EFFECT OF DIETARY SUPPLEMENTATION OF MICROALGAE EXTRACTS ON THE SKELETAL HEALTH OF ZEBRAFISH Danio rerio AND GILTHEAD SEABREAM Sparus aurata LARVAL STAGES: A COMPARATIVE STUDY

Introduction

S keletal anomalies are omnipresent in  most farmed fishes worldwide ,  raising  concerns for animal welfare  and causing economic losses for  the  aquaculture  industry

 . L arval n utrition has been recognized  as  one of the main factors leading to skeletal anomalies

 ,  thus the  development of  feeds supplemented with osteo-active compounds is seen as an important strategy to improve  the  skeletal status of farmed fish.  In this context, extracts from m arine organisms ,  a well-established  and  valuable source of  nutrients,  have  recently been found to contain bioactive compounds with osteogenic and mineralogenic activities that could be highly relevant to improve  skeletal health in  larvae of commercial species. Here, w e produced inert diets enriched with different concentrations of the ethanolic extracts from Skeletonema sp. and Tetraselmis sp. and tested them on larvae of the model species zebrafish (Danio rerio ) and the commercial species gilthead seabream (Sparus aurata), evaluating their effect on growth, survival and development of skeletal anomalies.

Materials and Methods

Extracts were prepared through the maceration of f reeze-dried biomass of Skeletonema sp. and Tetraselmis sp. (Necton S.A.) with 96% ethanol .  Ethanol extracts were coated on a commercial zebrafish diet  (Sparos Lda) at 0.5% and 2.5% and a commercial seabream diet at 0.5% . For zebrafish feeding trials , 5 days post-fertilization (dpf)  larvae were  maintained in 2 .5 L  tanks  in static conditions at 60 larvae/L until 20 dpf , then moved to 3 L tanks  in recirculating system at a  density  of 23 larvae/L . Experiments were conducted  in triplicates  for each of the 5 diets.  From 5 to 16 dpf,  zebrafish  larvae were co-fed with  the  supplemented inert diet and rotifers , gradually reducing the concentration of rotifers  to address the weaning from live feeds. F rom 17 dpf until the end of the trials, larvae were  fed  only  supplemented  inert diets . At 50 dpf, juvenile fish  were given a lethal anaesthesia and f inal survival, total length and dry weight were assessed.  Fish  were  sampled to asses c alcium and phosphorus content  and  for  double staining  with alizarin red S and alcian blue  for bony  and cartilaginous structures to evaluate skeletal anomalies. T he expression of marker genes of bone development, matrix mineralization and oxidative stress were assessed by qPCR.

 For the seabream trials,  larvae  of 30 days after hatching (dah)  were fed for 30 days with control diet or diets  supplemented  with 0.5% of Tetraselmis sp. or Skeletonema  sp. ethanolic  extracts. M icrodiets were formulated and processed to be isonitrogenous, and isoenergetic. Larvae were  maintained  in cylindroconical 100 L tanks in a semi-closed R AS with an initial density of 52 larvae/L and a p hotoperiod of  10h light:1 4h dark. E nvironmental parameters and mortality were monitored daily .  At the end of the trial larvae were euthanized, sampled and growth parameters assessed - dry w eight, length and condition factor.  Samples for oxidative status, digestive capacity, total mineral content, calcium and phosphorous ratio, mRNA levels of marker genes for bone formation,  matrix  mineralization, and oxidative stress were collected.  Some fish per condition were stained to reveal bone and cartilaginous structures and asses incidence of skeletal anomalies

Results and Discussion

In the past years, our laboratory has screened  a large number of extracts from microalgae for bone anabolic activity  using zebrafish in vivo tools and identified ethanolic extracts of  Skeletonema  sp. and Tetraselmis sp.  as  promising source of osteoactive compounds.  Although the use of zebrafish as model organism in aquaculture is a controversial topic due to physiological differences and evolutionary distances between this small freshwater cyprinid and commercial teleost species, its use in nutritional research has been recognized as a great opportunity and r esults obtained in zebrafish were shown to have predictive power of the effect  on  stress, immunological response and dietary toxicity towards species reared in aquaculture

.  Aiming at providing a comparative view on the potential application of microalgae extracts in fin fish nutrition, we produced inert diets enriched with different concentrations of  two promising osteogenic extracts ,  tested them on larval stages of zebrafish and  the  gilthead seabream,  evaluating their effect on growth, survival and the occurrence of skeletal anomalies . Regarding the feeding trials with seabream, results showed that dietary treatments had no impact on performance indicators like dry weight, length, or condition factor . For both species,  the impact  of the supplementation of the extracts on bone mineral content, expression of marker genes of bone growth and mineralization and incidence of skeletal anomalies revealed the advantages of microalgae extracts supplemented diets, and their potential for aquaculture applications.

Funding

W ork financed by  the European Maritime and Fisheries Fund (EMFF/FEAMP) through the National Operational Programme  MAR2020  (grant 16-02-01-FMP-0057/OSTEOMAR) , by the European Regional Development Fund (ERDF/FEDER) through the Transnational Cooperation Programme Atlantic Area  (grant EAPA/151/2016/BLUEHUMAN ),  by the  Marie Skłodowska-Curie innovative training network BIOMEDAQU ( grant H2020-MSCA-ITN /766347) and by National funds through the Portuguese Foundation for Science and Technology (grant UIDB/04326/2020).

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