During fish farming, fish are exposed to several stressor conditions that can lead to reduced growth, increased mortalities and large economical losses. These problems can be mitigated by the use of functional feeds, leading to a more sustainable aquaculture and improved animal welfare. The use of functional novel aquafeeds containing paraprobiotics (dead/inactivated probiotics) can be an important solutions to these problems. Lactiplantibacillus plantarum strain L-137 is a common bacterium found in fermented Southeast Asian dish made from fish and rice. Use of the heat-killed form of this bacterium (HK L-137) in aquaculture species is associated with improved growth performance and immunoregulation, representing a cost-effective way to prevent mortality. The effects of Feed LP20™ (20% HK L-137) were already proven in species such as Nile Tilapia, striped catfish and bighead catfish. Thus functional feeds containing HK L-137 also have potential to improve growth performance and health in Atlantic salmon.
Materials and methods
For the in vitro model, we used an intestinal epithelium cell line from rainbow trout (RTgutGC) stimulated with pre-digested HK L-137 to mimic in vivo gastro-intestinal digestion. We consider the distal intestine as a good model to study the effect of functional feeds, since this is where the active compound is in contact with the mucosa-associated lymphoid tissue (MALT) in this organ (GALT), which can coordinate both local and systemic immune responses.
For the in vivo dietary trial with Atlantic salmon, pre-smolts (average weight 27.3g) were fed ad libitum once a day (6 h) one of the five experimental diets (triplicate tanks, 55 fish per tank, 61 days): a commercial-like diet without any growth-promoting additives (control diet, CD); control diet supplemented with 20, 100 and 500 mg of Feed LP20™ kg-1 feed (LP20, LP100 and LP500, respectively); a positive control diet (MG) - control diet supplemented with 2 g of β-glucan kg-1 feed (Macrogard®).
Results and Discussion
In vitro results showed that the barrier function of the cell monolayer was strengthened, suggested by the reduced permeability of Lucifer yellow after 6 h exposure (Figure 1A). An improved barrier can improve digestive function and decrease the vulnerability to bacterial infection. At the protein production level (Figure 1B), there was an increased production of IL-1β both after 6 and 24 h after exposure and a decreased production of Anxa1 after 24 h exposure, indicating an activation of pro-inflammatory process. No difference in the expression of E-cadherin was observed.
At the in vivo dietary trial, HK L-137 did not compromise fish growth performance. Intestinal microbiota composition was slightly affected, but without relevant changes in alpha and beta-diversities. This was expected since the L. plantarum was inactivated and the inclusion levels were low, preventing colonization. Interestingly, indirect ELISA assay in distal intestine showed that fish fed higher inclusion level of HK L-137 also have a lower production of Anxa1 (Figure 2A), as seen in in vitro model, and no difference in production of IL-1β or E-cadherin. Fish fed LP500 also had a higher production of total IgM in plasma, which was not changed specifically by HK L-137 (Figure 2A). The increase of total IgM could be an increase in natural antibodies relevant for defense and homeostasis. Transcriptomic analysis in distal intestine tissue showed that increasing inclusion level of HK L-137 in diets induced an increase in differentially expressed genes. Lowest inclusion level (LP20) induced an up-regulation of 5 terms related with cell structure, adhesion, communication and proliferation and immune regulation (Figure 2B, terms in bold). The up-regulation of the same terms was conserved also in the higher inclusion levels, in addition to other terms related with barrier function, immune response and metabolism (Figure 2B). No differences were found between dietary groups regarding intestinal villi height.
In the current study, we investigated the potential of HK L-137 at both in vitro and in vivo levels. Our results suggest that HK L-137 has immunoproperties by modulating cytokines and effector molecules, suggesting its potential in functional feeds for salmonids. The similarity between the two models indicate that an in vitro model is indeed a useful tool to study the immunomodulatory properties of a novel fed component. This also is a faster, cheaper approach that also promote animal welfare. The current study was performed on healthy fish in fresh water deprived of major stressor factors. This creates a baseline for future work with HK L-137 in Atlantic salmon. A follow-up live pathogen challenge trial with salmon is on-going, where we are evaluating the ability of HK L-137 to reduce mortalities after seawater transfer.