Introduction
Gut health is crucial for aquaculture and a proper balance in intestine homeostasis and permeability has implications for fish feed efficiency, performance and health status . Over the last decades, innovation in aquaculture feed formulation has been directed towards functional feeds . Currently, macro- and microalgae aroused interest in their application as functional ingredients due to the biotherapeutic role they can play to enhance fish intestinal health, including modulation of the immune system , antioxidant status and gut integrity. Therefore, the objective of this study was to assess the effects of dietary inclusion of the microalgae Phaeodactylum tricornutum and/or the macroalgae Gracilaria gracilis on the gilthead seabream (Sparus aurata ) intestine recovery after provoking an insult based on the administration of soy saponins. P hysiological and genomic responses to macro-and microalgae inclusion on intestine homeostasis recovery and gut health were evaluated through the integration of data on plasma metabolic enzymes, and anterior intestine histology and gene expression analysis.
Material and methods
A control (CTRL), commercial-like diet, was formulated for gilthead seabream juveniles. Based on the CTRL formulation, three experimental diets were formulated by supplementation with microalgae (P . tricornutum; PHA) or macroalgae (G . gracilis; GRA) at 2.5%, or a blend of micro- and macroalgae at 5% (50:50; BLEND). T he experiment was conducted at the Ramalhete Station of the Centre of Marine Sciences of Algarve (CCMAR, Faro, Portugal). Gilthead seabream juveniles with a mean body weight of ~176 g were distributed into 15 flat-bottom 500 L tanks under natural photoperiod conditions.
To study the intestine recovery after an insult in gilthead seabream juveniles and the recovery through nutrition, five dietary treatments were evaluated: a positive control (PCTRL), where fish were assisted-fed with two empty gelatine capsules, and for the remaining treatments (NCTRL, PHA, GRA, and BLEND), fish were assisted-fed with two gelatine capsules filled with soy saponins (8 50 mg saponins). The assisted feeding procedure was performed after 24 h of fasting, and the capsules were gently inserted into the anesthetised fish’s stomach and pushed into the oesophagus (as tested preliminarly to avoid injury) in a 10 sec time-frame. Once recovered, the fish was transferred to the respective tank. After a period of 72 h without feeding, fish were fed their respective experimental diet for 20 days. Gilthead seabream from the positive (PCTRL) and negative control (NCTRL) were fed the CTRL diet during the experiment. Fish from PHA, GRA and BLEND treatments were fed the PHA diet containing microalgae, the GRA diet containing macroalgae, or the BLEND diet with the blend of micro- and macroalgae, respectively. The experimental diets were randomly assigned to triplicate tanks. Fish were fed by hand to apparent satiety. Water average temperature during the experiment was 15.2 ± 0.9 °C.
At the end of the trial, fish were fasted for 24 h. Fish from each replicate tank were bulk-weighed and counted. Three fish from each tank were euthanized. Blood was collected from three fish from each tank (n = 9 per treatment) and centrifuged . The collected plasma was used to study the enzymatic activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). The anterior intestine (AI) of each of these fish was dissected and approximately 1 cm was sampled for histological analysis. For gene expression, ~ 25 mg of the AI of each fish was used to analyse the gene expression of twelve genes related to antioxidant, immune system and epithelium permeability responses. Results from five genes are still under analysis.
Results and Discussion
The activity of ALT and AST was not affected by the treatments (p>0.05). The ratio ALT:AST that indicates possible liver damage and the ratio AST:ALT that identify a possible disorder in other organs were also not affected by the treatments. On the other hand, fish fed the BLEND diet presented a significant (p<0.05) lower level of ALP. L ower ALP levels could in some cases indicate a lower anti-inflammatory response.
Histological features of the AI showed a significant (p<0.05) increase in the n umber of mucosa cells in fish fed the NCTRL, PHA, GRA and BLEND diets, suggesting an intestinal recovery response . Fish fed PHA and GRA diets presented a significant (p<0.05) increase in mucosa vacuolation . A higher amount of cell vacuolation could be interpreted as a sign of intestine disruption .
Results from gene expression shed some light on intestine recovery responses in gilthead seabream juveniles. The expression of tight junction protein (tjp ) gene was significantly (p<0.05) higher in fish from NCTRL, PHA, GRA and BLEND. Occludin (ocl) expression was significantly higher in fish fed PHA, GRA and BLEND. The upregulation of tjp and ocl may imply a protective effect on the fish intestinal epithelial barrier . Catalase (cat) gene expression was significantly (p<0.05) higher in fish fed NCTRL, PHA, GRA and BLEND diets. There was a significant (p<0.05) upregulation of the g lutathione peroxidase (gpx ) expression in fish fed PHA diet. The upregulation of cat and gpx could be an indicator of oxidative stress response and a compensatory mechanism for reducing oxidative stress. T he levels of the immunoglobulin M (igm ) gene expression were significantly (p<0.05) upregulated in fish from PHA. Tumor necrosis factor alpha (tnf-α ) activity was significantly (p<0.05) higher in NCTRL, PHA, GRA and BLEND . Upregulation of igm and tnf-α expression may indicate an activation of the immune response. F ish fed BLEND showed a significant (p<0.05) upregulation of the proliferating cell nuclear antigen (pcna) . Pcna upregulation indicates possible cell proliferation and epithelial regeneration in the intestine .
In conclusion, these results may indicate a p ositive regulation of both PHA and BLEND diets in gilthead seabream gut health . PHA and BLEND diets may be used as functional diets to preserve intestinal homeostasis and accelerate the healing process acting as immunostimulants activating the immune system and alleviating cellular oxidative stress.
Acknowledgements
This project has received funding from the European Union’s Horizon 2020 Marie Skłodowska-Curie ITN Programme under grant agreement No. 956697 (EATFISH) and by the Portuguese Foundation for Science and Technology (Ministry of Science and Higher Education, Portugal) through UIDB/04326/2020, UIDP/04326/2020 and LA/P/0101/2020 to CCMAR and contract DL 57/2016/CP1361/CT0033 to CA. This abstract reflects the views only of the EATFISH consortium, and the European Union cannot be held responsible for any use which may be made of the information it contains.