins (SCPs) offer an alternative protein source for diet formulation for Zebrafish (Danio rerio) and other aquaculture models. In addition, the use of a single-cell bacterial protein source derived from multiple species provides a unique insight into the interplay among nutrients in the diet, microbial populations in the diet, and the gut microbiome in D. rerio. Objective: Our objective in this study was to evaluate the impact of dietary replacement of fish protein hydrolysate in a standard reference (SR) with a single-cell bacterial protein source on D. rerio gut microbiome. Methods: We investigated gut microbial compositions of D. rerio fed an open-formulation standard reference (SR) diet or a bacterial-sourced protein (BP) diet, utilizing microbial taxonomic co-occurrence networks, and predicted functional profiles. Results: Microbial communities in the SR diet were primarily composed of Firmicutes. In contrast, the BP diet was mainly composed of Proteobacteria. Alpha diversity revealed significant differences in microbial communities between the 2 diets, and between the guts of D. rerio fed either of the 2 diets. D. rerio fed with the SR diet resulted in abundance of Aeromonas and Vibrio. In contrast, D. rerio fed with a BP diet displayed a large abundance of members from the Rhodobacteraceae family. Taxonomic co-occurrence networks display unique microbial interactions, and key taxons in D. rerio gut samples were dependent on diet and gender. Predicted functional profiling of the microbiome across D. rerio fed SR or BP diets revealed distinct metabolic pathway differences. Female D. rerio fed the BP diet displayed significant upregulation of pathways related to primary and secondary bile acid synthesis. Male D. rerio fed the BP diet revealed similar pathway shifts and, additionally, a significant upregulation of the polyketide sugar unit biosynthesis pathway. Conclusions: The use of a BP dramatically affects the composition and activity of the gut microbiome. Future investigations should further address the interplay among biological systems and diet and may offer insights into potential health benefits in preclinical and translational animal models.

Replacement of Dietary Fish Protein with Bacterial Protein Results in Decreased Adiposity Coupled with Liver Gene Expression Changes in Female Danio rerio.

Background: Effective use of Danio rerio as a preclinical model requires standardization of macronutrient sources to achieve scientific reproducibility across studies and labs. Objective: Our objective was to evaluate a bacterial-based single-cell protein (SCP) for the production of open-source standardized diets with defined health characteristics for the zebrafish research community. Methods: We completed a 16-wk feeding trial using juvenile D. rerio 31 d postfertilization (10 tanks per diet and 14 D. rerio per tank) with formulated diets containing either a typical fish protein ingredient [standard reference (SR) diet] or a novel bacterial SCP source [bacterial protein (BP) diet]. At the end of the feeding trial, growth metrics, body composition, reproductive success, and bulk transcriptomics of the liver (RNAseq on female D. rerio with confirmatory rtPCR) were performed for each diet treatment. Results: D. rerio fed the BP diet had body weight gains equivalent to the D. rerio fed fish protein, and females had significantly lower total carcass lipid, indicating reduced adiposity. Reproductive success was similar between treatments, suggesting normal physiological function. Genes differentially expressed in female D. rerio fed the BP diet compared with females fed the SR diet were overrepresented in the gene ontologies of metabolism, biosynthesis of cholesterol precursors and products, and protein unfolding responses. Conclusion: Protein source substantially affected body growth metrics and composition as well as gene expression. These data support the development of an open-source diet utilizing an ingredient that correlates with improved health profiles and reduced variability in notable outcomes.

Replacement of Dietary Fish Protein With Bacterial Single Cell Protein Results in Decreased Adiposity Coupled With Liver Expression Changes in Female Danio Rerio.

Background: Effective use of Danio rerio as a preclinical model requires standardization of macronutrient sources to achieve scientific reproducibility across studies and labs. Our objective was to evaluate single cell protein (SCP) for production of open-source standardized diets with defined heath characteristics for the zebrafish research community. We completed a 16-week feeding trial using juvenile D. rerio 31 days post-fertilization (dpf) (10 tanks per diet, 14 D. rerio per tank) with formulated diets containing either a typical fish protein ingredient or a novel bacterial SCP source. At the end of the feeding trial, growth metrics, body composition, reproductive success, and bulk transcriptomics of the liver (RNAseq on female D. rerio only with confirmatory rtPCR) were performed for each diet treatment. Results: D. rerio fed the SCP containing diet had body weight gains equivalent to the D. rerio fed fish protein, and females had significantly lower total carcass lipid, indicating reduced adiposity. Reproductive success was similar between treatments. Genes differentially expressed in female D. rerio provided the bacterial SCP compared to females given fish protein were overrepresented in the gene ontologies of metabolism, biosynthesis of cholesterol precursors and products, and protein unfolding responses. Conclusion: These data support the development of an open-source diet utilizing an ingredient that correlates with improved health profiles and reduced variability in notable outcomes.

Body Metrics and the Gut Microbiome in Response to Macronutrient Limitation in the Zebrafish Danio rerio.

Background: Healthy and predictable physiologic homeostasis is paramount in animal models for biomedical research. Proper macronutrient intake is an essential and controllable environmental factor for maintaining animal health and promoting experimental reproducibility. Objective and Methods: Evaluate reductions in dietary macronutrient composition on body weight metrics, composition, and gut microbiome in Danio rerio. Methods: D. rerio were fed reference diets deficient in either protein or lipid content for 14 weeks. Results: Diets of reduced-protein or reduced-fat resulted in lower weight gain than the standard reference diet in male and female D. rerio. Females fed the reduced-protein diet had increased total body lipid, suggesting increased adiposity compared with females fed the standard reference diet. In contrast, females fed the reduced-fat diet had decreased total body lipid compared with females fed the standard reference diet. The microbial community in male and female D. rerio fed the standard reference diet displayed high abundances of Aeromonas, Rhodobacteraceae, and Vibrio. In contrast, Vibrio spp. were dominant in male and female D. rerio fed a reduced-protein diet, whereas Pseudomonas displayed heightened abundance when fed the reduced-fat diet. Predicted functional metagenomics of microbial communities (PICRUSt2) revealed a 3- to 4-fold increase in the KEGG (Kyoto Encyclopedia of Genes and Genomes) functional category of steroid hormone biosynthesis in both male and female D. rerio fed a reduced-protein diet. In contrast, an upregulation of secondary bile acid biosynthesis and synthesis and degradation of ketone bodies was concomitant with a downregulation in steroid hormone biosynthesis in females fed a reduced-fat diet. Conclusions: These study outcomes provide insight into future investigations to understand nutrient requirements to optimize growth, reproductive, and health demographics to microbial populations and metabolism in the D. rerio gut ecosystem. These evaluations are critical in understanding the maintenance of steady-state physiologic and metabolic homeostasis in D. rerio. Curr Dev Nutr 20xx;x:xx.

A modified standard American diet induces physiological parameters associated with metabolic syndrome in C57BL/6J mice.

Investigations into the causative role that western dietary patterns have on obesity and disease pathogenesis have speculated that quality and quantity of dietary fats and/or carbohydrates have a predictive role in the development of these disorders. Standard reference diets such as the AIN-93 rodent diet have historically been used to promote animal health and reduce variation of results across experiments, rather than model modern human dietary habits or nutrition-related pathologies. In rodents high-fat diets (HFDs) became a classic tool to investigate diet-induced obesity (DIO). These murine diets often relied on a single fat source with the most DIO consistent HFDs containing levels of fat up to 45-60% (kcal), higher than the reported human intake of 33-35% (kcal). More recently, researchers are formulating experimental animal (pre-clinical) diets that reflect mean human macro- and micronutrient consumption levels described by the National Health and Nutrition Examination Survey (NHANES). These diets attempt to integrate relevant ingredient sources and levels of nutrients; however, they most often fail to include high-fructose corn syrup (HFCS) as a source of dietary carbohydrate. We have formulated a modified Standard American Diet (mSAD) that incorporates relevant levels and sources of nutrient classes, including dietary HFCS, to assess the basal physiologies associated with mSAD consumption. Mice proffered the mSAD for 15 weeks displayed a phenotype consistent with metabolic syndrome, exhibiting increased adiposity, fasting hyperglycemia with impaired glucose and insulin tolerance. Metabolic alterations were evidenced at the tissue level as crown-like structures (CLS) in adipose tissue and fatty acid deposition in the liver, and targeted 16S rRNA metagenomics revealed microbial compositional shifts between dietary groups. This study suggests diet quality significantly affects metabolic homeostasis, emphasizing the importance of developing relevant pre-clinical diets to investigate chronic diseases highly impacted by western dietary consumption patterns.

High-throughput amplicon sequencing datasets of the metacommunity DNA of the gut microbiota of Zebrafish Danio rerio fed diets with differential quantities of protein and fat contents.

In this paper, we present high-throughput amplicon sequence (HTS) datasets of the gut microbiota of male and female Zebrafish Danio rerio fed diets consisting of sub-optimal and above-optimal quantities of proteins and fats. The HTS datasets were generated using an Illumina MiSeq targeting the V4 hypervariable segment of the 16S rRNA gene. The raw sequence reads were quality checked, demultiplexed into FASTQ files, denoised using DADA2 (q2-dada2 denoise-paired), and subsampled. Taxonomic ids were then assigned to amplicon sequence variants (ASVs) against the silva-138-99-nb-classifier for taxonomic output using the Quantitative Insights Into Microbial Ecology (QIIME2 v2021.4). The resultant taxa list was generated at the phylum level to confirm the applicability of the HTS dataset using the "qiime taxa collapse" command. These HTS datasets of the metagenome can be accessed through the BioSample Submission Portal (https://www.ncbi.nlm.nih.gov/bioproject/) under the BioProject IDs PRJNA772302 and PRJNA772305.

Effect of Long-Term Consumption of Poultry Egg Products on Growth, Body Composition, and Liver Gene Expression in Zebrafish, Danio rerio.

BACKGROUND: Poultry eggs are a low-cost, high-protein nutrient package that can be consumed as part of quality diets. However, consumption of poultry egg products is historically contentious, which highlights the importance of investigating impacts of long-term egg consumption on metabolic health. OBJECTIVE: Our study utilized the zebrafish, Danio rerio, a newly defined model of human metabolic health, to understand the metabolic consequence of consuming egg products in lieu of other well-described protein sources. METHODS: Reference diets were formulated to contain multisource protein with casein and fish protein hydrolysate (CON; control protein sources), the protein sources that have been historically utilized in numerous reference diets. These proteins were then partially replaced with either whole egg (WE; protein and lipid source), egg white (EW; protein source), wheat gluten (WG; cereal protein source), or a high-lipid-content diet containing a multisource protein with casein and fish protein hydrolysate (HFCON; isonitrogenous and isolipidic with the WE diet) in a 34-wk trial (n = 8 tanks, 10 fish per tank). Daily feeding was initiated at the early juvenile life stage and terminated at the late reproductive adult stage. RESULTS: The amino acid composition of control versus egg product diets did not vary substantially, although methionine and lysine were apparently limiting in fish fed WG. At termination, fish fed EW as the protein source had weight gain and body composition similar to those fed the CON diet. Fasting and postprandial blood glucose did not differ between any dietary treatment. Assessment of the liver transcriptome using RNAseq revealed no differential gene expression between zebrafish fed CON or WE diets. Zebrafish fed WG had lower weight gain in males. CONCLUSIONS: Long-term consumption of egg products promoted metabolic health equal to that of historically relevant proteins. These data support the value of egg products for maintaining long-term metabolic health in animal diets.