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. 2025 Nov 28;51(6):199. doi: 10.1007/s10695-025-01612-w

Dose-dependent benefits of dietary yeast cell wall supplementation on the growth performance and stress resilience of the European seabass (Dicentrarchus labrax)

Camille Houdelet 1,✉, Ana Teresa Gonçalves 2,3, Jorge Dias 2,3, Mark Rawling 4, Eric Leclercq 1
PMCID: PMC12662941  PMID: 41313476

Abstract

Preventive health management is central to modern aquaculture practices, within which functional feed additives are a key tool to optimising fish robustness and disease resilience. Despite being well documented, only few studies have assessed the dose–effect of graded levels of S. cerevisiae yeast cell wall (YCW) on fish performance and mucosal barrier immunity. A 9-week trial was conducted to assess the impact of graded levels of YCW on the performance and mucosal barriers of the European seabass under period: (1) non-challenge (43 days) then (2) with an applied husbandry stressors (20 days) in the form of high stocking density, low water oxygen level, and repetitive handling. The four experimental diets were produced by incorporating graded levels of YCW (0.0, control; 1.5; 3.0 or 4.5 kg/MT) which were top dressed on the basal diet prior extrusion. At the end of the period 1, fish supplemented with YCW at 4.5 kg/MT revealed a 5% increase in body-weight compared to the control. Likewise, throughout the experimental period SGR and FCR were also improved by YCW supplementation (+ 5.8% and + 8.3% with YCW4.5, respectively). Supplemented diets increased intraepithelial lymphocytes density and the highest level of intake was associated with increased skin mucus secretion (+ 31% with YCW 4.5 kg/MT). Finally, we observed a linear relationship between fecal calprotectin-like levels and the YCW gradient: a positive correlation during period 1 and a negative correlation during period 2. In contrast, YCW supplementation levels were characterized by a positive linear relationship with fecal mucins across both periods. These results provide novel insights on the benefits and associated mechanisms of YCW extracts and reveal an optimal dose response with inclusion levels between 3.0 and 4.5 kg/MT. This study demonstrates that YCW dietary inclusion enhances growth performance, feed utilization, and mucosal immune barrier function in European seabass, with improved stress resilience evidenced by biomarkers fecal calprotectin-like and fecal mucins.

Supplementary information

The online version contains supplementary material available at 10.1007/s10695-025-01612-w.

Keywords: European seabass, Functional ingredient, Performance, Dose–response, Calprotectin-like

Introduction

Farmed European seabass Dicentrarchus labrax (Linnaeus, 1758) are regularly exposed to biotic and abiotic stressors that affect their survival and growth performances (Islam et al. 2020a, 2020b). These environmental factors include sudden changes in water quality or temperature, exposure to pathogens or harmful non-infectious agents (Cheyadmi et al. 2023; Papandroulakis et al. 2014) and routine management stressors such as crowding, handling, and transportation (Barton 2002; Hurst 2007; Portz et al. 2006). The gut and skin mucosal surfaces are the first line of defence against pathogenic microorganisms and external threats (Peterson 2015). Disruption of these essential mucosal barriers undermines physiological homeostasis and compromises the coordinated network of innate and adaptive immune mechanisms that maintain immunity and tolerance. Such impairment facilitates microbial translocation leading to susceptibility to opportunistic infections and immune dysregulation, which compromises host health and performance. Accordingly, the preservation and rapid restoration of mucosal immune integrity are critical for resilience and disease resistance of fish under environmental and pathogenic stressors. Preventative health management is a cornerstone of modern aquaculture, with functional feed additives playing a key role in enhancing fish robustness and resilience to disease. Feeds supplemented with selected additives that provide health benefit beyond their basic nutrition have gained considerable attention (Shaikh 2022; Waagbø and Remø 2020). These additives include probiotics, prebiotics, enzymes, antioxidants, vitamins, organic acids and plant extracts. Indeed, a well-established class of functional additives is yeast cell wall (YCWs) extracts. YCWs are primarily composed of two bioactive molecules β‐1,3/1,6‐glucans (β-glucans) and mannan oligosaccharides (MOS) (Gow and Lenardon 2023). Numerous studies have demonstrated their beneficial effects on fish health and performance (Leclercq et al. 2020; Machuca et al. 2022; Meena et al. 2013; M. Rawling et al. 2023; Refstie et al. 2010; Van Doan et al. 2024). Yeast β−1,3/1,6-glucans are recognized by the host immune system as non-self molecules through signalling pathways involving pattern recognition receptors (PRRs) such as C-type lectin receptors (CLRs). Recognition triggers immune cell responsiveness, including classical macrophage activation, enhanced phagocytosis by phagocytic cells, and stimulation of other immunocompetent cells (Hadiuzzaman et al. 2022). The benefits of yeast β-glucans on fish health, immunity and pathogen resilience have been documented in particular in Atlantic salmon (Bridle et al. 2005; Refstie et al. 2010; Robertsen et al. 1990), rainbow trout (Guselle et al. 2007; Siwicki et al. 2004) and European seabass (Bonaldo et al. 2007). In comparison, yeast-derived MOS act as a ligand for PRRs like the endocytic mannose-receptor (MR), which is predominantly expressed on macrophages and dendritic cells (Ringø et al. 2010). The MR is a key receptor for augmentation of several key immune functions including both pro- and anti-inflammatory cytokine production, antigen processing, phagocytosis, cell migration and tolerization (Gazi and Martinez-Pomares 2009). These authors noted that MOS ligation is not a danger signal but is rather associated with a reduction of pro-inflammatory cytokine production thereby reducing excessive inflammation and so preserving mucosal barrier integrity (Sharma 2025). MOS supplementation has shown benefits across various aquaculture species notably Atlantic salmon (Leclercq et al. 2020; Refstie et al., 2010) and European seabass (Torrecillas et al. 2007; Torrecillas et al. 2011a, b) as reviewed Torrecillas et al. (2014) and Sharma (2025). These reviews concluded that yeast-derived MOS supplementation reinforces epithelial barriers, enhances feed efficiency and growth, and supports the functional integrity of enterocytes membranes. Additionally, these reviews report benefits of MOS on innate immunity and disease resistance, highlighting the promotion of gut and skin mucus excretion as primary mode of action against infection (Torrecillas et al. 2013; Torrecillas et al. 2011a, b). It is important to emphasize that yeast-based β−1,3/1,6-glucans and MOS are complex macromolecules within a YCW structural organisation. As such their biological activity are dependent upon the biochemical composition, the molecular structure and the architecture of the YCW (Hansen et al. 2021; Schiavone et al. 2019); themselves dependent upon the yeast strain, fermentation conditions and downstream processing applied. Accordingly, distinct YCWs can exhibit distinct biological effects that are not solely explained by their crude content of MOS or glucans (Rawling et al. 2023).

Fecal biomarkers are commonly applied as diagnostic health tools in mammals for both clinical and scientific purpose however their application in fish remains seldom. Fecal biomarkers offer several advantages including they are non-invasive, well-described, stable for analytical robustness and documented with threshold values available for diagnostic purposes (Kilburn-Kappeler and Aldrich 2023; Poudel et al. 2022; Wang et al. 2018). Among these biomarkers, mucins—O-glycosylated glycoproteins localized in mucosal surfaces are well-recognized in fish for their high susceptibility to environmental perturbations including pathogens, physical injuries and dietary modifications (Pérez-Sánchez et al. 2013). Another biomarker of importance is calprotectin (CP), a protein complex formed by two calcium binding proteins as shown in humans, S100A8 and S100A9. This immunomodulatory protein complex is constitutively produced and released by neutrophils in response to tissue damage, external stressors or potential pathogen (Jukic et al. 2021). Calprotectin serves as a ligand for various immune receptors, activating the NF-ĸB signaling pathway which drives innate immunity. Additionally, CP possesses biostatic properties, limiting bacterial growth through metal sequestration, and functions as an oxidant scavenger (Dhas et al. 2012). Under optimal physiological conditions CP contributes to immune homeostasis. However, during chronic inflammation, CP can perpetuate the inflammatory feedback loop, exacerbating tissue damage and impeding tissue remodeling. Consequently, CP serves as a key immunological biomarker and therapeutic target in chronic inflammatory disorders, such as human inflammatory bowel disease (IBD), where fecal CP concentrations can rise several-fold above physiological levels (Jukic et al. 2021). While fecal CP is well characterized in human medicine and increasingly documented in companion and terrestrial animals (Barbosa et al. 2021; Grellet et al. 2013; Riggers et al. 2023; Saco et al. 2025), its use in aquatic species remains seldom.

In the present study, we aimed to evaluate the dose–response of specific and fully characterized yeast cell wall compounds on the health and performance of the European seabass under both best and chronic stress conditions. In addition to conventional health and performance metrics, we assessed fecal biomarkers (fecal calprotectin-like and fecal mucins). These biomarkers were used to gain insights into underlying mechanisms and to evaluate their potential as non-invasive indicators of gut health in the context of microbial-based functional nutrition.

Material and methods

All experimental procedures and husbandry practices used in the present study were conducted in compliance with Portuguese Decreto-Lei nº. 113/2013, August 7th in accordance with EU regulation (EC Directive 86/609/EEC) and approved by the Animal Ethics and Welfare Committee of RIASEARCH Lda (Portugal).

Test compound and experimental feed

The test compound was a commercially available, single-strain S. cerevisiae yeast cell wall (YCW) compound up-cycled from a specific bioethanol fermentation plant (Optiwall; Lallemand SAS, Blagnac, France) containing 17% Mannans, 30% β-glucans and 35% proteins. This YCW product shows 30% of interaction with an Atomic Force Microscopy (AFM) tip functionalised with Concanavalin A (a lectin binding to α-mannose units) and mannan-chains of unfolded median length of 207 nm. AFM is used to measure specific interactions between biomolecules. A 30% interaction between the yeast cell wall surface and Concanavalin A indicates the presence of mannose on the surface, which is not homogeneous (only 30% of the surface). However, the unfolded mannan chains measure 207 nm. This means that mannans are not present across the entire surface (allowing access to the inner layer), but the mannan chains that are present on the surface are long.

Experimental diets were formulated and manufactured at SparosLda (Olhão; Portugal). A basal diet was formulated according to current commercial practices for the species life-stage in the West Mediterranean seabass industry (43% plant-meal; 20% fish-meal; 15% land-animal meals; 6.5% rapeseed-oil; 6% fish-oil; (Supplementary Table S1). From the basal formulation, four experimental diets were produced by incorporating graded levels of YCW (0.0, control; 1.5; 3.0 or 4.5 kg/MT) as top dressed onto the basal recipe prior extrusion. Oil was vacuum-coated post-extrusion, and two pellet sizes were manufactured (2.0 and 4.0 mm diameter). Experimental diets were packed in sealed plastic buckets and stored according to good feed storage practices until use. Diets proximate composition (46.3% protein; 16.4% crude fat; 21.1 MJ/kg) was analysed in accordance with official methods of Association of Official Analytical Chemists (AOAC): Moisture (AOAC 950.46), crude protein (AOAC 981.10), crude lipid (AOAC 950.54) and ash (AOAC 938.08).

System and fish

The experiment was performed at RIASEARCH (Murtosa, Portugual). Fish were grown in a fiberglass tank system (16 tanks, 400 L circular, self-cleaning central drain; equipped with aerators and oxygen-sensor) supplied with pumped ashore, pre-treated natural seawater at 5.5 L/min/tank. Salinity ranged between 36 ± 0.2 ppt and photoperiod was maintained at 14:10 (light:dark cycle) over the trial’s duration.

European seabass (n = 1200) were sourced locally from a single, size-graded commercial stock (AtlantikFish, Portugal), quarantined upon reception, randomly distributed into the experimental tanks (45 fish/tank) and given a 1-week acclimation period prior the start of the trial at an average initial body-weight (BWi) of 11.3 ± 0.6 g (BWi ± SD). The mean intra-tank and inter-tank coefficient of variation (CVintra and CVinter respectively) in BW were CVintra = 11.4 ± 1.9%; CVinter = 1.57% at the trial’s start.

Experimental design and rearing conditions

The trial tested four diets (YCW at 0.0, 1.5, 3.0 or 4.5 kg/MT) in quadruplicate with the test diets allocated to tanks by full randomization and hand-fed to apparent satiation three times daily over the full trial’s duration. The trial lasted a total of 63 days comprising two distinct experimental phases. During the first 43 days (Period 1; P1; non-challenging conditions), fish were reared under controlled, non-challenging environmental conditions selected to promote optimal zootechnical performance (water temperature 21.8 ± 0.4ºC; salinity 36.0 ± 0.2 ppt; dissolved oxygen 6.7 ± 0.3 mg/L (6.2 to 7.5 mg/L; > 90% saturation); maximum rearing density = 3.4 kg/m3). Subsequently during days 43 to 63 (Period 2; P2; Stressful conditions), fish were subjected to a combination of a chronic and repeated acute stressor as follow. First, tanks’ water level was reduced raising stocking density fourfold (3.3 ± 0.04 kg/m3 at the end of period 1, 13.1 ± 0.2 kg/m3 at the beginning of the period 2 and 16.1 ± 0.3 kg/m3 at the end of period 2) and reducing dissolved oxygen levels to 50% saturation over period 2 (from 6.7 ± 0.3 mg/L for the period 1 and 3.7 ± 0.2 for the period 2). Second, a repeated acute stressor was applied by subjecting the experimental fish to netting and 1 min air-dive twice weekly.

Data recording, sampling schedule and procedures

At day 0; 43 and 63, each tank was anesthetized (35 ppm, MS-222; Sigma Aldrich, Spain) and fish biomass bulk-weighted and the number of fish counted. Feed input to apparent satiation was recorded daily per tank. Any mortality was removed daily and measured for BW. At the start of the trial, 15 individual fish from the experimental stock were individually measured for BW and fork-length (FL). At day 43 and 63, 7 fish/tank were individually measured for BW and FL. Among these, 4 fish/tank were sampled for crude skin mucus, dorsal skin, distal intestine and fecal sample as follows.

First, crude skin mucus excretion was quantified using surgical cellulose sponge (SCS; Sugi® sponge strips, rectangular 35 × 7.5 mm, 2.63 cm2; #31003; Questalpha Verwaltungs GmbH; Eschenburg, Germany). Briefly, fish were individually netted-out from their experimental tank, anesthetized as previously mentioned, then carefully handled while allowing excess water dripping for 10 s. Using two forceps, a pre-weighed SCS strip was applied in contact with the right-flank dorsal skin, above the lateral line and below the dorsal fin for precisely 30 s during which a light pressure was applied to ensure full contact between the strip and the skin. For consistency, this manipulation was always performed by the same operator. The SCS was then immediately weighted on a precision scale (± 0.001 g) to determine the mass of the mucus absorbed per unit surface area (mg/cm2) of SCS; i.e. dorsal skin surface. The fish was then placed on a bench laying on its right-flank. Skin mucus was collected from the left-flank by gentle scrapping from the operculum to the anal pore using a smooth scraper and with care to avoid any contamination. The wiped-mucus was collected into a 2 ml Eppendorf, snap-frozen in liquid nitrogen then stored at −80ºC until further analysis. Standardized samples of skin from the dorsal regions and from the distal intestine (transversal section cleared of digesta and PBS-washed) were excised, placed into a vial pre-filled with 10% neutral-buffered formalin (1:10 ratio tissue: fixator) and stored at room temperature until histological processing. Feces were collected by dissection at the terminal section of the posterior intestine. Samples were placed into a 2 ml Eppendorf and stored at −20ºC until analysis.

Analytical protocols

Formalin-fixed skin and intestinal samples were processed following standard histological procedures. In brief, samples were dehydrated, embedded in paraffin wax for continuous transversal sectioning at 3 μm thickness and stained with either haematoxylin and eosin for determination of villus height, lamina propria width and intraepithelial leukocyte density (IELs) or alcian blue and van Gieson was used to determine goblet cell dynamics. Images were captured on a Leica DMD 108 digital microscope at × 400 magnification for measurement of parameters by image analysis (Image J 1.47v, National Institutes of Health, Bethesda, Maryland, USA). Goblet cells were also classified as acidic, neutral or basic phenotype based on their stained coloration using Ab-PAS (Blue, pink, purple, respectively). Skin sections were used to determine goblet cell density and goblet cell type across a 200 μm length of mucosal surface.

Skin mucus samples were thawed on ice, vortexed and protein concentration determined by colorimetric measurement at 550 nm following 30-min incubation Biuret solution (Catachem Spectrophotometer; Automated Total Protein Assay; V254-12. Lysozyme activity was determined based upon the lysis of Micrococcus lysodeikticus after (Ellis 1990). Briefly, 10 μl of mucus was added to 190 μl of M. lysodeikticus; at a concentration of 0.2 mg ml in 0.05 M Na2HPO4 (pH6.2). After mixing, the reduction in turbidity was measured between 0.5 and 15 min at 540 nm at 22 °C. One unit of lysozyme activity was defined as a decrease in absorbance of 0.001 unit min − 1. Fecal mucin contents were determined using a fluorometric Fecal Mucin assay kit (CSR-FFA-MU-K01E; Cosmo Bio USA) that discriminates O-linked glycoproteins (mucins) from N-linked glycoproteins. Fecal mucins were quantified and expressed as GalNAc (N-acetylgalactosamine) equivalents.

Fecal calprotectin-like level was determined using a Fish Calprotectin ELISA Kit (MBS010625; MyBioSource) according to the manufacturer’s instruction. Despite the commercially declared fish specificity, the kit usage for D. labrax was further standardized before application. Assay reactivity was tested using a surrogate positive sample obtained from fish with induced intestinal inflammation, serving as a replacement for a non-available commercial form of the analyte for the species. Briefly, different dosages (5 steps increments from 0.25% up to 1% inclusion besides the 0%) of DSS (dextran sodium sulfate) were included in a formulated feed, fish were fed the diets (in triplicate tanks per diet) and samples were collected after 5, 10 and 15 days of continuous feeding. For model optimization several parameters were considered throughout the trial, such as feed intake, growth performance, intestinal histopathology and inflammation related molecular markers, considering the recovery as well after insult (up to 4 weeks). Feces were collected by stripping at all points and calprotectin-like assay performed for all animals following fabricant instructions. The protein was detected in feces with a significant correlation (R2 of 0,61 and 0,76 with p < 0.05) with histopathological evidence of intestinal inflammation (including alteration in number of goblet cells, villi length and density, lymphocytes presence in lamina propria, resulting in a higher histopathological score). Linearity was confirmed using a dilution series of three separated surrogate positive samples with strongest signal (derived from different fish that received the highest dosage and longest administration period with confirmed inflammatory reaction by histology, and detected with the OD closest to the one of the highest standard), and this was repeated three times in separate days to assess linearity variation which was within acceptable limits (CV = 8,2% calculated on concentrations derived from the kit standard curve). The standard curve with the provided standards was always run in parallel, for OD comparison, and dilutions of surrogate positive samples fell within detection range. Accuracy was assessed by spike and recovery, using 10 µl of known concentration of the highest standard into the fish fecal negative control sample (n = 3, repeated 10 times, recovery of 91 ± 15%), whereas 10 µl of the surrogate positive sample mix (pool of three fish used for the linearity assay) was also spiked (n = 2 recovery of 90 ± 11%). Precision was assessed by measuring intra-assay variability (coefficient of variation per replicate wells in a single plate), as well as inter-assay variability by running the same set of samples 9–10 times divided by different plates and CVs were 12.5% and 9.8% respectively. Moreover, we have followed the recovery of the insulted animals for 4–6 weeks, and together with histopathology resolution, there was a progressive reduction of the calprotectin analyte levels.

Calculations and statistics

Mean body-weight (BW) was calculated as BW = BM/n with BM the total tank biomass and n the number of fish. Specific growth rate (SGR) was calculated as SGR (%/day) = 100 (eg—1); where g = (LnBWf − LnBWi)/MT; with BWf and BWi as final and initial BW (g) respectively over the period of interest and t the duration of the period (day). Feed Conversion Ratio (FCR) was calculated as FCR = FI/BG; where FI is the apparent feed intake (g) and BG the live biomass gain over the period. All data were analysed using R Studio version 2024.09.0.375 (Posit team 2024). The effects of diet and time were assessed using a generalized mixed model (GLMM) fitted with the glmmTMB package. The model included Diet, Time and their interaction as fixed effects, and Tank as a random effect to account for potential tank level variability. To ensure appropriate contrast coding for factorial effects, sum-to-zero contrasts (contr.sum) were applied for categorical variables, and polynomial contrasts (contr.poly) were used for trend analysis. Type III analysis of variance (ANOVA) was conducted to evaluate the significance of main effects and interactions. Estimated marginal means (EMMs) were computed using the emmeans package to explore the effects of Diet within each Time point. Pairwise comparisons between diet levels were performed with Sidack adjustment for multiple testing. Finally, to assess dose–response trends, polynomial contrasts (linear, quadratic and cubic) were applied based on the actual dietary dose level (0, 1.5, 3 and 4.5 kg/MT). A significance level of 5% (p < 0.05) was applied; data are presented as mean ± standard error of the mean (SEM) of replicates tanks unless otherwise stated. A tendency was considered between 0.05 and 0.10.

Results and discussion

Performance benefits

There was no difference in BW at the start of trial and no mortality over the duration of the trial. At the end of period 1 (P1; non-challenging conditions); despite a non-significant pairwise comparison for the BW factor, a significant linear contrast suggested a consistent directional effect across YCW levels supplementation, which may not be captured by individual pairwise test (linear contrast, p = 0.03, Fig. 1A). In parallel, at the end of period 1 (P1; non-challenging conditions) and compared to the Control; SGR was significantly higher at the higher level of supplementation tested (YCW4.5) with intermediary values measured at intermediary supplementation levels (p = 0.04, Fig. 1B). This result was confirmed by the significant linear trend describing a consistent increase of SGR with increasing YCW doses (linear contrast, p = 0.004, Fig. 1B). As expected, there was a significant negative effect, across dietary treatments, of the stress compared to the non-challenging rearing period for SGR (P1: 2.20 ± 0.03; P2: 1.04 ± 0.02; p < 2.2 × 10–16), FI (P1: 2.20 ± 0.04; P2 = 1.30 ± 0.04; p = 2.2 × 10–16) and FCR (P1: 1.09 ± 0.06; P2 = 1.31 ± 0.06; p = 2.0 × 10–16; Fig. 1B, C, D) together validating the deleterious impacts of the challenge model. At the end of the stress period (P2), SGR was no different to the control but significantly lower in the YCW3.0 compared to the other two supplemented diets. The complex trend observed was significantly associated with a cubic trend (cubic contrast, p = 0.0003, Fig. 1B). The opposite pattern of a cubic trend was measured for FCR (cubic contrast, p = 0.0001) with YCW1.5 and YCW4.5 showing enhanced FCR compared to the control (−11%, p = 0.02 and −15%, p = 0.0004, respectively, Fig. 1D). Holistically, the SGR data revealed a significant and positive dose–response across the experimental period for YCW dose, where SGR was significantly higher for the three YCW diets compared to the control (+ 2.8%, p = 0.03; + 2.8%, p = 0.02 and + 6.8%, p = 0.001 for YCW1.5, YCW3.0 and YCW4.0, respectively). Interestingly, this was concomitant with a significant reduction in feed intake (−2.6%, p = 0.03 and −3.1%, p = 0.02) for YCW3.0 and 4.5, respectively compared to the control, resulting in a clear dose-dependent improvement of FCR with increasing YCW level (−3.3%, p = 0.01; −5.8% p < 0.001 and −8.3%, p < 0.001 for YCW 1.5, 3.0 and 4.5, respectively, compared to the control). For these three parameters, a highly significant linear relationship was measured (p ≤ 0.001). A lower feed intake to apparent satiation alongside enhanced growth and FCR suggests enhanced feed utilization with increasing YCW intake. Such performance benefits and better feed utilization upon yeast-based MOS supplementation were previously reported in different finfish species such as the gilthead seabream (Gültepe et al. 2011), rainbow trout (Staykov et al. 2007) and European seabass (Torrecillas et al. 2007; Torrecillas et al. 2011a, b). In the later study, better feed utilization was supported by the observation of lower lipid vacuolization and more regularly shaped hepatocytes around the sinusoidal spaces. In contrast, other studies report increased microvilli length and density in adult rainbow trout (Dimitroglou et al. 2009) and gilthead seabream (Dimitroglou et al. 2010) but without improved performances upon MOS-supplementation.

Fig. 1.

Fig. 1

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A Body-weight, B specfic-growth-rate, C feed intake and D feed conversion ratio per experimental group over the non-challenging period (day 0 to 43) and stress period (day 43 to 63). Data shown as mean ± SEM of replicate tank (n = 4) with apparent feed intake measured daily per tank, total tank biomass measured at each time point and biomass mortality weighted when occurring. Different letters highlight significant differences between groups within time-points (p < 0.05). Asterisk highlights a tendency (p < 0.10)

Gut barrier function modulation

To better understand its effect on zootechnical performance, the study examined the putative dose–response of the test compound on gut immune barrier function. As observed through classical histology, the villi length (VL) of fish fed YCW1.5 showed longer villi compared to the control (382 ± 14 µm vs 332 ± 6; p = 0.01). There was no dietary effect on the density of mucus-excreting goblet cells (GC) in the gut nor in the skin; but a trend toward increased GC coverage (GCC) was noted at the highest YCW dose (YCW4.5) compared to the Control (Table 1). Further a significant curvilinear relationship, with GCC increasing across the YCW gradient (quadratic contrast, p = 0.02). This suggests a dose-dependent stimulatory effect of YCW on mucosal activity, potentially reflecting enhanced epithelial defence mechanism or increase mucus production to higher YCW inclusion level, as already observed in salmon (Leclercq et al. 2020; Refstie et al. 2010). Importantly, all supplemented diets showed a significant increase in intraepithelial leukocytes (IELs) density compared to the control (+ 39% to + 60%; p = 7.79 × 10–7; Table 1). IELs are an essential component of the gut associated lymphoid tissue (GALT) acting as first line of defence by surveying exogenous antigens and releasing endogenous signaling molecules upon activation. An increase in IELs was previously reported in the European seabass supplemented with a whole-YCW product (Torrecillas et al. 2013) and can be interpreted as indicating detection of the YCW microbial-patterns and reinforcement of the immune responsiveness of the epithelial barrier. Among various studies on European seabass and YCW derivative products, two key focusses can be distinguished. First fish-meal replacement with alternative ingredients (Oliva-Teles and Gonçalves 2001; Pelusio et al. 2023), second supplementation for beneficial health effects and performances (Rawling et al. 2019; Torrecillas et al. 2013; Torrecillas et al. 2011a, b). To the authors knowledge, only two studies investigated the dose effect of a specific MOS product; one assessing feed utilization, immunity and intestinal mucus production (Torrecillas et al. 2011a, b) and the other was focused on the resistance to infectious disease (Torrecillas et al. 2007). These two trials, conducted in bigger fish (60 and 33 g, respectively) also demonstrated a better growth and immune modulation with MOS incorporated at 4 kg/MT. Other parameters expected to determine the level of zootechnical benefits elicited upon yeast-based MOS supplementation includes the characteristics of the YCW, the duration of supplementation as well as the species and rearing conditions (Torrecillas et al. 2014). The present study quantifies the beneficial dose–response of YCW supplementation on the growth and feed performance of the European seabass in non-challenging and stressful-period which has not been previously described.

Table 1.

Histological measurements of (a) distal gut and (b) dorsal skin samples at the end of the non-challenging period (day 43) per experimental group. Data shown as mean ± SEM of replicate tank (n = 4) with 3 fish sampled/tank (12 fish/group). Different letters highlight significant differences between groups within time-points (p < 0.05)

Control YCW1.5 YCW3.0 YCW4.5
a. Gut
GCD (n/200 µm) 7.5 ± 1.1 8.0 ± 0.7 7.8 ± 0.6 9.7 ± 1.1
GCC (%) 2.2 ± 0.2 ab 2.1 ± 0.2 b 2.2 ± 0.2 b 3.0 ± 0.2 a
Villi-length (µm) 332 ± 6.5 b 382 ± 13.6 a 314 ± 16.3 b 334 ± 19.6 ab
LP width (µm) 5.1 ± 0.2 5.2 ± 0.2 5.3 ± 0.3 5.2 ± 0.2
IEL density (n/100 enterocytes) 23.5 ± 1.9 b 37.7 ± 2.0 a 32.6 ± 1.6 a 34.0 ± 1.5 a
b. Skin
GCD (n/200 µm) 12 ± 1.1 12.1 ± 1.2 12.2 ± 1.0 14.8 ± 2.0
GCC (%) 8.2 ± 0.6 b 9.5 ± 1.6 ab 11.9 ± 1.8 ab 11.9 ± 0.6 a
Neutral (%) 11.7 ± 3.7 12.1 ± 3.7 7.5 ± 2.4 7.9 ± 2.7
Acid (%) 39.5 ± 6.4 43.7 ± 6.2 42.1 ± 6.7 50.8 ± 4.7
Basic (%) 48.8 ± 8.3 44.2 ± 5.9 50.4 ± 6.3 41.2 ± 5.2
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GCD: goblet cell density, GCC: goblet cell coverage, LP: lamina propria, IEL: intraepithelial lymphocytes

A novelty of this study is the use of fecal markers (calprotectin-like and mucins) to evaluate the impact of YCW supplementation. After 43 days under non-challenging conditions (end P1), fecal CP-like was significantly higher in YCW4.5 compared to all the other groups (Control: 293 ± 6 ng/mg, YCW4.5: 371 ± 6 ng/mg, + 26.6%; p < 0.0001; Fig. 2A). Such diet effect on fecal CP-like indicates neutrophil activation; hence detection of the YCW microbial-patterns and initiation of innate immune cell responsiveness. Despite the lack of documented threshold values in our experimental species, we interpret this rise of 26.6% as being representative of a physiological adaptation indicative of heightened immune surveillance and activation, consistent with the enhanced immunocompetence and performance observed in the respective dietary group. This is further supported by human studies, where fecal CP levels must typically increase several-fold above baseline to indicate a pathological, pro-inflammatory state. For instance, fecal CP has been reported to rise approximately fivefold in patients with Parkinson’s disease (Mulak et al. 2019) and up to sixfold during inflammatory bowel disease (IBD) flares in adults (Pang et al. 2014). Concomitantly over period 1, a significant increase in fecal mucins was measured in all YCW-supplemented diet compared to the control (p = 0.008; Fig. 2B) indicating reinforcement of the protective gut mucosal barrier, confirming the immunomodulatory benefits of the test compound.

Fig. 2.

Fig. 2

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Levels of fecal A calprotectin-like and B mucins per experimental group. Data shown as mean ± SEM of replicate tank (n = 4). Different letters highlight significant differences between groups within time-points (p < 0.05)

At the end of period 2 and compared to period 1, there was a clear effect of time; i.e. of the stressors, on both fecal CP-like and fecal mucins in the Control (CP-like: + 39%, p < 2.2 × 10–16; mucins: + 115%, p < 2.2 × 10–16; Fig. 2A, B). Exposure to external stressors was previously documented to promote both pro-inflammation and mucins excretion as a defensive mechanism against external threats (Faught and Schaaf 2024). Interestingly in the supplemented groups, exposure to the stressors further promoted fecal mucins but mitigated fecal CP-like levels. Enhanced fecal mucin production in response to stress exposure suggests the potential for the test compound to confer a protective mucin-response when subjected to external stressors, which correlates with the documented benefits on mucosal immunity. Such reinforcement of the mucin-barrier and mucosal immunity in the YCW-groups is expected to reduce susceptibility to existing external stressors. This hypothesis was corroborated by the lower rise of fecal CP-like level in the supplemented groups at the end of period 2 (mean CP-like = 355 ± 7 to 361 ± 6 ng/mg in YCWs diets and 406 ± 6 ng/mg in the Control; p = 0.0001) as fecal CP-like was previously documented to increase upon stressor exposure. To date, only two studies have identified the S100a10b protein in zebrafish as functionally analogous to human calprotectin (CP) and examined its intestinal, but not fecal, expression in the context of infection (Farr et al. 2022; Nag et al. 2022). However, Orlandi and Harms 2025 reported the absence of a true CP ortholog in zebrafish. In this context, we assessed the immunoreactivity and diagnostic performance (sensitivity, specificity, accuracy) of a calprotectin-like protein in the studied fish species. Our findings confirm the presence of a calprotectin-like molecule in European seabass, supporting its potential as a biomarker of gut immune status and response to environmental stressors in the fish species.

Peripheral mucosal tissues

At the end of the non-challenging period (end of P1), crude skin mucus excretion was higher in the YCW4.5 group (+ 17%; p = 0.06, Table 2) in accordance with a significant increase in skin GCC (Control: 8.2 ± 0.6; YCW4.5: 11.9 ± 0.6; p = 0.0008, Table 1). Similarly following exposure to stressors (end of P2), crude skin mucus excretion was significantly higher in the YCW4.5 compared to the control group (+ 31%, p < 0.0001; Table 2) while intermediary values were measured at intermediary supplementation levels. For both periods, a positive linear relationship was highlighted between the quantity of mucus and the level of YCW supplementation (linear contrast, p = 0.04 and p = 0.0002, respectively). At the end of both periods, there was no diet effect on the protein concentration of the skin mucus but, interestingly, the lysozyme activity of the skin mucus was significantly higher in YCW3.0 compared to the control (Control: 28.2 ± 4.1 and 16.5 ± 2.1, YCW3.0: 48.3 ± 6.4 and 63.7 ± 8.2; p = 0.05 and p < 0.0001 for P1 and P2 respectively; Table 2). Increased skin mucus excretion in response to YCW-intake was previously documented in Atlantic salmon (Leclercq et al. 2020) and rainbow trout (Rodriguez-Estrada et al. 2013) but no such benefits were observed in the European seabass (Torrecillas et al. 2011a, b). Interestingly compared to the control, YCW3.0 had a higher lysozyme activity without effect on skin mucus excretion while YCW4.5 had, inversely, a higher mucus excretion without differences in lysozyme activity suggestive of a potential dilution or trade-off between mucus excretion (quantity) and concentration in anti-microbial activity (quality). Skin mucus is a biological barrier trapping and immobilizing pathogens before they enter in contact with epithelial surfaces. It is continually excreted and sloughed off, shedding adhering potential pathogens against host-invasion (Ángeles Esteban 2012). Our results indicate a benefit of YCW supplementation at 3.0 to 4.5 kg/MT feed on the skin mucosal barrier.

Table 2.

Skin mucus parameters measured at the end of the (a) non-challenging (day 43) and (b) stress (day 63) periods per experimental group. Data shown as mean ± SEM of replicate tank (n = 4) with 3 fish sampled/tank (12 fish/group). Different letters highlight significant differences between groups within time-points (p < 0.05)

Control YCW1.5 YCW3.0 YCW4.5
a. Non-challenging period
Quantity (mg/cm2) 18.2 ± 0.8 20.9 ± 0.9 19.4 ± 0.8 21.3 ± 0.9 *
Protein concentration (mg/mL) 0.39 ± 0.06 0.52 ± 0.13 0.33 ± 0.04 0.42 ± 0.04
Lysozyme activity (U/mg prot) 28.2 ± 4.1 ab 37.0 ± 6.8 ab 48.3 ± 6.4 a 24.4 ± 3.0 b
b. Stressful period
Quantity (mg/cm2) 21.1 ± 0.8 b 23.5 ± 0.8 ab 22.3 ± 0.8 b 26.3 ± 0.9 a
Protein concentration (mg/mL) 0.44 ± 0.06 0.39 ± 0.05 0.41 ± 0.01 0.42 ± 0.04
Lysozyme activity (U/mg prot) 16.5 ± 2.1 c 31.7 ± 5.2 bc 63.7 ± 8.2 a 31.8 ± 3.6 b
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Conclusion

In conclusion, the 9-week study documented a positive dose–response of a specific YCW ingredient on the growth and FCR of European seabass under field trial conditions combining a non-challenging and a stressful rearing period.

Using fecal biomarkers, the study documented the detection of the YCW at gut level and reinforcement of the gut mucosal immune barriers resulting in enhanced performance and reduced susceptibility to external stressors upon YCW intake. Benefits were also observed at the peripheral skin mucosal barrier in the form of a higher mucus excretion or antimicrobial activity. Together, the study demonstrates the potential of the test compound as a preventive health management tool in support of mucosal robustness and performance in the European seabass farming industry, which should now be validated by longer-term studies under commercial-like conditions.

Supplementary information

Below is the link to the electronic supplementary material.

Supplementary file 1 (DOCX 29.2 KB) (29.2KB, docx)

Acknowledgements

The authors acknowledge Marcelo Gindri for his help to improve the statistical analyses.

Author contributions

EL, ATG and JD contributed to the study conception and design. Material preparation, data collection and zootechnical analysis were performed by ATG and JD. Fecal calprotectin-like analysis and validation was conducted at the GreenCoLab laboratory (Faro, Portugual) by ATG. Histology and skin mucus analysis were conducted by MR. The first draft of the manuscript was written by CH and EL and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data availability

No datasets were generated or analyzed during the current study.

Declarations

Competing interests

The experiment was in part supported by Lallemand SAS with the aim of investigating the effect of a commercial postbiotic product and where CH and EL are employed. To mitigate this conflict of interest and secure the scientific objectivity of the study, the study was performed within an external Contract Research Organisation where experimental feed (including controls) was tested and reported blindly by the CRO staff and scientists.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Supplementary Materials

Supplementary file 1 (DOCX 29.2 KB) (29.2KB, docx)

Data Availability Statement

No datasets were generated or analyzed during the current study.

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