Zebrafish feed model Applications in aquaculture nutrition research

Zebrafish feed model: Applications in aquaculture nutrition research Mary Mitchell, JC Achenbach, Sean Tibbetts, Lee Ellis

What are Zebrafish? • Danio rerio – part of the minnow family • Small tropical freshwater fish • Native to South Asia • ~ 1. 5 – 2 inches • Popular aquarium fish 2

Zebrafish as a research model • • • 3 Broadly recognized research models Low-cost husbandry Prolific breeders and quick generation times Transparent embryos Sequenced genome Established transgenic lines

Zebrafish as a model for aquaculture nutrition research • Ulloa et. al, 2014 • High costs of testing novel compounds • Reduced feed requirements • Diets effect on molecular mechanisms • RNA sequencing • Nutritional genomics • In vivo response monitoring with fluorescently labelled proteins 4

Zebrafish Feed Model • Developed to assess the efficacy of various feed additives and diets on zebrafish • Two standardized purified diets • Control vs. Astaxanthin + diet (80 mg/kg feed) • Effects on growth and development • Development of Standardized measurements • Uptake • Tissue analysis • Whole body and tissue specific 5

Zebrafish Feed Model • Importance placed on reducing inter and intra-tank variability • Minimum densities while rearing prior to trial • 25 larval fish (5 dpf-30 dpf) per basket – 2 baskets/3 L tank • 15 fish/3 L tank >30 dpf • Highly selective of fish for trial • Size and health • Started with 130 AB line zebrafish • 65 fish per diet • 5 tanks with 13 fish each 6

Zebrafish Feed Model • Trial ran ~ 8 weeks • Started feeding fish trial diets at 38 dpf till 90 dpf • One week to acclimate to purified diets • Fed twice daily to satiation • 5 sampling events • • 7 Lethal and non-lethal sampling events 13 fish per diet randomly sampled each event Acquired weight and length measurements Dissection – brain, liver, gut, eyes, gills, spleen, kidneys, reproductive organs

Sampling Procedures 38 dpf 8 90 dpf

Results 0, 25 • Measured weight, standard length, and feed intake Weight (g) 0, 2 0, 15 0, 1 0, 05 0 38 dpf 45 dpf 59 dpf 73 dpf Days Post Fertilization 90 dpf Standard Length (cm) 3 2, 5 2 1, 5 1 0, 5 0 9 90 dpf

Results • Minimal feed requirements ~0. 3 g feed/fish for ~8 week trial 0, 14 Feed Consumed (g) 0, 12 0, 1 0, 08 Diet 1 0, 06 Diet 2 0, 04 0, 02 0 Apr 10 -Apr 23 10 Apr 24 - May 6 Time (days) May 7 - May 23

In conclusion • Zebrafish feed model could provide a low cost alternative to some current aquaculture feed trials • Develop make and test diets in house • Low husbandry and feed costs • Low degree of variance between clutches allows for identifying small differences in growth between groups • Serve as a pre-cursor to larger scale trials with intended species 11

QUESTIONS? 12