DIETARY ARA/DHA RATIOS AND CARBOHYDRATE LEVEL EFFECTS ON GILTHEAD SEA BREAM LIVER AND INTESTINE WELFARE AND GUT MICROBIOTA

Introduction

Presently, the utilization of plant feedstuffs and oils in commercial aquafeeds arise the necessity to evaluate the effects of  dietary essential fatty acids (EFA), such as arachidonic acid (ARA) and docosahexaenoic acid (DHA), and carbohydrate levels, and possible interactions between them in marine fish welfare .  For that purpose,  the effects of  dietary  ARA /DHA ratio and carbohydrate level on liver and intestine oxidative stress status,  liver  histology, and intestinal microbiota were evaluated in gilthead sea bream juveniles.

Material and methods                      
Four isoproteic (47% crude protein) and isolipidic (18% crude lipids) diets were formulated to include 18% or 0% gelatinized starch (HS and LS diets) and dietary ARA/DHA ratios of 2.3/0.3 or 0/2.6 (ARA and DHA diets) . Triplicate groups of fish (initial body weight = 47.5 g) were fed  each diet to satiety for 84 days . At the end of the trial,  the  liver and intestine of 3 fish from each tank were collected for the determination of oxidative stress parameters and liver histology .  Two other fish per tank were sampled under aseptic conditions for autochthonous (mucosa) microbiota characterization.

Results
No differences  in  growth performance were  observed between dietary treatments, but HS diets increased feed efficiency and protein efficiency ratio.  The ARA diets reduced liver and intestine lipidic peroxidation (LPO), and improved liver glutathione redox status (total, reduced (GSH) and oxidized glutathione (GSSG)) when combined with HS (Table 1) . In the intestine, ARA diets reduced GSSG content and oxidative stress index (OSI). DHA diets increased hepatic SOD and GR activities but in the intestine,  antioxidant enzymatic  activity was not affected by the dietary EFA ratios (Table 2) . HS diets increased liver OSI and reduced intestinal GSSG .   HS also decreased LPO values , but only in DHA diets (Table 1) . HS increased  liver  G6PDH, GR, and GPX (only  in the  DHA diets)  activities but in the intestine,  it decreased GR and SOD activities (Table 2) . Neither dietary EFA nor carbohydrates induced major histomorphology alterations in the distal intestine but HS, when combined with DHA, promoted hepatocyte hypertrophy and alterations in nuclei position (Fig. 1). Dietary ARA modified mucosa bacterial profile by reducing the number of operational taxonomic units, richness, and diversity, and promoted similarity between bacterial communities. In conclusion,  a  high dietary ARA/DHA ratio reduced oxidative stress (LPO) and hepatocyte histomorphological alterations.

Acknowledgments
This work was supported by the Fundação para a Ciência e a Tecnologia, Portugal and Fundo Europeu de Desenvolvimento Regional (FEDER), from COMPETE 2020-Programa Operacional Competitividade e Internacionalização (POCI) (Project Eicobream : PTDC/MAR-BIO/1949/2014). This research was partially supported by the Strategic Funding UIDB/04423/2020 and UIDP/04423/2020 through national funds provided by FCT. Magalhães, R. was supported by an FCT grant (SFRH/BD/115870/2016) and European social fund (ESF). Martins, N., Fontinha , F., and Moutinho, S., were supported by an FCT grant (SFRH/BD/137919/2018, 2020.07212.BD, SFRH/BD/138224/2018, respectively).