Introduction
Aquaculture is the fastest growing animal food-producing industry. Intensified production systems and climate change, facilitate the occurrence of disease outbreaks due to the favoring of stressed and immuno-compromised animals. Therefore, a suitable environmentally friendly solution is the use of feed additives with immunomodulatory properties that may be used as functional feeds to improve disease resistance (Dawood et al., 2021). A wide spectrum of phytogenics have been studied in aquafeeds due to their growth promoting, antimicrobial, immunostimulant, antioxidant and anti-inflammatory properties (Reverter et al. 2021). In this study, we have evaluated a feed additive rich in triterpenic compounds and polyphenols (AQUOLIVE®, NATAC Biotech SL, Spain) on the systemic immune response and disease resistance of Atlantic salmon (Salmo salar L.) smolts.
Materials and Methods
Atlantic salmon parrs were kept in tanks of 450 L connected to a water recirculation system (IRTAmar®) at an initial density of 2 kg m-3. Water temperature, pH and oxygen levels during the trial were 12 ± 0.1 °C, 7.4 ± 0.3 and 9.6 ± 0.2 mg/L. After 47 days, fish were smoltified during 10 days and transferred to 35 ppt, and kept at similar water temperatures than the parr phase during 76 days. Photoperiod was 24 h light and 0 h darkness. Two experimental diets were tested: D1, control diet (40% protein; 22.15% fat; 21.60 energy MJ/kg); D2, control diet supplemented with the feed additive AQUOLIVE® at 0.15% inclusion. This feed additive was obtained from olive tree containing 10% of olive bioactive compounds (8.0% triterpenic acid and 2% polyphenols). At the end of the trial, in order to investigate the immunomodulatory properties of the phytogenic tested against a bacterial infection, an in vivo challenge with Aeromonas salmonicida was performed (4 tank replicates; 8 fish per tank). In addition, total RNA was extracted from the head kidney (HK) of individual fish (n = 18 fish per dietary treatment) and its transcriptomic profiling analyzed by means of a microarray platform (Krasnov et al., 2011).
Results and Discussion
At the end of the trial (133 days), no differences in body weight were observed between fish fed the diet containing 0.15% AQUOLIVE® (252.3 ± 9.2 g) and the control group (240.2 ± 19.3 g). The transcriptomic profiling of the HK from smolts fed the control and AQUOLIVE® diets revealed a total of 1,027 differential expressed genes (DEGs). Among them, 805 DEGs were up-regulated (UP) and 222 genes were significantly down-regulated (DOWN). Furthermore, the ClueGO platform (Bindea et al., 2009) was used to identify the biological processes (BPs) linked to the above-mentioned DEGs. The enriched BPs were mainly associated with immune pathways. Particularly, “leukocyte activation” (34 UP and 14 DOWN genes), “granulocyte activation” (26 UP and 11 DOWN genes) and “neutrophil degranulation” (25 UP and 11 DOWN genes), indicating that the BPs identified were the primary actors of the innate immune response promoted by the tested functional additive in this lymphoid organ. In the case of granulocyte, neutrophils are one of the three types of granulocytes identified in fish, whereas neutrophilic granulocytes are the most abundant in salmonids (Rønneseth et al., 2006). As their main function is arriving first at the site of the infection and having a central role in host tissue protection by killing and degradation of microorganisms; therefore, neutrophils are an essential part of the innate immune system whose main activities are to phagocyte pathogens and stimulate lymphocytes and other immune cells (Secombes and Wang, 2012). Thus, the results of our functional analysis regarding leukocyte activation, granulocytes and neutrophils, in particular, might suggest and increased specific immune capacity promoted by the tested functional diet.
Among others BPs identified, the the “i-kappaB kinase/NF-kappaB signaling” pathway was evidenced. Nuclear factor kappa B (NF-κB) pathway is a nuclear transcription factor involved in the regulation of several cytokines, chemokines, antimicrobial peptides, and interferon-stimulated genes, being crucial for a multitude of important immnulogical transcriptional programs, including inflammatory responses to antigens by innate immune cells (Dorrington et al., 2019).
Results from the transcriptomic profiling of the HK were validated in an in vivo pathogenic challenge with A. salmonicida (intraperitoneal injection of 107 CFU/mL). The bacterial challenge lasted 15 days and at the end of the assay, cumulative survival was higher in fish fed the AQUAOLIVE diet (96.9 ± 6.4%) than in fish from the control group (60.7 ± 13.5%).
Conclusions
These results indicate that the dietary supplementation of the functional feed additive AQUOLIVE® at level of 0.15% enhanced the systemic immune response in Atlantic salmon smolts as the combined analysis of the HK transcriptomic profiling and results from the A. salmonicida challenge indicated.
Acknowledgements: this project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme (H2020, SME Instrument) under grant agreement No. 830202.
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