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HoloFood Salmon Experimental Design

Written by: Morten Limborg

Updated: 2023-04-21 17:18

In total three experimental trials were performed in the salmon system for the HoloFood project. Each trial has been designed to test the effect of novel sustainable feed additives to Atlantic salmon feed. We focused on two different types of additives that were tested in separate and independent trials.

The first additive we tested was fermented seaweed. Seaweed contains a large range of bioactive components as well as a dynamic and diverse microbial community on its surface biofilm (i.e. a natural probiotic package). The exact health effect of seaweed for salmon remains unknown. Fermenting the seaweed both stabilises the biomass and is expected to enrich the microbiome with Lactobacillus bacteria, which is well known to have a beneficial effect on gut health in warm-blooded animals, and here we hypothesise that this species may also be beneficial to teleost hosts.

We also tested the effect of replacing fish meal protein with blue mussel protein. The marine protein sources in the diets for salmonids have traditionally been fishmeal, but there is, however, an increasing pressure on the wild fish stock and at the same time an increasing demand from a rapidly growing sea farming industry. There is therefore an increasing need for alternative marine protein sources. Blue mussel meal can be one such source since it can be locally grown compared to wild resources that often also need to be shipped across continents. An overview of the experimental conditions and set-up is shown in the following paragraphs.

Experimental conditions

The experiments followed the EU principles for animal care and experimentation and experimental procedures were approved by the Food Authority in Norway as outlined in HoloFood Deliverables 13.3 and 13.4. All the staff involved in the experiment had appropriate training and qualifications to perform the required tasks. They had training in Quality Systems including data recording requirements and forms for completion of the study, QC techniques, relevant SOPs and specific protocol requirements prior to the experiment start.

All three experiments were conducted at LetSea land site aquaculture centre, at Bjørn, in Dønna, Norway, in cooperation with Lerøy Seafood Group. The fish tanks were all connected to a state-of-the-art flow-through water system with access to both freshwater and seawater. Fish were fed continually using 24- hours mechanical band feeders, based on well-established feeding protocols developed by LetSea AS. The daily feeding amount was regularly reassessed based on the overfeed amount from the previous days for each tank, adjusted for 10% overfeeding. All tanks are equipped with a collection system of uneaten feed, which allows ad libitum feeding and ensures 5 to 10% overfeeding, depending on the size of the fish. Throughout the duration of the trials, skilled staff from LetSea AS were responsible for the daily maintenance and monitoring of the experiment.

All three experimental trials included an initial 2-weeks acclimation period followed by an ~60 days long growth phase or until fish have at least doubled their average weight at the start of the trial, as a doubling in weight is generally thought of as being adequate to detect statistically significant differences in growth rate and other relevant performance metrics.

Experimental design

To avoid the possible influence of parental effects, all juvenile salmon were randomised before distribution in such a way that each replicate tank received the same composition of potential full and half siblings. Feeding treatments were randomly assigned to each tank and each tank was given a codename with no relation to the actual experimental groups (e.g. names of planets). Each dietary treatment had 2 or 3 replicate tanks following a standard dose - response design where the control and maximum concentration of the feed additive has three replicates and with two replicates for intermediate concentrations. Here, the experimental design differed from conventional studies in that we also aimed to increase inter-individual genetic variability, thus improving the likelihood of identifying interactions between animal and microbial genomic features. Therefore, a total of 60 salmon were sampled from each feed category at the end of the trial despite the different number of replicate tanks. We also sampled a reference group of 60 salmon at the start of the trial before administering different feed types.

Diets, additives and feeding

In the experimental trials, health performance and growth were assessed in fish fed with five distinct diets: 4 experimental diets with incremental doses of the respective additives, and a commercial control diet without any experimental additives. Each feeding group was organised in tanks, in duplicates or triplicates depending on the respective concentration of the feed additive in the dose-response study design. Prior to the start of each trial, during the acclimation period, all fish will be fed with “Energy X 75” (50 mg), a commercial feed from BioMar, which does not contain any of the experimental additives. During the experiment, a commercial feed from BioMar “Energy X 200” (50 mg) will be fed to the control group. All diets are in the form of small compact pellets, and the feed provided ad libitum in every tank.

Animal sampling

For all the samplings, each fish being sampled was euthanised beforehand using an overdose of Finquel (MS-222), following established ethical requirements fulfilling all EU standards. After euthanisation, the distal gut, gill tissue, liver tissue, and muscle samples were obtained. Tissues samples for the full holo-omics profiling were taken on day 0 (right before introducing the different diets) and on day 60 of the experiment. For this sampling, 6 sample types were sampled: gill, liver, muscle, distal gut epithelial tissue, distal gut epithelial cell scrapes, distal gut content, some of them will be sampled multiple times for different downstream purposes, giving a total of 11 samples per individual. Different aliquots were distributed and stored in adequate buffer solutions and/or in dry ice for downstream processes. The samples were transported to the corresponding destination and stored for long-term preservation at the most adequate temperature depending on the buffer and downstream purposes of each sample.