It is well-established that n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs), particularly eicosapentaenoic (C20:5n-3; EPA) and docosahexaenoic (C22:6 n-3; DHA) acids, have multiple beneficial effects in vertebrate health, such as promoting a correct neurophysiological development and in the prevention of cancers, cardiovascular, and inflammatory diseases (1–3). The main source for humans of these n-3 LC-PUFAs has traditionally been the dietary intake of fish and seafood. However, fish are also dependent on their dietary intake, since many marine fishes have a restricted ability to biosynthesize DHA and EPA due to their low capacity to desaturate and elongate linoleic (C18:2 n-6) and alpha-linolenic (C18:3 n-3) acids (4). Thus, n-3 LC-PUFAs have been usually incorporated to aquafeeds as fish oil. Nevertheless, the decreasing availability of fish oil to sustain the growth of aquaculture worldwide, coupled with its rising cost, have been a strong driver to search for functional ingredients to reduce fish oil in aquafeeds while maintaining fish health and welfare, and n-3 LC-PUFA levels within the recommended and expected range for the consumer.

During recent decades, the use of plant-based oils as an alternative source to fish oils for dietary energy has been widespread among aquafeed manufactures, given their lower cost and higher availability. The partial replacement of fish oil by vegetal oils does not usually affect fish performance in terms of growth and feed utilization (5, 6). Nonetheless, some studies have reported important changes in the fatty acid profile of the fillet, with a reduction in the levels of EPA and DHA and an increase in alpha-linolenic, linoleic, and oleic (C18:1 n-9) acids (5–7). Rendered fats from animals are another affordable and widely available resource, with a growing interest in its improved use in the context of a circular bioeconomy. Moreover, besides containing more n-3 LC-PUFAs and less n-6 PUFAs than plant-based oils (8), they have a higher level of saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), which are preferential substrates for metabolic energy production (β-oxidation), sparing LC-PUFAs from catabolism (9). For these reasons, rendered animal fats have been advocated as a better alternative, compared to vegetable oils, to partially replace fish oil in fish feeds. Nonetheless, the substitution of fish oil by rendered animal fats still leads to a reduction in the levels of DHA and EPA in the fillet, even if less marked than with vegetable oils, although it does not normally compromise fish performance (10). Furthermore, n-3 LC-PUFA are important regulators of metabolic pathways, with well-known hypolipidemic and hypocholesteremic effects in vertebrates (11), including fish (12). Therefore, fish oil replacement by either vegetal or land-based animal oils can cause important changes in lipid and energy metabolism (13, 14), which may lead to physiological disorders, such as a high accumulation of fat deposits within enterocytes, hepatic vacuolization and steatosis, and high levels of perivisceral fat (10, 15).

However, the reduction in fish oil levels in aquafeeds is inevitable, and although alternative sources of n-3 LC-PUFA are emerging (e.g., algal oils, heterotrophic single cell organisms, genetically modified oilseeds), available volumes and production costs are still not at a level that they could bridge the gap in the near future. Therefore, in the meantime, new strategies are required to mitigate some of the negative effects associated with the reduction in fish oils in fish diets. The present study explores the use of supplements with a lipotropic function. Several studies have demonstrated that the incorporation of ingredients with a lipotropic function in fish diets can regulate the fishes’ lipid profile, reducing physiological disorders such as inflammation and improving their performance (16, 17). Considering studies in higher vertebrates, spices are promising candidates as feed additives, which should be further explored in this context. Pungent vegetal substances are valued in cooking for their intense flavor and aroma and for their properties as food preservatives, but they also have many well-demonstrated physiological benefits (18, 19). For instance, mammalian studies have established that pungent spices act as hypocholesterolemic and hypotriglyceridemic agents (similarly to n-3 LC-PUFA in fish oils) and stimulate digestion, and having antioxidant, antimicrobial, and anti-inflammatory effects. Thanks to these properties, spices are also commonly used as nutraceuticals in humans, with many therapeutic and prophylactic applications, such as prevention or reduction in obesity, diabetes, and carcinogenesis, among others (18, 19).

The aim of this work is to offer a holistic insight of the effects of supplementing a reduced fish oil diet containing poultry fat as the major lipid source (high in SFAs) with a product based on a combination of capsicum, black pepper, and ginger oleoresins, and cinnamaldehyde, in terms of fish performance and overall physiological status, including nutrient metabolism, immune response, and gut microbial profile. The study was performed in gilthead seabream (Sparus aurata), since this is one of the most important farmed marine fish species in the Mediterranean area.

Present results revealed that the combination of capsicum, black pepper, and ginger oleoresins, and cinnamaldehyde promoted somatic growth in gilthead seabream (BW_f and SGR) at both dietary levels tested (0.1% and 0.15%). Such results are partially in accordance with those obtained when testing the same combination of pungent spices in broiler chickens, with production phase differences that can be attributed to inherent differences in species-specific physiology, production system, diet formulation, among other factors (41). In fish species, there is still scarce information on the pungent spices herein tested. However, a recent study was conducted in gilthead seabream under winter farming conditions (16 ± 2°C) and fed diets with a high content of SFAs (40%; 16% crude fat), in which the same blend of pungent spices was tested at different inclusion levels of 0.05%, 0.1%, and 0.15% (20). In this study, somatic growth presented a positive dose–response trend, but effects were not significant.

To our knowledge, there are no other studies in fish species testing the combined effect of all four spices evaluated herein. Nonetheless, the effects of capsicum, black pepper, and ginger (or their active principles) have been separately assessed on fish performance, with controversial results ( Supplementary Table S12 ). In this sense, some studies have reported that the dietary inclusion of capsicum did not lead to a significant improvement in BW nor SGR in gilthead seabream, blue streak hap (Labidochromis caeruleus), Mozambique tilapia (Oreochromis mossambicus), rainbow trout (Oncorhynchus mykiss), and jewel cichlid (Hemichromis guttatus) (56–60). On the other hand, Talebi et al. (61) showed that the dietary supplementation of red bell pepper (Capsicum annum) at an inclusion level of 44 or 55 mg/kg for 20–60 days significantly increased the BW and total length, but not the SGR in rainbow trout (Oncorhynchus mykiss). When supplementing fish diets with black pepper or its extracts, growth was not enhanced in African catfish (Clarias gariepinus), rainbow trout, and common carp (Cyprinus carpio) (62–64), but weight gain was significantly increased in rohu fish (Labeo rohita) when adding the extract to the diet at an inclusion level of 1% or 2% (65). Among the four spices tested in the current study, ginger has been the most studied regarding its growth enhancing effect in teleosts (66–70, among others). Reviewing the literature, different results have been reported, even in the same species, depending on the study. Such changes in growth performance may be attributed to the specific conditions and design of each nutritional assay, namely, feeding period duration, basal diet formulation, supplement dosage, and form (e.g., dried powder, essential oil, or oleoresin), and to the physiological condition of the fishes. In the case of cinnamaldehyde, despite the absence of differences in the growth of Nile tilapia (Oreochromis niloticus) when supplementing the diets with this active principle (1 and 2 mL/kg) reported by Amer et al. (71), many studies have demonstrated its potential in improving growth performance in different fish species, such as Nile tilapia, grass carp (Ctenopharyngodon idella), tongue sole (Cynoglossus semilaevis), and fat greenling (Hexagrammos otakii) (72–75).

Regarding somatic indices, the fact that there were no significant differences in Fulton’s condition factor among dietary treatments suggested that the tested combination of pungent spices did not compromise the overall body condition of gilthead seabream juveniles (76). Furthermore, the HSI was also not altered, in line with the absence of inflammation upon histological evaluation and the absence of differences in the activity of hepatic metabolic stress and oxidative stress enzymes, which confirmed the good health condition of the liver. On the other hand, PVFI was significantly decreased by more than 20% in fish fed the SPICY_0.1% diet. In this regard, the diminishment of the perivisceral fat levels usually has a positive effect on the consumer’s perception (i.e., visual impact, more pleasant smell) and extends the shelf-life of the edible fraction (77, 78). Despite the reduced PVFI, the proximate macronutrient composition and fatty acid profile of the liver and fillet in gilthead seabream fed the SPICY_0.1% diet were very conserved.

When supplementing the diet with the combination of pungent spices at 0.15%, the hepatic levels of DHA and n-3 PUFAs significantly increased. These results are highly remarkable, since n3-PUFAs, and especially DHA, are essential to ensure optimal fish growth, development, and reproduction, and to enhance their immune response (4). Regarding the fillet, the DHA/EPA ratio was significantly increased in fish fed the SPICY_0.1% diet in comparison to the control group, reaching values closer to those expected in gilthead seabream juveniles fed conventional diets without fish oil replacement (79, 80). This was associated with a slight numerical increase in DHA and decrease in EPA levels in the fillet of fish fed the SPICY_0.1% diet. However, the reason why the combination of pungent spices increased the DHA/EPA ratio is presently unclear. In the previous study testing the same combination of spices in gilthead seabream (20), DHA/EPA ratio in fillet was not significantly affected, and both DHA and EPA were numerically increased by the combination of pungent spices (although DHA had a higher numerical increase and, hence, DHA/EPA was slightly raised). The main effect previously described was a significant increase in the n-3 PUFA levels in fillet and a numerical reduction in n-6 PUFA in seabream fed a diet supplemented with 0.15% of the spicy additive (20; Patent Number WO/2022/117810). Since gilthead seabream has a restricted ability to synthesize DHA and EPA (4), it is unlikely that the observed differences are caused by changes in LC-PUFA biosynthesis pathways. Supporting this, the expression of the genes encoding the enzymes that elongate PUFAs (elovl4, elovl5, and probably elovl1; 81) was not upregulated in fish fed the SPICY_0.1% diet, although the higher increase in hepatic levels of DHA was observed in fish fed the SPICY_0.15%, diet, for which gene expression was not assayed. Another possibility is that the increase in the levels of n-3 PUFAs could be caused by a higher capacity of absorption and assimilation of n-3 PUFAs (specifically DHA) from the diet, conferred by the combination of pungent spices, and related to the increased bile-salt-activated lipase activity, as further discussed below. However, a more likely hypothesis is that these fatty acids were better conserved and deposited in fish tissues. There is good evidence suggesting that animal fats can promote LC-PUFA sparing, associated with their high SFA content, which are preferentially used as metabolic energy sources (82, 83). On the other hand, spices, including the ones used in the combination tested herein, are well-known promotors of fat oxidation in mammals (84). Hence, the combination of pungent spices could have further potentiated the oxidation of dietary SFAs and MUFAs for energy purposes and the sparing of LC-PUFAs, with its resulting increased tissue deposition. Furthermore, in line with the present results, DHA has been shown to be more efficiently retained than EPA in Atlantic salmon tissues (85), which has been explained by the higher complexity of DHA catabolism as compared to EPA, since DHA oxidation requires an extra step involving the peroxisomes (86).

Under the current experimental conditions, the supplementation of the diet with the combination of pungent spices at 0.1% reduced the FCR, in spite of the absence of significant differences in feed intake and lipid and protein ADCs. A similar decrease in FCR was reported in the study of Morais et al. (20) when supplementing gilthead seabream diets with 0.1% and 0.15% of this additive during winter. While none of the aforementioned studies testing the effect of capsicum in fish diets described an amelioration of FCR (56, 58–60), some of the works with black pepper or ginger and many of those with cinnamaldehyde supplementation did (63, 65, 66, 68, 70, 72, 74, 75). Therefore, the decrease in FCR in fish fed the SPICY_0.1% diet might be partly attributed to the active principles of ginger and black pepper and to cinnamaldehyde or to the synergetic effect of the four spices present in the tested product. One of the possible mechanisms by which the combination of pungent spices decreased FCR values may lie, at least partly, in the induction of the bile-salt-activated lipase in the AI, as its activity was higher when supplementing the diets with the tested combination.

When evaluating the histological organization of the liver and AI, none of the fish presented an excessive fat accumulation (score of 5—steatosis; Figure 1 ), nor were there signs of inflammation or physiological alterations, the presence of which normally indicates disorders associated with unbalanced dietary conditions (87). The most effective dose of the tested additive for reducing hepatic lipid deposits was 0.1%, while in the intestine, both inclusion levels followed a similar dynamic in terms of regulation of intracellular fat deposits. In this sense, it is well-established in mammals that several different spices have an effect on the regulation of body adiposity through different mechanisms (18, 19). One of these mechanisms is the stimulation of lipid digestion, which may occur through two different pathways, as discussed in Platel and Srinivasan (18). The first is by stimulating the activity of digestive pancreatic and intestinal enzymes, whereas the second pathway is associated with the potential of the spices to induce a higher secretion of BAs from the liver into the bile, and subsequently to the intestine. Bile acids allow fat emulsification and provide a higher action surface for lipases through the formation of micelles, and being necessary for the activation of bile-salt-activated lipase (88). In the current experiment, we did not report statistical variation on lipid ADC values among experimental groups, which might be related to the relatively high water temperature (22.5°C ± 0.5°C), not imposing a major challenge for lipid digestibility. Nevertheless, the numerical increase in the levels of BAs (T-CA, T-CDCA, and their sum) in the AI of fish fed the SPICY_0.1% diet and the higher activity of the bile-salt-activated lipase at both dietary inclusion levels indicated a stimulatory effect towards hepatic production and/or secretion of BAs by the tested combination of spices. To our knowledge, this is currently the only study that has evaluated the fish BA profile under dietary supplementation with any of the spices herein tested. Drawing on the existing mammalian literature, some studies on rats have reported an induced synthesis and secretion of BAs into bile after dietary supplementation with capsaicin (the pungent active principle of capsicum) or ginger, the latter also increasing the bile flowrate. By contrast, dietary supplementation with piperine (the active principle of black pepper) and with cinnamon (the main source of cinnamaldehyde) did not show a stimulatory effect on BA synthesis and secretion (18, 19). However, when the four spices were combined in a mixture, along with others, and supplemented in rat diets, the secretion of BAs and bile flowrate markedly increased, even though the higher increase was shown when the mixture did not contain cinnamon (89). In the current study, the apparently increased secretion of BAs may be associated with a higher synthesis of BAs in the liver, considering the significantly upregulated expression of cholesterol 7-alpha-monooxygenase (cyp7a1) in fish fed the SPICY_0.1% diet. Indeed, CYP7A1 is the first rate-limiting enzyme in the classic pathway of the biosynthesis of BAs from cholesterol.

Considering the results on growth and feeding performance, DHA/EPA ratio of the fillet, vacuolization levels in the liver and AI, the activity of bile-salt-activated lipase, and the BA profile, the best dietary inclusion level of the combination of pungent spices under the present experimental conditions was 0.1%. Hence, samples from the control and SPICY_0.1% diets fed fish were selected for analyzing the gene expression patterns of the liver and AI, and the gut microbial communities. Assuming a higher synthesis and secretion of BAs in fish fed the SPICY_0.1% diet, these BAs might be responsible for the 2-h postprandial hepatic downregulation of lipoprotein lipase (lpl) and upregulation of fatty acid synthase (fasn). In this regard, we previously carried out an assay feeding gilthead seabream juveniles with the same basal diet used in this study but supplemented with a blend of BAs (31), and the expression patterns of lpl and fasn followed similar trends to the ones of the current work. Regarding LPL, this enzyme hydrolyzes the triacylglycerides from chylomicrons and very low-density lipoproteins circulating in the plasma into glycerol and free fatty acids (90). In this way, LPL facilitates the posterior incorporation of these fatty acids into the tissues, which are stored as an energetic reservoir in the form of triacylglycerides. Thus, lpl downregulation may be in line with the decreased PVFI and lower levels of fat deposits in the liver. On the other hand, FASN is a key lipogenic enzyme involved in de novo fatty acid synthesis, which catalyzes the conversion of malonyl-CoA and acetyl-CoA into palmitic (C16:0) acid (90). Furthermore, Dorn et al. (91) suggested a correlation between fasn expression and hepatocellular lipid accumulation. Thus, the upregulation of fasn could be a counter-regulatory mechanism to maintain a balance on the hepatic fat deposits and on the levels of fatty acids regarding their lower incorporation caused by reduced expression of lpl (31). In this sense, many studies in fish have reported an inverse regulation between the expression patterns of lpl and fasn in the liver (92–94, among others). It might be worthwhile to further examine the mechanisms underlying the transcription of these two biomarkers of lipid metabolism in fish from a deeper molecular perspective. Interestingly, in the current study, the supplementation of the diet with the combination of pungent spices also led to a hepatic upregulation of the de novo lipogenic biomarkers stearoyl-CoA desaturase 1b (scd1b) and elongation of very long chain fatty acids 6 (elovl6). SCD1 metabolizes palmitic and stearic (C18:0) acid into palmitoleic (C16:1 n-7) and oleic (C18:1 n-9) acid, respectively (95), while ELOVL6 elongates SFAs and MUFAs with 12, 14, and 16 carbons (96). Assuming that activity and gene expression matched, the upregulation of elovl6 and scd1b may be part of the mechanism, in which fasn is involved, to stabilize hepatic fatty acid levels in response to their lower incorporation linked to lpl downregulation and/or be a strategy to prevent accumulation of palmitic (C16:0) acid triggered by fasn upregulation. The expression changes in the abovementioned biomarkers of lipid metabolism were accompanied by the downregulation of peroxisome proliferator-activated receptor β (pparβ) in fish fed the SPICY_0.1% diet. The role of PPARβ in lipid metabolism is not yet fully elucidated, and the change reported in this work was not very pronounced given its low expression levels. However, PPARβ may stimulate the expression of genes involved in fatty acid oxidation, and its ligands are likely fatty acids (97, 98), so a possibility is that pparβ is downregulated due to the presumed lower hepatic accumulation of fatty acids linked to lpl downregulation, which would also match the lower PVFI observed in fish fed the SPICY_0.1% diet.

Regarding the gut microbial composition, several studies have highlighted that the most dominant bacterial phyla of the gut microbial communities in gilthead seabream are Firmicutes, Proteobacteria, Bacteroidota, and Actinobacteriota (44, 99), in line with the results obtained in the present work. Under current conditions, the gut microbial community composition was very conserved, with only an increase in the relative abundance of Chloroflexi in the PI of fish fed the SPICY_0.1% diet. This is a widespread and metabolically diverse phylum of bacteria that has been reported as part of fish microbiota (44, 100, 101), but whose role remains unknown. No differences were found in the values of the F/B ratio, which is a well-documented factor whose changes have been used as a biomarker of intestinal dysbiosis in fishes (102). Our F/B values remain within the results reported for farmed fish, which vary widely depending on the rearing conditions and experimental diets utilized (103, 104). On the other hand, the absence of differences in beta diversity (Weighted UniFrac) indicated that all samples were similar in terms of their phylogenetic features, and therefore, the addition of pungent spices to the diet did not pose a threat in terms of dysbiosis. Regarding alpha diversity metrics, there were no differences in estimated richness (Chao1 and ACE indices) or phylogenetic diversity (Faith index), but there was an increased value of Simpson’s diversity index in the PI of fish fed the SPICY_0.1% diet with respect to the control group. Both the Shannon and Simpson’s indices are estimators of species richness and evenness, but while the former puts more weight on species richness, the latter puts more weight on species evenness (51). Therefore, the variation reported with Simpson’s Index is more likely due to the differential abundances of species among dietary treatments. Such changes in abundance may be attributed to the potential antimicrobial effect on some bacterial strains of the spices present in the additive (105–108). Specifically, in the current study, there was a reduction in relative abundance of the genera Campylobacter, Corynebacterium, and Peptoniphilus in the PI using the pungent spices supplement at a dietary inclusion level of 0.1%. On the other hand, such changes in abundance may also be related to the presumed higher secretion of bile stimulated by the spices and/or to the higher levels of BAs found in the intestine of fish fed the SPICY_0.1% diet. Furthermore, when supplementing the diets with the combination of pungent spices in the present work, there was an increase in the relative abundance of unassigned genera belonging to the family Lachnospiraceae ( Supplementary Table S9 ), some members of whom possess bile acids-inducible genes, which encode for enzymes involved in the metabolism of primary BAs into secondary BAs (109). In addition, Prevotella, which is a non-bile-acid-resistant genus, tended to be present at a lower relative abundance in fish fed the SPICY_0.1% diet than the control group (p = 0.051), which can be seen as another evidence of an increase in the secretion of bile acids.

As clearly reflected by the PLS-DA representation, the overall gene expression profile from the intestine of fed animals (2 h postprandial) did not show any significant differences among dietary groups ( Supplementary Table S11 ). Only the expressions of interleukin-1 beta (il-1β) and C–C chemokine receptor type 9 (ccr9) showed a substantial increase in fish fed the SPICY_0.1% diet (p < 0.1). IL-1β is a pro-inflammatory cytokine produced by several cell types in response to different processes, such as the activation of pattern recognition receptors (PRRs) by pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs), among others. Additionally, IL-1β is responsible for a cascade of effects on different members of this cytokine family, leading to signal transduction and activation of the nuclear factor (NF)-kB pathway (110). Furthermore, CCR9 is a cell marker found in a wide range of immune cells (B cells, T cells, monocytes, macrophages, and dendritic cells) that drives their migration to the gut-associated lymphoid tissue (GALT), where these cells may play a regulatory role in inflammation (111). Considering the absence of signs of intestinal inflammation under histological analysis and the absence of expression changes on other pro-inflammatory cytokines (tnf-α, il-6, il-8, il-15, or il-34) and on the measured PRRs, the upregulation of il-1β and ccr9 may be attributed to a mild innate immune system priming. Since CCR9 has both pro- and anti-inflammatory functions (111), it is difficult to know whether the upregulation of ccr9 was also associated with the pro-inflammatory effect that IL-1β may have on the intestine, or it was part of an anti-inflammatory response to maintain homeostasis counteracting IL-1β-induced pro-inflammatory response. In any case, as mentioned above, no signs of inflammation or physiological disorders were observed with any of the dietary treatments under current experimental conditions, meaning that during the feeding period, the supplementation of pungent spices did not compromise the intestinal health of the fish.

Regarding the gene expression of biomarkers of inflammation in 48-h fasted fish, an anti-inflammatory effect of the tested additive was indicated by the downregulation of interleukin-15 (il-15), interleukin-34 (il-34), cluster of differentiation 4-1 (cd4-1), cluster of differentiation 8 beta (cd8b), and CD302 antigen (cd302). While the role of IL-15 and IL-34 in the fish immune response is yet unclear, many studies have suggested that, as in mammals, these cytokines may be associated with an inflammatory response (110). In mammals, IL-15 induces the proliferation of B cells, T cells, and natural killer (NK) cells (112), and IL-34 stimulates the proliferation and differentiation of monocytes and macrophages and can promote the proliferation of CD8⁺ T regulatory cells (113). Thus, the downregulation of both cytokines may be in line with the lower expression of the cell markers cd4 and cd8b, which are commonly found on the surface of different T-cell subtypes (114). Similarly, the C-type lectin receptor CD302 is expressed in monocytes, macrophages, granulocytes, and dendritic cells and is involved in cell adhesion and migration and in endocytosis and phagocytosis (115). Some in vitro assays using cells from ayu (Plecoglossus altivelis) and Nile tilapia have also unraveled the phagocytic and bactericidal activity of CD302 (116, 117). In this sense, the downregulation of il-15, il-34, cd4-1, cd8b, and cd302 may be related to an anti-inflammatory effect of the tested combination of pungent spices. This has been seen previously when each of the spices was tested individually in mammals (107, 118) and is also in line with the anti-inflammatory effect that ginger and cinnamaldehyde apparently have in the intestine of rohu fish and bacteria-infected zebrafish (Danio rerio), respectively (68, 119). Although black pepper and capsicum have also been reported to act as immunostimulants in fish, such as rainbow trout and rohu fish, respectively (65, 120, 121), their effects on inflammation have not yet been elucidated.

In addition to the potential anti-inflammatory response, the dietary supplementation with the combination of pungent spices may also have an effect on intestinal epithelial integrity. In particular, there was an upregulation of proliferating cell nuclear antigen (pcna), which may suggest a higher cell proliferation (122) and, in line with it, a downregulation of the coxsackievirus and adenovirus receptor homolog (cxadr), whose expression has been inversely correlated with the rate of cell proliferation (123). Based on these discussed results, a possibility is that such enhanced functionality may modulate the potential entry of PAMPs through the enterocytes, which could be the cause of the lower expression of the PRR cd302 by the leukocytes of the GALT. Consequently, this would lead to a lower immune response as indicated by the lower expression of cd4-1 and cd8b cell markers and reduced secretion of the pro-inflammatory cytokines IL-15 and IL-34. Hence, the reduced levels of expression of pro-inflammatory biomarkers may be the consequence of a reduced exposure to foreign molecules due to the increased permeability induced by pcna upregulation rather than an orchestrated activation of an anti-inflammatory response. Regardless of the cause of the downregulation of il-15, il-34, cd4-1, cd8b, and cd302, the results suggest that the tested additive enhanced the health status of the intestine under short-term fasting conditions, since the SPICY_0.1% diet prevented intestinal inflammation that can affect the fish intestinal motility and deregulate digestion and absorption of nutrients, leading to a lower utilization of the feed and to physiological disorders (124). In this sense, some strategies currently used in aquaculture, such as the replacement of fish oil or the use of high-fat diets, have been correlated with inflammation and excessive lipid accumulation in digestive tissues and with a lower non-specific immunity and disease resistance (16, 125, 126). Therefore, the tested combination of pungent spices may be used as a potential tool to aid in the prevention of intestinal inflammation, enhance peristalsis, and optimize nutrient digestion and absorption. However, further studies are needed to confirm the anti-inflammatory effect in the intestine of the combination of spices tested herein. Overall, to complete the detailed understanding of the mechanisms underlying the above-mentioned pathways, the invaluable help of omics sciences (i.e., RNA-seq or microarray analyses) may be needed in order to further track the gene expression of key biomarkers, such as some anti-inflammatory cytokines (TGF-β, IL-4/13A, and IL-4/13B).

Although there was no suggestion of potential anti-inflammatory effect of the combination of pungent spices until feeding cessation, this was probably because the transit of feed along the gastrointestinal tract had a greater impact on gene expression than the differential ingredient formulation of the experimental diets themselves. Regardless, based on our above-discussed results, we recommend that the incorporation of the combination of pungent spices in the diet be continuous whenever the purpose is to promote lipid metabolism to reduce body fat content and improve the health status of the fish, accompanied by an increase in fish growth and feed utilization. In this sense, the regulatory effect that the combination of spices had on the expression of hepatic biomarkers related to lipid metabolism practically disappeared after the 48-h fasting period ( Supplementary Table S10 ), except for the upregulation of sterol regulatory element-binding proteins 1 (srebp1) and peroxiredoxin 5 (prdx5). SREBP1 is a membrane transcription factor that has been related to lipid homeostasis through the regulation of de novo lipogenesis and fatty acid synthesis in fish liver (81), which may be activated after feed deprivation to maintain a balance on the levels of hepatic fatty acids. Albeit the function of PRDX5 in fish still needs to be further studied, recent works in mammals have elucidated its role in preventing adipogenesis by maintaining the intracellular redox balance and avoiding fat deposition through inhibition of fatty acid synthesis and acceleration of their oxidation (127, 128). Thus, the decreased fatty acid production triggered by prdx5 upregulation in the liver of 48-h fasted fish fed the SPICY_0.1% diet may be in line with the downregulation in the intestine of the liver-type fatty acid-binding protein (fabp1) and the intestinal fatty acid-binding protein (fabp2) ( Supplementary Table S11 ), both involved in fatty acid uptake, transport, and metabolism (129).

This study showed that under the regimen of a high saturated fat diet (with poultry fat as the major lipid source), the supplementation of the feed with 0.1% of a product containing a combination of capsicum, black pepper, and ginger oleoresins, and cinnamaldehyde improved the growth performance and FCR in gilthead seabream. In addition, the SPICY_0.1% diet reduced the levels of fat deposits in the visceral cavity, liver, and intestine and increased the DHA/EPA ratio in the fillet to a range closer to the levels commonly found in fillet of farmed gilthead seabream fed a conventional fish-oil-based diet. Moreover, the SPICY_0.1% diet was able to increase the activity of the bile-salt-activated lipase and regulate the gene expression of biomarkers of lipid metabolism. In this sense, the upregulation of cyp7a1, which is involved in the synthesis of primary BAs, which may stimulate lipid digestion, and the downregulation of lpl, which hydrolyzes plasmatic triacylglycerides releasing free fatty acids that can be incorporated into the tissues, was noteworthy. There was no great impact in the structure and composition of the gut microbial communities, albeit the relative abundance of the phylum Chloroflexi increased, while the relative abundances of the genera Campylobacter, Corynebacterium, and Peptoniphilus decreased. In addition, when supplementing the basal diet with the combination of pungent spices at 0.1%, there was a downregulation of the PRR cd302, the immune cell markers cd4-1 and cd8b, and the pro-inflammatory cytokines il-15 and il-34 in 48-h fasted fish. On the other hand, the supplementation of the diet with the pungent spices at an inclusion level of 0.15% also had a positive effect on growth performance, in the reduction in lipid vacuolization within the enterocytes, and in the activity of the bile-salt-activated lipase in the intestine. Moreover, the SPICY_0.15% diet showed higher levels of DHA and total n-3 PUFAs in the liver than the control group. Therefore, the supplementation of fish feeds containing reduced levels of fish oil, especially when fish oil is substituted by oils containing high levels of SFAs, with the combination of pungent spices, may provide a functional solution to optimize the usage of fish oil as a strategic ingredient in the aquafeed industry, even though the optimal inclusion level might need to be optimized depending on the species and diet composition.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

The animal study was approved by Ethical Committee of the Institute of Agrifood Research and Technology and the Generalitat of Catalunya. The study was conducted in accordance with the local legislation and institutional requirements.

SM and EG conceptualized the research. EG designed the nutritional assay. AR, IS, PH, and JP processed the samples. AR, PH, JC-G, JP, MV, JP-S, and EG analyzed the data. AR, KA, DF, JC-G, JP, JP-S, SM, and EG interpreted the results. AR wrote the original draft of the manuscript. EG supervised the study and the writing of the original draft and acquired the funding. All authors contributed to the article and approved the submitted version.