The method by which natural immunostimulants are included in the diet of gilthead seabream (Sparus aurata) modulates their physiological effects on mucosal tissues

The development of the aquaculture industry is conditioned to the establishment of new strategies that guarantee the health status and welfare of fish. The use of phytogenic additives as natural immunostimulants is increasing due to the beneficial properties attributed to them. Elucidating the bioactivity of the additives and the appropriate method of inclusion in aquafeeds is essential to determine their potential application and ensure optimal administration and assimilation by the animals, as some technologies in the production of diets can cause the degradation or alteration of feed additives. In this study, the effects of the method of inclusion in the diet of a mixture of natural additives (garlic essential oil, carvacrol, and thymol) were evaluated in the gilthead seabream ( Sparus aurata ). For this purpose, the phytogenic blend was differentially included in the diet either by coating (PAL diet) or before the extrusion of pellets (PAS diet) at an equivalent concentration. After 78 days of administration of supplemented diets, growth performance, plasma homeostasis (biochemical parameters), digestive function (enzyme activities), as well as mucosal health status in intestine, skin, and gills (histological and biochemical parameters related to tissue integrity, immune system and oxidative stress), were assessed as an approach to immune responses and welfare of fish. In general, neither experimental aquafeed had a negative effect on the physiological status of fish, as growth performance was similar among groups. However, some significant differences were detected in plasma and mucosa-associated lymphoid tissues (MALT) derived from the method of including the additive in the diet. PAL-fed fish presented an improved intestinal structure and functionality compared to PAS-fed fish, as the absorptive ratio in the anterior region and the number of goblet cells were higher. Furthermore, the PAS diet altered digestive capacity by increasing leucine aminopeptidase activity in the anterior region of the intestine; as well as plasma homeostasis, which led to higher glucose levels. These findings suggest that coating is a better option than extrusion for including these additives in aquafeed for seabream. • Carvacrol, thymol and garlic were distinctly included in the diet of Sparus aurata . • The intestine was the main mucosa-associated lymphoid tissue affected (MALT). • Inclusion by coating improved gut functionality compared to inclusion by extrusion. • Inclusion by extrusion altered foregut absorptive ratio and digestive capacity. • Coating is a better way to include these additives in seabream aquafeeds.