Introduction
Fish health and welfare are great concerns to the aquaculture industry. In this regard, the search for sustainable feed compounds with bioactive properties, which contribute to maintaining fish health, is highly relevant. Seaweeds are important natural resources for the food and feed industry worldwide and volumes of cultivated seaweeds a re increasing over the years. Seaweeds contain bioactive compounds such as laminarin and fucoidan which have shown antioxidant, immunostimulant and anti-bacterial properties in several mammalian and fish cells. Few studies have evaluated effects of laminarin in salmon cells , and so far, no studies have been done to evaluate laminarin as a bioactive compound in diets for salmon with the aim to improve/enhance immune responses.
To assess whether laminarin has immune-modulating activity on salmon cells, and to reduce the use of experimental animals, a “stepwise approach” was used. Step 1 involved an in vitro screening of differentially processed laminarin on the viability and on immune responses of salmon cells . Step 2 will involve an in vivo salmon trial evaluating the effects of different dietary inclusion levels of laminarin o n growth performance and immune-related parameters. Together, this work will contribute to setting a basis for the inclusion of laminarin in salmon feed, with an overall aim to support and improve fish health and welfare.
Methods
Laminaria hyperborea biomass was processed by water extraction (50 oC, 4 h). A precipitate consisting of enriched laminarin was formed after storage of extract at 4 º C for several days. After extensive washing in 4 ºC ion-free water and in 50 % ethanol, three fractions were made by acid hydrolysis for further testing: L1, hydrolyzed 1 h, L2, hydrolyzed 3 h, and L3, control fraction without hydrolysis. To evaluate their effects in Atlantic salmon-isolated leukocytes from spleen and head kidney (HK) , viability and bioactivity assays were performed using the three laminarin fractions. For the viability assay, 100, 250 and 500 µg mL-1 were used, and the cell viability was measured after 6, 24, 48 and 72 h. For the bioactivity assay, spleen and HK cells were incubated with laminarin fractions at 250 µg mL-1 for 6 and 24 h, and a panel of immune-related biomarkers were measured by qPCR.
Results and discussion
None of the fractions exhibited a toxic effect on spleen and head kidney (HK) cells; L1 even showed a positive effect on cell viability in cells from both organs at 250 µg mL-1 . Regarding bioactive properties on salmon leukocytes , L1 elicited an up-regulation of tnfa , il1b , inos , ifng, cath-2 , tgfb and sod in both organs, which is similar to a mammalian macrophage type 1 profile (M1), evidencing a short-term inflammatory response that decreased 24 h after the exposure to laminarin. Moreover, in HK, L3 induced an up-regulation of tgfb , while no significant gene expression modulation was observed with L2. On the contrary, in spleen L2 and L3 showed an up-regulation of ifng , il1b, in os and tgfb, suggesting a differential response between these two organs.
Conclusions
Results show that laminarin from L. hyperborea has immuno-modulating capacity on salmon leukocytes . Therefore, laminarin could be a promising functional component for aquafeeds, especially when exposed to a low processing level such as in fraction L1. Further work is being carried out to elucidate whether laminarin may exert further immune effects on other salmon cells. B ased on the promising results obtained in Step 1, Step 2 , which will focus on including laminarin in salmon diets, is now being planned , with a future view to its application in novel functional feeds for the salmon aquaculture industry.
Acknowledgements: This work was supported by The Norwegian Seaweed Biorefinery Platform (The Research Council of Norway; grant number 294946) and Foods of Norway, a Center for Research-based Innovation (The Research Council of Norway; grant number 237841/030).
References