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

Zebrafish as a research model Broadly recognized research models Low-cost husbandry Prolific breeders and quick generation times Transparent embryos Sequenced genome Established transgenic lines their use as model research organisms especially for developmental and genetic studies has increased significantly since the 90s. Some of the reasons for their popularity among researchers is their prolific breeding, fast generation times – they reach sexual maturation ~3months, transparent embryos – easily monitor development, their sequenced genome and the presence of 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 How can these benefits translate into aquaculture, this paper published in 2014 outlines some of the major areas a zebrafish model could benefit the field of aquaculture. Their small size reduces housing and diets costs which can be quite significant when testing novel compounds. They are also easy to handle – making breeding and experimentation less time consuming. With a fully mapped genome and many established transgenic lines (with many different fluorescently labelled proteins) it is possible to identify specific effects a diet may have on molecular mechanisms (nutrigenomics) or how an individuals genetics could affect their response to a certain diet (nutrigenetics)

Zebrafish Feed Model Developed to assess the efficacy of various feed additives and diets on zebrafish Two standardized purified diets Control vs. Astaxanthin + diet (80mg/kg feed) Effects on growth and development Development of Standardized measurements Uptake Tissue analysis Whole body and tissue specific We have started developing a zebrafish feed model in which we can effectively test feed additives in diets and assess the associated growth, development, uptake and tissue distribution of the additives. Our first test diet used astaxanthin as the feed additive, understanding zebrafish would metabolize it differently than salmon, we were looking to establish a model as well as sound sampling, rearing and uptake analysis techniques.

Zebrafish Feed Model Importance placed on reducing inter and intra-tank variability Minimum densities while rearing prior to trial 25 larval fish (5dpf-30dpf) per basket – 2 baskets/3L tank 15 fish/3L tank >30dpf 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 Due to the small size of the zebrafish it is even more important in this model that we reduce as much inter and intra tank variability as possible, this allows us to measure even small growth differences between groups. We did this through established zebrafish husbandry techniques and kept to minimum densities prior to

Zebrafish Feed Model Trial ran ~ 8 weeks 5 sampling events Started feeding fish trial diets at 38dpf till 90dpf One week to acclimate to purified diets Fed twice daily to satiation 5 sampling events 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 We ran our feed trial for almost 8 weeks, we chose to end the trial at ~90 days due to the fish reaching sexual maturation at about this point and there starts to be significant differentiation between the size of the males and the females. We did both lethal and non-lethal sampling throughout this trial – the main difference between the two methods is the dose of tricaine, otherwise they were completed in similar ways. We had 5 sampling events throughout the trial. Day 1 and Day 8 were non-lethal sampling events in order to achieve a good base line for growth, after that 3 lethal sampling events occurred bi-weekly, and during the final sampling we also dissected ~6 fish/diet to analyze any tissue specific distribution of the feed additive

38dpf 90dpf Sampling Procedures We used a tarred piece of mesh as the weighing vehicle for the fish which allowed us to blot off most of the excess water giving us the most accurate measurement possible, since the fish weights are so small especially ~30dpf its important that the weighing technique reduces the amount of excess water left on the fish – this is more important during non-lethal sampling as we want to get the fish in the recovery tank as quickly as possible and can’t spend a lot of time letting them dry. photos were taken of the fish at this point to analyze for standard length using software. After lethal sampling events the fish were flash frozen as either a whole specimen or as dissected organs. 38dpf 90dpf

Results Measured weight, standard length, and feed intake Some of the variability in weight especially in our model would be the weight difference between males and females, during our 4th sampling it was evident there were

Results Minimal feed requirements ~0.3g feed/fish for ~8 week trial Pre-weighed the daily feed for each tank each day, then weighed the residual to determine feed intake, Fed to satiation but within a 5 min time frame as zebrafish will continue to eat

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

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