Standardized Reference Diets for Zebrafish: Addressing Nutritional Control in Experimental Methodology.

The ideal of experimental methodology in animal research is the reduction or elimination of environmental variables or consistency in their application. In lab animals, diet has been recognized as a very influential response variable. Reproducibility in research using rodents required the development of a unique diet of consistent ingredient and nutrient composition to allow for cross-comparisons of lab results, spatially and temporally. These diets are commonly referred to as standard reference diets (SRDs). The established validity of published nutritional requirements combined with the cooperation of commercial partners led to species-specific reference diets commonly used by the research community. During the last several decades, zebrafish (Danio rerio) have become a widespread alternative animal model, but specific knowledge of their nutrition is lacking. We present a short-term approach for developing an SRD for zebrafish, similar to that eventually attained for rodents over decades. Imminent development of an open-formulation, commercially produced SRD for zebrafish will notably advance translational biomedical science.

Microbial Composition and Genes for Key Metabolic Attributes in the Gut Digesta of Sea Urchins Lytechinus variegatus and Strongylocentrotus purpuratus Using Shotgun Metagenomics.

This paper describes the microbial community composition and genes for key metabolic genes, particularly the nitrogen fixation of the mucous-enveloped gut digesta of green (Lytechinus variegatus) and purple (Strongylocentrotus purpuratus) sea urchins by using the shotgun metagenomics approach. Both green and purple urchins showed high relative abundances of Gammaproteobacteria at 30% and 60%, respectively. However, Alphaproteobacteria in the green urchins had higher relative abundances (20%) than the purple urchins (2%). At the genus level, Vibrio was dominant in both green (~9%) and purple (~10%) urchins, whereas Psychromonas was prevalent only in purple urchins (~24%). An enrichment of Roseobacter and Ruegeria was found in the green urchins, whereas purple urchins revealed a higher abundance of Shewanella, Photobacterium, and Bacteroides (q-value < 0.01). Analysis of key metabolic genes at the KEGG-Level-2 categories revealed genes for amino acids (~20%), nucleotides (~5%), cofactors and vitamins (~6%), energy (~5%), carbohydrates (~13%) metabolisms, and an abundance of genes for assimilatory nitrogen reduction pathway in both urchins. Overall, the results from this study revealed the differences in the microbial community and genes designated for the metabolic processes in the nutrient-rich sea urchin gut digesta, suggesting their likely importance to the host and their environment.

Feed Intake as an Estimation of Attractability in Pacific White Shrimp Litopenaeus vannamei.

A simple bioassay that quantifies feed intake as an estimation of relative attractability of feeds containing different ingredients in the Pacific white shrimp Litopenaeus vannamei is described. Fish meal (FM), fish protein hydrolysate (FPH), squid meal (SqM) and casein (CN) were assessed at the same dietary level for their relative influence on feed intake rates of Litopenaeus vannamei. A bland diet containing 92% whole wheat grain meal, 6% diatomaceous earth and 2% alginate with a known low attractability was used as the standard control or base diet. Ingredients were added to the bland base control diet at a level of 3% as fed. Shrimp were stocked into 80 L glass tanks (n= 20 per tank) in a recirculating aquaculture system. Tanks were randomly assigned to one of five diet treatments (3tanks/treatment). Experiments measuring the attractability of each feed were conducted twice daily at 0900 hours and 1330 hours over a five day period. For each experiment, 40 feed pellets (ca. 1 g) corresponding to the assigned treatment were provided to each tank. To calculate the rate of feed intake, pellets remaining in each tank were counted at six minute intervals for a seventy-two minute period. Differences in rate of feed intake among diets were evaluated using Cox Regression Analysis. This attractability assay required only small amounts of ingredients and incorporated ingredients into a bland feed, which significantly reduces the influence from other ingredients or compound in the pellets. All of the test protein ingredients, especially SqM, in the feeds significantly increased the feed consumption rate. The diet containing SqM was consumed at a significantly higher rate than those containing casein and FM but not FPH. FPH and CN containing diets were not significantly different but consumed at a higher rate than the diet containing FM. Results of these trials indicate that the presence of certain ingredients can increase feed intake, thereby increasing nutrient availability of the diets. This reported method to determine consumption of diets containing certain ingredients can be considered as a valid method to estimate attractability.

PRODUCTION AND ECONOMIC OPTIMIZATION OF DIETARY PROTEIN AND CARBOHYDRATE IN THE CULTURE OF JUVENILE SEA URCHIN Lytechinus variegatus.

Juvenile Lytechinus variegatus (ca. 3.95± 0.54 g) were fed one of 10 formulated diets with different protein (ranging from 11- 43%) and carbohydrate (12 or 18%; brackets determined from previous studies) levels. Urchins (n= 16 per treatment) were fed a daily sub-satiation ration equivalent to 2.0% of average body weight for 10 weeks. Our objective was (1) to create predictive models of growth, production and efficiency outcomes and (2) to generate economic analysis models in relation to these dietary outcomes for juvenile L. variegatus held in culture. At dietary protein levels below ca. 30%, models for most growth and production outcomes predicted increased rates of growth and production among urchins fed diets containing 18% dietary carbohydrate levels as compared to urchins fed diets containing 12% dietary carbohydrate. For most outcomes, growth and production was predicted to increase with increasing level of dietary protein up to ca. 30%, after which, no further increase in growth and production were predicted. Likewise, dry matter production efficiency was predicted to increase with increasing protein level up to ca. 30%, with urchins fed diets with 18% carbohydrate exhibiting greater efficiency than those fed diets with 12% carbohydrate. The energetic cost of dry matter production was optimal at protein levels less than those required for maximal weight gain and gonad production, suggesting an increased energetic cost (decreased energy efficiency) is required to increase gonad production relative to somatic growth. Economic analysis models predict when cost of feed ingredients are low, the lowest cost per gram of wet weight gain will occur at 18% dietary carbohydrate and ca. 25- 30% dietary protein. In contrast, lowest cost per gram of wet weight gain will occur at 12% dietary carbohydrate and ca. 35- 40% dietary protein when feed ingredient costs are high or average. For both 18 and 12% levels of dietary carbohydrate, cost per gram of wet weight gain is predicted to be maximized at low dietary protein levels, regardless of feed ingredient costs. These models will compare dietary requirements and growth outcomes in relation to economic costs and provide insight for future commercialization of sea urchin aquaculture.

Optimizing dietary levels of menhaden and soybean oils and soybean lecithin for pre-gonadal somatic growth in juveniles of the sea urchin Lytechinus variegatus.

Dietary lipids serve as important sources of energy and essential fatty acids for aquatic animals. Sources of animal and plant oils are increasingly limited as well as expensive, and dietary requirements associated with the inclusion of these oils must be carefully evaluated to facilitate sustainable and affordable formulations. In this study, we investigated quantities of menhaden oil (MO) with and without soybean lecithin or soybean oil (SO) to determine appropriate levels for optimal somatic growth for pre-gonadal juvenile Lytechinus variegatus. We prepared semi-purified diets that varied in neutral lipid content (0, 2, 4, or 8% dry matter) and soy lecithin (0 or 2%) and exchanged lipids reciprocally with purified starch while holding constant all other nutrients. We maintained laboratory-reared juvenile L. variegatus (average initial wet weight 82 ± 0.7 mg, mean ± SE , n = 9 treatment-1) in recirculating seawater systems and fed each daily a sub-satiation ration for five weeks. We assessed wet weights and test diameters every two weeks and at the end of the experiment (5 wk). Level of MO with or without soybean lecithin did not significantly affect wet weight gain; however, increasing levels of SO in the diet reduced wet weight gain and dry matter production efficiency and increased feed conversion ratio. Dry gut weight was positively correlated with level of MO. Lipid level in the gut increased with increasing dietary lipid level, regardless of source. These data suggest the composition of the SO is inhibitory for either nutrient absorption or metabolic processes associated with growth at this life stage. Diets containing total lipid levels of approximately 5 to 6% that include sources of n-3 fatty acids may support optimal growth for pre-gonadal juvenile L. variegatus.

New antithrombotic agents in the ambulatory setting.

PURPOSE OF REVIEW: Many patients presenting for surgical or other procedures in an ambulatory setting are taking new antiplatelet or anticoagulant agents. This review assesses how the novel features of these new agents affect the management of antithrombotic therapy in the ambulatory setting. RECENT FINDINGS: There have been very few studies investigating the relative risks of continuing or ceasing new antithrombotic agents. Recent reviews indicate that the new antithrombotic agents offer greater efficacy or ease of administration but are more difficult to monitor or reverse. They emphasize the importance of assessing the bleeding risk of the procedure, the thrombotic risk if the agent is ceased, and patient factors that increase the likelihood of bleeding. The timing of cessation of the agent, if required, depends on its pharmacokinetics and patients' bleeding risks. Patients at high risk of thrombotic complications may require bridging therapy. Once agreed upon, the perioperative plan should be made clear to all involved. SUMMARY: As there are few clinical studies to guide management, clinicians must make rational decisions in relation to continuing or ceasing new antithrombotic agents. This requires knowledge of their pharmacokinetics, and a careful multidisciplinary assessment of the relative thrombotic and bleeding risks in individual patients.

Protein absorption in the zebrafish gut is regulated by interactions between lysosome rich enterocytes and the microbiome.

Dietary protein absorption in neonatal mammals and fishes relies on the function of a specialized and conserved population of highly absorptive lysosome rich enterocytes (LREs). The gut microbiome has been shown to enhance absorption of nutrients, such as lipids, by intestinal epithelial cells. However, whether protein absorption is also affected by the gut microbiome is poorly understood. Here, we investigate connections between protein absorption and microbes in the zebrafish gut. Using live microscopy-based quantitative assays, we find that microbes slow the pace of protein uptake and degradation in LREs. While microbes do not affect the number of absorbing LRE cells, microbes lower the expression of endocytic and protein digestion machinery in LREs. Using transgene assisted cell isolation and single cell RNA-sequencing, we characterize all intestinal cells that take up dietary protein. We find that microbes affect expression of bacteria-sensing and metabolic pathways in LREs, and that some secretory cell types also take up protein and share components of protein uptake and digestion machinery with LREs. Using custom-formulated diets, we investigated the influence of diet and LRE activity on the gut microbiome. Impaired protein uptake activity in LREs, along with a protein-deficient diet, alters the microbial community and leads to increased abundance of bacterial genera that have the capacity to reduce protein uptake in LREs. Together, these results reveal that diet-dependent reciprocal interactions between LREs and the gut microbiome regulate protein absorption.

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.

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.

Tenth Aquatic Models of Human Disease Conference 2022 Workshop Report: Aquatics Nutrition and Reference Diet Development.

Progress in biomedical research requires rigorous studies and reproducible outcomes. However, despite recent achievements, standard reference diets (SRDs) for aquatic model organisms, vital for supporting scientific rigor and reproducibility, are yet to be adopted. At this workshop, we presented findings from a 7-month diet test study, tightly coordinated and conducted across three aquatic research facilities: Zebrafish International Resource Center (ZIRC), Kent and Sharpton laboratories (Oregon State University), and Xiphophorus Genetic Stock Center (XGSC, Texas State University). We compared the impact of two commercial diets and a suggested zebrafish SRD on general fish husbandry, microbiome composition, and health in three fish species (zebrafish, Xiphophorus, and Medaka), and three zebrafish wild-type strains. We reported outcomes, gathered community feedback, and addressed the aquatic research community's need for SRD development. Discussions underscored the influence of diet on aquatic research variability, emphasizing the need for SRDs to control cross-experiment and cross-laboratory reproducibility. Species-specific reference diets are essential for model organism health and consistent research 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.

EFFECT OF DIETARY PROTEIN AND CARBOHYDRATE LEVELS ON WEIGHT GAIN AND GONAD PRODUCTION IN THE SEA URCHIN LYTECHINUS VARIEGATUS.

Adult Lytechinus variegatus were fed eight formulated diets with different protein (ranging from 12 to 36%) and carbohydrate (ranging from 21 to 39 %) levels. Each sea urchin (n = 8 per treatment) was fed a daily sub-satiation ration of 1.5% of average body weight for 9 weeks. Akaike information criterion analysis was used to compare six different hypothesized dietary composition models across eight growth measurements. Dietary protein level and protein: energy ratio were the best models for prediction of total weight gain. Diets with the highest (> 68.6 mg P kcal--1) protein: energy ratios produced the most wet weight gain after 9 weeks. Dietary carbohydrate level was a poor predictor for most growth parameters examined in this study. However, the model containing a protein × carbohydrate interaction effect was the best model for protein efficiency ratio (PER). PER decreased with increasing dietary protein level, more so at higher carbohydrate levels. Food conversion ratio (FCR) was best modeled by total dietary energy levels: Higher energy diets produced lower FCRs. Dietary protein level was the best model of gonad wet weight gain. These data suggest that variations in dietary nutrients and energy differentially affect organismal growth and growth of body components.

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.

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.

Construction and characterization of a portable sound booth for onsite voice recording.

The negative effects of environmental noise on sound recordings are recognized in the professional literature. Sound booths and anechoic chambers are examples of controlled acoustical environments widely used in research. However, both enclosures are expensive, require substantial space, and are not portable. Our research has been directed to measuring vocal endurance and voice characteristics of singers before and after sustained voice use. Our desire to acquire high-quality onsite recordings necessitated the development of a portable recording environment. In this article, we report the design, construction, and acoustic characterization of a prototype portable sound box (PSB) to acquire high-quality voice recordings in a controlled, portable acoustical measurement. Simulations were conducted to model the intended use of the PSB by voice users, using two acoustic characterization procedures. The first method showed higher intensity variations by region and depth as frequency changed. For the modified method, intensity response was more uniform and displayed less variation with frequency change. Both methods enabled us to 1) refine the onsite recording procedure, 2) provide insight into potential sources of analysis errors, and 3) develop detailed analysis of frequency intensity response affected by equipment variability. We found that it is possible to construct a PSB for onsite high-quality voice recording.

The effects of dietary saturated fat source on weight gain and adiposity are influenced by both sex and total dietary lipid intake in zebrafish.

The effects of saturated fat intake on obesity and cardiovascular health remain inconclusive, likely due in part to their varied nature and interactions with other nutrients. Investigating the synergistic effects of different saturated fat sources with other dietary lipid components will help establish more accurate nutritional guidelines for dietary fat intake. Over the past two decades, zebrafish (Danio rerio) have been established as an attractive model system to address questions regarding contributions of dietary lipid intake to diet-induced obesity in humans. The goal of the present study was to assess interactions of three different saturated fat sources (milk fat, palm oil, and coconut oil) with sex and total dietary lipid intake on weight gain and body composition in adult zebrafish. Larvae were raised on live feeds until 28 days post fertilization, and then fed a formulated maintenance diet until three months of age. An eight-week feeding trial was then initiated, in which zebrafish were fed nine experimental low- and high-fat diets varying in saturated fatty acid and long-chain polyunsaturated fatty acid content, in addition to a low-fat and high-fat control diet. At termination of the feeding trial, each treatment was evaluated according to body mass, moisture content, and adiposity. Sex and diet significantly interacted in their effects on body mass (P = 0.026), moisture content (P = 0.044), and adiposity (P = 0.035). The influence of saturated fat source on body mass was observed to be dependent on intake of total dietary lipid. In females, all three saturated fat sources had similar effects on adiposity. From these observations, we hypothesize that impacts of saturated fat intake on energy allocation and obesity-related phenotypes are influenced by both sex and intake of other dietary lipid components. Our results suggest that current nutritional guidelines for saturated fat intake may need to be re-evaluated and take sex-specific recommendations into consideration.

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.

Both Dietary Ratio of n-6 to n-3 Fatty Acids and Total Dietary Lipid Are Positively Associated with Adiposity and Reproductive Health in Zebrafish.

BACKGROUND: Controversial findings have been reported in human and animal studies regarding the influence of n-6 (ω-6) to n-3 (ω-3) fatty acid ratios on obesity and health. Two confounding factors may be related to interactions with other dietary lipid components or sex-specific differences in fatty acid metabolism. OBJECTIVE: This study investigated main and interactive effects of total dietary lipid, ratio of n-6 to n-3 fatty acids, and sex on growth, adiposity, and reproductive health in wild-type zebrafish. METHODS: Male and female zebrafish (3 wk old) were fed 9 diets consisting of 3 ratios of n-6 to n-3 fatty acids (1.4:1, 5:1, and 9.5:1) varied within 3 total lipid amounts (80, 110, and 140 g/kg) for 16 wk. Data were then collected on growth, body composition (determined by chemical carcass analysis), and female reproductive success (n = 32 breeding events/diet over 4 wk). Main and interactive effects of dietary lipid and sex were evaluated with regression methods. Significant differences within each dietary lipid component were relative to the intercept/reference group (80 g/kg and 1.4:1 ratio). RESULTS: Dietary lipid and sex interacted in their effects on body weight (P = 0.015), total body length (P = 0.003), and total lipid mass (P = 0.029); thus, these analyses were stratified by sex. Female spawning success decreased as dietary total lipid and fatty acid ratio increased (P = 0.030 and P = 0.026, respectively). While total egg production was not associated with either dietary lipid component, females fed the 5:1 ratio produced higher proportions of viable embryos compared with the 1.4:1 ratio [median (95% CI): 0.915 (0.863, 0.956) vs 0.819 (0.716, 0.876); P < 0.001]. CONCLUSIONS: Further characterization of dietary lipid requirements will help define healthy balances of dietary lipid, while the sex-specific responses to dietary lipid identified in this study may partially explain sex disparities in the development of obesity and its comorbidities.

Current basis and future directions of zebrafish nutrigenomics.

This review investigates the current state of nutrigenomics in the zebrafish animal models. The zebrafish animal model has been used extensively in the study of disease onset and progression and associated molecular changes. In this review, we provide a synopsis of nutrigenomics using the zebrafish animal model. Obesity and dyslipidemia studies describe the genomics of dietary-induced obesity in relation to high-fat/high-calorie diets. Inflammation and cardiovascular studies describe dietary effects on the expression of acute inflammatory markers and resulting chronic inflammatory issues including atherosclerosis. We also evaluated the genomic response to bioactive dietary compounds associated with metabolic disorders. Carbohydrate metabolism and ß-cell function studies describe the impacts of high-carbohydrate dietary challenges on nutritional programming. We also report tumorigenesis in relation to dietary carcinogen exposure studies that can result in permanent genomic changes. Vitamin and mineral deficiency studies demonstrate transgenerational genomic impacts of micronutrients in the diet and temporal expression changes. Circadian rhythm studies describe the relation between metabolism and natural temporal cycles of gene expression that impacts health. Bone formation studies describe the role of dietary composition that influences bone reabsorption regulation. Finally, this review provides future directions in the use of the zebrafish model for nutrigenomic and nutrigenetic research.