Introduction
Exercise promotes health in mammals (Pascoe et al., 2014). In farmed fish, the physiological effects of swimming activity are, however, overlooked. Swimming is a fundamental behavioral element contributing to the fitness of most fish species. Reduced swimming performance has been associated with lower disease resistance and poorer welfare (Castro et al., 2013). The current study aims to fulfill the lack of knowledge on swimming physiology by evaluating the impact of induced swimming activity on the welfare of gilthead seabream (Sparus aurata).
Materials and Methods
Forty eight seabream (total length: 13.3 ± 0.3 cm; total weight: 57.9 ± 1.8 g) were individually subjected to a 6-h exercise in a swimming tunnel on continuous or discontinuous flow patterns, as follows: i) continuous low (CL, 0.8 BL s-1); ii) continuous high (CH, 2.3 BL s-1); iii) discontinuous low (DL, 0.2-0.8 BL s-1); iv) discontinuous high (DH, 0.8-2.3 BL s-1) velocities and v) control non-exercised group (CTRL). At the end of the trial, fish were euthanized, and gills and head-kidney (HK) were sampled and frozen at -80ºC. Total RNA was extracted, purified, and complementary DNA (cDNA) was synthesized from 1 µg of RNA using reverse transcriptase enzyme SuperScriptIV with an oligodT18 primer. Expression of inflammatory (tnf-α, il-1β, il-6, and nf-κb) and immune-related genes (igf-1, ctsd, and lyz) were analyzed by RT-qPCR and normalized with elongation factor 1 beta (ef1β). Results were expressed as mean ± standard error of the mean (SEM) and data were analyzed by Two-way ANOVA followed by Tukey’s multiple comparison test to determine differences between the type of flow and velocity. The level of significance used was p < 0.05.
Results and Discussion
TNFα (tumor necrosis factor alpha), IL-1β (interleukin 1 beta) and IL-6 (interleukin 6) are cytokines considered important components in the inflammatory response in fish and are key molecules in the immunomodulatory cascade in fish gills following an environmental challenge and regulating the host defense against pathogens mediating the innate immune response (Secombes et al., 2001). Additionally, NF-κB (nuclear factor kappa b) is a transcription factor with a fundamental role in the immediate early pathogen response and in promoting inflammation (Cheng et al., 2014). The expression of il-1β, il-6, and nf-κb genes in gills were up-regulated in fish swimming in DL velocity condition when compared to CTRL and DH velocity conditions. However, no significant variations were found in the relative expression of immune-related genes in the HK. Higher mRNA levels of il-1β and il-6 genes in HK of fish swimming in DL and CL patterns may suggest that a longer swimming period at low velocity may be required to induce significant differences compared to CTRL. Results suggest that swimming condition at DL velocity triggers in the gills a similar response to the inflammatory process and reveals how this load may regulate the immune response in this tissue. Although the relative expression of igf-1 and ctsd genes in gills were increased, they did not display significant changes between experimental groups. IGF-1 (insulin-like growth factor-I) and CTSD (cathepsin D) are involved in somatic growth and innate immune response respectively (Mommsen, 2001; Subramanian et al., 2007). It is plausible to infer that a longer swimming period may require to be applied to induce significant differences in the mRNA levels for both genes.
Conclusions
Induced swimming activity under a discontinuous low velocity may stimulate an inflammatory response in the gills of gilthead seabream. This suggests that the gills may play a key role in the immune response in addition to the well-described functions (gas and ions exchanges). It is plausible to infer that swimming conditions, often overlooked in aquaculture, may be correlated to how farmed and wild fish cope differently with immune challenges. Considering the current results, induced swimming may be an adequate tool for improving the health and fitness of farmed fish.
Acknowledgments
This work was supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) through the structural funds (UID/Multi/04423/2019) to the Interdisciplinary Centre of Marine and Environmental Research (CIIMAR). C. Espírito Santo is supported by BYT-PhD grant UI/BD/150911/2021 from FCT.
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