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The experimental design and fish use protocol were approved by the Institutional Animal Care and Use Committee (IACUC) of Dartmouth College. Also, we conducted all experiments in accordance with relevant guidelines and regulations. We euthanized the fish by single cranial pithing in the nutritional feeding experiment.
Diet formulation for nutritional feeding experiment
We incorporated N. oculata defatted biomass to replace different percentages of FM and whole cell Schizochytrium sp. to replace all FO in three tilapia experimental diets for a nutritional feeding trial. These three diet formulations were based on our previous digestibility data for N. oculata defatted biomass and whole cell Schizochytrium sp.17,30,33, and a prior study showing potential to replace all FO with whole cell Schizyochytrium sp.30. We compared these three experimental diets to a reference diet (served as control diet) containing FMFO at levels found in commercial tilapia feed. All diets were iso-nitrogenous (37% crude protein) and iso-energetic (12 kJ/g). Microalgae inclusion diets used N. oculata defatted biomass to replace 33% (33NS), 66% (66NS), and 100% (100NS) of the FM and whole cell Schizochytrium sp. to replace all FO in the test diets (33NS, 66NS, 100NS). Thus N. oculata comprised 3%, 5% and 8% of the diet by weight, respectively, and Schizochytrium sp. made up 3.2% of the diet by weight. We produced the diets in accordance with our previous work17,30,36. We obtained dried Schizochytrium sp. from ALGAMAC, Aquafauna Bio-marine, Inc., Hawthorne, CA, USA; and menhaden FO from Double Liquid Feed Service, Inc., Danville, IL, USA. Qualitas Health Inc., which markets EPA-rich oil extracted from N. oculata as a human supplement39 and seeks uses for tons of under-utilized defatted biomass from its large-scale production facilities, donated the N. oculata defatted biomass. Supplementary Table S8 reports proximate compositions and amino acid profiles of N. oculata defatted biomass and Schizochytrium sp.; total fatty acid profile by percentage of the defatted biomass and Schizochytrium sp ingredients reported in Supplementary Table S9; and macromineral and trace element composition of both ingredients reported in Supplementary Table S10. The formula, proximate analysis, and amino acid profiles of four dietary treatments reported in Table 1. The fatty acid profiles reported in Supplementary Table S11 and the macrominerals and trace elements of the four experimental diets reported in Supplementary Table S7.
Table 1 Formulation (g/100 g diet) and essential amino acids (% in the weight of diet) of four experimental diets for juvenile tilapia.
Full size table

Experimental design and sampling to evaluate tilapia growth on N. oculata defatted biomass and Schizochytrium sp. Diets
We conducted the feeding experiment using a completely randomized design of four diets × three replicates tanks in recirculating aquaculture systems (RAS). Four hundred eighty Nile tilapia (mean initial weight 34.5 ± 2.06 g) were put into randomized groups of 40, bulk weighed, and transferred to a tank. Tilapia had been acclimated to the FMFO containing reference diet for 7 days prior to distribution. The initial stocking density remained within levels recommended to avoid physiological stress on tilapia ( More

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