Are We There Yet? Zebrafish Nutrition Stephen A. Watts, Louis D’ Abramo, Susan Farmer, Lacey Dennis, Daniel Smith and Mickie L. Powell University of Alabama at Birmingham and Mississippi State University

Why Nutrition? ZF have been used extensively as a drug and disease model ZF are a useful toxicology model Despite their widespread use the nutritional requirements have not been determined

Aquaculture-Grow large fish fast. Aquarists-Color, Reproduction Research-Growth, Overall health, High fecundity.

Ingredients are practical Possible anti-nutritional factors No indication of quantities Use of lakes and dyes Use of preservatives

There are no standards among diets in zebrafish studies!!

Where do we start?

Examples of Nutrition Studies The need to develop standardized diets to support zebrafish (Danio rerio) research is supported by the knowledge that specific dietary ingredients, nutrients, or antinutritional factors in diets have been shown to affect development and growth of adult D. rerio and their offspring. In this study, there were seven dietary treatments consisting of five commercially available diets and two laboratory-prepared diets, three replicates per treatment. Fish were fed ad libitum twice daily for 9 weeks. At 9 weeks, both weight and length were recorded to determine condition indices. D. rerio fed one of the laboratory-prepared diets had significantly higher weights than individuals fed any of the other diets and exhibited significantly higher lengths than those fed five of the six remaining diets. Although there were significant differences in general growth demographics (length=weight) after the 9-week feeding trial, no significant differences in overall health of D. rerio were observed for the different dietary treatments as determined by statistical analysis of condition factor indices (K¼[weight100]=length3). The success achieved with the laboratory-prepared diets represents the foundation for establishing an open-formulation nutritional standard to ensure that the D. rerio model for research does not generate confounding research results caused by nutritional vagaries. Sadasivam Kaushik,1 Ioanna Georga,2 and Giorgos Koumoundouros2,3 (France & Greece) Although zebrafish is used as a major model species for understanding a number of biological functions and mechanisms involved, there is practically no information on the nutritional requirements of this cyprinid. The rearing practices, especially with regard to feeds and feeding, also vary considerably. To obtain base line information on the nutrient requirements and to develop a standard diet, we initiated the present work of feeding zebrafish larvae with a formulated feed right from first feeding onward. Growth of zebrafish fed the compound feed was very good, reaching a total length of 23 ± 4mm in 9 weeks with a survival rate of 89% ± 4%. We present the first ever published data on whole-body composition in terms of essential amino acids (ideal protein profile), total, neutral and polar fatty acid profiles, minerals, and trace elements. Results obtained here show clearly that zebrafish can be reared with formulated feed right from mouth opening without resorting to any live prey.

Considerations in the Use of Formulated Diets Age: Larval vs juvenile vs adult (breeding) Physical form of the diet Feed Frequency Protein source/quality Carbohydrate Lipid and Fatty Acids Gut Retention Time Bacteria Culture Container

Methods Fish were co-cultured with enriched rotifers for the first 5 days post hatch and fed Artemia for 11 days prior to stocking. At 21 days post hatch 15 fish were stocked randomly into 2.8 liter tanks and maintained on a recirculating zebrafish system. For each diet, fish were fed a ration in excess of 5% of their body weight per day, divided into two feedings. Fish were photographed and weighed every two weeks to measure growth and adjust feed rations.

UAB Z-12 Diet Formulation Ingredient % casein - vitafree 25.00 fish protein hydrolysate 20.00 wheat starch 9.60 wheat gluten 7.00 alginate 5.38 soy protein isolate 5.00 dextrin menhaden fish oil 4.67 soy lecithin (refined) 4.00 vitamin premix mineral premix 3.00 corn oil 2.33 canthaxanthin (10%) 2.31 potassium phosphate monobasic 1.15 alpha cellulose 1.00 glucosamine 0.25 betaine 0.15 cholesterol 0.12 ascorbylpalmitate 0.04 Total 100.00 UAB Z-12 Diet Formulation Chemically defined semi purified~25% lipid <5% fiber ~5% ash Samples being analyzed for full nutritional profiles

Week 10 Artemia Gemma

Diet Body Condition Index Artemia 0.98 Gemma 1.09 Z-12 1.19 Ziegler 1.24 Otohime Tetramin 1.32 High BCI Low BCI

Can one nutrient affect experimental outcomes?

Protein Mixed source vs sole source

Amino Acid Analysis MIXED FPI CAS SOY WG Cysteine 0.400 0.190 0.530 1.010 Methionine 1.080 1.160 1.320 0.560 0.730 Lysine 2.930 3.200 3.610 2.700 0.660 Alanine 1.894 2.915 1.427 1.932 1.249 Arginine 2.090 2.748 1.642 3.187 1.547 Aspartic Acid 3.284 3.849 3.322 4.996 1.509 Glutamic Acid 9.631 5.626 10.120 8.388 18.060 Glycine 2.072 3.984 0.876 1.848 1.622 Isoleucine 2.023 1.709 2.394 2.132 1.763 Leucine 3.631 2.866 4.354 3.500 3.293 Serine 2.140 1.804 2.461 2.103 2.177 Threonine 1.706 1.744 1.917 1.612 1.217 Valine 2.424 2.019 3.016 2.196 1.868 Histidine 1.120 0.940 1.337 1.110 0.975 Phenylalanine 2.130 1.573 2.387 2.325 2.600 Tyrosine 1.877 1.574 2.372 1.473 1.491 Taurine 0.098 0.367 0.010 Tryptophan 0.506 0.374 0.620 0.582 0.467 Amino Acid Analysis

µCT of Mixed Protein and Soy Protein Diets

Bone Alterations

Conclusions All diets supported growth and survival. However, commercial diets contain undefined ingredients. The consequence: Nutrients and nutrient source affect outcomes. Any health or disease-related outcomes will be affected by diet. Interpretation of experimental results must be made within the context of a defined nutritional history, or lack thereof. Microbiome?

Acknowledgments UAB NORC Aquatic Animals Research Core (NIH P30DK056336). Jeff Barry, Adele Fowler, Chris Taylor, Michael Williams, Karen Jensen, Marlee Hayes ACLAM