Introduction
Animal welfare is becoming increasingly important in aquaculture, particularly in intensified production systems. Welfare and stress are closely linked, as reduced stress levels are generally indicative of improved rearing conditions for fish (Balasch & Tort 2019). Among the strategies proposed to improve welfare in aquaculture, environmental enrichment (EE) has shown to provide multiple benefits, including reduced aggression and abnormal behavior, improved growth and survival, lower cortisol, enhanced brain function and cognition (Zhang et al., 2020; Arechavala-Lopez et al., 2022). Similarly, adaptogens have shown potential as effective tools to improve fish welfare. Cannabidiol (CBD), a non-psychoactive compound of Cannabis sativa, has been demonstrated to have anti-inflammatory, neuroprotective and anxiolytic properties (Hudson et al., 2019; Mortuza et al., 2023). The present study evaluated the potential effect of dietary CBD supplementation (0.001%) and aeration modification as an occupational EE or, as a novelty, their combination. After 38 days fish were subjected to a confinement stress challenge. Key performance and welfare indicators, such as survival, selected stress and immunological parameters, lipid mediators and the expression of several stress-response and appetite-regulating related genes were analysed in European sea bass (Dicentrarchus labrax).
Materials and Methods
Four treatments were evaluated in juvenile of European sea bass over 38 days: control (-CBD/-EE), dietary cannabidiol supplementation (0.001%; +CBD/-EE), EE using a microbubble curtain (-CBD/+EE), and their combination (+CBD/+EE). Growth performance was evaluated followed by a confinement stress challenge with sampling at 2, 24, and 168 h post challenge. Blood samples were collected to determine plasma cortisol and serum lysozyme activity. Head kidney was analysed for lipid mediators using UPLC-MS/MS. The telencephalon, hypothalamus and pituitary gland were collected to evaluate the expression of stress-response related genes (crf, gr2, pomc and hrt1a) and appetite-regulating related genes (agrp1, agrp2, cart2, cnr1, cnr2, npy and mc4r) by RT-qPCR. Survival was also recorded and analysed using Kaplan-Meier survival curve.
Results and Discussion
The utilization of microbubble curtain system as EE improved growth performance and feed efficiency, while also increasing dissolved oxygen levels, whereas dietary CBD had no effect on growth. Conversely, all welfare-promoting strategies increased survival compared with the control group under confinement stress. This result indicates that fish reared under improved environmental or nutritional conditions developed a greater capacity to cope with subsequent stress. Fish exposed to CBD, EE or both showed significantly lower basal plasma cortisol concentrations (ng mL-1) than controls, suggesting an improved physiological state before the stress challenge. Likewise, CBD-fed fish also maintained lower cortisol levels during both acute and chronic stress, indicating rapid anxiolytic effects and improved recovery via modulation of the HPI axis. At the immune stress parameters, CBD supplementation enhanced basal serum lysozyme activity (U mg-1) and promoted a faster post-stress stabilization, suggesting improved innate immunity and a more efficient return to homeostasis in treated fish. Lipid mediator analyses further suggest that these treatments modulated inflammatory regulation. The higher expression of ARA derived lipid mediators in the combined group suggests that CBD and EE may synergistically promote a resolution-oriented inflammatory response, facilitating the clearance of stress-induced damage rather than fostering chronic pro-inflammatory activation. Combined treatment also increased EPA derived lipid mediators often associated with anti-inflammatory pathways. Although their specific physiological roles remain unclear, CBD and environmental enrichment may help to promote a more balanced inflammatory response to stress in European sea bass. The analyses of gene expression in the brain revealed tissue-specific modulation of neuroendocrine pathways related to stress perception, behavioural regulation, and feeding control. In the telencephalon, treatment effects were detected in genes related to stress regulation, cannabinoid and serotonergic signalling, and appetite control. In the hypothalamus, several stress-axis genes and appetite-related genes were modulated, indicating that welfare-promoting conditions affected both stress and metabolic control. In the pituitary, the combined treatment downregulated key stress-associated genes. The pattern across brain regions points to a shift toward lower HPI-axis activity and a more stable neuroendocrine state under welfare-promoting conditions in European sea bass. Overall, the present study demonstrates that EE through microbubble-curtain aeration and dietary cannabidiol supplementation can improve stress resilience, physiological homeostasis, and overall rearing performance in juvenile European seabass.
References
Arechavala-Lopez, P., D��az-Gil, C., Saraiva, J. L., Moranta, D., Castanheira, M. F., Nu��ez-Vel��zquez, S., Ledesma-Corvi, S., Mora-Ruiz, M.R., & Grau, A. (2019). Effects of structural environmental enrichment on welfare of juvenile seabream (Sparus aurata). Aquaculture Reports, 15, 100224. https://doi.org/10.1016/j.aqrep.2019.100224
Balasch, J. C., & Tort, L. (2019). Netting the stress responses in fish. Frontiers in Endocrinology, 10, 62. https://doi.org/10.3389/fendo.2019.00062
Hudson, R., Renard, J., Norris, C., Rushlow, W. J., & Laviolette, S. R. (2019). Cannabidiol counteracts the psychotropic side-effects of δ-9-tetrahydrocannabinol in the ventral hippocampus through bidirectional control of erk1–2 phosphorylation. Journal of Neuroscience, 39(44), 8762-8777. https://doi.org/10.1523/JNEUROSCI.0708-19.2019
Mortuza, A., Fahim, N., Ahmed, M., & Mustafa, A. (2023). Effects of CBD (Cannabidiol) on the physiology of Nile tilapia (Oreochromisn niloticus) as a chronic stress mitigating agent In-vivo. Plos one, 18(9), e0290835. https://doi.org/10.1371/journal.pone.0290835
Zhang, Z., Xu, X., Wang, Y., & Zhang, X. (2020). Effects of environmental enrichment on growth performance, aggressive behavior and stress-induced changes in cortisol release and neurogenesis of black rockfish Sebastes schlegelii. Aquaculture, 528, 735483. https://doi.org/10.1016/j.aquaculture.2020.735483