# Temporal hedonic regulation of feeding behavior in rainbow trout: a role for the opioid system under plant-based diets from first feeding onward

Elisabeth Plagnes-Juan, Maud Martinat, Elodie Baranek, Anne Surget, Cécile

Heraud, Jérôme Roy

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Elisabeth Plagnes-Juan, Maud Martinat, Elodie Baranek, Anne Surget, Cécile Heraud, et al.. Temporal hedonic regulation of feeding behavior in rainbow trout: a role for the opioid system under plant-based diets from first feeding onward. Water Biology and Security, 2025, pp.100529. 10.1016/j.watbs.2025.100529. hal-05406131

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Research Article

# Temporal hedonic regulation of feeding behavior in rainbow trout: a role for the opioid system under plant-based diets from first feeding onward

Elisabeth Plagnes-Juan, Maud Martinat, Elodie Baranek, Anne Surget, Cécile Heraud, Jérôme Roy*

Université de Pau et des Pays de l'Adour, INRAE, NUMEA, Aquapôle, 64310, Saint-Pée-sur-Nivelle, France

ARTICLE INFO

Keywords:

First feeding

Aquaculture

Plant-based diet

Hedonic regulation

Feeding behavior

Brain

ABSTRACT

In rainbow trout (*Oncorhynchus mykiss*), early exposure to plant-based diets devoid of fishmeal and fish oil may alter feed intake and affect both nutrient sensing and reward pathways. While homeostatic regulation of feeding is well studied, the potential role of the opioid system in hedonic control during early development remains unexplored. This study investigates, for the first time, the involvement of the opioid system in the regulation of feeding behavior in fry and juveniles fed a plant-based diet (V diet) compared to a conventional diet (C diet) containing fishmeal and fish oil from first feeding to 8 months. Fish were sampled at 5 and 30 Day (fry) and 8 months (juveniles) after feeding initiation. At each age, fish were fasted for 16 h, sampled, re-fed and sampled again at 20 min and 6 h (4 h for juveniles). Expression of opioid receptors (*oprd1*, *oprk1*, *oprm1*, and *ogf1*) and precursors (*pdyn* and *penka*) was measured in the whole brain (fry) and telencephalon (juveniles). Gene expression changes were minimal at early stages but became more pronounced at 8 months, particularly in those fed the V diet. Significant differences were observed for *pdyn*, *penka*, *oprd1*, and *oprk1* expression levels, as well as increased serotonin turnover in the V diet group at 30 days and 8 months. Body weight was also lower in the V diet group at all ages. These findings highlight a delayed but diet-dependent modulation of the opioid system, suggesting its involvement in hedonic regulation under plant-based diets in rainbow trout.

1. Introduction

Understanding the regulation of feeding behavior in fishes remains a challenging task, particularly in aquaculture species where individual physiological responses to feeding can vary widely. In farming conditions, fish are typically fed in groups, and feed intake is measured at the tank level. This approach masks individual variability and prevents the detection of heterogeneous feeding responses. As a result, molecular analyses conducted after sampling often reveal significant inter-individual variability within the same group, complicating the interpretation of data related to appetite regulation. As in mammals (Blouet and Schwartz, 2010), the homeostatic regulation of feed intake in fishes is primarily orchestrated by the hypothalamus, specifically the nucleus lateralis tuberalis (an analog of the arcuate nucleus in mammals) (Soengas et al., 2018). This brain region harbors specific neuronal populations that express nutrient sensors and hormone receptors, enabling them to regulate feeding behavior. These neurons produce

orexigenic neuropeptides such as neuropeptide Y (Npy) and agouti-related peptide (Agrp), which stimulate appetite, as well as anorexigenic neuropeptides such as pro-opiomelanocortin (Pomc) and cocaine- and amphetamine-regulated transcript (Cart), which suppress it (Morton et al., 2014). In fishes, due to the lack of specific antibodies for these neuronal markers, expression analysis is typically conducted via qPCR. The mRNA levels of these neuropeptides in fishes are responsive to their feeding status (Bodas et al., 2023), indicating their pivotal role in the regulation of feed intake (Volkoff, 2016). However, they are expressed by a small neuronal population within the hypothalamus, an area that itself constitutes less than 10 % of the total brain volume. Consequently, some target genes may be expressed at very low levels, with cycle threshold (Ct) values approaching the detection limit (Ct 30–33), particularly in small-bodied life stages such as fry. This presents a technical limitation in accurately quantifying appetite-related gene expression.

Despite these challenges, understanding the biological mechanisms

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: jerome.roy@inrae.fr (J. Roy).</footnote>

<footnote>Peer review under the responsibility of Editorial Office of Water Biology and Security.</footnote>
controlling feed intake in farmed fishes is critical for improving adaptation to aquaculture conditions. This need is particularly urgent in the context of the industry's growing reliance on sustainable feed formulations (Naylor et al., 2009). Traditional aquafeeds, which are rich in fishmeal (FM) and fish oil (FO), raise increasing ecological and economic concerns. In 2022, approximately 18 million tonnes of these marine-derived ingredients were produced globally, a substantial portion of which was used in aquaculture. Projections by the Food and Agriculture Organization (FAO) indicate that the demand for aquaculture products is expected to increase by 22 % by 2030, further intensifying pressure on wild fish stocks (FAO, 2024). Consequently, there is a pressing need to develop alternative feed sources that reduce or eliminate the use of FM and FO, while maintaining optimal fish health and performance (FAO, 2024), contributing to a more environmentally friendly aquaculture model (Cottrell et al., 2020).

Among aquaculture species, rainbow trout (Oncorhynchus mykiss) is of particular interest due to its economic value and its well-characterized physiology. While plant-based diets offer a more sustainable alternative, full replacement of FM and FO with plant ingredients has been associated with significant drawbacks, especially during early development (Lazzarotto et al., 2018). Several studies have shown that total replacement of FM/FO with plant-based ingredients can impair growth and survival, particularly during the early life stages (Baranek et al., 2024; Callet et al., 2018; Fernández-Maestú et al., 2025; Heraud et al., 2022; Martinat et al., 2025; Panserat et al., 2009). Recently, long-term experiments from first feeding to eight months of age have indicated that reduced performance in rainbow trout may be linked to altered feeding behavior (Baranek et al., 2023) and a decreased preference for fish-free diets (Roy et al., 2020, 2022). At the juvenile stage, whether after a single meal or following long-term exposure to plant-based feeds, disruptions in nutrient sensing and dysregulation of neural pathways involved in appetite and reward have been documented (Baranek et al., 2023; Heraud et al., 2022). Notably, diet composition can influence hedonic perception in rainbow trout, i.e., the sensation of pleasure (by quantification of serotonin turnover in the telencephalon) experienced during feed consumption, which can in turn enhance feed intake (Baranek et al., 2023, 2024). Thus, beyond neuropeptide-based homeostatic regulation, food intake may also be modulated by the hedonic properties of the diet.

Homeostatic signals are driven by the physiological need to maintain energy balance, while hedonic signals are associated with sensory pleasure and palatability (Hernández Ruiz de Eguilaz et al., 2018). This distinction gives rise to what is known as “hedonic feeding behavior.” In both mammals and fishes, hedonic regulation is mediated by neural reward circuits, including the serotonin and dopamine systems (Tavares et al., 2020). These circuits can override homeostatic signals and lead to the consumption of highly palatable foods, even in the absence of energy deficits (Lutter and Nestler, 2009; Rossi and Stuber, 2018). In mammals, the opioid system is a central component of this reward circuitry and is critically involved in the hedonic aspects of feeding (Berthoud et al., 2017; Le Merrer et al., 2009). In fishes, mounting evidence supports the existence of an opioid system, with components including opioid receptors and precursors identified in several teleost species (Bao et al., 2019; Demin et al., 2018; Larhammar et al., 2015; Vallarino et al., 2012). In rainbow trout, following the identification of opioid receptor mRNA relative abundance in the brain (Rosengren et al., 2018), studies have indicated that the opioid system may lay a role in feeding regulation, particularly under acute feeding conditions using palatable, high-fat diets in juvenile rainbow trout (Díaz-Rúa et al., 2022). However, most existing studies have focused on short-term effects in juvenile rainbow trout, with little attention paid to the early feeding phase in fry a critical period for establishing feed acceptance and long-term performance (Geurden et al., 2013). The complex processes that govern food perception, and consequently the palatability of food, can undergo changes based on early life experiences, such as the acquisition of knowledge about food and its reward value (Birch, 1999; Ventura and

Worobey, 2013). The lack of data on the molecular regulation of feeding in rainbow trout fry limits our understanding of how early development of sensory and reward pathways may influence later phenotypes.

To address this knowledge gap, this study aims to investigate, for the first time at the alevin stage in a farmed fish species, the regulation of the opioid system in rainbow trout fed either a plant-based diet (referred to as the V diet) or a commercial like diet containing fishmeal and fish oil (referred to as the C diet). Our objectives are to evaluate early and long-term effects of these diets on the expression of genes involved in the opioid system, which plays a key role in the hedonic regulation of feeding behavior. By focusing on the developmental stages from first feeding to juvenile, we seek to better understand how diet composition influences molecular pathways linked to nutrient perception and reward, ultimately contributing to improved sustainable aquaculture practices.

## 2. Materials and methods

### 2.1. Experimental diet design

Diets were manufactured at INRAE's experimental facilities at Donzacq (permit no. A40-228.1, Landes, France) using a twin-screw extruder (BC 45, Clextral, Firminy, France) after formulation using Allix® software (A-Systems, City, France). The pellets had a diameter of 0.5 until 5 mm and a length of 0.5/5 mm depending of fish size during the experiment. Details about the ingredients and composition of the experimental diets are given in Table 1 (first feeding to 1 month and 20g to 8 months). The experiment was conducted with two different diets: a controldiet (C diet) containing a mix of FM (32 % at the alevin stage and 27 % at the juvenile stage), FO (11.4 % at the alevin stage and 12.1 % at the juvenile stage), and plant ingredients; and a V diet, completely free from FM and FO, which were replaced by a blend of plant ingredients from soybean meal, peas, and corn gluten to replace FM, and palm, rapeseed, sunflower, and linseed oil to replace FO. Diets were isoen energetic and were formulated to cover the nutritional requirements of the fish (NRC, 2011). Diets were also isoproteic between feeding periods (Fig. 1) and almost to be isolipidic. In order to avoid exceeding anti-nutrient threshold levels, we used a blend of wheat gluten, soybean meal, whole wheat, corn gluten meal, soy protein, and peas as protein sources. L-lysine, L-methionine, dicalciumphosphate, and soy-lecithin were added to all diets to correct the deficiency in essential amino acids, phosphorous, and phospholipids. Mineral and vitamin premixes were also added to each diet.

### 2.2. Proximate composition

Nutrient composition, crude protein, crude lipids, gross energy, ash content, starch content, and fatty acid profils of the diets were analyzed as previously described (Baranek et al., 2023; Martinat et al., 2025) and following the Official Methods of Analysis of AOAC International (Laitimer, 2023). Briefly, the nutrient composition of the diets was analyzed after 24 h of drying at 105 °C. Gross energy was determined using an adiabatic bomb calorimeter (IKA, Heitersheim Grißheimer, Germany). Ash content was measured by combustion in a muffle furnace (550 °C for 8 h). Crude protein was quantified using the Kjeldahl method (Kjeldahl, 1883) and crude lipids using the Soxhlet method.

### 2.3. Experimental design

Genetically all-female rainbow trout fry came from the same parental stock (INRAE Fish Farm of Lees-Athas, permit no. A64.104.1, Aspe Valley, France). Swimming fry with an initial body weight of 108 ± 0.1 mg were transferred to a tank system at the INRAE's facilities of Donzacq (permit no. A40-228.1, Landes, France) for the feeding experiment. Fry were randomly allocated to five tanks per dietary treatment, with 150 fish per tank; this density minimized the influence of tank size
<table><caption>**Table 1**

Ingredients and composition of the experimental diets.</caption><thead><tr><th rowspan="2">Ingredient (%)</th><th colspan="2">DIET (first-feeding to 30 days)</th><th colspan="2">DIET (20g-8 months)</th></tr><tr><th>Control diet (C)</th><th>Plant-based diet (V)</th><th>Control diet (C)</th><th>Plant-based diet (V)</th></tr></thead><tbody><tr><td>Fish meal</td><td>32.0</td><td>0.0</td><td>27.0</td><td>0.0</td></tr><tr><td>Extruded whole wheat</td><td>8.6</td><td>2.0</td><td>22.5</td><td>17.0</td></tr><tr><td>Corn gluten</td><td>11.9</td><td>25.6</td><td>8.7</td><td>17.0</td></tr><tr><td>Wheat gluten</td><td>7.0</td><td>17.0</td><td>7.0</td><td>17.0</td></tr><tr><td>Pea meal</td><td>17.7</td><td>25.5</td><td>3.0</td><td>6.0</td></tr><tr><td>Soybean meal</td><td>9.0</td><td>23.3</td><td>-</td><td>-</td></tr><tr><td>Gelatinized wheat starch</td><td>2.2</td><td>0.0</td><td>-</td><td>-</td></tr><tr><td>Extruded peas</td><td>-</td><td>-</td><td>7.0</td><td>9.5</td></tr><tr><td>White lupin seed meal</td><td>-</td><td>-</td><td>2.5</td><td>4.5</td></tr><tr><td>Rapeseed meal</td><td>0.8</td><td>3.0</td><td>0.8</td><td>3.0</td></tr><tr><td>Sunflower lecithin</td><td>0.0</td><td>3.0</td><td>-</td><td>-</td></tr><tr><td>Soy lecithin</td><td>-</td><td>-</td><td>0.0</td><td>2.0</td></tr><tr><td>L-lysine</td><td>0.0</td><td>1.0</td><td>0.5</td><td>1.7</td></tr><tr><td>L-methionine</td><td>0.4</td><td>1.0</td><td>0.0</td><td>0.4</td></tr><tr><td>CaHPO<sub>4</sub>.2H<sub>2</sub>O</td><td>0.0</td><td>1.5</td><td>0.0</td><td>1.4</td></tr><tr><td>Mineral premix<sup>a</sup></td><td>1.5</td><td>1.5</td><td>1.0</td><td>1.0</td></tr><tr><td>Vitamin premix<sup>b</sup></td><td>1.5</td><td>1.5</td><td>1.0</td><td>1.0</td></tr><tr><td>Fish oil</td><td>11.40</td><td>0.0</td><td>4.8</td><td>0.0</td></tr><tr><td>Palm oil</td><td>0.0</td><td>8.30</td><td>2.0</td><td>5.0</td></tr><tr><td>Rapeseed oil</td><td>3.80</td><td>1.80</td><td>-</td><td>-</td></tr><tr><td>Sunflower oil</td><td>-</td><td>-</td><td>4.9</td><td>2.7</td></tr><tr><td>Linseed oil</td><td>0.0</td><td>4.00</td><td>0.0</td><td>10.8</td></tr><tr><td>DHA marine oil (from Polaris)<sup>c</sup></td><td>-</td><td>-</td><td>7.3</td><td>0.0</td></tr><tr><td colspan="5">Composition (% of DM)</td></tr><tr><td>Dry matter (%)</td><td>94.47</td><td>96.03</td><td>97.30</td><td>96.55</td></tr><tr><td>Crude protein</td><td>54.54</td><td>52.29</td><td>42.62</td><td>41.44</td></tr><tr><td>Crude lipid</td><td>17.03</td><td>20.61</td><td>22.64</td><td>22.2</td></tr><tr><td>Ash</td><td>9.96</td><td>7.24</td><td>7.04</td><td>4.47</td></tr><tr><td>Energy (kJg-1 DM)</td><td>24.37</td><td>24.33</td><td>24.29</td><td>24.81</td></tr></tbody></table>

<sup>a</sup> Mineral premix: (g or mg/kg diet): calcium carbonate (40 % Ca), 2.15 g; magnesium oxide (60 % Mg), 1.24 g; ferric citrate, 0.2 g; potassium iodide (75 % I), 0.4 mg; zinc sulphate (36 % Zn), 0.4 g; copper sulphate (25 % Cu), 0.3 g; manganese sulphate (33 % Mn), 0.3 g; dibasic calcium phosphate (20 % Ca, 18 % P), 5 g; cobalt sulphate, 2 mg; sodium selenite (30 % Se), 3 mg; KCl, 0.9 g; NaCl, 0.4 g (UPAE, INRA)

<sup>b</sup> Vitamin premix: (IU or mg/kg diet): DL-a tocopherol acetate, 60 IU; sodium menadione bisulphate, 5 mg; retinyl acetate, 15,000 IU; DL-cholecalciferol, 3000 IU; thiamin, 15 mg; riboflavin, 30 mg; pyridoxine, 15 mg; B12, 0.05 mg; nicotinic acid, 175 mg; folic acid, 500 mg; inositol, 1000 mg; biotin, 2.5 mg; calcium pantothenate, 50 mg; choline chloride, 2000 mg (UPAE, INRAe).

<sup>c</sup> Omegavie® DHA oil (min 70 %): concentrated marine oil produced mainly from anchovy and sardine oil. The crude oil is refined, then purified and concentrated. This oil contains marine DHA omega 3 fatty acids under triglycerides form. From POLARIS, Quimper, France.

(depending of fish size) on feeding behaviour. Water flow was set to ensure a dissolved oxygen concentration greater than 90 % of saturation. Fish were exposed to natural photoperiod conditions and the water temperature was 17 ± 1 °C. During the experiment, the water conditions were as follows: dissolved oxygen was 9 mg/L, ammonia levels were below 0.01 mg/L, nitrite levels were below 0.04 mg/L, and nitrate concentration was approximately 17 ppm. The flow rate in each tank was 0.3 L/s, resulting in a complete water renewal of each tank 6 times per hour. During the first 30 days, all fish were fed by hand eight times a day with C or V diet until apparent satiety (two times for 8 months). At 5 and 30 Day and 8 months, fish were starved for 16 h to exclude effects of recent feeding and sampled (16 h starvation condition). Then the trout were fed once either the C diet or the V diet and sampled twice as described below.

For tissue sampling, fish (n = 6 per time point and dietary treatment for qPCR analysis and 10 for HPLC analysis) were first anesthetized by

immersing them in a benzocaine solution (30 mg/L) then euthanized in a bath containing 60 mg/L of benzocaine. The whole brain for fry (5 and 30 days) and the telencephalon for juveniles (8 months) were sampled after 16 h starvation and 20 min and 6 h after the meal for fry (4 h after the meal for juveniles). To minimize stress on the fish during sampling, we sampled one tank per diet for each sampling point. The tissues were immersed in liquid nitrogen to avoid degradation, and frozen at -80 °C until mRNA or metabolite extraction.

After 5 days, 30 days, and 8 months, fish biomass and the quantity of feed distributed per tank were measured to calculate mean body weight (g), estimated daily absolute feed intake (mg of feed for fry and g of feed for juveniles, calculated per fish per day), and estimated feed efficiency (g of body weight/g of given feed).

## 2.4. Indolamine and catecholamine release measurement using UHPLC-FL

Indolamine and catecholamine polar metabolites were extracted from the whole brain of fry (5 and 30 days on the experimental diets) and the telencephalon of juveniles (8 months on the experimental diets) (n = 10 per age and diet) as described in Heraud et al. (2022). The entire protocol is described in Baranek et al. (2023).

Whole brain samples for fry or telencephalon for juveniles (n = 10) were analyzed by UHPLC (ACQUITY H-Class PLUS, Waters) equipped with a thermostatted autosampler (4 °C) and a fluorescence detector (ACQUITY Multi-λ, Waters). Data acquisition and quantification were performed using Empower™ Pro software. All reagents and standards were from Merck (Darmstadt, Germany); solvents were MS-grade (ADL & Prochilab, France). Whole brains or telencephalon were homogenized using a Precellys® 24 system (Bertin Technologies) in 20 mmol/L phosphate buffer with 1 mmol/L EDTA (pH 6.5 ± 0.05) at a 1:5 (w/v) ratio. After centrifugation (14,000 g, 20 min, 4 °C), the supernatant was deproteinized with 10 % metaphosphoric acid (v/v). Following a second centrifugation (14,000 g, 5 min, 4 °C), the supernatant was filtered using 0.22 µm PVDF filters and centrifuged again. Samples were stored at -20 °C until analysis. Chromatographic separation was carried out on a Luna PFP(2) column (150 × 4.6 mm, 3 µm, Phenomenex) at 30 °C. Injection volume was 10 µL; flow rate 0.4 mL/min. The quaternary solvent system included phosphate buffer (A), methanol (B), and ultrapure water (C). A detailed linear gradient elution profile was used (see supplemental methods).

Metabolites were detected using two excitation/emission settings: 285/355 nm for serotonergic compounds (5-HT, 5-HIAA) and 280/330 nm for catecholamines (L-DOPA, HVA). Metabolites of interest were identified by comparing retention times to standards. Quantification was based on integrating peak areas and comparing them to standard calibration curves ($R^2$ > 0.995) of each metabolite of interest. Calibration curves were linear from 0.05 to 55 pmol per injection for each standard. Normalization was made using the total protein quantification using the bicinchoninic acid method (#BCA1-1 KT, Sigma-Aldrich).

In our study, the turnover of indolamine serotonin (5-HT) was assessed by calculating the ratio between its concentration and that of its direct degradation product, 5-hydroxyindoleacetic acid (5-HIAA). The turnover of catecholamin dopamine was assessed by the ratio of the direct precursor 3,4-dihydroxyphenylalanine (L-DOPA) to one of its degradation products, homovanillic acid (HVA). The assessment of turnover using these ratios in rainbow trout has been reported elsewhere (Bacqué-Cazenave et al., 2020; Baranek et al., 2023, 2024; Heraud et al., 2022).

## 2.5. Total RNA isolation and real-time quantitative PCR

Gene expression analyses were conducted on the whole brains of fry (5 and 30 days) and the telencephalon of juveniles (8 months) to assess selected transcripts involved in the opioid system, including opioid receptor delta 1 (oprd1), kappa 1 (oprk1), and mu 1 (oprm1), and opioid
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Levels of mRNA-encoding genes of the opioid system in the whole brain of fry fed the C or V diet for 5 days</figcaption></figure>

Relative gene expression measured by RT-qPCR of opioid precursors (*penka* and *pdyn*) and receptors (*ogf1*, *oprd1*, *opkr1*, and *oprm1*) of fry fed the C or V diet for 5 days and then sampled after 16 h fasting, fed once, and sampled again after 20 min and 6 h. Data are fold change $2^{-\Delta\Delta C_t}$ vs 16 h of fasting of C diet for all genes. Two-way ANOVA, Tukey's post-hoc test when appropriate.

growth factor 1 (*ogf1*), and opioid precursors such as prodynorphin (*pdyn*) and proenkephalin A (*penka*). Total RNA was isolated from the brain or telencephalon (n = 6 per time point and dietary treatment) using the TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) with Pellicellys® 24 (Bertin Technologies, Montigny-le-Bretonneux, France) following the instructions of the manufacturer, as previously described (Roy et al., 2023). The quantities of extracted RNA were measured using a spectrophotometer (ND-1000, Nanodrop, Thermo) and the quality of extracted RNA was estimated using the 260 nm:280 nm OD ratio (purity

of DNA and RNA) and 260 nm:230 nm OD ratio (purity of solution to avoid phenol contamination). For gene expression analysis, only total RNA with a 260 nm:280 nm OD ratio exceeding 1.8 and a 260 nm:230 nm OD ratio exceeding 2.0 were utilized. One microgram of total RNA was reverse-transcribed into cDNA using the Super-Script III RNAse H-Reverse transcriptase kit (Invitrogen) and random primers (Promega, Chartonnières-les-Bains) (Table 2) following the manufacturer's recommendations. Each sample was reverse-transcribed in duplicate. The LightCycler® 480 system (Roche Diagnostics) was used to measure

<caption>**Table 2**</caption>

Nucleotide sequences of the PCR primers used to evaluate the mRNA abundance of transcripts by real-time quantitative PCR (5'-3' sens).

<table><thead><tr><th>Transcript</th><th>Forward Primer</th><th>Reverse Primer</th><th>Accession Number</th><th>Efficiency</th></tr></thead><tbody><tr><td><strong>Reference</strong></td><td></td><td></td><td></td><td></td></tr><tr><td><em>eef1a1</em></td><td>TCCTCTTGGTCGTTTCGCTG</td><td>ACCCGAGGGACATCCTGTG</td><td>AF498320</td><td>1.91</td></tr><tr><td><em>Luciferase</em></td><td>CATTCTTCGCCAAAAAGCACTCTG</td><td>AGCCCATATCCTTGTCGTATCCC</td><td></td><td>1.87</td></tr><tr><td><strong>Opioid system</strong></td><td></td><td></td><td></td><td></td></tr><tr><td><em>penka</em></td><td>GCGACTTGGTAAGCACCGGG</td><td>GCGCACAGTCCTTCTCGCAG</td><td>XM_021613078.2</td><td>2.13</td></tr><tr><td><em>pdyn</em></td><td>ATGCGCGCAACAAATCCTCA</td><td>TCTCTGGCGTGGACCGTGG</td><td>XM_021615876.2</td><td>2.06</td></tr><tr><td><em>ogf1</em></td><td>CCGGACGATTTAGTGTGCGT</td><td>CCTTGGCAGCCCTCATGTTCC</td><td>XM_021615595.2</td><td>2.01</td></tr><tr><td><em>oprd1</em></td><td>AGAGGATCCCATGTCCCCAAT</td><td>GCCAGAGCCAGGTTGAAGATG</td><td>XM_021562945.2</td><td>2.11</td></tr><tr><td><em>opkr1</em></td><td>CATCTTCGGCTTCGTGGCCC</td><td>GGGGGCGCCAGCATCTTAAC</td><td>XM_036986795.1</td><td>1.97</td></tr><tr><td><em>oprm1</em></td><td>CATGCGCACGCTCTTCAGGC</td><td>TCTGCCTGTCATGTTCACCGC</td><td>XM_021581332.2</td><td>2.03</td></tr></tbody></table>

eef1a1, elongation factor1; penka, proenkephalin; pdyn, prodynorphin; ogf1, opioid growth factor receptor; oprd1, delta-type opioid receptor; oprk1, kappa-type opioid receptor 1; oprm1, mu-type opioid receptor.
mRNA levels via real-time quantitative PCR (RT-qPCR). The reaction mixture was 6 µL and consisted of 2 µL of diluted cDNA (1:10), 0.24 µL of each primer (10 µmol/L), 3 µL of LightCycler® 480 SYBR Green I Master mix (Roche Diagnostics), and 0.52 µL of DNAse/RNAse-free water. Thermal cycling was initiated with incubation at 95 °C for 10 s for the initial denaturation of the cDNA and hot-start Taq-polymerase activation, followed by 45 cycles of a three-step amplification program (10 s at 95 °C, 10 s at 60 °C, and 15 s at 72 °C). Cycle thresholds greater than 32 cycles were not considered. Melting curves were controlled systematically (temperature gradient: 0.11 °C/s from 65 °C to 95 °C) at the end of the last amplification cycle to ensure that only one fragment was amplified. During a run, each sample was deposited twice, and two negative controls were added: a pool of samples without reverse transcriptase, and a pool of samples without cDNA. The efficiency of all qPCR reactions was 96–100 % and was systematically monitored for each PCR run. The sequences of amplicons were assessed via sequencing.

Data were extrapolated from standard curves and normalized to the housekeeping elongation factor 1α gene (eef1α) for data from the telencephalon (juveniles at 8 months). For the whole brain (fry at 5 and 30 days), eef1α was not stable and luciferase was used for normalization. Luciferase control RNA (Promega) was added during the reverse-transcription step (1 pg/µg of RNA) to each sample to allow for data normalization. Relative expression of the target genes was determined using the ΔΔCT method (Pfaffl, 2001). The values for each group are expressed as fold changes relative to the C diet 16 h starvation and are presented as mean ± standard error (SE) for all genes. Primer sequences used to amplify all paralog genes simultaneously were previously validated and their accession numbers are presented in Table 2.

## 2.6. Statistical analysis

All statistical analyses were performed using R software (version 3.6.1; R Core Team) (R: The R Project for Statistical Computing). The tank was the experimental unit for data on growth and Anova was used for that analysis (Table 3). Individual fish were the experimental unit for the ratios of indolamine and catecholamine (Table 3) and gene expression. Analyses were carried out on untransformed data as criteria for normality of distribution and homeogeneity of variance were fulfilled (Shapiro-Wilk and Levene's test, respectively). The effect of the two factors, diet and time, was assessed using a two-way analysis of variance (ANOVA) with an interaction. When a significant interaction was observed, a Tukey's post-hoc test was realized.

# 3. Results

## 3.1. Husbandry parameters and neurotransmitter activity

Table 3 summarizes the body weight, daily food intake, and feed efficiency of rainbow trout fed either the C or V diet from first feeding to 5 days, 30 days, and 8 months.

After only 5 days of feeding, fry consuming the V diet exhibited 8 % lower growth than those on the C diet. This gap increased to 17 % after 30 days and reached 33 % after 8 months, with an average body weight of 229 g compared to 344 g for rainbow trout fed the C diet.

The amount of feed distributed during the initial feeding phase (less than one month, 5 and 30 days) was difficult to quantify precisely. However, after 30 days, although feed efficiency remained unchanged, daily feed intake was 16.3 % lower in fry fed the V diet. After 8 months, feed efficiency was still the same, but estimated daily absolute feed intake (from first feeding to 8 months) was 33 % lower in rainbow trout fed the V diet.

The activity of 5-HT and L-DOPA pathways can be assessed by the ratio of their respective degradation products: 5-HIAA for 5-HT and HVA for L-DOPA (Table 3). While these ratios were the same after 5 days of feeding, the 5-HIAA/5-HT ratio was 44.5 % higher after 30 days (whole brain) and 29.7 % higher after 8 months (telencephalon) in rainbow trout fed the V compared to the C diet. Conversely, the HVA/L-DOPA ratio was 74.8 % higher after 30 days (whole brain) in rainbow trout fed the C compared to the V diet.

## 3.2. Influence of diet on the opioid system gene expression in the whole brain of fry after 5 days

The mRNA levels of opioid receptors (oprd1, oprk1, oprml, and ogf1) and precursors (pdyn and penka) in the whole brain of fry fed the C or V diet for 5 days and then sampled after 16 h fasting, fed once, and sampled again after 20 min and 6 h are presented in Fig. 1.

The mRNA levels of opioid precursor genes did not vary by diet or by feeding status. However, the mRNA levels of the opioid receptor gene oprk1 was up-regulated in the V diet at 20 min after being fed (p-value: 1.57×10⁻²).

## 3.3. Influence of diet on the opioid system gene expression in the whole brain of fry after 30 days

The mRNA levels of opioid receptors (oprd1, oprk1, oprml, and ogf1)

<table><caption>**Table 3**
Zootechnical performance and indolamine/catecholamine ratio of rainbow trout from first feeding to 5 days, 30 days, and 8 months on each diet.</caption><thead><tr><th></th><th>Age</th><th>Control diet (C)</th><th>Plant-based diet (V)</th><th>Statistical results</th></tr></thead><tbody><tr><td rowspan="3">Body weight (g)</td><td>5 days</td><td>0.128 ± 0.00</td><td>0.118 ± 0.02</td><td>1.45 × 10<sup>-2</sup></td></tr><tr><td>30 days</td><td>0.596 ± 0.00</td><td>0.494 ± 0.02</td><td>9.82 × 10<sup>-3</sup></td></tr><tr><td>8 months</td><td>344 ± 4.49</td><td>228.9 ± 1.39</td><td>6.46 × 10<sup>-4</sup></td></tr><tr><td rowspan="3">Food intake (mg or g feed/fish/day)</td><td>5 days</td><td>N/A</td><td>N/A</td><td>na</td></tr><tr><td>30 days</td><td>9.30 ± 0.11 (mg)</td><td>7.79 ± 0.13 (mg)</td><td>9.68 × 10<sup>-3</sup></td></tr><tr><td>8 months</td><td>2.09 ± 0.01 (g)</td><td>1.38 ± 0.03 (g)</td><td>6.275 × 10<sup>-7</sup></td></tr><tr><td rowspan="3">Feed efficiency (g of body weight/g of feed)</td><td>5 days</td><td>N/A</td><td>N/A</td><td>na</td></tr><tr><td>30 days</td><td>1.99 ± 0.09</td><td>1.94 ± 0.06</td><td>n.s</td></tr><tr><td>8 months</td><td>1.09 ± 0.02</td><td>1.05 ± 0.02</td><td>n.s</td></tr><tr><td rowspan="3">Serotonin turnover (ratio 5-HIAA/5-HT)</td><td>5 days</td><td>0.36 ± 0.07</td><td>0.76 ± 0.22</td><td>n.s</td></tr><tr><td>30 days</td><td>0.40 ± 0.05</td><td>0.61 ± 0.07</td><td>3.18 × 10<sup>-2</sup></td></tr><tr><td>8 months</td><td>0.17 ± 0.02</td><td>0.28 ± 0.04</td><td>1.49 × 10<sup>-2</sup></td></tr><tr><td rowspan="3">Dopamine turnover (ratio HVA/L-DOPA)</td><td>5 days</td><td>2.88 ± 0.53</td><td>2.60 ± 0.59</td><td>n.s</td></tr><tr><td>30 days</td><td>8.07 ± 0.88</td><td>2.84 ± 0.99</td><td>1.24 × 10<sup>-3</sup></td></tr><tr><td>8 months</td><td>1.22 ± 0.14</td><td>1.78 ± 0.30</td><td>n.s</td></tr></tbody></table>

n.s = not significant; N/A: not applicable.
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Levels of mRNA-encoding genes of the opioid system in the whole brain of fry fed the C or V diet after 30 days
Relative gene expression measured by RT-qPCR of opioid precursors (*penka* and *pdyn*) and receptors (*ogf1*, *oprd1*, *opkr1*, and *oprm1*) in the whole brain of fry fed for 30 days and then sampled after 16 h fasting, fed once, and sampled again after 20 min and 6 h. Data are fold change $2^{-\Delta\Delta C_t}$ vs 16 h of fasting of C diet for all genes. Two-way ANOVA, Tukey's post-hoc test when appropriate; different letters indicate a significant difference (*P* < 0.05) between diet and time (n = 6).</figcaption></figure>

and precursors (*pdyn* and *penka*) in the whole brain of fry fed the C or V diet for 30 days and then sampled after 16 h fasting, fed once, and sampled again after 20 min and 6 h are presented in Fig. 2.

After 30 days of feeding, the mRNA levels of the *pdyn* opioid precursor gene was up-regulated in the V diet at 20 min after the meal and remained higher 6 h after the meal (*p-value*: 9.92×10⁻³). The mRNA levels of opioid receptor genes only varied for *oprm1*, with a significant interaction showing an up-regulation 20 min after the meal in fish fed the C diet compared to the V diet after 16 h of fasting. Additionally, 6 h after the meal, fish fed the V diet showed an upregulation of *oprm1* compared to fish fed the C diet (*p-value*: 3.11×10⁻²).

### 3.4. Influence of diet on the opioid system gene expression in the telencephalon of juveniles after 8 months

The mRNA levels of opioid receptors (*oprd1*, *opkr1*, *oprm1*, and *ogf1*) and precursors (*pdyn* and *penka*) in the telencephalon of juveniles fed the C or V diet for 8 months and sampled after 16 h of fasting, fed once, then sampled again 20 min and 4 h after the meal are presented in Fig. 3.

After 8 months of feeding, the mRNA levels of the *penka* and *pdyn* precursor genes varied by diet and time (diet x time; *p-value*: 1.1×10⁻² and *p-value*: 3.6×10⁻³, respectively) showing a down-regulation 4 h after the meal in fish on the C diet compared to both diets after 16 h of fasting and the C diet 20 min after the meal. For the *pdyn* gene, there was also an upregulation in the V diet 4 h after the meal compared to the C

diet 20 min and 4 h after the meal.

For the mRNA level of the *oprm1* gene, there was a downregulation in fish fed the C diet 4 h after the meal compared to all others diet and time points except in fish fed the V diet 20 min after the meal (diet x time: *p-value*: 1.3×10⁻⁴).

For the mRNA level of the *ogf1* gene, there was an up-regulation in fish fed the V diet at 6 h after the meal compared to fish fed the V diet and either fasted for 16 h or sampled 20 min after the meal and compared to fish fed the C diet and sampled 6 h after the meal (diet x time: *p-value*: 1×10⁻⁴).

For the mRNA level of the *oprd1* gene, there was a downregulation in fish fed the C diet 6 h after the meal compared to all others diet and time points except for fish fed the V diet 20 min after the meal. It was also upregulated in the fish fed the C diet 20 min after the meal compared to fish fed the V diet 20 min after the meal (diet x time: *p-value*: 5.4×10⁻⁴).

For the mRNA level of the *oprk1* gene, there was an up-regulation in fish fed the V diet 6 h after the meal compared to fish fed the C diet 6 h after the meal (diet x time: *p-value*: 6×10⁻³).

### 4. Discussion

Our study investigated, for the first time in rainbow trout, the temporal dynamics of the opioid system in response to a plant-based diet from the first feeding until the juvenile stage (8 months), with a particular focus on the regulation of feeding behavior. Our results
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Levels of mRNA encoding genes of opioid system in the telencephalon of juveniles fed the C or V diet after 8 months
Relative gene expression measured by RT-qPCR of opioid precursors (penka and pdyn) and receptors (ogf1, oprd1, opkr1 and oprm1) in the telencephalon of juveniles fed for 8 months and then sampled after 16 h fasting, fed once, and sampled again after 20 min and 4 h. Datas are fold change 2<sup>-ΔΔCT</sup> vs 16 h of fasting of C diet for all genes. Two-way ANOVA Tukey's post-hoc test when appropriate; different letters indicate a significant difference (*P* < 0.05) between diet and time (n = 6).</figcaption></figure>

provide new insights into how dietary composition, particularly the replacement of marine-derived ingredients with plant-based ones, can shape neurobiological and behavioral responses during early development and the involvement of the opioid system in these effects.

These results align with studies in mammals, where opioid receptor signaling is tightly associated with hedonic evaluation and palatability of food (Kelley, 2004; Le Merrer et al., 2009; Nogueiras et al., 2012). The upregulation of opioid precursors and receptors we observed in rainbow trout juveniles fed the V diet resembles the increased activation of opioid circuits seen in rodents exposed to palatable diets (Berthoud et al., 2017).

In mammals, the brain opioid system (endogenous peptides and their receptors) plays a role in the general regulation of food intake and specifically in rewarding palatable food intake (Nathan and Bullmore, 2009). In particular, this is one of the key systems that regulate hedonic overeating (binge eating) (Stein et al., 2007; Valbrun and Zvonarev, 2020). In mammals, the administration of opioid receptor agonists increases food intake, while opioid receptor antagonists decrease it (Kelley, 2004). It has been theorized that opioids specifically mediate palatability or the hedonic evaluation of food (Cooper and Kirkham, 1993), effects mediated by complex interactions between opioids and other neural systems, notably the dopaminergic system, which plays a crucial role in reward and motivation (Smith and Berridge, 2007).

In our study, from the earliest ages, rainbow trout exposed to the plant-based V diet exhibited lower growth compared to those fed the fishmeal-based C diet, and this difference increased throughout the

study until 8 months of age. Interestingly, the molecular responses of the opioid system were initially limited, suggesting that the pathways involved in the hedonic regulation of feeding may be underdeveloped or only partially functional at early feeding. At this stage, only *oprk1* showed differential expression, which might reflect higher sensitivity of this receptor to dietary components or a compensatory mechanism in response to reduced palatability. These results are consistent with previous studies in mammals, where opioid receptors show higher sensitivity to diet and play a role in the regulation of food intake (Bodnar, 2017).

As development progressed, specifically at 30 days post-feeding, the expression of *pdyn* and changes in serotonin turnover indicated a shift in hedonic regulation. Increased 5-HIAA/5-HT ratios in the V diet group suggest an altered serotonergic tone, potentially reflecting negative feedback in response to reduced food palatability (Bacqué-Cazenave et al., 2020). This age marked the divergence in neurochemical response between the two diets and supports the idea that hedonic systems begin to modulate feeding behavior as early as one month post-feeding. These results are similar to those observed in mammals, where hedonic systems play a crucial role in the regulation of food intake and motivation (Berthoud and Morrison, 2008).

At the juvenile stage (after 8 months of feeding), the neurobiological responses were more pronounced. All the opioid-related genes studied (*penka*, *pdyn*, *ogf1*, *opkr1*, *oprd1*, and *oprm1*) in the telencephalon were affected by diet, which is a brain region associated with reward processing and behavioral regulation (Nogueiras et al., 2012). These results
indicate maturation and functional engagement of the opioid system under nutritional pressure. The upregulation of multiple opioid genes in the V diet group might represent a compensatory mechanism to maintain feeding motivation despite reduced palatability of this diet (Branek et al., 2023; Heraud et al., 2022). These results are consistent with studies in mammals, where the upregulation of opioid genes is observed in response to dietary challenges (Bodnar, 2019).

This is further supported by the consistent increase in serotonin turnover in the brain, reinforcing the hypothesis of central and peripheral regulation of feeding behavior through hedonic mechanisms (Bacqué-Cazenave et al., 2020). Although the role of serotonin in appetite suppression is well established, its interaction with opioid signaling might underlie more complex behavioral changes in response to dietary challenges (Berthoud and Morrison, 2008). Altogether, our results are consistent with the integrative model proposed by Nogueiras et al. (2012), in which the opioid system serves as a bridge between sensory input, palatability, and the motivation to feed, and can be modulated by long-term diet exposure. Our findings support this framework and highlight rainbow trout as a relevant model to explore neurobiological processes in relation to sustainable aquaculture feeds.

The involvement of the opioid system in feeding behavior has been well documented in mammals, where it modulates pleasure and motivation associated with eating (Giuliano and Cottone, 2015). In fish, evidence is more limited, but our study contributes to a growing body of work suggesting similar functional roles. For instance, studies in fishes have shown that opioids play a role in the stress response and pain modulation (Rodrigues et al., 2019; Soengas et al., 2018). In fish, opioid receptors are widely distributed in the central nervous system, particularly in regions such as the hypothalamus and telencephalon, which are involved in the regulation of food intake and reward processing (Soengas et al., 2018). Additionally, a study in juvenile rainbow trout showed significant changes in the expression of opioid-related genes in response to feeding (Díaz-Rúa et al., 2022). In particular, the administration of a palatable high-fat diet led to a decrease in the abundance of *penka* mRNA and an increase in the abundance of *oprd1* and *oprk1* mRNA in the telencephalon, suggesting that the opioid system is involved in the hedonic regulation of food intake but not in the homeostatic regulation of food intake (through fasted/fed/refed fish in this study).

The dynamic regulation observed here in response to plant-based diets highlights the potential for early dietary experience to shape long-term neurobiological trajectories, a concept consistent with nutritional programming (Delgado et al., 2017). Furthermore, the progressive engagement of the opioid system over time highlights the importance of considering the developmental stage when evaluating dietary impacts. Early-life exposure to specific dietary cues may influence not only immediate food intake and growth but also the maturation of neurobiological systems involved in reward and motivation. These results are consistent with studies in mammals, where early-life exposure to specific diets can influence food preferences and behaviors in the long term (Berthoud and Morrison, 2008). From an applied perspective, these results highlight the need to consider both the nutritional adequacy and sensory properties of alternative feeds in aquaculture. In light of the increasing interest in sustainable aquaculture, future research could also explore the use of functional plant-based ingredients, such as medicinal plants or phytogenic additives, not only for their immunosup timulatory or antioxidant properties but also for their potential impact on feed perception and neurobiological regulation of appetite. While our study focused on the effect of whole plant-based diets on the opioid system, it would be valuable to assess whether specific bioactive compounds, such as polyphenols, fruits, herbal extract, flavonoids, or essential oils, can modulate reward-related pathways in fishes. These functional ingredients have shown promise in other species (e.g., *Micropterus salmoides*, *Cyprinus carpio*, *Salmo salar*, *Danio rerio*) (Ahmadifar et al., 2019, 2021, 2022; Yue et al., 2024), and their role in sensory and hedonic regulation in fish nutrition remains an open and stimulating research avenue.

## 5. Conclusion

Our study highlights a temporal modulation of the opioid system in rainbow trout subjected to a plant-based diet, with potential implications for understanding hedonic regulation in fish. The dynamic regulation observed underscores the potential for early dietary experience to shape long-term neurobiological trajectories, a concept consistent with nutritional programming. Similarly, our study provides new data on the role of the opioid system in the regulation of food intake and reward in fishes, with important implications for aquaculture and fish welfare. Future research should aim to characterize the precise neurochemical pathways involved, explore the potential role of other modulatory systems such as endocannabinoids, and evaluate the long-term consequences of early dietary exposure on the behavior and physiology of fishes. These studies could also include genetic approaches to identify the molecular mechanisms underlying these neurobiological responses.

### CRediT authorship contribution statement

**Elisabeth Plagnes-Juan:** Writing – review & editing, Writing – original draft, Software, Formal analysis, Data curation. **Maud Martinet:** Writing – original draft, Methodology, Data curation. **Elodie Branek:** Writing – original draft, Software, Data curation. **Anne Surget:** Formal analysis, Data curation. **Cécile Heraud:** Writing – original draft, Software, Formal analysis, Data curation. **Jérôme Roy:** Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.

### Data availability

All data supporting the findings in this study are available within the article. Raw phenotypic datasets are available from the Recherche Data Gouv public repository entitle "DHA-enriched plant-based diets on rainbow trout (Oncorhynchus mykiss) broodstock: impact on early offspring performance", https://doi.org/10.57745/H1DSXG. Correspondence and material requests should be addressed to Jérôme Roy (jerome.roy@inrae.fr).

### Ethics approval and consent to participate

The experiment was conducted according to the guidelines of the National Legislation on Animal Care of the French Ministry of Research (decree no. 2013-118, 1 February 2013) and in accordance with European Union legal frameworks relating to the protection of animals used for scientific purposes (Directive 2010/63/EU). The experiment was conducted at the INRAE's NuMeA facilities (https://doi.org/10.15454/GPYD-AM38) and the long-term experiment was approved by the ethical committee (C2EA-73) of INRAE "Comité d'éthique Aquitaine poissons oiseaux" (INRAE agreement no. 21699, 19 December 2019). All efforts were made to minimize the number of fish used and their suffering. The scientists in charge of the experiment received training and personal authorization.

### Funding

This research was funded by the French National Research Agency (grant no. ANR-21-CE20-0009-01 "FEEDOMEGA").

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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