Introduction
Due to the growing demand driven by aquaculture expansion, the supply of marine-derived ingredients for aquafeeds can no longer keep pace, prompting a shift toward alternative raw materials. This transition has led to increasingly complex feed formulations, which may impact fish growth, nutrient absorption, and overall health. In this context, maintaining gut health is vital, as disruptions in gut function can impair nutrient utilization and immune function, raising the risk of disease outbreaks. To overcome these challenges, protein hydrolysates (PH), rich in essential amino acids (AAs) and bioactive peptides (<10 kDa), have emerged as promising functional ingredients in aquafeeds, providing benefits beyond their nutritional value, enhancing animal resilience. Serotonin (5-hydroxytryptamine, 5-HT), a neurotransmitter mainly produced in the gut, regulates key physiological processes, including appetite, gut motility, and mucus secretion. Certain AAs have been shown to modulating serotonergic activity, although underlying mechanisms remain unclear. Moreover, PH have shown potential in modulating intestinal physiology and integrity through the regulation of serotonin metabolism. A better understanding of serotonin metabolism, and how it can be influenced by diet, is crucial for developing effective nutritional strategies to preserve gut health, under ever evolving aquafeed formulation. Thus, this study hypothesizes that dietary PHs can improve gut physiology and barrier integrity in gilthead seabream (Sparus aurata) through modulation of the serotonergic metabolism.
Material & Methods
Four diets were formulated: a commercial-like diet (CTRL) and three experimental diets (INSECT, FISH, SWINE), in which 3% of high-quality commercial PH was replaced with locally sourced agri-food-derived PHs. Juvenile fish were fed these diets thrice daily for 88 days and, at the end of the growth trial, anterior intestine samples were collected. Gut integrity was evaluated histologically using both light and transmission electron microscopy (TEM). Gene expression analyses in the anterior intestine targeted key serotonin-related genes (involved in 5-HT synthesis, transport, and receptor activity), alongside markers of gut integrity and immune response.
Results & Discussion
All diets were well accepted, with no differences in voluntary feed intake. Fish fed the INSECT and SWINE diets exhibited significantly higher final body weight compared to CTRL. Histomorphological parameters of the anterior intestine assessed under light microscopy, such as absorption area, submucosa thickness, villus length, goblet cell (GC) quantification and size, revealed no significant differences among treatments. Histomorphology analyses were further supported by TEM analyses, that revealed no observable differences in the epithelial cells, presenting a regular morphology, with an intact epithelium barrier. Enterocytes showed a distinct brush border, orderly nuclear alignment, and a tall columnar morphology, and intact intercellular junctions, with no signs of disruption. Moreover, the expression of tjp1, a gene that encodes a tight junction protein involved in regulating the movement of macronutrients across epithelial cells, did not differ among treatments, supporting observed gut morphology and cellular architecture. Fish fed the SWINE diet showed higher expression of slc6a4a, a gene associated with serotonin transport. Additionally, muc2 and il6 were also upregulated in these fish. This suggests that an increased re-uptake of serotonin may be associated with increased mucus production and modulation of immune and inflammatory response. These findings are consistent with other animal models, where serotonin has been shown to stimulate intestinal mucin secretion. In contrast, INSECT fed fish also showed an upregulation of muc2 expression, but serotonin related genes remained unaffected. Notably, despite a trend (p = 0.054) toward fewer GCs, which remained unchanged in size, muc2 expression was upregulated in fish fed the INSECT and SWINE diets. Thus, this could reflect a compensatory mechanism where increased transcriptional activity helps sustain mucus production despite fewer GCs. Gene expression results will be further discussed to clarify the links between gut integrity, immune response and 5-HT system response.
Conclusion
This study showed that PHs derived from swine by-products and insect meal promote growth in gilthead seabream while preserving intestinal integrity. All PH supported a healthy intestinal architecture, with enterocytes maintaining normal morphology and tissue organization. Although a slight reduction in goblet cell number was observed, the concurrent upregulation of muc2 expression in INSECT and SWINE suggests an adaptive mechanism that maintains mucosal protection and secretory function. Moreover, fish fed the SWINE diet showed higher expression of genes related with serotonin transport, suggesting a potential modulatory effect on serotonin-mediated gut functions. These findings point to a broader impact of this PH on gut homeostasis and neuroimmune communication. Collectively, these results highlight the value of PHs, particularly SWINE and INSECT, as functional ingredients in aquafeeds, with the potential to support not only growth but also gut health and integrity. Ongoing molecular analyses will further clarify the mechanisms by which PHs influence serotonin pathways and contribute to long-term gut well-being in aquaculture species.
Acknowledgments
This work was supported by the Blue Bioeconomy Pact (C644915664-00000026), under WP6 FEED (Pep4Fish project) through international funds provided by the European Union. A. Alves-de-Oliveira acknowledges FCT for the PhD grant (2023.02611.BD).