1. 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

2. 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

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

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

7. There are no standards among diets in zebrafish studies!!

8. Where do we start?

9. 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.

10. 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

11. 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.

13. 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

17. Week 10
Artemia
Gemma

18. 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

19. Can one nutrient affect experimental outcomes?

20. Protein
Mixed source vs sole source

21. 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

22. µCT of Mixed Protein and Soy Protein Diets

23. Bone Alterations

24. 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?

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