# Shrimp Hydrolysate-Based Palatability Enhancer: A Circular Economy and Cost-Effective Strategy to Reduce Fish in Fish Out Ratio in Marine Fish Species

Mikaël Herault, Buddhi Gunathilaka, Vincent Fournier, Hervé Le Bris, Kyeong-jun Lee, Bastien Sadoul

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Mikaël Herault, Buddhi Gunathilaka, Vincent Fournier, Hervé Le Bris, Kyeong-jun Lee, et al., Shrimp Hydrolysate-Based Palatability Enhancer: A Circular Economy and Cost-Effective Strategy to Reduce Fish in Fish Out Ratio in Marine Fish Species. 2025. [hal-05247252](https://hal.science/hal-05247252v1)

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SHRIMP HYDROLYSATE-BASED PALATABILITY ENHANCER: A CIRCULAR ECONOMY AND COST-EFFECTIVE STRATEGY TO REDUCE FISH IN FISH OUT RATIO IN MARINE FISH SPECIES

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Mikaël Herault<sup>a,b*</sup>, Buddhi E. Gunathilaka<sup>c</sup>, Vincent Fournier<sup>a</sup>, Hervé Le Bris<sup>b</sup>, Kyeong-Jun

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Lee<sup>c</sup>, Bastien Sadoul<sup>b</sup>

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<sup>a</sup>SYMRISE AQUA FEED, ZA du Gohelis, Elven 56250, France

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<sup>b</sup>DECOD, Institut Agro Rennes Angers, INRAE, Ifremer, Rennes, 35042, France

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<sup>c</sup>Department of Marine Life Sciences, Jeju National University, Jeju 63243, South Korea

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*Corresponding author: Tel: +33 749 874 588

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E-mail address: [mikael.herault@agrocampus-ouest.fr](mailto:mikael.herault@agrocampus-ouest.fr) (M. Herault)

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**Abstract**

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Reducing the reliance of the aquaculture industry on wild fish resources remains a key challenge. Generally obtained from food coproducts, protein hydrolysates have emerged as promising functional and sustainable protein sources that can help compensate for the limitations of alternatives to marine proteins. In this study, we evaluated their use as a palatability enhancer (PE) by top-coating them directly on the feed, as a liquid and at low dose.

We tested a PE made primarily from a shrimp liquid hydrolysate, as a cost-effective dietary solution to replace significant amounts of fish meal (FM) with plant proteins in juvenile red sea bream diets. The experimental feeds consisted in a high FM diet (HFM, 30% FM), a low FM diet (LFM, 15% FM) and two additional diets with 2% PE top-coated (HFM+PE and LFM+PE). Six replicate tanks, each containing fish weighing 27.2 ± 0.2 g, were provided with one of the test diets twice daily until apparent satiety for a duration of 15 weeks. Half of the
tanks were exposed to an intermittent stress consisting in a 1 min net-chasing, 1 hour before their first meal. After the nutritional trial, individuals were subjected to an *Edwarselia tarda* challenge through injections. The basal diets, LFM and HFM, achieved comparable growth and feed efficiency. The use of LFM, therefore, mechanically reduced wild fish use by 25% if referring to the eFIFO index (Kok et al., 2020). PE supplementation in both diets resulted in enhanced fish growth and health performance, and improved feed use. Consequently, we estimated that PE use can significantly, and additionally, reduce by 6% the use of wild fish biomass in these conditions. The beneficial effects of PE on the growth performance were stable despite the stress challenge, which led however to overall strong reduction in growth performances in all groups. Survival was significantly increased by PE addition in response to the bacterial challenge compared to the LFM diet. All these results were supported by underlying physiological results. We concluded that liquid hydrolysate PE represent a great potential for feed formulators to cost-effectively reduce pressure on wild resources, while maintaining high performance of their feed.

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40 **Keywords:** Fish meal replacement, eFIFO, Repeated stress, Disease challenge, Red seabream.

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42 **1. Introduction**

The production of key aquaculture-fed species grew by 10.5% annually, on average, from 2000 to 2020, whereas fish meal (FM) use in aquaculture feeds rose by 2.46% per year over this interval (FAO, 2022). In response to resource limitations and sustainability challenges, the aquafeed industry has investigated and adopted novel protein ingredients, enabling substantial FM replacement. Consequently, FM inclusion in marine fish diets dropped from 50 % down to 14 % between 1997 and 2017 (Naylor et al., 2021), resulting in an economic Fish In Fish Out

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ratio (eFIFO) of 0.94 (Kok et al. 2020) making, based on this calculation, marine fish farming a net fish producer overall. During this period, however, aquaculture's reliance on FM also rose, making it the primary consumer of FM worldwide – its share of global FM volumes increased from 42% in 2000 to 83% in 2020 (FAO, 2022).

Soybean meal has emerged as the primary replacement for fishmeal, thanks to its advantageous nutritional properties, high production capacity, worldwide accessibility, and cost-effectiveness (Gatlin et al., 2007; Boyd and McNevin, 2022). As a premium alternative to soybean meal due to its enhanced digestibility and lower levels of anti-nutritional compounds, soy protein concentrate (SPC) is often included in carnivorous fish feeds (Refstie et al., 2001; Gyan et al. 2019). Substituting FM with SPC has demonstrated satisfactory zootechnical outcomes in multiple fish species, provided that essential dietary nutrients are adequately supplied and a certain level of palatability is retained (Bureau et al., 1998; Gunathilaka et al., 2021). Nevertheless, underlying negative effects on fish welfare and immunity were highlighted, when investigated, resulting in a higher susceptibility to bacterial diseases (Khosravi et al., 2015b, Herault et al., 2023).

The global production of sparids exceeded 485,000 metric tons in 2020 (Fishstat, 2023), with the largest share through species like the Gilthead seabream (*Sparus aurata*), which alone accounted for approximately 282,100 metric tons (FAO, 2022). Sparidae production is driven by the demand for carnivorous species that require high-quality diets, often rich in FM. Among these, red seabream, a member of the Sparidae family, is an important species within East Asia. It ranked as the third most commonly farmed fish during the 2000s in South Korea and, in Japan, it continues to hold the position of second most farmed fish (Tabata and Taniguchi, 2000; FAO, 2022).
Earlier studies on red seabream showed that dietary FM could be partially substituted with SPC at levels ranging from 25 to 50% without compromising health performance. This was achieved by 5% Aquatic Product Hydrolysates-based supplementation into low FM diets (Khosravi et al., 2015b; Gunathilaka et al., 2021; Herault et al., 2023). Despite these promising results, the main limit to a generalized use of APH in aquafeed industry is the cost and availability.

One potential strategy to enhance the cost-effectiveness of APH and broaden their adoption within the industry would be to use them in liquid form at reduced dosages. Top-coating is also the preferred application mode to increase diet palatability, making palatable compounds more readily available to fish taste receptors (Kolkovski, 2006; Fournier, 2013; Tola et al., 2019; Rigos et al., 2001). Nevertheless, they are usually added to lower extent when top-coated as a liquid (~2-3%) compared to powder inclusion (~2-5%); consequently, lower fish zootechnical and health performance are expected because of reduced addition of functional components. This is corroborated by recent studies, which demonstrated a positive correlation between the dietary concentrations of bioactive peptides, or APH, and the final fish zootechnical or health performance (Gunathilaka et al., 2020; Wang et al., 2023). To our knowledge, however, there is a limited number of studies investigating the real impact of hydrolysate-based products top-coated on fish meal substituted diets, and at low doses (Tola et al., 2022).

We therefore propose to explore the potential of a formulated palatability enhancer (PE) made from a shrimp liquid hydrolysate as a cost-effective dietary solution to replace half of the FM with plant proteins in the diet of red seabream juveniles in controlled or unstressed conditions. Resulting FM consumption and eFIFO will be assessed for each experimental group while a repeated daily stress will be implemented at regular intervals to mimic husbandry stress found in fish farming operations. The performance indicators and physiological biomarkers used were
95 identical to those in Herault et al. (2023, Table 4) to ensure that the proposed dietary
96 optimization did not negatively impact the fish physiological processes and to make easier the
97 interpretation of any changes observed in the performance indicators.

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2. Materials and Methods

100 All the procedures used during this nutritional and health trial adhered to the guidelines
101 established by the Institutional Animal Care and Use Committee of Jeju National University
102 (approval number: 2021-0041) and utilized a methodology adapted from Herault et al. (2023).

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2.1. *Experimental diets*

104 Four test diets were prepared to have equivalent nitrogen and energy contents on dry matter
105 basis (46.8% crude protein and 20.7 kJ.g⁻¹, respectively). The reference diet included 30% FM
106 of fair average quality (brown FM, 67.7% crude protein) and was designated as the high FM
107 (HFM) diet (Table 1). For the low FM (LFM) formulation, half of the FM in the HFM diet was
108 replaced by SPC, wheat, and corn gluten meals while essential nutrients – such as amino acids,
109 lipids, omega-3 fatty acids, phosphorus, and calcium – were adjusted accordingly. The dry
110 ingredients were mixed thoroughly, followed by the addition of fish oil, soybean oil, and 15-
111 20% double-distilled water. The resulting dough was pelletized using a pelletizer (SP-50, Gum
112 Gang Engineering, Daegu, Korea), yielding pellets with diameters of 2-3mm and lengths of 3-
113 4mm. These pellets were freeze-dried at -40 °C for 24 h before being coated with a 50%
114 mixture fish and soybean oils (Table 1). Two experimental diets were prepared in addition by
115 top-coating HFM and LFM at 2% with Extrapal Shrimp, a palatability enhancer provided by
116 Symrise Aqua Feed (part of Taste, Nutrition & Health segment of Symrise group, Elven,
117 France). Extrapal Shrimp (Table 3) is made of a shrimp hydrolysate derived from
118 enzymatically treated cephalothorax co-products of farmed white shrimp (*Litopenaeus vannamei*), combined with sensory flavors and stabilized with phosphoric acid to achieve a pH
119 of 3.5. This 2% dietary supplementation of Extrapal Shrimp results in a nutritional addition of
120 0.45% dry matter and 0.25% crude protein. Supplemented diets can therefore be considered iso
121 nutrients compared to their control. Diets were stored at -20 °C until used. Details on the
122 proximate composition and amino acid profiles of each diet are provided in Tables 1 and 2.
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2.2. *Fish and feeding trial*

125 Juveniles of red seabream were obtained from a local hatchery and subsequently reared at the
126 Marine Sciences Institute of Jeju National University, South Korea. Upon arrival, the fish
127 underwent a 30-minute oxytetracycline treatment (100 ppm bath). For an acclimation period
128 of two weeks, the fish were fed a commercial diet (VOGUELUCK, Jeju, South Korea; 52%
129 crude protein, 8% crude lipid).

130 A total of 960 fish (mean initial body weight: 27.2 ± 0.11 g) were distributed randomly among
131 24 circular tanks (40 fish per 240 L tank), each receiving a continuous flow of sand-filtered
132 seawater at 3 L/min. Aeration in each tank ensured dissolved oxygen levels of 7.5 ± 0.6 ppm.
133 During the summer season, water temperatures ranged from 20.5°C to 29.7°C, reflecting
134 natural coastal conditions. Six tanks were assigned randomly to each dietary treatment. Fish
135 were fed the test diets to visual satiety twice daily (at 09:30 and 18:30) for 15 weeks. Uneaten
136 feed was collected 20-30 minutes after feeding, dried at 125°C for 4 hours, and weighed. Feed
137 intake was calculated by adjusting for the initial moisture content and subtracting the uneaten
138 feed weight from the total feed offered. For the stress protocol, fish in three tanks per diet (n=3)
139 were exposed to daily stress, consisting of one minute of net chasing at 08:30 every morning.
140 This procedure was repeated for 7 days every two weeks, mirroring intermittent farm-related
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stressors. Compared to a daily stress model used in a previous study (Herault et al., 2023), this
approach imposed less frequent stress. Fish were weighed in bulk before and after each 7-day
stress period, occurring weekly except during weeks 1, 4, 7, 10, and 13.

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## 2.3. Calculations of zootechnical & environmental parameters

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The zootechnical performance indicators were calculated as follows:

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$$ \text{Specific growth rate (\%d)} = [\ln(\text{final body weight (g)}) - \ln(\text{initial body weight(g)})] / \\ \text{duration of feeding trial (d)} \times 100 $$

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$$ \text{Feed intake (g)} = \text{total feed provided (g)} - \text{uneaten feed (including moisture content (g))} $$

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$$ \text{Economic feed conversion ratio (eFCR)} = \frac{\text{crude feed distributed}}{\text{final biomass (g) – initial biomass (g)}} $$

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$$ \text{Condition factor} = (\text{Fish weight (g)} / \text{Fish length(cm)}^3) \times 100 $$

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$$ \text{Fish Meal Consumption (kg of FM / kg of produced fish)} = \text{eFCR} \times \text{dietary FM content (\%)} $$

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In addition to FM consumption (FMC), we propose calculating the economic Fish in Fish Out (eFIFO) ratio to place greater emphasis on the limiting feed ingredient, whether FM or FO, that contributes the most to FIFO ratio (Kok et al., 2020).

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$$ \text{Economic fish in fish out ratio (eFIFO)} = eFCR \times (\% \text{ dietary FM content} \times Pm) + (\% \text{ dietary FO content} \times Po) $$
where Pm and Po are economic embodiments of FM and FO respectively, calculated based on the following formula:

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$$ \text{Pm or Po} = \frac{\text{EVm or EVo}}{(\text{EVm} \times \text{FmY} + \text{Evo} \times \text{FoY})} \text{ where EVm or EVo are economic values of FM, or FO (cost / mT). FmY and FoY are embodiments of FM and FO, which are usually 22.5\% and 5\%, respectively (Tacon and Metian, 2008). When writing this manuscript, FAQ65} $$

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FM and pelagic FO prices were 1,800 and 3,700 US$/mT respectively resulting in Pm and Po
at 1,95 and 4,01 respectively.

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## 2.4. Sample collection and analysis

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At the end of the feeding trial, following a 24-hour fasting period, each fish was individually
weighted. 9 fish were then randomly selected, and identified from 1 to 9 (Table 4), for tissue
sampling. These selected fish were euthanized immediately using a 500-ppm solution of 2-
phenoxyethanol.

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All the procedures used in this study to collect and analyze samples have been well described
in our previous research (Herault et al., 2023), with the controlled physiological biomarkers
listed in Table 4. We propose here to adapt this table to summarize sampled fish allocation and
tissues, and methods or commercial kits used for biomarker analysis (Table 4).

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## 2.5. Bacterial challenge

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Following the nutritional study, 15 individuals in each tank (total of 45 per statistical group) were randomly selected and intraperitoneally injected with 0.1mL of a suspension of *Edwardsiella tarda* (ATCC 15947, American type culture collection) at a concentration of 1×10⁶ CFU mL⁻¹. The *E. tarda* broth was prepared at the required concentration according to the method outlined by Bui et al. (2014). The fish were then placed into twenty-four 120L fiberglass aquarium. No mortality or abnormal behavior was observed during the first five days. Therefore, fish were re-injected with 0.1 mL of 1×10⁸ CFU mL⁻¹ *E.tarda* suspension from the same strain on day six of the trial. After the second injection, fish were kept in the same tanks and mortality was recorded for 11 days, when the survival rates remain steady. The injected fish were not fed, and the water temperature was maintained at 25°C throughout the bacterial challenge trial. Tanks were siphoned daily, with 75% of the water replaced.
## 2.6. Estimation of apparent digestibility coefficients

Apparent digestibility coefficients (ADCs) were estimated in specific dedicated trials, each of them lasting 10 days and repeated twice (n = 3).

The full procedure was previously described in Herault et al. (2023). In brief, modified Guelph system (Cho et al., 1982) was used to collect fish feces while 1% chromic oxide ($Cr_2O_3$) (Sigma-Aldrich, St. Louis, USA) was added to experimental feeds as an inert indicator of digestibility. Eighty red seabreams of 100g BW (bulk mean weight of 8kg) were distributed in each of the 4 fecal collection tank (300L). Chromic oxide levels were then analyzed following the procedure described by Divakaran et al. (2002) and applying following formula:

$$ ADC \ of \ nutrients \ or \ dry \ matter \ (%) = 100 - 100 \times (\% \ Cr_2O_3 \ in \ diet / \% \ Cr_2O_3 \ in \ faeces) \times (\% \ nutrients \ in \ faeces / \% \ nutrients \ or \ dry \ matter \ in \ diet) $$

## 2.7. Statistical analysis

All measurements were analyzed at the tank level, using the average value of all individuals within the tank. All statistical methods used in this study were previously described in Herault et al. (2023).

# 3. Results

## 3.1. Effects of dietary FM level and PE application on growth performance in control and stressed conditions

Red seabream responded well to the tested diets, including LFM, leading to consistent growth across all groups (Figure 1) and high survival rates exceeding 95%. The substitution of FM did not result in significant differences in growth and feed efficiency, whatever the stress
conditions (p > 0.05, Figures 2A and 2B). However, PE supplementation showed improved growth and feed performance for the two basal diets (p < 0.001), with increased effects for LFM dietary groups (p < 0.05 for the PExFM interaction). These effects were observed in stressed and controlled conditions (p > 0.05 for the SxPE interaction). PE supplementation also resulted in higher individual feed intake (Figure 2C; p < 0.05) especially for LFM dietary groups (p < 0.001 for PExFM interactions). The chasing stress significantly reduced the survival and growth performance of all groups (p < 0.01). Stress induction and PE supplementation showed effects on fish growth after 3 weeks of feeding trial, with each 1-week period of daily net chasing increasing the amplitude of these differences (Figure 1). FM replacement and chasing stress negatively impacted fish IGF-I mRNA expression (p ≤ 0.001) while PE dietary supplementation positively impacted it (p < 0.05 and non-significant interactions, Figure 2D).

No significant change was observed in the proximate compositions of the fish whole-body, or carcass, regardless of the PE supplementation or chasing stress (Tables S1 and S2). Similar to growth performance, fish condition factor was negatively impacted by chasing stress while it was improved by PE dietary coating (p < 0.001, Table S3). Repeated periods of chasing stress significantly lowered fish somatic indexes and liver fat content (p < 0.01). Dietary changes did not impact fish somatic indices nor viscera or liver fat contents (Table S3). Fish exposed to chasing stress conditions or receiving the FM substituted diets showed lower water retention in muscle tissue (p < 0.05, Table S3).

## 3.2. LFM and PE on the reduction of wild fish use

Because of little growth differences, 50% FM replacement with plant proteins led to a FM consumption (FMC) divided by around 2 (Figure 3A). PE supplementation significantly
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reduced FMC (p < 0.01), while stress increased FMC (p < 0.001), but both to a much lower

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extend. Similarly, 50% FM replacement resulted in a 30% reduction of eFIFO approximately

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(Figure 3B, p < 0.001). Stress and PE supplementation impacted eFIFO in the same direction

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as for FMC (p ≤ 0.001).

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### 3.3. Underlying digestive and stress physiology

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Dietary FM replacement significantly decreased ADC of proteins and amino acids but not of

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dry matter (Table 5). Dietary PE supplementation resulted in enhanced ADCs, regardless of

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FM levels. Interestingly, the LFM diet top-coated with 2% PE resulted in significantly higher

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ADC than the ones observed for HFM dietary group. In most cases, the availability of

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individual amino acids was enhanced by the dietary PE supplementation compared to the levels

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of the non-supplemented LFM and HFM diets (Table S4).

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While FM reduction only affected fish gut EH (p < 0.05, Table 6), repeated periods of chasing

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stress resulted in compromised status of fish gut morphometry, with significant impairment in

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EH and GC counts, and numerical decrease in VL and ID (p ≤ 0.065). Whatever the basal diet

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or stress conditions, PE dietary coating resulted in significantly improved values of all fish gut

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morphometric parameters (p < 0.01, Table 6). The Spearman correlation coefficient analysis

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revealed a significant negative correlation (p < 0.05, Table S7) between fish FCR values and

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their gut morphometrics, with the exception of EH (p > 0.05). It also indicated that all fish gut

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morphometric parameters analyzed in this study were strongly correlated among themselves (p

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≤ 0.001).

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Morphologies of fish intestine and liver tissues are illustrated with Figure S1 and S2,

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respectively. Higher liver vacuolation was observed in LFM non coated groups (Figure S2, b
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and f) while the lowest vacuolation was shown in the fish groups fed PE supplemented diets, regardless of stress conditions (Figure S2, c, d, g and h).

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Both plasma and muscle cortisol were strongly affected by repeated stress periods (p < 0.001, Table 7). However, fish tissues were not affected the same ways with 6-10 times higher cortisol levels in muscle of the stressed groups, while differences observed for circulating plasma cortisol were limited to 2-3 times the observed basal values. FM reduction also significantly increased cortisol levels in fish muscle (p < 0.05) but not in fish plasma. Fish receiving the PE supplement in the LFM diet exhibited muscular cortisol levels (p < 0.05), similar to those observed in groups receiving HFM, irrespective of stress conditions.

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Plasma levels of metabolic indicators were not influenced by feed changes, except for slightly lower circulating TG values observed in LFM groups (p < 0.05, Table S5). Repeated periods of stress showed a significant reduction of all fish circulating fat components (TG, cholesterol, LDL, p < 0.01, Table S5) except for HDL which increased. No effects on glucose and protein levels were observed.

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Haematocrit and haemoglobin levels were not impacted by the stress nor PE coating or FM reduction (p > 0.05, Table S6). However significant interactions (p < 0.01, Table S6) were shown for Ht between PE coating and FM reduction, indicating a differential trend of Ht values, following PE coating when associated to LFM groups.

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### 3.4. Survival to the bacterial challenge

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The survival kinetics and log-rank analysis clearly highlighted 3 distinct statistical groups (p < 0.001, Figure 4). In the first group, highest survival rates were observed for the non-stressed fish fed HFM basal diets or with PE dietary supplementations. Oppositely, the lowest survival
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rate, i.e. almost 7 times lower than the average one observed for the first statistical group, was
seen for the fish fed LFM control diet in stress conditions. Between these two groups, survival
rates at the end of the bacterial challenge were approximately 30% for LFM, and stressed fish
receiving either HFM basal diet or PE supplementation.

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### 3.5. *Underlying immune physiology*

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Except for lysozyme activity, all the measures of non-specific immune response were reduced in repeated stress conditions (Table 8). The dietary top coating with PE resulted in a substantial enhancement of all innate immune responses whatever the stress conditions (Table 8, SxPE interactions > 0.05), with the exception of myeloperoxidase levels. Dietary FM reduction resulted in significantly lower levels of lysozyme activities and circulating Ig. Significant interactions, between PE and FM were also observed for these two parameters, when fish groups received the LFM diet top-coated with PE.

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All markers associated with antioxidative capacities were substantially impacted by periodical repeated stress, resulting in a simultaneous decrease in TAC and an elevation in malondialdehyde muscle concentrations (Table 9). While FM reduction mostly impacted SOD, TAC and MDA (p < 0.05), dietary PE coating resulted in improved values of all parameters reflecting fish antioxidative status, with significant differences compared to LFM fish groups (p < 0.05) but not for HFM ones (Table 9).

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## **4. Discussion**

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295 4.1. *FM reduction preserves fish zootechnical performance but compromises stress levels and resistance to bacterial challenges.*

297 Consistent with previous findings (Herault et al., 2023), the reduction of dietary FM from 30 to 15% did not impair fish growth or FCR, despite a reduction in FI and IGF-I levels. However, the digestibility trial revealed a significantly lower ADC of protein (p < 0.05) in the LFM group, after 10 days of feeding, compared to the HFM group. This suggests that fish fed the LFM diet may exhibit an adaptive capacity, enabling them to compensate during the 15 weeks of feeding.
301 This compensation likely reflects the ability of the fish to adjust their digestive processes or metabolism over time, allowing for improved nutrient utilization despite the initial reduction in protein digestibility. This phenomenon was also observed in gilthead seabream in pilot scale trials implemented by Benedito-Palos et al. (2016) and Simo-Mirabet et al. (2018). However, Kokou et al. (2016) did not observe this trend, maybe because SPC was the only source of plant protein for FM replacement.
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308 Similarly to Herault et al. (2023), 50% FM replacement with plant proteins resulted in noticeable immunodeficiencies and reduced antioxidative defenses. Similarly to previous studies, FM reduction had no significant effects on plasmatic cortisol values (Ribeiro et al., 2015; Sadoul et al., 2016). However, low FM diets showed, in our current study, elevated levels of muscle cortisol, supposed to be more integrative. This suggests that replacing FM with plant proteins can sometimes be perceived by fish as a long-term nutritional stressor (Bonaldo et al., 2015), even though fish zootechnical parameters remain unaffected. Long term exposures to cortisol are known to be deleterious (Yada and Tort, 2016) and resulting immunodeficiencies are likely to make fish less resistant to any infection process from an opportunistic pathogen as illustrated in this study by the increased mortality kinetics observed in LFM fish group.
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This study confirmed that acceptable zootechnical performance can be achieved with red seabream sub-adults fed a diet containing only 15% fishmeal (FM). However, this comes at the cost of silently compromised fish welfare and health, possibly leading, like any significant stressor, to severe losses if a pathogen infection occurs.

## 4.2. *Intermittent repeated stress periods result in decreased fish performance and health.*

Compared to a previous study (Herault et al., 2023) where a 1 min net chasing stress was repeated daily for 15 weeks, the impact of the same stressor on fish zootechnical and health performance remained very severe in the current study despite of 2-weeks resting periods following the 1-week daily repeated stress.

Deleterious impact of repeated stress on fish welfare, feeding motivation and resulting compromised growth and health performance have been well described in the literature (Bernier et al. 2004; Sadoul and Vijayan, 2016; Yada and Tort, 2016).

In this research, the declines in zootechnical and health performance caused by intermittent repeated stress were similar for all diets, illustrated by very few interactions between FM replacement and stress conditions. This suggests an additive detrimental effects of dietary and handling stressors, also illustrated by survival kinetics. Such additive effects were previously observed despite a more pronounced stressor (Herault et al. 2023). Consequently, these results demonstrate that the diet, including PE addition, cannot mitigate absolute effects of stress.

## 4.3. *PE supplementation as an effective mitigator of reduced fish meal in the diet.*

The tested PE, combining shrimp hydrolysate and sensory flavors, aimed to enhance dietary palatability in marine fish. This was overall not the case for HFM diet, in both stressed and control diet, illustrated by similar food intake levels. However, the supplementation with PE increased feed intake and growth rates in red seabream groups fed the LFM diet, restoring the levels observed for the HFM groups. Similarly, Tola et al. (2019) reported improved FCR in
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red seabream on an SPC-based diet with 0.5% L-glutamic acid and 1.8% fish hydrolysate. Also,

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Tola et al. (2022) found that a 2.5% liquid PE made from tuna hydrolysate enhanced the protein

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efficiency ratio (PER) in Asian seabass fed a 40% SBM diet.

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As observed in Herault et al. (2023), these improvements in FCR were likely related to better

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gut morphology (VL, EH, ID, GC) compared to the LFM groups. Rahimnejad et al. (2023)

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confirmed positive effects on the gut morphology of pikeperch (*Sander lucioperca*) using the

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same PE, while Khosravi et al. (2018) saw similar results in olive flounder (*Paralichthys*

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olivaceus) with various protein hydrolysates at higher doses. Notably, ADC for DM, CP, and

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amino acids improved within 10 days of PE supplementation, indicating rapid gut remodeling.

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Calo et al. (2024) also reported improved ADC for crude protein and energy in rainbow trout

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(Oncorhynchus mykiss) supplied with a LFM feed containing umami PE but saw no FCR

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enhancement. Their study, along with Tola et al. (2019, 2022), did not find changes in stomach

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or intestine digestive functions while the impact of PE supplementation on fish microbiota

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would deserve further investigations.

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Fish supplemented with 2% PE showed enhanced health status, including significantly

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enhanced non-specific immune responses, antioxidants capacities and reduced levels of stress

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markers compared to the LFM diet, restoring values observed for the HFM groups. Khosravi

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et al. (2015) also reported improved non-specific immunity in red seabream and olive flounder

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fed HFM diets with 2% top-coated tuna or krill hydrolysates. In their study, possibly due to

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higher dietary FM levels (52 and 50% respectively), only red seabream receiving the tuna

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hydrolysate PE supplementation showed a significantly higher survival at the end of 21 days

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of bacterial injection challenge with 1 x 10⁵ CFU/mL⁻¹.
364 Dietary supplementation of a shrimp hydrolysate-based PE helped fish to cope with reduced
365 FM content, and mitigated bacterial challenge impacts in LFM groups through a significant
366 reduction of fish mortality. This is consistent with a variety of research on hydrolysates use in
367 aquatic species (Siddik et al., 2021). However, despite the well-documented benefits of
368 functional peptides, such as anti-microbial peptides (AMP), which have been shown to be
369 effective at low doses (Wang et al., 2023), it remains challenging to conceive such exhaustive
370 list of benefits with a liquid product (PE) applied at maximum 0.45% dry matter or 0.25%
371 crude protein. A recent study (Herault et al., unpublished), which followed similar
372 methodology in red seabream juveniles, showed a higher global performance resulting from
373 the dietary top-coating versus inclusion of the shrimp hydrolysate-based PE. These results
374 suggest that top-coating application of a PE makes it more available to fish gustative receptors
375 compared to when it is included in the feed matrix. The full mechanistics between a higher
376 feeding motivation, and enhanced fish health performance would deserve further
377 investigations. Part of it could rely on lower expressed cortisol levels, known to be immune-
378 suppressant when chronically high (Yada and Tort, 2016) and a higher contribution to fish
379 optimum nutritional requirements.

380 As described in Fournier (2013), PE dietary dosage can be reduced with fish pellet size due to
381 a lower volume-specific surface area. This will result in even lower environmental and
382 economic cost-in-use as most of aquafeed volumes consist in pellet size higher than 5mm
383 length.

384 Ensuring optimal feed palatability for juvenile fish by applying a 2% top-coating of shrimp
385 hydrolysate liquid PE has resulted in restored zootechnical and health performance,
386 compensating for the reduced FM content. Interestingly, zootechnical performances of PE
387 supplementation outperformed the HFM diet for both diets (HFM and LFM). Similar effects,
more pronounced were observed with a 5% supplementation of powdered APH (Herault et al., 2023). However, the difference in efficiency is counterbalanced by the lower economic and environmental costs of the PE tested in the present study.

### 4.4. Assessment of the potential sparing of wild fish using a shrimp hydrolysate-based PE.

In 2019, 22.52 million tons of fish - 86% being pelagic fish - were reportedly used as animal feed (Tacon et al., 2023) with 10% of them used -as FM and FO- in marine fish diets (Naylor et al., 2021). As mentioned before, a typical marine fish diet formulation currently contains 14% FM (Naylor et al., 2021) but this number hides great disparities between species, growth stages, markets but also feed manufacturers. While it is admitted that most of fish feed volumes consists in feed sizes longer than 5mm, shorter size feeds are containing higher contents of fish meal due to both higher nutritional requirements and conservatism among feed producers. In our research, we effectively decreased the proportion of FM derived from wild fish to 15% in a 3 to 4 mm diet produced for sub-adult red seabream.

Considering that the applied stress condition is more representative of commercial fish farming, we can assume that the FMC ratio to produce 1kg of crude fish biomass can reasonably be reduced to a maximum of 0.26 instead of 0.50 for red seabream fed HFM diet (Figure 3A). This 52% FMC reduction can be considered as realistic, and conservative, as fish groups receiving LFM+PE outperformed fish groups supplied with HFM control diet. Extending this FMC ratio to the entire Sparidae farming industry, which is mostly represented by gilthead seabream (282,1 kT in 2020; FAO 2022), and assuming similar nutritional properties as in red sea bream, it could result in a saving of more than 67.7 kT of FM derived from wild sources. A rough extrapolation to the entire marine fish farming industry would result in a saving of 594.8 kT per year (Naylor et al., 2021).
412

eFIFO can be considered as the most relevant indicator for assessing the sparing of wild fish biomass (Kok et al., 2020) in aquafeed industry. Because eFIFO accounts for the levels of oil included in the diet, and integrates that fish oil as the limiting ingredient on the market, the FM replacement resulted in a 25% eFIFO reduction in the stressed fish group while PE supplementation resulted in an additional 6% reduction. As a result, the eFIFO required to produce 1 kg of fish, from 27 to 125 g, was reduced from 2.05 down to 1.41 in farm-alike stress conditions.

413

414

415

416

417

418

419

This value, estimated for juveniles, can be compared with the 0.94 eFIFO calculated by Kok et al. (2020) for the entire marine fish farming industry. We can reasonably assume that this could also be reduced by 31% by generalizing the practice of PE coating on diets where 50% of FM is substituted. This would further support marine fish aquaculture in being a net fish producer. Extrapolating it to Sparidae, or whole marine fish industry, this would respectively represent a sparing of 87.5 kT and 768.3 kT of wild fish biomass.

420

421

422

423

424

425

Last but not least, it is interesting to note that a more ambitious study has recently been conducted in red seabream by Takakuwa et al. (2023) who fully substituted dietary FM with a mix of plant proteins and land-based proteins, during a full production cycle in commercial conditions. They observed a moderate, but significant, loss of growth that they explained with a lower feed intake and bile acid production. We may imagine that these issues would have been mitigated with the adequate use of a PE.

426

427

428

429

430

431

432

## 5. Conclusions and perspectives

433

This research supports that it is possible to obtain fully satisfying zootechnical and health performance in red seabream juvenile fed diets containing 47% plant protein and only 15% of FM, when top-coated with liquid PE formulated mainly from a sustainable source of shrimp

434

435
436 hydrolysate made from coproducts. However, we also confirmed, that it is mandatory to
437 supplement FM-substituted diets with a functional ingredient in order to offset the underlying
438 effects of plant proteins silently affecting fish welfare and health defenses.

439 Additionally, PE supplementation was essential to maintain satisfying fish performance under
440 intermittent periods of repeated husbandry stress, more representative of what fish would
441 normally experience in commercial farming operations.

442 Our results also suggest that FM content in diets could be further reduced, as juvenile fish fed
443 the PE-supplemented diet outperformed the control group. This would allow the Sparidae, and
444 possibly other marine fish, farming industries to further progress on their reliance on wild fish.

445 Such supplementations can therefore help alleviate concerns regarding aquaculture's use of
446 wild fish, though it is important to consider other potential environmental impacts. Therefore,
447 additional research is required to comprehensively evaluate the environmental footprint of
448 using these types of PE in aquaculture.

449

450 Declaration of competing interest and funding source

451 This study was supported by Symrise Aqua Feed (Taste, Nutrition & Health division of
452 Symrise Group), Elven, France and Basic Science Research Program through the National
453 Research Foundation of Korea (NRF) funded by the Ministry of Education
454 (2021R1A2C2008384).

455 Mikaël Herault and Vincent Fournier are working as Performance Measurement Manager and
456 R&D Manager at Symrise Aqua Feed.

457 Buddhi E. Gunathilaka and Kyeong-Jun Lee declare no conflict of interests.

458 Hervé Le Bris and Bastien Sadoul declare no conflict of interests.

459
460 Author contributions

461 Mikaël Herault participated to the conceptualization, formal analysis, and writing - original draft of the study. Vincent Fournier participated to the conceptualization of the study and funding acquisition. Bastien Sadoul participated to the formal analysis and writing - original draft of the study. Hervé Le Bris participated to the writing - original draft of the study. Buddhi E. Gunathilaka participated to the data curation, formal analysis and investigation of the study.

466 Kyeong-Jun Lee participated to the conceptualization, project administration and supervision of the study. All coauthors contributed to the writing - reviewing & editing.

468

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684
685 https://doi.org/10.1080/10888700701729106
<table><caption>**Table 1.** Formulation and proximate composition of the experimental diets for red seabream (*Pagrus major*) (%, dry matter basis)</caption><thead><tr><th rowspan="2">Ingredients</th><th colspan="4">Experimental diet</th></tr><tr><th>HFM<sup>1</sup></th><th>LFM<sup>2</sup></th><th>HFM+PE</th><th>LFM+PE</th></tr></thead><tbody><tr><td>FM FAQ65<sup>3</sup></td><td>30.00</td><td>15.00</td><td>30.00</td><td>15.00</td></tr><tr><td>PE<sup>4</sup> / coating</td><td></td><td></td><td>2.00</td><td>2.00</td></tr><tr><td>Soy protein concentrate<sup>5</sup></td><td>14.33</td><td>20.20</td><td>14.33</td><td>20.20</td></tr><tr><td>Wheat Gluten<sup>6</sup></td><td>9.00</td><td>12.00</td><td>9.00</td><td>12.00</td></tr><tr><td>Corn gluten meal<sup>7</sup></td><td>10.00</td><td>15.00</td><td>10.00</td><td>15.00</td></tr><tr><td>Wheat flour<sup>8</sup></td><td>22.00</td><td>21.00</td><td>22.00</td><td>21.00</td></tr><tr><td>Fish oil<sup>9</sup></td><td>4.95</td><td>6.85</td><td>4.95</td><td>6.85</td></tr><tr><td>Soybean oil<sup>10</sup></td><td>4.98</td><td>3.94</td><td>4.98</td><td>3.94</td></tr><tr><td>Mineral Mix<sup>11</sup></td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td></tr><tr><td>Vitamin Mix<sup>12</sup></td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td></tr><tr><td>Starch<sup>13</sup></td><td>2.14</td><td>1.04</td><td>2.14</td><td>1.04</td></tr><tr><td>Choline chloride<sup>14</sup></td><td>0.50</td><td>0.88</td><td>0.50</td><td>0.88</td></tr><tr><td>L-Lysine<sup>15</sup></td><td>0.00</td><td>0.37</td><td>0.00</td><td>0.37</td></tr><tr><td>L-Methionine<sup>16</sup></td><td>0.00</td><td>0.08</td><td>0.00</td><td>0.08</td></tr><tr><td>Taurine<sup>17</sup></td><td>0.10</td><td>0.14</td><td>0.10</td><td>0.14</td></tr><tr><td>Mono-calcium phosphate<sup>18</sup></td><td>0.00</td><td>1.50</td><td>0.00</td><td>1.50</td></tr><tr><td colspan="5"><em>Proximate composition (%, dry matter)</em></td></tr><tr><td>Crude protein</td><td>50.4</td><td>49.4</td><td>50.1</td><td>49.2</td></tr><tr><td>Crude lipid</td><td>13.3</td><td>12.5</td><td>13.6</td><td>13.0</td></tr><tr><td>Crude ash</td><td>7.07</td><td>6.31</td><td>7.14</td><td>6.22</td></tr><tr><td>Gross energy (MJ.kg<sup>-1</sup>)</td><td>21.78</td><td>21.59</td><td>21.84</td><td>21.65</td></tr><tr><td>Moisture</td><td>9.67</td><td>8.41</td><td>9.81</td><td>9.65</td></tr></tbody></table>

¹High fish meal

²Low fish meal

³Fish meal fair average quality (DM basis, 69.2% protein and 8.3% lipid), Orizon S.A., Co., Ltd., Chile.

⁴Palatability enhancer - Extrapal L410 (crude basis, 12.8% protein and 1.3% lipid), Symrise Aqua Feed, Ecuador.

⁵Soy protein concentrate (DM basis, 72.0% protein and 0.14% lipid), CJ CheilJedang Co., Ltd., Seoul, South Korea.

⁶Wheat gluten (DM basis, 80.7% protein and 1.5% lipid), Royal Ingredients Group, Alkmaar, Netherlands.

⁷Corn gluten meal (DM basis, 68.9% protein and 11.9% lipid), Daebong LF Co., Jeju, South Korea.

⁸Wheat flour (DM basis, 12.8% protein and 6.2% lipid), CJ CheilJedang Co., Ltd., Seoul, South Korea.

⁹Fish oil, E-wha Oil Industry, Busan, South Korea.

¹⁰Soybean oil, Ottogi Co., Ltd., Anyang, South Korea.

¹¹Mineral premix (g kg⁻¹ of mixture): MgSO₄·7H₂O, 80.0; NaH₂PO₄·2H₂O, 370.0; KCl, 130.0; Ferric citrate, 40.0; ZnSO₄·7H₂O, 20.0; Ca-lactate, 356.5; CuCl, 0.2; AlCl₃·6H₂O, 0.15; Na₂Se₂O₃, 0.01; MnSO₄·H₂O, 2.0; CoCl₂·6H₂O, 1.0.

¹² Vitamin premix (g kg⁻¹ of mixture): L-ascorbic acid, 121.2; DL-α tocopheryl acetate, 18.8; thiamin hydrochloride, 2.7; riboflavin, 9.1; pyridoxine hydrochloride, 1.8; niacin, 36.4; Ca-D-pantothenate,
12.7; myo-inositol, 181.8; D-biotin, 0.27; folic acid, 0.68; p-aminobezoic acid, 18.2; menadione, 1.8; retinyl acetate, 0.73; cholecalciferol, 0.003; cyanocobalamin, 0.003.

¹³Starch, Samyang Co., Ltd., Seoul, South Korea.

¹⁴Choline chloride, Solton-Biochem Co., Ltd., Cheonan, South Korea.

¹⁵L-Lysine, Ajinomoto Amino Acid Co., Ltd., Shanghai, China.

¹⁶L-Methionine, Evonik Pharmaceutical Co., Ltd., China.

¹⁷Taurine, Qianjiang Yongan Pharmaceutical Co., Ltd., Qianjiang, China.

¹⁸Mono-calcium phosphate, Sigma-Aldrich, Missouri, USA.

<table><caption>**Table 2.** Amino acid contents (% of the ingredient as dry matter basis) of the four experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) for red seabream.</caption><thead><tr><th rowspan="2"></th><th colspan="4">Experimental diets</th></tr><tr><th>HFM</th><th>LFM</th><th>HFM+PE</th><th>LFM+PE</th></tr></thead><tbody><tr><td colspan="5"><em>Non-essential amino acids</em></td></tr><tr><td>Aspartic acid</td><td>3.35</td><td>2.77</td><td>3.64</td><td>2.67</td></tr><tr><td>Alanine</td><td>2.29</td><td>1.97</td><td>2.69</td><td>1.85</td></tr><tr><td>Serine</td><td>1.85</td><td>1.81</td><td>2.08</td><td>1.72</td></tr><tr><td>Glutamic acid</td><td>9.54</td><td>9.64</td><td>10.5</td><td>9.21</td></tr><tr><td>Proline</td><td>2.78</td><td>2.53</td><td>2.99</td><td>2.63</td></tr><tr><td>Glycine</td><td>1.87</td><td>1.50</td><td>2.07</td><td>1.43</td></tr><tr><td>Tyrosine</td><td>1.22</td><td>1.19</td><td>1.45</td><td>1.12</td></tr><tr><td colspan="5"><em>Essential amino acids</em></td></tr><tr><td>Arginine</td><td>2.37</td><td>2.04</td><td>2.63</td><td>1.93</td></tr><tr><td>Threonine</td><td>1.58</td><td>1.35</td><td>1.79</td><td>1.29</td></tr><tr><td>Valine</td><td>2.07</td><td>1.77</td><td>2.34</td><td>1.68</td></tr><tr><td>Phenylalanine</td><td>2.05</td><td>1.96</td><td>2.32</td><td>1.85</td></tr><tr><td>Isoleucine</td><td>1.87</td><td>1.63</td><td>2.1</td><td>1.56</td></tr><tr><td>Leucine</td><td>3.56</td><td>3.68</td><td>4.22</td><td>3.46</td></tr><tr><td>Histidine</td><td>1.13</td><td>0.90</td><td>1.23</td><td>0.86</td></tr><tr><td>Lysine</td><td>1.99</td><td>1.49</td><td>2.23</td><td>1.41</td></tr><tr><td>Methionine</td><td>0.93</td><td>0.81</td><td>1.10</td><td>0.76</td></tr><tr><td>Taurine</td><td>0.35</td><td>0.24</td><td>0.40</td><td>0.23</td></tr></tbody></table>

<table><caption>**Table 3.** Chemical compositions of Extrapal Shrimp, a palatability enhancer (PE) formulated from a shrimp liquid hydrolysate (from product technical data sheets).</caption><thead><tr><th></th><th>PE</th></tr></thead><tbody><tr><td>Dry matter, DM (%)</td><td>≥ 21</td></tr><tr><td>Protein (%)</td><td>≥ 11</td></tr><tr><td>Lipid (% DM)</td><td>≤ 5</td></tr><tr><td>Ash (% DM)</td><td>≤ 8</td></tr><tr><td>Soluble nitrogen (% N)</td><td>91.1</td></tr><tr><td>Molecular weight repartition (% of peptides)</td><td></td></tr><tr><td>Peptides > 30,000 Da</td><td>&lt;0.1</td></tr></tbody></table>
<table><tr><td>Peptides 20,000 - 30,000 Da</td><td>&lt;0.1</td></tr><tr><td>Peptides 10,000 - 20,000 Da</td><td>&lt;0.1</td></tr><tr><td>Peptides 5,000 - 10,000 Da</td><td>&lt;0.5</td></tr><tr><td>Peptides 1,000 - 5,000 Da</td><td>8</td></tr><tr><td>Peptides 500 - 1,000 Da</td><td>9</td></tr><tr><td>Peptides &lt; 500 Da</td><td>83</td></tr></table>
<table><caption>**Table 4.** Physiological biomarkers used and methods</caption><thead><tr><th>Category</th><th>Physiological biomarker</th><th>Acronym</th><th>Number of samples per tank</th><th>Sampled fish ID</th><th>Sampled tissues</th><th>Methods</th></tr></thead><tbody><tr><td rowspan="2">Zootechnical</td><td>Insulin Growth Factor-I</td><td>IGF-1</td><td>3</td><td>4, 5, 6</td><td>L</td><td>Gunathilaka et al. (2021), primers: Hossain et al. (2016)</td></tr><tr><td>Condition Factor</td><td>CF</td><td>2</td><td>7, 8</td><td>WB</td><td>x</td></tr><tr><td rowspan="7">Metabolic</td><td>Proximate composition</td><td>DM</td><td>3, pooled</td><td>4, 5, 6</td><td>WB</td><td>AOAC (1995)</td></tr><tr><td>Proximate composition</td><td>DM</td><td>3, pooled</td><td>7, 8, 9</td><td>C</td><td>AOAC (1995)</td></tr><tr><td>Liver lipid</td><td>LL</td><td>2, pooled</td><td>7, 8, 9</td><td>L</td><td>AOAC (1995)</td></tr><tr><td>Viscera lipid</td><td>VL</td><td>2, pooled</td><td>1, 2</td><td>V</td><td>AOAC (1995)</td></tr><tr><td>Muscle water retention</td><td>MWR</td><td>2, pooled</td><td>1, 2</td><td>M</td><td>AOAC (1995)</td></tr><tr><td>Hepato-somatic index</td><td>HSI</td><td>2</td><td>7, 8, 9</td><td>L</td><td>x</td></tr><tr><td>Viscera-somatic index</td><td>VSI</td><td>2</td><td>7, 8, 9</td><td>V</td><td>x</td></tr><tr><td rowspan="6">Blood biochemistry</td><td>Glucose</td><td>-</td><td>3</td><td>1, 2, 3</td><td>P</td><td>Gunathilaka et al. (2021)</td></tr><tr><td>Triglyceride</td><td>TG</td><td>3</td><td>1, 2, 3</td><td>P</td><td>CK#2200-225 from Biovision Inc. (Moutainview, USA)</td></tr><tr><td>Total cholesterol</td><td>-</td><td>3</td><td>1, 2, 3</td><td>P</td><td>CK#1010-225 from Biovision Inc. (Moutainview, USA)</td></tr><tr><td>High density lipoprotein</td><td>HDL</td><td>2</td><td>1, 2, 3</td><td>P</td><td>CK#K613-100 from Biovision Inc. (Moutainview, USA)</td></tr><tr><td>Low density lipoprotein</td><td>LDL</td><td>2</td><td>1, 2, 3</td><td>P</td><td>CK#K613-100 from Biovision Inc. (Moutainview, USA)</td></tr><tr><td>Total protein</td><td>-</td><td>3</td><td>1, 2, 3</td><td>P</td><td>Gunathilaka et al. (2021)</td></tr><tr><td rowspan="6">Immune – non-specific</td><td>Antiprotease</td><td>Ap</td><td>3</td><td>1, 2, 3</td><td>P</td><td>Ellis (1990) with slight modification (Magnadóttir et al., 1999)</td></tr><tr><td>Lysozyme</td><td>LYS</td><td>3</td><td>4, 5, 6</td><td>S</td><td>Khosravi et al. (2015a)</td></tr><tr><td>Myeloperoxidase</td><td>MPO</td><td>3</td><td>4, 5, 6</td><td>S</td><td>Quade and Roth (1997)</td></tr><tr><td>Respiratory burst activity</td><td>NBT</td><td>3</td><td>4, 5, 6</td><td>S</td><td>NBT, Anderson and Siwicki, 1995</td></tr><tr><td>Total immunoglobulin</td><td>Ig</td><td>3</td><td>1, 2, 3</td><td>P</td><td>Siwicki and Anderson (1993)</td></tr></tbody></table>
<table><caption>**Table 4 (continued).** Physiological biomarkers used and methods</caption><thead><tr><th>Category</th><th>Physiological biomarker</th><th>Acronym</th><th>Number of samples per tank</th><th>Sampled fish ID</th><th>Sampled tissues</th><th>Methods</th></tr></thead><tbody><tr><td rowspan="6">Health</td><td>Catalase</td><td>CAT</td><td>2</td><td>4, 5</td><td>S</td><td>CK#K773-100 from Biovision Inc. (Moutainview, USA)</td></tr><tr><td>Super oxide dismutase</td><td>SOD</td><td>3</td><td>4, 5, 6</td><td>S</td><td>CK#19160 from Sigma</td></tr><tr><td>Glutathione peroxidase</td><td>GPx</td><td>3</td><td>4, 5, 6</td><td>S</td><td>CK#K762-100 from Biovision Inc. (Moutainview, USA)</td></tr><tr><td>Total antioxidant capacity</td><td>TAC</td><td>2</td><td>1, 2</td><td>M</td><td>CK#CS0790 from Sigma</td></tr><tr><td>Malondialdehyde</td><td>MDA</td><td>2</td><td>1, 2</td><td>M</td><td>CK#K739-100 from Biovision Inc. (Moutainview, USA)</td></tr><tr><td>Cortisol</td><td></td><td>3</td><td>1, 2, 3</td><td>P/M</td><td>CK# Fish CSB-E08487f from Cusabio</td></tr><tr><td rowspan="5">Gut morphometrics</td><td>Hematocrit</td><td>Ht</td><td>3</td><td>1, 2, 3</td><td>HB</td><td>Gunathilaka et al. (2021)</td></tr><tr><td>Hemoglobin</td><td>Hg</td><td>3</td><td>1, 2, 3</td><td>P</td><td>Gunathilaka et al. (2021)</td></tr><tr><td>Villi length</td><td>VL</td><td>3</td><td>7, 8, 9</td><td>V</td><td>Gunathilaka et al., 2020</td></tr><tr><td>Enterocyte height</td><td>EH</td><td>3</td><td>7, 8, 9</td><td>V</td><td>Gunathilaka et al., 2020</td></tr><tr><td>Intestinal diameter</td><td>ID</td><td>3</td><td>7, 8, 9</td><td>V</td><td>Gunathilaka et al., 2020</td></tr><tr><td></td><td>Goblet cell count</td><td>GC</td><td>3</td><td>7, 8, 9</td><td>V</td><td>Gunathilaka et al., 2020</td></tr></tbody></table>

HB: heparinized blood, P: plasma, S: Serum

M: Muscle, V: viscera, L: Liver

WB: Whole Body, C: carcass

CK: commercial kit
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Mean weight kinetics of red seabream juveniles fed experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) for 15 weeks and submitted to control or periodical stress conditions (net chased daily during periods identified with the horizontal black line). Statistical significance is indicated with asterisks (1-way and 2-way ANOVA) and NS when *p* > 0.05).</figcaption></figure>

<figure><img src="image_3.png" /></figure>
**Figures 2.** Zootechnical parameters of red seabream groups fed the experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) for 15 weeks and submitted to control or periodical stress conditions (1 week daily net chasing every 2 weeks, S) 15 weeks feeding trial. **A.** Specific Growth Rates (%/d), **B.** Economic feed conversion ratio **C.** Feed intakes (g/fish), **D.** Liver insulin-like growth factors I (relative expression of mRNA). Statistical significance is indicated with asterisks (2-way ANOVA) and NS when $p > 0.05$).

<figure><img src="image_4.png" /></figure>

**Figures 3.** Fisheries resources efficiency metrics of red seabream groups after 15 weeks fed on experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) and submitted to control or periodical stress conditions (1 week daily net chasing every 2 weeks, S). **A.** Fish meal consumption (FMC, kg of FM/kg of produced biomass), **B.** Economic fish in fish out ratio (eFIFO). Statistical significance is indicated with asterisks (2-way ANOVA) and NS when $p > 0.05$).

**Table 5.** Apparent digestibility coefficients (%, of ADC) for protein and dry matter of the four experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) for red seabream.

<table><thead><tr><th></th><th>ADCd<sup>1</sup></th><th>ADCp<sup>2</sup></th><th>ADCa<sup>3</sup></th></tr></thead><tbody><tr><td>HFM</td><td>62.2±1.28<sup>b</sup></td><td>86.7±0.45<sup>b</sup></td><td>81.7±3.69<sup>b</sup></td></tr><tr><td>LFM</td><td>61.3±1.36<sup>b</sup></td><td>82.5±0.61<sup>c</sup></td><td>72.1±1.44<sup>a</sup></td></tr><tr><td>HFM+PE</td><td>69.2±3.00<sup>a</sup></td><td>90.8±0.90<sup>a</sup></td><td>87.5±2.17<sup>b</sup></td></tr><tr><td>LFM+PE</td><td>73.0±2.62<sup>a</sup></td><td>91.3±0.85<sup>a</sup></td><td>82.9±2.12<sup>b</sup></td></tr></tbody></table>
Factorial ANOVA (P>F)

<table><tr><td>PE coating</td><td>0.001</td><td>0.000</td><td>0.000</td></tr><tr><td>FM reduction</td><td>0.303</td><td>0.003</td><td>0.001</td></tr><tr><td>Interactions PExFM</td><td>0.105</td><td>0.001</td><td>0.124</td></tr></table>

Data are presented as mean of triplicate tanks ± S.D. Values with different superscripts in the same column are significantly different (P < 0.05). ¹Apparent digestibility coefficients of dry matter (%); ²Apparent digestibility coefficients of protein (%);³ Apparent digestibility coefficients of total amino acids (%) based on their dietary ponderation.

<table><caption>**Table 6.** Morphometric parameters of red seabream intestine fed the four experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) and submitted to control or periodical stress conditions (1-week daily net chasing every 2 weeks, S) for 15 weeks.</caption><thead><tr><th></th><th>VL<sup>1</sup></th><th>EH<sup>2</sup></th><th>ID<sup>3</sup></th><th>GC<sup>4</sup></th></tr></thead><tbody><tr><td>HFM</td><td>1456±38<sup>ab</sup></td><td>57.7±2.38<sup>a</sup></td><td>3512±402</td><td>876±8.8<sup>abc</sup></td></tr><tr><td>LFM</td><td>1196±84<sup>b</sup></td><td>47.8±1.49<sup>c</sup></td><td>2855±101</td><td>855±55<sup>bc</sup></td></tr><tr><td>HFM+PE</td><td>1395±65<sup>ab</sup></td><td>54.6±1.43<sup>ab</sup></td><td>3516±206</td><td>910±54<sup>abc</sup></td></tr><tr><td>LFM+PE</td><td>1572±117<sup>a</sup></td><td>56.5±3.08<sup>a</sup></td><td>3715±325</td><td>974±23<sup>a</sup></td></tr><tr><td>HFM-S</td><td>1224±149<sup>b</sup></td><td>48.7±3.90<sup>bc</sup></td><td>2891±521</td><td>835±32<sup>c</sup></td></tr><tr><td>LFM-S</td><td>1203±68<sup>b</sup></td><td>47.6±2.26<sup>c</sup></td><td>2804±309</td><td>809±17<sup>c</sup></td></tr><tr><td>HFM+PE-S</td><td>1379±157<sup>ab</sup></td><td>53.2±1.58<sup>abc</sup></td><td>3343±428</td><td>884±53<sup>abc</sup></td></tr><tr><td>LFM+PE-S</td><td>1445±36<sup>ab</sup></td><td>52.3±0.53<sup>abc</sup></td><td>3471±144</td><td>959±24<sup>ab</sup></td></tr><tr><td colspan="5">Factorial ANOVA (P>F)</td></tr><tr><td>Chasing stress</td><td>0.056</td><td>0.005</td><td>0.065</td><td>0.049</td></tr><tr><td>PE coating</td><td>0.001</td><td>0.005</td><td>0.002</td><td>0.000</td></tr><tr><td>FM reduction</td><td>0.840</td><td>0.045</td><td>0.460</td><td>0.143</td></tr><tr><td>Interactions SxPE</td><td>0.653</td><td>0.460</td><td>0.648</td><td>0.430</td></tr><tr><td>Interactions SxFM</td><td>0.485</td><td>0.210</td><td>0.378</td><td>0.952</td></tr><tr><td>Interactions PExFM</td><td>0.010</td><td>0.019</td><td>0.069</td><td>0.006</td></tr></tbody></table>

Data are presented as mean of triplicate tanks ± S.D. Values with different superscripts in the same column are significantly different (P < 0.05). The lack of superscript letter indicates no significant differences among treatments. ¹Villi length (µm); ²Enterocyte height (µm); ³Intestinal diameter (µm) and ⁴Goblet cell count.

<table><caption>**Table 7.** Muscle and plasma cortisol levels of red seabream fed the four experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) and submitted to control or periodical stress conditions (1-week daily net chasing every 2 weeks, S) for 15 weeks.</caption><thead><tr><th>Muscle cortisol</th><th>Plasma cortisol</th></tr></thead></table>
<table><tbody><tr><td>HFM</td><td>3.37±2.03<sup>b</sup></td><td>5.08±1.00</td></tr><tr><td>LFM</td><td>13.60±0.64<sup>a</sup></td><td>6.06±1.16</td></tr><tr><td>HFM+PE</td><td>4.59±1.10<sup>b</sup></td><td>5.05±0.93</td></tr><tr><td>LFM+PE</td><td>4.27±1.00<sup>b</sup></td><td>5.49±1.04</td></tr><tr><td>HFM-S</td><td>31.1±6.48<sup>b</sup></td><td>14.3±4.00</td></tr><tr><td>LFM-S</td><td>83.6±21.1<sup>a</sup></td><td>16.3±0.36</td></tr><tr><td>HFM+PE-S</td><td>44.4±12.7<sup>b</sup></td><td>12.0±2.02</td></tr><tr><td>LFM+PE-S</td><td>34.5±10.2<sup>b</sup></td><td>13.2±1.26</td></tr><tr><td>Factorial ANOVA (P>F)</td><td></td><td></td></tr><tr><td>Chasing stress</td><td>0.000</td><td>0.000</td></tr><tr><td>PE coating</td><td>0.041</td><td>0.052</td></tr><tr><td>FM reduction</td><td>0.017</td><td>0.123</td></tr><tr><td>Interactions SxPE</td><td>0.181</td><td>0.113</td></tr><tr><td>Interactions SxFM</td><td>0.119</td><td>0.537</td></tr><tr><td>Interactions PExFM</td><td>0.002</td><td>0.656</td></tr></tbody></table>

Data are presented as mean of triplicate tanks ± S.D. Values with different superscripts in the same column are significantly different (P < 0.05) – cortisol values were distinguished for the 1-way ANOVA between stressed and non-stressed dietary groups due to very different values between the 2 stress conditions groups. Cortisol (ng mL⁻¹).

<table><caption>**Table 8.** Non-specific immune response parameters of red seabream fed the four experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) and submitted to control or periodical stress conditions (1-week daily net chasing every 2 weeks, S) for 15 weeks.</caption><thead><tr><th></th><th>AP<sup>1</sup></th><th>LYS<sup>2</sup></th><th>MPO<sup>3</sup></th><th>NBT<sup>4</sup></th><th>Ig<sup>5</sup></th></tr></thead><tbody><tr><td>HFM</td><td>15.0±0.5<sup>abc</sup></td><td>5.65±0.56<sup>abc</sup></td><td>0.41±0.04<sup>ab</sup></td><td>1.06±0.06</td><td>22.7±2.87<sup>bcd</sup></td></tr><tr><td>LFM</td><td>14.4±1.2<sup>bcd</sup></td><td>4.24±0.39<sup>bc</sup></td><td>0.38±0.01<sup>abcd</sup></td><td>1.01±0.05</td><td>19.0±0.95<sup>d</sup></td></tr><tr><td>HFM+PE</td><td>16.2±0.6<sup>ab</sup></td><td>6.28±0.62<sup>a</sup></td><td>0.43±0.05<sup>a</sup></td><td>1.08±0.02</td><td>27.0±0.42<sup>a</sup></td></tr><tr><td>LFM+PE</td><td>16.8±1.5<sup>a</sup></td><td>6.37±0.10<sup>a</sup></td><td>0.41±0.01<sup>abc</sup></td><td>1.08±0.02</td><td>25.8±1.15<sup>ab</sup></td></tr><tr><td>HFM-S</td><td>12.5±0.6<sup>d</sup></td><td>5.78±0.61<sup>abc</sup></td><td>0.32±0.05<sup>bcd</sup></td><td>0.99±0.02</td><td>21.0±2.19<sup>cd</sup></td></tr><tr><td>LFM-S</td><td>12.6±0.9<sup>d</sup></td><td>4.04±0.50<sup>c</sup></td><td>0.34±0.01<sup>bcd</sup></td><td>0.99±0.05</td><td>18.8±0.93<sup>d</sup></td></tr><tr><td>HFM+PE-S</td><td>12.8±0.4<sup>cd</sup></td><td>6.19±1.37<sup>a</sup></td><td>0.32±0.01<sup>cd</sup></td><td>1.02±0.03</td><td>24.2±0.24<sup>abc</sup></td></tr><tr><td>LFM+PE-S</td><td>13.6±0.6<sup>cd</sup></td><td>5.96±0.45<sup>ab</sup></td><td>0.31±0.03<sup>d</sup></td><td>1.04±0.01</td><td>25.1±0.93<sup>abc</sup></td></tr><tr><td>Factorial ANOVA (P>F)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Chasing stress</td><td>0.000</td><td>0.601</td><td>0.000</td><td>0.007</td><td>0.034</td></tr><tr><td>PE coating</td><td>0.002</td><td>0.000</td><td>0.795</td><td>0.015</td><td>0.000</td></tr><tr><td>FM reduction</td><td>0.519</td><td>0.006</td><td>0.298</td><td>0.533</td><td>0.016</td></tr><tr><td>Interactions SxPE</td><td>0.100</td><td>0.693</td><td>0.106</td><td>0.812</td><td>0.499</td></tr><tr><td>Interactions SxFM</td><td>0.476</td><td>0.542</td><td>0.259</td><td>0.323</td><td>0.122</td></tr><tr><td>Interactions PExFM</td><td>0.163</td><td>0.011</td><td>0.610</td><td>0.265</td><td>0.027</td></tr></tbody></table>
Data are presented as mean of triplicate tanks ± S.D. Values in the same row having different superscript letters are significantly different (P < 0.05). ¹Antiprotease (% inhibition); ²Lysozyme activity (µg mL⁻¹); ³Myeloperoxidase level; ⁴Nitro blue tetrazolium activity and ⁵Total immunoglobulin (mg mL⁻¹).

<table><caption>**Table 9.** Antioxidant enzyme activities, muscle antioxidant capacity and malondialdehyde level of red seabream fed the four experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) and submitted to control or periodical stress conditions (1-week daily net chasing every 2 weeks, S) for 15 weeks.</caption><thead><tr><th></th><th>CAT<sup>1</sup></th><th>SOD<sup>2</sup></th><th>GPx<sup>3</sup></th><th>TAC<sup>4</sup></th><th>MDA<sup>5</sup></th></tr></thead><tbody><tr><td>HFM</td><td>39.2±3.30</td><td>68.3±1.22<sup>ab</sup></td><td>120±1.04<sup>ab</sup></td><td>1.91±0.15<sup>ab</sup></td><td>0.46±0.05<sup>c</sup></td></tr><tr><td>LFM</td><td>38.3±2.90</td><td>63.1±2.04<sup>bcd</sup></td><td>118±3.44<sup>bc</sup></td><td>1.67±0.06<sup>cd</sup></td><td>0.77±0.03<sup>a</sup></td></tr><tr><td>HFM+PE</td><td>40.1±4.25</td><td>70.4±0.98<sup>a</sup></td><td>128±3.04<sup>a</sup></td><td>2.00±0.07<sup>a</sup></td><td>0.54±0.06<sup>bc</sup></td></tr><tr><td>LFM+PE</td><td>42.3±3.95</td><td>70.5±1.77<sup>a</sup></td><td>127±2.85<sup>a</sup></td><td>2.05±0.09<sup>a</sup></td><td>0.50±0.02<sup>bc</sup></td></tr><tr><td>HFM-S</td><td>34.6±2.53</td><td>61.3±3.25<sup>cd</sup></td><td>113±4.73<sup>bc</sup></td><td>1.63±0.06<sup>cd</sup></td><td>0.78±0.01<sup>a</sup></td></tr><tr><td>LFM-S</td><td>33.9±0.31</td><td>59.0±1.99<sup>d</sup></td><td>111±4.56<sup>c</sup></td><td>1.56±0.03<sup>d</sup></td><td>0.82±0.12<sup>a</sup></td></tr><tr><td>HFM+PE-S</td><td>39.0±5.58</td><td>67.2±1.01<sup>abc</sup></td><td>118±1.14<sup>bc</sup></td><td>1.83±0.02<sup>abc</sup></td><td>0.70±0.12<sup>ab</sup></td></tr><tr><td>LFM+PE-S</td><td>38.5±4.11</td><td>66.6±3.16<sup>abc</sup></td><td>118±1.90<sup>bc</sup></td><td>1.77±0.06<sup>bcd</sup></td><td>0.68±0.07<sup>ab</sup></td></tr><tr><td colspan="6">Factorial ANOVA (P>F)</td></tr><tr><td>Chasing stress</td><td>0.030</td><td>0.000</td><td>0.000</td><td>0.000</td><td>0.000</td></tr><tr><td>PE coating</td><td>0.030</td><td>0.000</td><td>0.000</td><td>0.000</td><td>0.008</td></tr><tr><td>FM reduction</td><td>0.969</td><td>0.031</td><td>0.349</td><td>0.038</td><td>0.053</td></tr><tr><td>Interactions SxPE</td><td>0.481</td><td>0.253</td><td>0.412</td><td>0.762</td><td>0.734</td></tr><tr><td>Interactions SxFM</td><td>0.677</td><td>0.515</td><td>0.963</td><td>0.594</td><td>0.093</td></tr><tr><td>Interactions PExFM</td><td>0.578</td><td>0.059</td><td>0.450</td><td>0.046</td><td>0.008</td></tr></tbody></table>

Data are presented as mean of triplicate tanks ± S.D. Values in the same row having different superscript letters are significantly different (P < 0.05). ¹Catalase activity (mU ml⁻¹); ²Superoxide dismutase (% inhibition); ³Glutathione peroxidase activity (mU ml⁻¹); ⁴Total antioxidant capacity (mM) and ⁵Malondialdehyde level (nmol mg⁻¹).
<figure><img src="image_5.png" /><figcaption>**Figure 4.** Survival of red seabream fed the four experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) and submitted to control or periodical stress conditions (1-week daily net chasing every 2 weeks, S) after challenge with *E. tarda*. At the beginning, fish were injected with *E. tarda* suspension containing 1 x 10⁶ CFU mL⁻¹. Different letters besides the legend denote significant differences between diet x stress groups at the *P* < 0.001 level (Log-rank analysis with Bonferroni post-hoc analysis).</figcaption></figure>

Appendices

<table><caption>**Table S1.** Whole-body proximate composition (%, dry matter) of red seabream fed the four experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) and submitted to control or periodical stress conditions (1-week daily net chasing every 2 weeks, S) for 15 weeks.</caption><thead><tr><th></th><th>Dry matter</th><th>Crude protein</th><th>Crude lipid</th><th>Crude ash</th></tr></thead><tbody><tr><td>HFM</td><td>35.8±1.51</td><td>17.9±0.22</td><td>12.3±0.87</td><td>4.16±0.65</td></tr><tr><td>LFM</td><td>34.6±1.43</td><td>19.7±1.79</td><td>12.9±2.20</td><td>4.13±1.01</td></tr><tr><td>HFM+PE</td><td>34.4±1.44</td><td>18.9±1.89</td><td>12.0±1.67</td><td>4.12±0.56</td></tr><tr><td>LFM+PE</td><td>34.6±0.99</td><td>19.6±0.70</td><td>12.6±1.65</td><td>4.48±0.77</td></tr><tr><td>HFM-S</td><td>35.6±1.26</td><td>18.6±1.49</td><td>12.6±1.66</td><td>4.46±0.50</td></tr><tr><td>LFM-S</td><td>35.3±1.05</td><td>18.7±1.79</td><td>12.0±1.75</td><td>4.43±0.96</td></tr><tr><td>HFM+PE-S</td><td>35.1±0.46</td><td>19.1±1.70</td><td>12.3±2.19</td><td>4.35±0.71</td></tr><tr><td>LFM+PE-S</td><td>34.9±1.49</td><td>19.2±1.28</td><td>12.3±1.06</td><td>4.25±0.68</td></tr><tr><td colspan="5">Factorial ANOVA (P>F)</td></tr><tr><td>Chasing stress</td><td>0.379</td><td>0.836</td><td>0.850</td><td>0.625</td></tr><tr><td>PE coating</td><td>0.260</td><td>0.444</td><td>0.817</td><td>0.991</td></tr><tr><td>FM reduction</td><td>0.481</td><td>0.271</td><td>0.848</td><td>0.860</td></tr></tbody></table>
<table><tr><td>Interactions SxPE</td><td>0.787</td><td>0.964</td><td>0.800</td><td>0.621</td></tr><tr><td>Interactions SxFM</td><td>0.828</td><td>0.369</td><td>0.526</td><td>0.717</td></tr><tr><td>Interactions PExFM</td><td>0.477</td><td>0.678</td><td>0.842</td><td>0.796</td></tr></table>

Data are presented as mean of triplicate tanks ± S.D.

<table><caption>**Table S2.** Whole-body eviscerated carcass proximate composition (%, dry matter) of red seabream fed the four experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) and submitted to control or periodical stress conditions (1-week daily net chasing every 2 weeks, S) for 15 weeks.</caption><thead><tr><th></th><th>Dry matter</th><th>Crude protein</th><th>Crude lipid</th><th>Crude ash</th></tr></thead><tbody><tr><td>HFM</td><td>36.3±1.23</td><td>19.0±1.22</td><td>11.3±1.17</td><td>4.69±0.33</td></tr><tr><td>LFM</td><td>36.0±1.27</td><td>19.5±0.76</td><td>11.0±0.99</td><td>4.83±0.79</td></tr><tr><td>HFM+PE</td><td>35.4±1.85</td><td>20.1±0.63</td><td>11.6±0.75</td><td>4.66±0.96</td></tr><tr><td>LFM+PE</td><td>35.5±0.65</td><td>19.2±1.68</td><td>11.5±1.15</td><td>4.68±0.68</td></tr><tr><td>HFM-S</td><td>36.3±0.56</td><td>18.9±0.82</td><td>11.7±1.19</td><td>4.64±0.31</td></tr><tr><td>LFM-S</td><td>36.2±0.84</td><td>19.9±1.75</td><td>11.4±0.92</td><td>4.79±0.25</td></tr><tr><td>HFM+PE-S</td><td>35.8±1.32</td><td>20.1±1.10</td><td>11.1±0.79</td><td>4.82±0.36</td></tr><tr><td>LFM+PE-S</td><td>36.1±1.43</td><td>20.2±1.08</td><td>11.4±0.95</td><td>4.76±0.40</td></tr><tr><td colspan="5">Factorial ANOVA (P>F)</td></tr><tr><td>Chasing stress</td><td>0.599</td><td>0.485</td><td>0.908</td><td>0.883</td></tr><tr><td>PE coating</td><td>0.331</td><td>0.236</td><td>0.846</td><td>0.982</td></tr><tr><td>FM reduction</td><td>0.995</td><td>0.767</td><td>0.779</td><td>0.781</td></tr><tr><td>Interactions SxPE</td><td>0.673</td><td>0.738</td><td>0.425</td><td>0.709</td></tr><tr><td>Interactions SxFM</td><td>0.883</td><td>0.462</td><td>0.798</td><td>0.942</td></tr><tr><td>Interactions PExFM</td><td>0.686</td><td>0.207</td><td>0.602</td><td>0.731</td></tr></tbody></table>

Data are presented as mean of triplicate tanks ± S.D.

<table><caption>**Table S3.** Viscera proximate composition, muscle water retention and liver lipid content (%, wet basis) of red seabream fed the four experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) and submitted to control or periodical stress conditions (1-week daily net chasing every 2 weeks, S) for 15 weeks.</caption><thead><tr><th></th><th>CF<sup>1</sup></th><th>HSI<sup>2</sup></th><th>VSI<sup>3</sup></th><th>VL<sup>4</sup></th><th>MWR<sup>5</sup></th><th>LL<sup>6</sup></th></tr></thead><tbody><tr><td>HFM</td><td>1.93±0.03<sup>b</sup></td><td>1.45±0.17</td><td>7.52±0.95</td><td>39.6±2.32</td><td>73.1±0.79<sup>a</sup></td><td>29.1±7.06</td></tr><tr><td>LFM</td><td>1.95±0.07<sup>ab</sup></td><td>1.52±0.02</td><td>7.46±0.54</td><td>39.1±4.04</td><td>73.0±0.45<sup>ab</sup></td><td>28.1±1.87</td></tr><tr><td>HFM+PE</td><td>2.00±0.06<sup>ab</sup></td><td>1.44±0.07</td><td>7.14±0.10</td><td>41.6±3.25</td><td>73.4±0.79<sup>a</sup></td><td>30.8±3.90</td></tr><tr><td>LFM+PE</td><td>2.09±0.07<sup>a</sup></td><td>1.44±0.22</td><td>7.10±0.84</td><td>41.1±3.03</td><td>73.2±0.85<sup>a</sup></td><td>27.1±6.19</td></tr><tr><td>HFM-S</td><td>1.92±0.07<sup>bc</sup></td><td>1.26±0.09</td><td>6.19±0.12</td><td>41.6±3.69</td><td>73.1±0.68<sup>a</sup></td><td>23.6±1.75</td></tr><tr><td>LFM-S</td><td>1.87±0.03<sup>b</sup></td><td>1.28±0.17</td><td>6.13±0.06</td><td>40.8±1.93</td><td>71.0±0.68<sup>b</sup></td><td>23.5±3.04</td></tr><tr><td>HFM+PE-S</td><td>1.91±0.03<sup>b</sup></td><td>1.21±0.07</td><td>7.07±0.62</td><td>41.7±1.33</td><td>73.0±0.80<sup>ab</sup></td><td>23.9±1.58</td></tr><tr><td>LFM+PE-S</td><td>1.93±0.03<sup>b</sup></td><td>1.21±0.07</td><td>6.18±0.57</td><td>39.3±4.07</td><td>72.8±0.61<sup>ab</sup></td><td>23.5±1.76</td></tr></tbody></table>
Factorial ANOVA (P>F)

<table><tr><td>Chasing stress</td><td>0.001</td><td>0.001</td><td>0.001</td><td>0.897</td><td>0.037</td><td>0.004</td></tr><tr><td>PE coating</td><td>0.005</td><td>0.311</td><td>0.839</td><td>0.440</td><td>0.083</td><td>0.894</td></tr><tr><td>FM reduction</td><td>0.362</td><td>0.670</td><td>0.277</td><td>0.286</td><td>0.049</td><td>0.420</td></tr><tr><td>Interactions SxPE</td><td>0.084</td><td>0.862</td><td>0.095</td><td>0.184</td><td>0.318</td><td>0.957</td></tr><tr><td>Interactions SxFM</td><td>0.084</td><td>0.813</td><td>0.383</td><td>0.862</td><td>0.146</td><td>0.515</td></tr><tr><td>Interactions PExFM</td><td>0.150</td><td>0.670</td><td>0.413</td><td>0.572</td><td>0.141</td><td>0.648</td></tr></table>

Data are presented as mean of triplicate tanks ± S.D.

¹Condition factor, ²Hepatosomatic index = (Liver weight/ Fish weight) x 100, ³Viscerosomatic index = (Viscera weight/ Fish weight) x 10; ⁴Viscera crude lipid (%); ⁵Muscle water retention (%) and ⁶Liver crude lipid (%).

<table><caption>**Table S4.** Apparent digestibility coefficients (%, of ADC) for amino acid composition of the four experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) and submitted to control or periodical stress conditions (1-week daily net chasing every 2 weeks, S) for red seabream.</caption><thead><tr><th rowspan="2"></th><th colspan="4">Experimental diets</th></tr><tr><th>HFM</th><th>LFM</th><th>HFM+PE</th><th>LFM+PE</th></tr></thead><tbody><tr><td colspan="5"><em>Non-essential amino acids</em></td></tr><tr><td>Aspartic acid</td><td>82.5±4.16<sup>a</sup></td><td>72.1±3.01<sup>b</sup></td><td>86.3±2.21<sup>a</sup></td><td>82.1±1.97<sup>a</sup></td></tr><tr><td>Alanine</td><td>77.4±2.88<sup>b</sup></td><td>66.4±0.52<sup>c</sup></td><td>85.6±2.20<sup>a</sup></td><td>78.9±2.77<sup>b</sup></td></tr><tr><td>Serine</td><td>78.3±3.11<sup>b</sup></td><td>70.4±0.70<sup>c</sup></td><td>86.0±3.34<sup>a</sup></td><td>81.5±2.82<sup>ab</sup></td></tr><tr><td>Glutamic acid</td><td>83.7±4.28<sup>a</sup></td><td>74.2±1.72<sup>b</sup></td><td>88.8±2.68<sup>a</sup></td><td>84.8±2.27<sup>a</sup></td></tr><tr><td>Proline</td><td>91.6±6.4</td><td>80.0±6.42</td><td>91.3±1.75</td><td>89.1±6.53</td></tr><tr><td>Glycine</td><td>80.8±3.12<sup>a</sup></td><td>68.8±1.43<sup>b</sup></td><td>86.7±2.43<sup>a</sup></td><td>82.6±2.35<sup>a</sup></td></tr><tr><td>Tyrosine</td><td>77.4±3.04<sup>b</sup></td><td>68.2±0.95<sup>c</sup></td><td>87.1±2.82<sup>a</sup></td><td>81.6±2.88<sup>ab</sup></td></tr><tr><td colspan="5"><em>Essential amino acids</em></td></tr><tr><td>Arginine</td><td>81.0±3.28<sup>b</sup></td><td>71.3±0.53<sup>c</sup></td><td>88.2±3.01<sup>a</sup></td><td>83.0±2.95<sup>ab</sup></td></tr><tr><td>Threonine</td><td>78.6±3.28<sup>b</sup></td><td>69.1±0.60<sup>c</sup></td><td>85.6±2.91<sup>a</sup></td><td>80.7±2.81<sup>ab</sup></td></tr><tr><td>Valine</td><td>80.5±3.99<sup>a</sup></td><td>70.2±1.07<sup>b</sup></td><td>86.8±2.03<sup>a</sup></td><td>81.2±2.50<sup>a</sup></td></tr><tr><td>Phenylalanine</td><td>78.7±3.44<sup>b</sup></td><td>70.3±1.19<sup>c</sup></td><td>86.2±2.36<sup>a</sup></td><td>81.0±2.67<sup>ab</sup></td></tr><tr><td>Isoleucine</td><td>80.0±4.13<sup>a</sup></td><td>70.0±1.30<sup>b</sup></td><td>86.5±2.12<sup>a</sup></td><td>81.0±2.64<sup>a</sup></td></tr><tr><td>Leucine</td><td>77.2±3.51<sup>b</sup></td><td>70.6±0.93<sup>c</sup></td><td>86.0±2.22<sup>a</sup></td><td>80.7±2.67<sup>ab</sup></td></tr><tr><td>Histidine</td><td>83.0±3.36<sup>a</sup></td><td>73.5±0.51<sup>b</sup></td><td>88.5±2.30<sup>a</sup></td><td>83.2±2.70<sup>a</sup></td></tr><tr><td>Lysine</td><td>86.1±4.11<sup>a</sup></td><td>76.5±2.44<sup>b</sup></td><td>88.9±1.73<sup>a</sup></td><td>84.9±0.72<sup>a</sup></td></tr><tr><td>Methionine</td><td>79.0±3.60<sup>b</sup></td><td>71.4±0.24<sup>c</sup></td><td>87.2±2.58<sup>a</sup></td><td>82.3±2.58<sup>ab</sup></td></tr></tbody></table>

Data are presented as mean of triplicate tanks ± S.D. with different superscripts in the same row are significantly different (P < 0.05).
<figure><img src="image_6.png" /><figcaption>**Figures S1.** Intestine morphology of the red seabream fed the four experimental diets (high or low - H and L- fish meal -FM- with or without palatability enhancer -PE-) and submitted to control or periodical stress conditions (1-week daily net chasing every 2 weeks, S) for 15 weeks (A. villus height; magnification, x4). (a) HFM, (b) LFM, (c) HFM+PE, (d) LFM+PE, (e) HFM-S, (f) LFM-S, (g) HFM+PE-S, (h) LFM+PE-S</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figures S2.** Liver morphology of the red seabream fed the four experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) and submitted to control or periodical stress conditions (1-week daily net chasing every 2 weeks, S) for 15 weeks (A- blood vessels, B-vacuoles; magnification, x10). (a) HFM, (b) LFM, (c) HFM+PE, (d) LFM+PE, (e) HFM-S, (f) LFM-S, (g) HFM+PE-S, (h) LFM+PE-S</figcaption></figure>
<table><caption>**Table S5.** Biochemical parameters of red seabream fed the four experimental diets for 15 weeks.</caption><thead><tr><th></th><th>Glucose<sup>1</sup></th><th>Triglyceride<sup>2</sup></th><th>T. Protein<sup>3</sup></th><th>Cholesterol<sup>4</sup></th><th>HDL<sup>5</sup></th><th>LDL<sup>6</sup></th></tr></thead><tbody><tr><td>HFM</td><td>58.1±5.54<sup>ab</sup></td><td>136±22.8<sup>a</sup></td><td>3.70±0.48</td><td>179±12.8<sup>ab</sup></td><td>82.5±4.27</td><td>58.4±5.92<sup>ab</sup></td></tr><tr><td>LFM</td><td>50.5±12.3<sup>b</sup></td><td>107±11.2<sup>abc</sup></td><td>3.44±0.39</td><td>167±8.83<sup>abc</sup></td><td>83.1±2.28</td><td>51.8±10.9<sup>abc</sup></td></tr><tr><td>HFM+PE</td><td>58.6±3.68<sup>ab</sup></td><td>118±13.5<sup>ab</sup></td><td>3.36±0.46</td><td>179±12.6<sup>ab</sup></td><td>82.9±2.74</td><td>58.4±20.0<sup>ab</sup></td></tr><tr><td>LFM+PE</td><td>56.6±3.38<sup>ab</sup></td><td>119±16.6<sup>ab</sup></td><td>3.65±0.69</td><td>192±11.3<sup>a</sup></td><td>81.9±1.07</td><td>61.5±10.7<sup>a</sup></td></tr><tr><td>HFM-S</td><td>50.7±5.88<sup>b</sup></td><td>88.7±4.40<sup>bc</sup></td><td>3.50±0.23</td><td>161±7.41<sup>bc</sup></td><td>85.2±2.08</td><td>33.0±3.01<sup>bc</sup></td></tr><tr><td>LFM-S</td><td>68.3±3.30<sup>a</sup></td><td>84.9±5.87<sup>bc</sup></td><td>3.61±0.24</td><td>150±1.69<sup>c</sup></td><td>85.8±1.29</td><td>30.7±1.72<sup>bc</sup></td></tr><tr><td>HFM+PE-S</td><td>59.2±0.57<sup>ab</sup></td><td>88.7±13.2<sup>bc</sup></td><td>3.88±0.34</td><td>162±10.7<sup>bc</sup></td><td>87.2±1.13</td><td>25.7±8.25<sup>c</sup></td></tr><tr><td>LFM+PE-S</td><td>54.9±5.09<sup>ab</sup></td><td>71.6±3.91<sup>c</sup></td><td>3.43±0.56</td><td>151±5.47<sup>c</sup></td><td>84.1±2.29</td><td>27.4±4.28<sup>c</sup></td></tr><tr><td colspan="7">Factorial ANOVA (P>F)</td></tr><tr><td>Chasing stress</td><td>0.435</td><td>0.000</td><td>0.723</td><td>0.000</td><td>0.006</td><td>0.000</td></tr><tr><td>PE coating</td><td>0.889</td><td>0.424</td><td>0.938</td><td>0.143</td><td>0.870</td><td>0.950</td></tr><tr><td>FM reduction</td><td>0.751</td><td>0.047</td><td>0.704</td><td>0.197</td><td>0.450</td><td>0.799</td></tr><tr><td>Interactions SxPE</td><td>0.334</td><td>0.743</td><td>0.672</td><td>0.192</td><td>0.752</td><td>0.210</td></tr><tr><td>Interactions SxFM</td><td>0.064</td><td>0.752</td><td>0.629</td><td>0.164</td><td>0.591</td><td>0.859</td></tr><tr><td>Interactions PExFM</td><td>0.179</td><td>0.502</td><td>0.986</td><td>0.138</td><td>0.178</td><td>0.390</td></tr></tbody></table>

Data are presented as mean of triplicate tanks ± S.D. Values with different superscripts in the same column are significantly different (P < 0.05). The lack of superscript letter indicates no significant differences among treatments.; ¹Glucose (mg dL⁻¹); ²Triglyceride (mg dL⁻¹); ³Total protein (g dL⁻¹); ⁴Total cholesterol (mg dL⁻¹); ⁵High-density lipoprotein (mg dL⁻¹) and ⁶Low-density lipoprotein (mg dL⁻¹).

<table><caption>**Table S6.** hematological Ht and Hb levels of red seabream fed the four experimental diets (high or low -H and L- fish meal -FM- with or without palatability enhancer -PE-) and submitted to control or periodical stress conditions (1-week daily net chasing every 2 weeks, S) for 15 weeks.</caption><thead><tr><th></th><th>Ht<sup>2</sup></th><th>Hb<sup>3</sup></th></tr></thead><tbody><tr><td>HFM</td><td>39.1±0.69<sup>ab</sup></td><td>4.93±0.15</td></tr><tr><td>LFM</td><td>36.1±2.22<sup>ab</sup></td><td>4.39±0.53</td></tr><tr><td>HFM+PE</td><td>36.8±2.83<sup>ab</sup></td><td>4.54±0.24</td></tr><tr><td>LFM+PE</td><td>40.6±4.17<sup>a</sup></td><td>4.58±0.45</td></tr><tr><td>HFM-S</td><td>39.6±0.96<sup>ab</sup></td><td>4.54±0.81</td></tr><tr><td>LFM-S</td><td>33.6±1.71<sup>b</sup></td><td>4.02±0.16</td></tr><tr><td>HFM+PE-S</td><td>38.9±1.17<sup>ab</sup></td><td>4.64±0.26</td></tr><tr><td>LFM+PE-S</td><td>38.8±3.10<sup>ab</sup></td><td>4.56±0.42</td></tr><tr><td colspan="3">Factorial ANOVA (P>F)</td></tr><tr><td>Chasing stress</td><td>0.645</td><td>0.326</td></tr><tr><td>PE coating</td><td>0.097</td><td>0.548</td></tr><tr><td>FM reduction</td><td>0.178</td><td>0.129</td></tr><tr><td>Interactions SxPE</td><td>0.528</td><td>0.230</td></tr></tbody></table>
<table><tr><td>Interactions SxFM</td><td>0.087</td><td>0.871</td></tr><tr><td>Interactions PExFM</td><td>0.004</td><td>0.153</td></tr></table>

Data are presented as mean of triplicate tanks ± S.D. Values with different superscripts in the same column are significantly different (P < 0.05) – cortisol values were distinguished for the 1-way ANOVA between stressed and non-stressed dietary groups due to very different values between the 2 stress conditions groups. ¹Cortisol (ng mL⁻<fnref n="1" />); ²Hematocrit (%) and ³Hemoglobin (g dL⁻<fnref n="1" />).

<table><caption>**Table S7.** Spearman correlation coefficients between fish gut morphometrics and FCR (n=24 individuals, p-values are mentioned below the coefficients).</caption><thead><tr><th></th><th>FCR<sup>1</sup></th><th>EH<sup>2</sup></th><th>GC<sup>3</sup></th><th>ID<sup>4</sup></th><th>VL<sup>5</sup></th></tr></thead><tbody><tr><td>FCR</td><td></td><td>-0.3514</td><td>-0.6540</td><td>-0.4161</td><td>-0.4158</td></tr><tr><td></td><td></td><td><strong>0.092</strong></td><td><strong>0.002</strong></td><td><strong>0.046</strong></td><td><strong>0.046</strong></td></tr><tr><td>EH</td><td>-0.3514</td><td></td><td>0.4933</td><td>0.7790</td><td>0.8049</td></tr><tr><td></td><td></td><td><strong>0.092</strong></td><td><strong>0.018</strong></td><td><strong>0.000</strong></td><td><strong>0.000</strong></td></tr><tr><td>GC</td><td>-0.6540</td><td>0.4933</td><td></td><td>0.5026</td><td>0.6797</td></tr><tr><td></td><td></td><td><strong>0.002</strong></td><td><strong>0.018</strong></td><td><strong>0.016</strong></td><td><strong>0.001</strong></td></tr><tr><td>ID</td><td>-0.4161</td><td>0.7790</td><td>0.5026</td><td></td><td>0.8216</td></tr><tr><td></td><td></td><td><strong>0.046</strong></td><td><strong>0.000</strong></td><td><strong>0.016</strong></td><td><strong>0.000</strong></td></tr><tr><td>VL</td><td>-0.4158</td><td>0.8049</td><td>0.6797</td><td>0.8216</td><td></td></tr><tr><td></td><td></td><td><strong>0.046</strong></td><td><strong>0.000</strong></td><td><strong>0.001</strong></td><td><strong>0.000</strong></td></tr></tbody></table>

<footnote n="1">¹Feed Conversion Ratio; ²Enterocyte height (µm); ³Goblet cell count.; ⁴Intestinal diameter (µm) and ⁵Villi length (µm).</footnote>