# Temporal dynamics of oxylipins and antioxidant metabolites in rainbow trout muscle are driven by diet type rather than methylmercury or selenomethionine supplementation

Marius Bidon, Cécile Heraud, Claudia Marchán-Moreno, Laurence Larroquet, Guillaume Reversat, Claire Vigor, Camille Oger, Jean-Marie Galano, Thierry Durand, Zoyne Pedrero, et al.

► To cite this version:

Marius Bidon, Cécile Heraud, Claudia Marchán-Moreno, Laurence Larroquet, Guillaume Reversat, et al. Temporal dynamics of oxylipins and antioxidant metabolites in rainbow trout muscle are driven by diet type rather than methylmercury or selenomethionine supplementation. Free Radical Biology and Medicine, 2025, 239, pp.417-431. 10.1016/j.freeradbiomed.2025.07.043. hal-05225823

HAL Id: hal-05225823

https://univ-pau.hal.science/hal-05225823v1

Submitted on 27 Aug 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

# Temporal dynamics of oxylipins and antioxidant metabolites in rainbow trout muscle are driven by diet type rather than methylmercury or selenomethionine supplementation

Marius Bidon<sup>a</sup>, Cécile Heraud<sup>a</sup>, Claudia Marchán-Moreno<sup>b</sup>, Laurence Larroquet<sup>a</sup>, Guillaume Reversat<sup>c</sup>, Claire Vigor<sup>c</sup>, Camille Oger<sup>c</sup>, Jean-Marie Galano<sup>c</sup>, Thierry Durand<sup>c</sup>, Zoyne Pedrero-Zayas<sup>b</sup>, Stéphanie Fontagné-Dicharry<sup>a</sup>, Jérôme Roy<sup>a,∗</sup>

## ARTICLE INFO

### Keywords:

- Mercury

- Selenium

- Tuna by-products

- Oxylipins

- Oncorhynchus mykiss

## ABSTRACT

Tuna by-products in aquafeed pose challenges due to heavy metals like mercury (Hg), which has pro-oxidant effects. However, they also contain selenium (Se), an antioxidant oligoelement. This study assessed the oxidative impact of these elements in juvenile rainbow trout over a 6-month feeding trial. Fish were fed plant-based or tuna-based diets, with or without supplementation of methylmercury (MeHg) and selenomethionine (SeMet). Muscle samples were collected at 0, 21, 84, and 168 days to analyze fatty acid profiles, non-enzymatic oxylipins, and antioxidant metabolites (glutathione, vitamin E). Tuna-fed fish exhibited higher oxylipin levels and lower antioxidant metabolites compared to plant-fed fish. Notably, oxylipin levels remained elevated at 84 and 168 days in tuna-fed fish, correlating with a decrease in the GSH:GSSG ratio. In contrast, plant-fed fish showed a transient increase in oxylipins at 84 days, which normalized by day 168, aligning with a temporary drop in the GSH:GSSG ratio. MeHg and SeMet supplementation did not alter these trends. Surprisingly, these findings, along with a previous study, suggest that prolonged oxidative stress in tuna-fed fish is more closely linked to Se bio-accumulation than Hg. Future work should investigate dietary Se concentration and form to better understand these results.

## 1. Introduction

The muscle of fish is the edible portion, and that of trout (*Oncorhynchus mykiss*) is known to contain high levels of long chain polyunsaturated fatty acids (PUFA), notably from the ω-3 series [1]. These ω-3 PUFA confer a plethora of benefits for human health [2]. However, PUFA, being highly unsaturated fatty acids characterised by double bonds in their structures, are susceptible to oxidation, resulting in the formation of non-enzymatic oxylipins [3,4], which can contribute to fish health issues [5] and diminish the health benefits of fish consumption for humans. Non-enzymatic lipid oxidation represents a phenomenon that can occur under conditions of oxidative stress, wherein substantial quantities of free radicals are produced [3,4]. These radicals are able to interact with PUFA by abstracting a hydrogen atom from one of

methylene close to the double bonds. The primary products of lipid oxidation include conjugated dienes and lipid hydroperoxides, which subsequently give rise to secondary products of low molecular weight, including aldehydes, alkanes, alcohols, and acids [6–8]. A direct method of measuring lipid peroxidation is to quantify the primary products derived from oxidized lipids. However, this approach presents a significant challenge due to the labile nature of these products. Consequently, the detection of lipid peroxidation typically relies on indirect methods, such as the thiobarbituric acid reactive substances (TBARS) method, which measures secondary products of oxidation or end products derived from hydroperoxides, such as malondialdehydes [9]. In the present study, we propose to measure the secondary products resulting from non-enzymatic lipid peroxidation of fatty acids, with a particular focus on phytoprostane and phytofuranes derived from α-linolenic acid

<footnote><sup>a</sup> Université de Pau et des Pays de l'Adour, INRAE, NUMEA, Aquapôle, 64310, Saint-Pée-sur-Nivelle, France</footnote>

<footnote><sup>b</sup> Université de Pau et des Pays de l'Adour, E2S UPPA, CNRS, IPREM, Institut des Sciences Analytiques et de Physico-chimie pour l'Environnement et les matériaux, Pau, France</footnote>

<footnote><sup>c</sup> Institut des Biomolécules Max Mousseron (IBMM), Pôle Chimie Balard Recherche, UMR 5247 CNRS, Université de Montpellier, ENSCM, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: jerome.roy@inrae.fr (J. Roy).</footnote>
(ALA, 18:3 ω-3), neuroprostane from docosahexaenoic acid (DHA, 22:6 ω-3) and isoprostane from arachidonic acid (ARA, 20:4 ω-6).

In order to disrupt the chain reaction that leads to lipid oxidation, antioxidant molecules such as vitamin E [10] and glutathione [11,12], among others, are essential. These antioxidant molecules have been reported to decrease in parallel with an increase in lipid peroxidation over time in fish [13,14]. It should be noted that these studies were conducted over an extended period of time, up to three years. In addition to those time effects, diet composition also has a major influence on lipid peroxidation levels in fish. In two major aquaculture species, rainbow trout and European sea bass (Dicentrarchus labrax), an increase of fat and protein dietary inclusions lead to a higher susceptibility to muscle oxidation [15]. Furthermore, in rainbow trout, after 21 days of feeding, muscle fatty acid composition reflects the fatty acid composition of the diet [16] and, consequently, the potential lipid peroxides resulting from the oxidation of these specific fatty acids [17].

The significant advancement of aquaculture, one of the most rapidly expanding food-producing sectors globally [18], has resulted in the diversification of aquafeed ingredients utilized by this industry. Consequently, plant and fish by-products are now employed as ingredients in aquafeed, representing a challenge for the aquaculture sector [19]. Among the available fish by-products, tuna by-products constitute a significant biomass that is increasingly used as an aquafeed ingredient [19]. However, contaminants, particularly heavy metals such as mercury (Hg), are present in tuna by-products [20]. Rainbow trout exposed to plant or tuna-based diets supplemented with MeHg and Se, for 7, 21, 84 and 168 days showed a bioaccumulation of Hg in their muscle, the edible part of the fish, over the course of the dietary exposure [21]. Hg was mainly found in the form of methylmercury (MeHg), an organic form of Hg, and complexed with antioxidant molecules such as glutathione [22], potentially reducing the antioxidant capacity of the muscle and promoting lipid peroxidation. Indeed, MeHg has been demonstrated to exert pro-oxidative effects [23] and to promote lipid peroxidation in fish [24]. Thus, the presence of dietary MeHg might influence time effects observed in fish muscle on lipid peroxide formation and the antioxidant status.

However, tuna by-products contain high levels of selenium (Se) (8.0 µg g⁻¹ of wet weight) [21]. Se is an element that has been reported to counteract the pro-oxidant effects of MeHg in fish [25]. Disruption of Se biochemistry is one of the mechanisms by which Hg exerts its toxicity [26]. Because of this close relationship between Hg and Se, several toxicity assessment criteria have been developed that take into account not only the dietary concentration of MeHg but also Se, for a better evaluation of the toxicological consequences of Hg [27]. Indeed, a Se:Hg molar ratio greater than one is associated with lower Hg toxicity [28]. However, it has recently been emphasised that the assessment of Hg toxicity by evaluating the Se:Hg molar ratio should be accompanied by the assessment of Hg and Se speciation for a proper safety assessment of Hg toxicity [29]. Juvenile rainbow trout exposed to plant- or tuna-based diets exhibited varying tissue accumulations of selenocompounds, depending on the dietary Se source. Fish fed plant-based diets were exposed to low basal Se levels, while those fed tuna-based diets received high basal Se levels, with approximately 80 % of total Se originating from the natural content of tuna by-products. Notably, selenoneine was detected in several tissues, including muscle, but exclusively in fish fed the tuna-based diets [22], reinforcing the hypothesis that selenoneine is one of the major Se forms present in tuna by-products. Moreover, among different dietary forms of added dietary Se, trout bioaccumulate more Se in their muscle when it is supplied as SeMet rather than sodium selenite [21]. Interestingly, Se bioaccumulation in the muscle of fish fed plant- or tuna-based diets supplemented with SeMet was not linear over the time course of the dietary exposure, with a higher increase in Se bioaccumulation after 84 days, followed by a decrease after 168 days of feeding. This observation could underline the lower protection afforded by Se against the pro-oxidant effects of Hg in fish muscle after 168 days. Finally, Marchán-Moreno et al. [21] showed an interesting effect of

SeMet and MeHg supplements in a diet with a high basal Se content (tuna-based diet), with a delay in Hg bioaccumulation in fish muscle compared to fish fed a diet supplemented with MeHg only. These interactions observed on Hg and Se muscle bioaccumulation upon MeHg and SeMet dietary supplementation could influence the temporal effects on antioxidant defences and lipid peroxide formation in fish muscle. However, to the best of our knowledge, no study has evaluated the effects of dietary MeHg and its interaction with SeMet over time on lipid profile, oxylipins and antioxidant metabolite levels in the light of previously obtained data on Se and Hg bioaccumulation in muscle.

The aim of our study was to evaluate the effects of feeding duration (21, 84 and 168 days) with different dietary protein sources containing different levels of MeHg and SeMet on the muscle of juvenile rainbow trout, with particular emphasis on changes in fatty acid profile, oxylipin levels resulting from non-enzymatic oxidation of PUFA, and levels of antioxidant molecules such as glutathione and major vitamin E isoforms. The fish were fed two types of diet: a low Se-plant based diet (0.25 µg Se. g⁻¹ of wet weight and no detectable amount of Hg) or a high Se tuna-based diet (8.0 µg Se.g⁻¹ of wet weight and 0.3 µg Hg.g⁻¹ of wet weight), supplemented with MeHg and SeMet (see Ref. [21]). The level of MeHg supplementation used in this study was chosen to approximate environmentally relevant exposure scenarios. Mercury concentrations can reach up to 2 ppm in the organs and blood of tuna [30] which are used in the production of tuna by-products. Therefore, a concentration of 2 ppm MeHg in fishmeal represents a realistic exposure level in aquaculture. The 2 ppm SeMet supplementation was selected as it remains below the established safe feed limit of 2.8 ppm for salmon (Salmo salar), at which no selenium-related toxic effects, particularly oxidative stress or disruptions in lipid metabolism, have been reported [31]. At this supplementation level, the dietary Se:Hg molar ratio exceeds 1, a threshold above which selenium has been shown to mitigate mercury toxicity [27]. This enabled us to investigate the potential protective role of SeMet against dietary MeHg in aquafeeds.

## 2. Material and methods

### 2.1. Ethics statement

A 6-month feeding trial was realized on rainbow trout juveniles at the INRAE fish farm facilities in Donzacq (permit no. A40-228.1, Landes, France, https://doi.org/10.15454/GPYD-AM38), following the European Directive 2010/63/EU and the guidelines of the National Legislation of Animal Care used for scientific purposes, established by the French ministry of research (decree no. 2013-118, February 1, 2013). This trial was approved by the ethical committee of animal experimentation C2EA-73 and the French Ministry of Higher Education and Research, project saisine n°APAFIS#27846-2020102812241350 v2.

### 2.2. Experimental diets, feeding trial and sampling

A full description of the feeding trial settings is given elsewhere [32]. The experimental diets consisted of 6 diets, 3 of which were based on plant-derived proteins and the other 3 diets were based on proteins derived from tuna by-products (Table 1): a control plant and tuna-based diet (PC and TC, respectively) unsupplemented with methylmercury(II) chloride (MeHg, Sigma-Aldrich, Saint-Quentin-Fallavier, France) and L-selenomethionine (SeMet, Excential Selenium 4000, Orffa Breda, Netherlands), a MeHg-supplemented plant (PH, 2 mg Hg.kg diet⁻¹) and tuna-based diet (TH, 1.6 Hg.kg diet⁻¹), a SeMet and MeHg-supplemented plant (PHO, 1.5 mg Se.kg diet⁻¹ and 2 mg Hg.kg diet⁻¹) and tuna-based diet (THO, 1.5 mg Se.kg diet⁻¹ and 1.6 mg Hg.kg diet⁻¹). The final values of Se and Hg measured in diets are detailed in Table 1 ranging from 0.3 to 9.0 mg Se.kg diet⁻¹ and from 0 to 1.9 mg Hg. kg diet⁻¹. The only significant effect on growth performance was a reduction in mean body weight in fish fed a plant-based diet
<table><caption>**Table 1**
Plant and tuna-based diet compositions.</caption><thead><tr><th rowspan="2">Diet</th><th colspan="3">Plant-based diets</th><th colspan="3">Tuna-based diets</th></tr><tr><th>PC</th><th>PH</th><th>PHO</th><th>TC</th><th>TH</th><th>THO</th></tr></thead><tbody><tr><td><strong>Analytical composition</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Dry matter (DM, %)</td><td>97.4</td><td></td><td></td><td>97.7</td><td></td><td></td></tr><tr><td>Crude protein (% DM)</td><td>49.3</td><td></td><td></td><td>51.2</td><td></td><td></td></tr><tr><td>Total lipid (% DM)</td><td>22.0</td><td></td><td></td><td>19.1</td><td></td><td></td></tr><tr><td>Gross energy (kJ/g DM)</td><td>24.8</td><td></td><td></td><td>24.1</td><td></td><td></td></tr><tr><td>Starch (% DM)</td><td>8.2</td><td></td><td></td><td>11.6</td><td></td><td></td></tr><tr><td>Ash (% DM)</td><td>6.7</td><td></td><td></td><td>7.8</td><td></td><td></td></tr><tr><td><strong>Lipid profile (%)</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>14:0</td><td>3.67</td><td></td><td></td><td>2.89</td><td></td><td></td></tr><tr><td>16:0</td><td>15.63</td><td></td><td></td><td>16.82</td><td></td><td></td></tr><tr><td>18:0</td><td>3.07</td><td></td><td></td><td>4.43</td><td></td><td></td></tr><tr><td>Sum saturated<sup>a</sup></td><td>23.61</td><td></td><td></td><td>25.83</td><td></td><td></td></tr><tr><td>16:1</td><td>3.57</td><td></td><td></td><td>3.21</td><td></td><td></td></tr><tr><td>18:1</td><td>28.66</td><td></td><td></td><td>29.13</td><td></td><td></td></tr><tr><td>Sum monounsaturated<sup>b</sup></td><td>33.71</td><td></td><td></td><td>33.80</td><td></td><td></td></tr><tr><td>18:2 ω-6</td><td>15.46</td><td></td><td></td><td>10.37</td><td></td><td></td></tr><tr><td>ARA (20:4 ω-6)</td><td>0.45</td><td></td><td></td><td>0.85</td><td></td><td></td></tr><tr><td>Sum ω-6<sup>c</sup></td><td>16.26</td><td></td><td></td><td>12.22</td><td></td><td></td></tr><tr><td>ALA (18:3 ω-3)</td><td>9.42</td><td></td><td></td><td>10.15</td><td></td><td></td></tr><tr><td>EPA (20:5 ω-3)</td><td>7.48</td><td></td><td></td><td>5.49</td><td></td><td></td></tr><tr><td>DHA (22:6 ω-3)</td><td>3.44</td><td></td><td></td><td>7.16</td><td></td><td></td></tr><tr><td>Sum ω-3<sup>d</sup></td><td>22.67</td><td></td><td></td><td>24.79</td><td></td><td></td></tr><tr><td>Saturated/<br>polyunsaturated<sup>e</sup></td><td>0.58</td><td></td><td></td><td>0.67</td><td></td><td></td></tr><tr><td>ω-3/ω-6</td><td>1.39</td><td></td><td></td><td>2.03</td><td></td><td></td></tr><tr><td><strong>Vitamin E (mg.kg of diet<sup>-1</sup>)</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>α-tocopherol</td><td>4.35</td><td>6.03</td><td>3.70</td><td>0.00</td><td>0.13</td><td>0.00</td></tr><tr><td>γ-tocopherol</td><td>28.33</td><td>30.61</td><td>21.97</td><td>12.82</td><td>12.00</td><td>11.09</td></tr><tr><td>δ-tocopherol</td><td>0.63</td><td>0.63</td><td>0.50</td><td>0.30</td><td>0.47</td><td>0.27</td></tr><tr><td><strong>Elemental composition (mg.kg diet<sup>-1</sup> wet weight)</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Mercury</td><td>–</td><td>1.8</td><td>1.9</td><td>0.3</td><td>1.9</td><td>1.8</td></tr><tr><td>Selenium</td><td>0.3</td><td>0.3</td><td>2.0</td><td>7.5</td><td>7.2</td><td>9.0</td></tr><tr><td>Se:Hg molar ratio</td><td>–</td><td>0.4</td><td>2.6</td><td>62.7</td><td>9.9</td><td>13.0</td></tr></tbody></table>

PC, plant-based diet not supplemented; PH, plant-based diet supplemented with MeHg; PHO, plant-based diet supplemented with both MeHg and SeMet; TC, tuna-based diet not supplemented; TH, tuna-based diet supplemented with MeHg; THO, tuna-based diet supplemented with both MeHg and SeMet; ALA, alpha-linolenic acid; ARA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid.

a sum saturated includes 12:0, 14:0, 15:0, 16:0, 17:0, 18:0, 20:0, 22:0, 24:0.

b sum monounsaturated includes 14:1, 16:1, 17:1, 18:1, 20:1, 22:1.

c sum ω-6 includes 18:2 ω-6, 18:3 ω-6, 20:2 ω-6, 20:3 ω-6, 20:4 ω-6, 22:4 ω-6, 22:5 ω-6.

d sum ω-3 includes 18:3 ω-3, 18:4 ω-3, 20:3 ω-3, 20:4 ω-3, 20:5 ω-3, 21:5 ω-3, 22:5 ω-3.

e polyunsaturates include ω-6 fatty acids, ω-3 fatty acids, 14 PUFA, 16:2 ω-4, 16:3 ω-4, 16:4 ω-1, 18:2 ω-4, 18:3 ω-4, 18:4 ω-1.

supplemented with MeHg (PH) compared to the control diet (PC) at 21 days [32]. No differences were observed between other dietary groups at 21 or 168 days (Supplementary Fig. 1A and 1B). Samplings were realized after 0, 21, 84 and 168 days of feeding. Fish muscle was collected at the dorsal fin from each dietary group and snap-frozen in liquid nitrogen. Samples were then stored at -80 °C until analysis. Prior to chemical and biochemical analysis frozen muscle tissues were reduced into powder in liquid nitrogen using a grinder. Muscle Se bioaccumulation followed a similar trend in all dietary groups, increasing from 21 to 84 days before decreasing at 168 days [21]. Fish fed the PHO diet accumulated more Se than those on the THO diet, after 84 days of feeding (Supplementary Fig. 1C and 1D). Fish fed a plant-based diet exhibited higher muscle Hg bioaccumulation than those fed a tuna-based diet [21]. A gradual increase in Hg concentrations was observed in fish fed plant-based diets supplemented with MeHg (PH, PHO), whereas those fed tuna-based diets supplemented with MeHg (TH, THO) displayed a different bioaccumulation pattern, with a significant increase occurring only after 168 days. A delay in Hg bioaccumulation was also observed

within the tuna-based diet group, with significant increases detected after 21 days for the TH diet and 84 days for the THO diet compared to the control (Supplementary Fig. 1E and 1F).

## 2.3. Fatty acid analysis

The lipid content and fatty acid profiles were determined in diets (approx. 2.5 g) (Table 1) and frozen crushed muscles (n = 6; approx. 5 g per sample) using gas chromatography coupled with flame ionization detector (GC-FID), as previously described [33]. Briefly, total lipid was extracted according to Folch et al. [34] using dichloromethane instead of chloroform. Total lipids were transesterified using boron trifluoride and methanol (14%) [35]. Fatty acid methyl esters were analyzed through GC-FID. Standard mixtures (Sigma, St-Louis, MO, USA) were used to identify fatty acids with Varian Star Chromatography Software (Star Software, version 5) for chromatogram analysis. Fatty acids were expressed as a percentage of the fatty acid of interest relative to the total of identified fatty acid methyl esters. Derived parameters included the ratio of saturated to PUFA, ω-3 to ω-6 fatty acid ratio, and an unsaturation index calculated as: unsaturation index = sum ((percentage fatty acids of interest) × (number of double bonds in fatty acids of interest)).

## 2.4. Determination of selected non-enzymatic lipid oxidation mediators in muscle

Non-enzymatic lipid metabolites were measured on frozen crushed muscles (n = 6; 200 mg per sample) at the Institut des Biomolécules Max Mousseron (IBMM, Montpellier, France) by liquid chromatography with tandem mass spectroscopy (microLC MS/MS). For the extraction of non-enzymatic lipid oxylipins, trout samples were prepared as previously published [36]. Briefly, organic extracts of muscle were obtained using methanol, butylated hydroxytoluene (1%, w/v in methanol) and chloroform solutions. Extracts were evaporated under N₂ stream and then hydrolyzed with KOH (1 M in H₂O) and formic acid (40 mM) solutions. Samples were subjected to solid phase extraction on a Biotage® Extrahera™. The samples were then injected and analyzed using a microLC-MS/MS 5500 QTrap system. Data processing of the mass spectrometry results was carried out using MultiQuant 3.0 software (Sciex Applied Biosystems). Results are expressed in pg.mg of sample⁻¹.

## 2.5. Vitamin E levels determination

The concentrations of main forms of vitamin E (α, γ, and δ-tocopherol) in diets (approx. 100 mg) (Table 1) and frozen crushed muscles (n = 6, approx. 100 mg per sample) were measured using high-performance liquid chromatography (HPLC) coupled with fluorescence detection. Tocopherols were extracted according to Folch et al. [34] using dichloromethane instead of chloroform. Samples were evaporated using N₂ stream and then reconstituted in methanol. Separation was carried out on a Phenomenex Luna® PFP (2) 100 Å, 3 µm, 100 × 2 mm column. The solvent used for separation was a methanol and water solution (93:7, v/v). Separation was performed with an isocratic mobile phase (methanol/H₂O 93:7, v/v) at a flow rate mode at 0.5 mL min⁻¹. Detection was done using fluorescence (Waters 2475 fluorescence detector, Waters, Saint-Quentin en Yvelines, France) at 295/330 nm (excitation/emission). Tocopherol forms were identified based on their standard retention times. The concentrations of different forms of tocopherols are expressed in nmol.g⁻¹ of sample. Our approach employed a Folch extraction without saponification, allowing the quantification of the free and biologically active forms of tocopherols. In contrast, the acetylated form (tocopheryl acetate), which was added to the diet, requires saponification prior to measurement and was therefore not detected.
## 2.6. Glutathione levels determination

Total levels of reduced (GSH) and oxidized (GSSG) glutathione were measured on frozen crushed muscles (n = 6, approx. 500 mg per sample) using HPLC coupled with UV detector as previously described [37]. Briefly, frozen crushed muscles were homogenized with zirconium beads using Precellys® (Precellys® 24, Bertin Technologies, Montigny-le-Bretonneux, France) in 20 mM of phosphate buffer (20 mM, EDTA 1 mM, pH 6.5). Then deproteinization was realized using metaphosphoric acid (10 %). Deproteinization of samples was realized using metaphosphoric acid (10 %). Sample were centrifugated (14 000 g, 5

min) and then filtered using polyvinylidene fluoride 0.22 µm. Separation was performed on a Symmetry Shield RP18 column using gradient elution: 0–10 min 99.5 % (A), 0.5 % (D); 12–15 min 40 % (A), 60 % (B); 17–30 min 99.5 % (A), 0.5 % (D) with 20 mM phosphate buffer solution, pH 2.7 (A), acetonitrile (B), ultrapure water (C), and methanol (D). Detection of metabolites was performed at 210 nm based on their standard retention times. Total protein content in samples was determined through a colorimetric BCA assay (#BCA1-1 KT, Sigma-Aldrich, Saint-Louis, MO, USA), using 5 µL of homogenized muscle sample diluted in ultra-pure water (1:25). Absorbance was measured at 562 nm. The concentrations of the different metabolites are expressed in pg.mg⁻¹

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** PCA biplots of all parameters measured at all time points (21, 84 and 168 days) in fish fed plant- or tuna-based diets (A); in Control (not supplemented), MeHg (supplemented with MeHg), MeHgSeMet (supplemented with MeHg and SeMet) (B); in both based diets (plant and tuna) after 0, 21, 84 and 168 days (C). Arrows represent the 5 most contributing variables for the representation of the dataset: monounsaturates, sum of monounsaturated fatty acid; saturates, sum of saturated fatty acid; 12:0, fatty acid 12:0; Sum.FA: sum of fatty acids; PUFA.n-6: sum of PUFA ω-6; 22:6.n-3, fatty acid 22:6 ω-3; 17:0, fatty acid 17:0; 16:1, fatty acid 16:1; n3/n6, ratio sum polyunsaturated fatty acid ω-3/sum polyunsaturated fatty acid ω-6; saturates. Ellipses represent a 95 % confidence intervals around 42 individuals in plant- and tuna-based diets (A); around 28 individuals in control, MeHg and MeHg/SeMet-supplemented groups (B) and around 23 individuals at 21 days, 25 individuals at 84 days and 36 individuals for 168 days. Too few individuals were used at day 0 (n = 3) to display an ellipse, only the barycenter for day 0 was shown (C).</figcaption></figure>
of proteins.

## 2.7. Statistical analysis

For visual data exploration, PCA was realized using the R package factoextra. For the temporal clustering analysis, Z-score normalization was first applied to the data, and clusters were identified using K-means clustering. Data are presented as mean ± standard error of the mean (SEM). A one-way ANOVA was realized to evaluate the effect of the based-diets and the feeding duration. A two-way ANOVA was performed to evaluate the effects of (i) feeding duration, (ii) dietary supplementations with SeMet and MeHg, and (iii) their interaction within each basal diet. Prior to performing one- and two-way ANOVA, data were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene's test). When significant main effects or interactions were observed, a *post-hoc* Tukey test was conducted to identify specific differences. All statistical analyses were performed using R software (version 3.6.1; R Core Team, 2008), and differences were considered significant at *p-value* < 0.05.

# 3. Results

## 3.1. Effects of dietary base

Principal component analysis (PCA) revealed a clear separation between fish fed plant- and tuna-based diets, primarily driven by fatty acid-related variables. The sum of ω-6 PUFA was the main contributing variable (Fig. 1A). Dietary base significantly influenced muscle fatty acid profiles: fish fed tuna-based diets had higher levels of saturates, monounsaturates, and ω-3 PUFA which resulted in higher saturates/ polyunsaturates and ω-3/ω-6 ratios. Whereas fish fed plant-based diets showed higher total ω-6 PUFA. However, no differences between the insaturation index was observed between both based groups (Supplemental Table 1). These trends mirrored the fatty acid composition of the respective based diets (Table 1). On the parameter of interests, fish fed plant-based diets had higher total fatty acids, while those fed tuna-based diets exhibited higher levels of ARA and DHA (Table 2), consistent with the dietary profiles (Table 1). Among oxylipins, 4 derived from ALA (9-F₁t-PhytoP, ent-16-F₁t-PhytoP, 9-epi-9-F₁t-PhytoP, 9-L₁-PhytoP), 2 from ARA (5-F₂c-IsoP, PGF₂), and 3 from DHA (4(RS)-4-F₄t-NeuroP, 10(S)-10-F₄t-NeuroP, 10(R)-10-F₄t-NeuroP) were elevated in the muscle of fish fed tuna-based diets (Table 2). Antioxidant α-, β-, and γ-tocopherol were higher in fish fed plant-based diets. The GSH/GSSG ratio was also higher in this group, although levels of GSH and GSSG were greater in tuna-fed fish (Table 2).

## 3.2. Effects of dietary supplementation

PCA analysis showed no separation between groups based on dietary supplementation, with substantial overlap of confidence ellipses (Fig. 1B). As expected, supplementation had no effect on muscle fatty acid profiles (Supplemental Table 2). Effects were limited to antioxidant markers: δ-tocopherol levels were reduced in fish fed a diet supplemented with SeMet and MeHg compared to the control. GSSG levels were lower in group fed with a MeHg-supplemented diet, compared to the control. Consequently, the GSH/GSSG ratio was elevated in fish fed MeHg and SeMet/MeHg-supplemented diets (Table 3).

## 3.3. Effects of timing

PCA revealed a clear separation between fish sampled at days 0 and 21 and those sampled at later time points, indicating marked differences in the underlying data. In contrast, samples from days 84 and 168 were spatially adjacent, as shown by overlapping confidence ellipses, suggesting greater similarity between these groups (Fig. 1C). Fatty acid profile parameters were the main contributors to the PCA, with the ω-3/

<table><caption>**Table 2**
Based-diet effects on selected parameters.</caption><thead><tr><th>Based-diet</th><th>Plant</th><th>Tuna</th><th>p-value</th></tr></thead><tbody><tr><td><strong>Fatty acid profile</strong></td><td></td><td></td><td></td></tr><tr><td>Total fatty acid (%)</td><td>7.19 ± 0.21<sup>a</sup></td><td>6.37 ± 0.18<sup>b</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>ALA (%)</td><td>6.14 ± 0.11</td><td>6.21 ± 0.13</td><td>0.72</td></tr><tr><td>ARA (%)</td><td>0.57 ± 0.01<sup>b</sup></td><td>0.86 ± 0.01<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>DHA (%)</td><td>8.10 ± 0.17<sup>b</sup></td><td>10.66 ± 0.14<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td><strong>Lipid peroxides levels</strong></td><td></td><td></td><td></td></tr><tr><td>Peroxides derived from ALA (pg·mg<sup>-1</sup>)</td><td></td><td></td><td></td></tr><tr><td>ent-16A-9-epi-ST-D<sup>14</sup>-10-PhytoF</td><td>2.46 ± 0.10</td><td>2.45 ± 0.10</td><td>0.96</td></tr><tr><td>ent-16B-9-epi-ST-D<sup>14</sup>-10-PhytoF</td><td>4.92 ± 0.25</td><td>5.07 ± 0.20</td><td>0.65</td></tr><tr><td>ent-16-epi-16-F<sub>1</sub>t-PhytoP</td><td>1.31 ± 0.08</td><td>1.54 ± 0.10</td><td>0.07</td></tr><tr><td>9-F<sub>1</sub>t-PhytoP</td><td>2.02 ± 0.12<sup>b</sup></td><td>2.42 ± 0.15<sup>a</sup></td><td><strong>0.04</strong></td></tr><tr><td>ent-16-F<sub>1</sub>t-PhytoP</td><td>1.13 ± 0.07<sup>b</sup></td><td>1.39 ± 0.09<sup>a</sup></td><td><strong>0.02</strong></td></tr><tr><td>9-epi-9-F<sub>1</sub>t-PhytoP</td><td>2.17 ± 0.10<sup>b</sup></td><td>2.53 ± 0.13<sup>a</sup></td><td><strong>0.04</strong></td></tr><tr><td>16-B<sub>1</sub>t-PhytoP</td><td>6.36 ± 0.31</td><td>6.88 ± 0.40</td><td>0.30</td></tr><tr><td>9-L<sub>1</sub>t-PhytoP</td><td>5.69 ± 0.31<sup>b</sup></td><td>6.98 ± 0.46<sup>a</sup></td><td><strong>0.02</strong></td></tr><tr><td>Peroxides derived from ARA (pg·mg<sup>-1</sup>)</td><td></td><td></td><td></td></tr><tr><td>5-F<sub>2c</sub>-IsoP</td><td>1.31 ± 0.06<sup>b</sup></td><td>1.73 ± 0.11<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>PGF<sub>2</sub></td><td>0.26 ± 0.01<sup>b</sup></td><td>0.38 ± 0.01<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>Peroxides derived from DHA (pg·mg<sup>-1</sup>)</td><td></td><td></td><td></td></tr><tr><td>4(RS)-4-F<sub>4t</sub>-NeuroP</td><td>1.32 ± 0.05<sup>b</sup></td><td>1.76 ± 0.06<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>10(S)-10-F<sub>4t</sub>-NeuroP</td><td>0.54 ± 0.02<sup>b</sup></td><td>0.69 ± 0.02<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>10(R)-10-F<sub>4t</sub>-NeuroP</td><td>0.97 ± 0.03<sup>b</sup></td><td>1.24 ± 0.03<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td><strong>Antioxidant molecules</strong></td><td></td><td></td><td></td></tr><tr><td>Vitamin E metabolites (nmol·g<sup>-1</sup>)</td><td></td><td></td><td></td></tr><tr><td>α-tocopherol</td><td>23.49 ± 1.01<sup>a</sup></td><td>14.73 ± 0.78<sup>b</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>γ-tocopherol</td><td>12.05 ± 1.29<sup>a</sup></td><td>3.23 ± 0.33<sup>b</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>δ-tocopherol</td><td>0.71 ± 0.06<sup>a</sup></td><td>0.53 ± 0.04<sup>b</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td><strong>Glutathione metabolites</strong></td><td></td><td></td><td></td></tr><tr><td>GSH (pg/g of proteins<sup>-1</sup>)</td><td>37.24 ± 1.75<sup>b</sup></td><td>43.36 ± 1.94<sup>a</sup></td><td><strong>0.02</strong></td></tr><tr><td>GSSG (pg/g of proteins<sup>-1</sup>)</td><td>1.15 ± 0.09<sup>b</sup></td><td>2.30 ± 0.17<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>Ratio GSH/GSSG</td><td>60.53 ± 9.52<sup>a</sup></td><td>29.23 ± 4.22<sup>b</sup></td><td><strong>&lt;0.01</strong></td></tr></tbody></table>

Values are represented as mean ± SEM. In both based-diets, parameters related to fatty acid profile, oxylipins and GSH metabolites *n* = 54 individuals. For tocopherol isoforms measurement *n* = 68 individuals. Plant: plant-based diets; Tuna: tuna-based diets. Values not sharing the same superscript letter are considered as significantly different, based on one-way ANOVA (significance *p-value* < 0.05) followed by Tukey's *post-hoc* test.

ω-6 ratio and the percentage of 17:0 fatty acid as the main contributing variables. The sum of saturated fatty acids increased at days 84 and 168 compared to days 0 and 21. Sum of monounsaturates decreased after 21 days compared to 0 day and decrease to a higher extent after 84 and 168 days, compared to 0 and 21 days. Sum of PUFA ω-3 increased gradually increased between 0 and 84 days, then declined by day 168 to levels similar to day 21. As a result, saturates/polysaturates ratio was higher at 168 days, compared to 21 and 84 days, whereas the insaturation index was lower at 168 days, compared to 21 and 84 days (Supplemental Table 3). Total fatty acids were higher at days 0 and 168 compared to days 21 and 84. ALA levels rose steadily from day 0 to day 84 and remained high at day 168. Although ARA levels showed a significant time effect, Tuckey *post-hoc* analysis revealed no differences between time points. DHA levels significantly declined at day 168 compared to day 21 (Table 4). Among the 8 oxylipins derived from ALA oxidation, 4 (9-F₁t-PhytoP, 9-epi-9-F₁t-PhytoP, 16-B₁t-PhytoP, 9-L₁-PhytoP) increased at days 84 and 168 compared to days 0 and 21. Two (ent-16-epi-16-F₁t-PhytoP, ent-16-F₁t-PhytoP) increased at days 84 and 168 relative to day 21. Ent-16B-9-epi-ST-Δ<sup>14</sup>-10-PhytoF increased at day 84 compared to days 0 and 21, and ent-16A-9-epi-ST-Δ<sup>14</sup>-10-PhytoF increased at day 84 relative to day 21. Among ARA-derived oxylipins, 5-F₂c-IsoP increased at day 84 compared to days 0 and 21, and further increased at day 168, compared to 0, 21 and 84 days. PGF₂ levels were higher at days 84 and 168 compared to days 0 and 21. For DHA-derived oxylipins, 4(RS)-4-F₄t-NeuroP increased at days 84 and 168 relative to day 21; 10(S)-10-F₄t-NeuroP and 10(R)-10-F₄t-NeuroP increased at day 84 compared to day 21 (Table 4). Regarding antioxidants, α-tocopherol was higher at days 0 and 84 compared to days 21 and 168. γ-tocopherol
<table><caption>**Table 3**
Supplementation effects on selected parameters.</caption><thead><tr><th>Based-diet<br>supplementation</th><th>Control</th><th>+MeHg</th><th>+MeHg/SeMet</th><th>p-value</th></tr></thead><tbody><tr><td><strong>Fatty acid profile</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Total fatty acid<br>(%)</td><td>6.86 ± 0.27</td><td>6.71 ± 0.26</td><td>6.78 ± 0.23</td><td>0.92</td></tr><tr><td>ALA (%)</td><td>6.21 ± 0.15</td><td>6.13 ± 0.14</td><td>6.19 ± 0.15</td><td>0.94</td></tr><tr><td>ARA (%)</td><td>0.71 ± 0.03</td><td>0.72 ± 0.03</td><td>0.71 ± 0.03</td><td>0.97</td></tr><tr><td>DHA (%)</td><td>9.34 ± 0.29</td><td>9.49 ± 0.27</td><td>9.32 ± 0.28</td><td>0.91</td></tr><tr><td><strong>Lipid peroxides levels</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Peroxides derived from ALA (pg.mg<sup>-1</sup>)</td><td></td><td></td><td></td><td></td></tr><tr><td>ent-16A-9-epi-<br>ST-D<sup>14</sup>-10-<br>PhytoF</td><td>2.33 ± 0.09</td><td>2.51 ± 0.10</td><td>2.53 ± 0.13</td><td>0.48</td></tr><tr><td>ent-16B-9-epi-<br>ST-D<sup>14</sup>-10-<br>PhytoF</td><td>4.86 ± 0.22</td><td>5.12 ± 0.25</td><td>5.02 ± 0.29</td><td>0.81</td></tr><tr><td>ent-16-epi-16-<br>F<sub>1</sub><sub>1</sub>-PhytoP</td><td>1.27 ± 0.08</td><td>1.51 ± 0.09</td><td>1.50 ± 0.12</td><td>0.20</td></tr><tr><td>9-F<sub>1</sub><sub>1</sub>-PhytoP</td><td>1.94 ± 0.12</td><td>2.35 ± 0.13</td><td>2.40 ± 0.19</td><td>0.11</td></tr><tr><td>ent-16-F<sub>1</sub><sub>1</sub>-<br>PhytoP</td><td>1.14 ± 0.08</td><td>1.31 ± 0.08</td><td>1.33 ± 0.11</td><td>0.33</td></tr><tr><td>9-epi-9-F<sub>1</sub><sub>1</sub>-<br>PhytoP</td><td>2.12 ± 0.11</td><td>2.45 ± 0.12</td><td>2.51 ± 0.17</td><td>0.14</td></tr><tr><td>16-B<sub>1</sub><sub>1</sub>-PhytoP</td><td>5.94 ± 0.36</td><td>6.98 ± 0.46</td><td>6.95 ± 0.45</td><td>0.16</td></tr><tr><td>9-L<sub>1</sub>-PhytoP</td><td>5.63 ± 0.42</td><td>6.70 ± 0.49</td><td>6.69 ± 0.51</td><td>0.22</td></tr><tr><td><strong>Peroxides derived from ARA (pg.mg<sup>-1</sup>)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>5-F<sub>2c</sub>-IsoP</td><td>1.52 ± 0.12</td><td>1.48 ± 0.12</td><td>1.56 ± 0.12</td><td>0.89</td></tr><tr><td>PGF<sub>2</sub></td><td>0.30 ± 0.02</td><td>0.32 ± 0.02</td><td>0.33 ± 0.02</td><td>0.45</td></tr><tr><td><strong>Peroxides derived from DHA (pg.mg<sup>-1</sup>)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>4(RS)-4-F<sub>4t</sub>-<br>NeuroP</td><td>1.47 ± 0.07</td><td>1.55 ± 0.07</td><td>1.59 ± 0.08</td><td>0.52</td></tr><tr><td>10(S)-10-F<sub>4t</sub>-<br>NeuroP</td><td>0.59 ± 0.02</td><td>0.62 ± 0.03</td><td>0.65 ± 0.04</td><td>0.32</td></tr><tr><td>10(R)-10-F<sub>4t</sub>-<br>NeuroP</td><td>1.06 ± 0.04</td><td>1.12 ± 0.05</td><td>1.14 ± 0.05</td><td>0.34</td></tr><tr><td><strong>Antioxidant molecules</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Vitamin E metabolites (nmol.g<sup>-1</sup>)</td><td></td><td></td><td></td><td></td></tr><tr><td>α-tocopherol</td><td>18.72 ± 1.31</td><td>19.49 ± 1.34</td><td>19.24 ± 1.40</td><td>0.91</td></tr><tr><td>γ-tocopherol</td><td>7.42 ± 1.24</td><td>6.56 ± 1.12</td><td>9.36 ± 1.77</td><td>0.34</td></tr><tr><td>δ-tocopherol</td><td>0.72 ± 0.07<sup>a</sup></td><td>0.66 ± 0.06<sup>ab</sup></td><td>0.46 ± 0.04<sup>b</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td><strong>Glutathione metabolites</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>GSH (pg/g of<br>proteins<sup>-1</sup>)</td><td>39.14 ± 2.45</td><td>38.78 ± 2.24</td><td>43.15 ± 2.22</td><td>0.34</td></tr><tr><td>GSSG (pg/g of<br>proteins<sup>-1</sup>)</td><td>2.06 ± 0.15<sup>a</sup></td><td>1.39 ± 0.17<sup>b</sup></td><td>1.74 ± 0.24<sup>ab</sup></td><td><strong>0.05</strong></td></tr><tr><td>Ratio GSH/GSSG</td><td>22 ± 2<sup>b</sup></td><td>59 ± 5<sup>a</sup></td><td>54 ± 12<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr></tbody></table>

Values are represented as mean ± SEM. For fatty acid profile, oxylipins and GSH metabolites parameters *n* = 36 per supplementation. For tocopherol parameters *n* = 48 per supplementation. Control: diets not supplemented, +MeHg: diets supplemented with MeHg; +MeHg/SeMet: diets supplemented with MeHg and SeMet. Values not sharing the same superscript letter are considered as significantly different, based on one-way ANOVA (significance *p*-value < 0.05) followed by Tukey's post-hoc test.

increased progressively from day 21 to day 168. δ-tocopherol levels were elevated at days 84 and 168 relative to days 0 and 21, with higher values at day 84, compared to 168 days. GSH decreased at day 84 compared to all other time points. GSSG increased at day 168 compared to days 0 and 21. The GSH/GSSG ratio declined from day 0 to day 84 and remained stable at day 168, compared to 84 days (Table 4).

### 3.4. Effects of dietary base on temporal responses

As previously shown, fish fed different based-diets exhibited marked dissimilarities. Therefore, temporal changes in all measured parameters were explored within each dietary group. Significant variations occurred between days 0, 21, and 84. In contrast, fewer changes were observed between days 84 and 168, as indicated by the flatter trends during this period for both diets (Fig. 2A and B). The dynamic of all

parameters was clustered by based-diet (Fig. 2C–F). Some clusters exhibited opposite temporal trends between based-diets. In the plant-based diet, Cluster 2 showed a gradual increase over time, while Cluster 1 and 2 in the tuna-based diet exhibited a gradual decrease. Cluster 1 in the plant-based group decreased from day 0–84, then increased at day 168. Conversely, Cluster 4 in the tuna-based group increased until day 84, then decreased by day 168. Cluster 3 in the plant-based diet increased between days 0 and 21, followed by a gradual decline, whereas in the tuna-based diet, Cluster 3 decreased initially (0–21 days), then increased. Some trends were shared: Cluster 1 in the plant-based diet and Clusters 1 and 2 in the tuna-based diet all decreased from day 0–84. From day 84–168, values increased in the plant-based diet and decreased in the tuna-based diet. Cluster 2 in the plant-based group and Cluster 4 in the tuna-based group both increased from day 0–84, but after 84 days, the plant-based increased and the tuna-based diets decreased (Fig. 2C–F). In the plant-based diet, Cluster 2 (average pattern: 168 > 84 > 21 > 0) included the sum of ω-3 and ω-6 PUFA, ALA, ALA-derived oxylipins (16-B<sub>1</sub><sub>1</sub>-PhytoP, 9-L<sub>1</sub>-PhytoP), one ARA-derived oxylipin (5-F<sub>2c</sub>-IsoP), and antioxidant-related compounds (GSSG, δ- and γ-tocopherol) (Supplemental Table 4). In contrast, Clusters 1 and 2 (average pattern: 0 > 21 > 84 > 168) of the tuna-based diet were associated with the sum of monounsaturated fatty acids, ω-6 PUFA, and antioxidant markers such as GSH, the GSH/GSSG ratio, and α-tocopherol (Supplemental Table 5). Cluster 1 (average pattern: 0 > 21 > 168 > 84) in the plant-based diet included sum of monounsaturated fatty acids, GSH, and the GSH/GSSG ratio. Its opposite, Cluster 4 (average pattern: 84 > 168 > 21 > 0) in the tuna-based diet, was characterized by sum of saturated fatty acids. ω-3 PUFA, ARA, ALA, and DHA levels. Cluster 3 (average pattern: 21 > 84 > 168 > 0) in the plant-based diet contained ARA, five ALA-derived oxylipins (9-F<sub>1</sub><sub>1</sub>-PhytoP, 9-epi-9-F<sub>1</sub><sub>1</sub>-PhytoP, ent-16A-9-epi-ST-Δ<sup>14</sup>-10-PhytoF, ent-16B-9-epi-ST-Δ<sup>14</sup>-10-PhytoF, ent-16-F<sub>1</sub><sub>1</sub>-PhytoP, ent-16-epi-16-F<sub>1</sub><sub>1</sub>-PhytoP), and one DHA-derived oxylipin (10(S)-10-F<sub>4t</sub>-NeuroP). In the tuna-based diet, all measured oxylipins were grouped in Cluster 3 (average pattern: 168 > 84 > 0 > 21), along with GSSG, δ- and γ-tocopherol (Supplemental Table 4–5).

### 3.5. Effects of timing and supplementation in plant- and tuna-based diets

The effects of time and supplementation were analyzed within each diet type. As observed in the other PCA, time was the primary factor separating groups, particularly between days 21 and 84, exceeding the effect of supplementation in both based-diets (Fig. 3A and B). In the plant-based diet, the main contributors to the PCA were 22:4 ω-6 and 22:5 ω-3, while in the tuna-based diet, the GSH/GSSG ratio and 17:1 were most contributing. Although time significantly affected saturated fatty acids in the plant-based group, Tukey's post-hoc test revealed no significant differences between time points (Supplemental Table 6). In contrast, sum of saturated fatty acids gradually increased in the tuna-based group (Supplemental Table 7). In fish fed the plant-based diet, the sum of unsaturated fatty acids in muscle decreased at days 84 and 168. In the tuna-based group, a decrease was observed only at day 84, compared to 21 days. The total ω-6 content increased in the plant-based group at days 84 and 168, while it decreased at day 21 in the tuna-based group. The sum of ω-3 fatty acids increased at day 84 compared to day 21. By day 168, no significant differences were detected in either group relative to day 21. The saturates/polyunsaturates ratio decreased at day 84 compared to day 21 in the plant-based diet, while it increased progressively in the tuna-based diet. In the plant-based group, the ω-3/ω-6 ratio declined at day 168 compared to days 21 and 84. In contrast, it increased in the tuna-based group at days 84 and 168, with a greater increase observed at day 84. The unsaturation index decreased at day 168 compared to days 21 and 84 in fish fed the tuna-based diet. The percentage of total lipids in muscle increased at day 168 compared to days 21 and 84 in both based-diet groups. In fish fed the plant-based diet, DHA levels decreased at days 84 and 168, while in the tuna-based group, the decrease was observed only at day 168, compared to
<table><caption>**Table 4**
Timing effects on selected parameters.</caption><thead><tr><th>Timing</th><th>Day 0</th><th>Day 21</th><th>Day 84</th><th>Day 168</th><th>p-value</th></tr></thead><tbody><tr><td><strong>Fatty acid profile</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Total fatty acid (%)</td><td>8.54 ± 0.40<sup>a</sup></td><td>5.93 ± 0.15<sup>b</sup></td><td>6.39 ± 0.18<sup>b</sup></td><td>8.03 ± 0.25<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>ALA (%)</td><td>3.28 ± 0.00<sup>c</sup></td><td>5.00 ± 0.04<sup>b</sup></td><td>6.71 ± 0.05<sup>a</sup></td><td>6.81 ± 0.06<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>ARA (%)</td><td>0.49 ± 0.01<sup>a</sup></td><td>0.67 ± 0.02<sup>a</sup></td><td>0.75 ± 0.03<sup>a</sup></td><td>0.72 ± 0.03<sup>a</sup></td><td><strong>0.02</strong></td></tr><tr><td>DHA (%)</td><td>8.89 ± 0.42<sup>ab</sup></td><td>10.11 ± 0.24<sup>a</sup></td><td>9.50 ± 0.31<sup>ab</sup></td><td>8.53 ± 0.25<sup>b</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td><strong>Lipid peroxides levels</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Peroxides derived from ALA (pg.mg<sup>-1</sup>)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ent-16A-9-epi-ST-D<sup>14</sup>-10-PhytoF</td><td>2.14 ± 0.22<sup>ab</sup></td><td>2.14 ± 0.10<sup>b</sup></td><td>2.76 ± 0.14<sup>a</sup></td><td>2.47 ± 0.11<sup>ab</sup></td><td><strong>0.02</strong></td></tr><tr><td>ent-16B-9-epi-ST-D<sup>14</sup>-10-PhytoF</td><td>3.63 ± 0.32<sup>b</sup></td><td>4.10 ± 0.19<sup>b</sup></td><td>5.87 ± 0.27<sup>a</sup></td><td>4.98 ± 0.27<sup>ab</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>ent-16-epi-16-F<sub>1</sub>r-PhytoP</td><td>1.02 ± 0.09<sup>ab</sup></td><td>1.03 ± 0.08<sup>b</sup></td><td>1.58 ± 0.09<sup>a</sup></td><td>1.64 ± 0.12<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>9-F<sub>1</sub>r-PhytoP</td><td>1.42 ± 0.13<sup>b</sup></td><td>1.57 ± 0.12<sup>b</sup></td><td>2.51 ± 0.14<sup>a</sup></td><td>2.56 ± 0.19<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>ent-16-F<sub>1</sub>r-PhytoP</td><td>0.95 ± 0.09<sup>ab</sup></td><td>0.87 ± 0.07<sup>b</sup></td><td>1.40 ± 0.08<sup>a</sup></td><td>1.49 ± 0.11<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>9-epi-9-F<sub>1</sub>r-PhytoP</td><td>1.64 ± 0.13<sup>b</sup></td><td>1.67 ± 0.10<sup>b</sup></td><td>2.70 ± 0.11<sup>a</sup></td><td>2.66 ± 0.16<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>16-B<sub>1</sub>r-PhytoP</td><td>4.13 ± 0.52<sup>b</sup></td><td>3.90 ± 0.17<sup>b</sup></td><td>7.52 ± 0.30<sup>a</sup></td><td>8.37 ± 0.38<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>9-L<sub>1</sub>r-PhytoP</td><td>3.71 ± 0.51<sup>b</sup></td><td>3.38 ± 0.16<sup>b</sup></td><td>7.22 ± 0.34<sup>a</sup></td><td>8.27 ± 0.46<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>Peroxides derived from ARA (pg.mg<sup>-1</sup>)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>5-F<sub>2c</sub>-IsoP</td><td>0.73 ± 0.10<sup>c</sup></td><td>0.86 ± 0.05<sup>c</sup></td><td>1.68 ± 0.08<sup>b</sup></td><td>2.02 ± 0.10<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>PGF<sub>2</sub></td><td>0.21 ± 0.02<sup>b</sup></td><td>0.25 ± 0.01<sup>b</sup></td><td>0.36 ± 0.02<sup>a</sup></td><td>0.34 ± 0.02<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>Peroxides derived from DHA (pg.mg<sup>-1</sup>)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>4(RS)-4-F<sub>4t</sub>-NeuroP</td><td>1.57 ± 0.20<sup>ab</sup></td><td>1.24 ± 0.05<sup>b</sup></td><td>1.71 ± 0.06<sup>a</sup></td><td>1.66 ± 0.08<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>10(S)-10-F<sub>4t</sub>-NeuroP</td><td>0.56 ± 0.06<sup>ab</sup></td><td>0.54 ± 0.03<sup>b</sup></td><td>0.67 ± 0.02<sup>a</sup></td><td>0.64 ± 0.04<sup>ab</sup></td><td><strong>0.02</strong></td></tr><tr><td>10(R)-10-F<sub>4t</sub>-NeuroP</td><td>1.12 ± 0.12<sup>ab</sup></td><td>1.00 ± 0.03<sup>b</sup></td><td>1.22 ± 0.04<sup>a</sup></td><td>1.10 ± 0.05<sup>ab</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td><strong>Antioxidant molecules</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Vitamin E metabolites (nmol.g<sup>-1</sup>)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>α-tocopherol</td><td>29.28 ± 1.63<sup>a</sup></td><td>15.17 ± 0.70<sup>b</sup></td><td>28.16 ± 1.12<sup>a</sup></td><td>13.83 ± 0.68<sup>b</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>γ-tocopherol</td><td>5.00 ± 0.34<sup>bc</sup></td><td>0.58 ± 0.04<sup>c</sup></td><td>10.51 ± 1.17<sup>b</sup></td><td>14.77 ± 1.57<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>δ-tocopherol</td><td>0.32 ± 0.03<sup>c</sup></td><td>0.24 ± 0.01<sup>c</sup></td><td>0.98 ± 0.06<sup>a</sup></td><td>0.74 ± 0.03<sup>b</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td><strong>Glutathione metabolites</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>GSH (pg/g of proteins<sup>-1</sup>)</td><td>61.48 ± 0.81<sup>a</sup></td><td>47.52 ± 1.33<sup>a</sup></td><td>26.51 ± 1.79<sup>b</sup></td><td>47.16 ± 1.72<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>GSSG (pg/g of proteins<sup>-1</sup>)</td><td>0.15 ± 0.01<sup>b</sup></td><td>1.22 ± 0.15<sup>b</sup></td><td>1.77 ± 0.16<sup>ab</sup></td><td>2.18 ± 0.24<sup>a</sup></td><td><strong>&lt;0.01</strong></td></tr><tr><td>Ratio GSH/GSSG</td><td>400 ± 22<sup>a</sup></td><td>76 ± 12<sup>b</sup></td><td>19 ± 2<sup>c</sup></td><td>40 ± 8<sup>c</sup></td><td><strong>&lt;0.01</strong></td></tr></tbody></table>

Values are represented as mean ± SEM. At day 0 *n* = 3 for parameters related to fatty acid profile and GSH metabolites, *n* = 4 for tocopherol and *n* = 6 for oxylipins. At latter time points for fatty acid profile, oxylipins and GSH metabolites *n* = 36. For tocopherol *n* = 54 at day 21 and 84, *n* = 36 at day 168. Values not sharing the same superscript letter are considered as significantly different, based on one-way ANOVA (significance *p-value* <0.05) followed by Tukey's *post-hoc* test.

the other time points. ALA levels increased at days 84 and 168 in both groups compared to day 21. Although time significantly affected ARA levels in the plant-based group, Tukey's *post-hoc* test did not detect differences between time points. In the tuna-based group, ARA levels increased at days 84 and 168, with a higher increase at day 84 (Supplemental Table 6–7). Oxylipins derived from ALA showed distinct patterns across diets. In the plant-based group, *ent-16A-9-epi-ST-Δ<sup>14</sup>-10-PhytoF*, *ent-16B-9-epi-ST-Δ<sup>14</sup>-10-PhytoF*, and 9-F<sub>1</sub>r-PhytoP, 9-epi-9-F<sub>1</sub>r-PhytoP increased at day 84, then returned to baseline levels (21 days) by day 168. In the tuna-based group, *ent-16B-9-epi-ST-Δ<sup>14</sup>-10-PhytoP* and 9-epi-9-F<sub>1</sub>r-PhytoP increased at both 84 and 168 days, *ent-16A-9-epi-ST-Δ<sup>14</sup>-10-PhytoP* increase only at day 168, compared to 21 days and 9-F<sub>1</sub>r-PhytoP showed a gradual increase. For *ent-16-epi-16-F<sub>1</sub>r-PhytoP* and *ent-16-F<sub>1</sub>r-PhytoP*, increases were observed at day 84 in the plant-based group, with no further changes after 168 days. In the tuna-based group, *ent-16-epi-16-F<sub>1</sub>r-PhytoP* and *ent-16-F<sub>1</sub>r-PhytoP* increased over time. Additionally, 16-B<sub>1</sub>r-PhytoP and 9-L<sub>1</sub>-PhytoP increased at days 84 and 168 in the plant-based group, and showed a continuous increase in the tuna-based group. Regarding ARA-derived oxylipins, PGF<sub>2</sub> levels increased at day 84 only in the plant-based group, while in the tuna-based group, PGF<sub>2</sub> increase at both 84 and 168 days. The 5-F<sub>2c</sub>-IsoP oxylipin increased at days 84 and 168 in the plant-based group, and gradually in the tuna-based group. For DHA-derived oxylipins, in plant-based diet group 4(RS)-4-F<sub>4t</sub>-NeuroP increased at day 84 only. The 10(S)-10-F<sub>4t</sub>-NeuroP and 10(R)-10-F<sub>4t</sub>-NeuroP oxylipins increased at day 84, followed by a decrease at day 168. In the tuna-based group, all three oxylipins increased continuously over time (Tables 5 and 6). Concerning antioxidant molecules, after 84 days, α-tocopherol concentrations increased in muscle of fish fed the plant-based diet, but declined at day 168 to levels below those at days 21 and 84. A similar pattern was

observed in the tuna-based group, although levels at day 168 returned to those seen at day 21. In both diets, γ-tocopherol levels increased progressively over time. The δ-tocopherol levels also increase at days 84 and 168 compared to day 21, with higher values at day 84. GSH concentrations declined at day 84 but returned to baseline at day 168 (equivalent to 21 days) in both diet groups. In contrast, GSSG levels progressively increased only in fish fed the tuna-based diet. As a result, the GSH:GSSG ratio decreased at day 84 in both groups compared to day 21, but remained reduced at day 168 only in the tuna-based group (Tables 5 and 6).

In fish fed tuna-based diets, SeMet and MeHg supplementation had no effect on fatty acid of interests. Similarly, no impact was observed on non-enzymatic peroxide levels in fish fed plant-based diets (Table 5). In contrast, a dietary supplementation effect was detected for 9-epi-9-F<sub>1</sub>r-PhytoP and 9-L<sub>1</sub>r-PhytoP in fish fed tuna-based diets, although Tukey's *post-hoc* test showed no significant differences between groups. After 168 days of feeding, 16-B<sub>1</sub>-PhytoP concentrations were significantly higher in fish fed MeHg-supplemented TH and THO diets compared to the control (Table 6). An interaction between time and diet was observed for α-tocopherol levels in fish fed the PC, PH, and PHO diets, but no significant differences were detected at specific time points or over time. In contrast, after 84 days, fish fed the THO diet showed higher α-tocopherol levels than those fed the control diet or the TH diet. After 84 days, γ-tocopherol levels decreased in fish fed the PH diet compared to the PC. Fish fed a PHO diet had increased γ-tocopherol levels relative to fish fed a PH and PC diets. A similar increase was observed in fish fed the THO diet, after 84 days of feeding, compared to those on the control diet TC or the TH diet. At day 168, γ-tocopherol remained elevated in fish fed TH and THO diets relative to TC. Regarding δ-tocopherol, fish fed the PH diet for 84 days showed reduced levels compared to the PC.
A mean Z-score - Plant-based

<figure><img src="image_5.png" /></figure>

B mean Z-score - Tuna-based

<figure><img src="image_6.png" /></figure>

C Clusters - Plant-based

<figure><img src="image_7.png" /></figure>

D Clusters - Tuna-based

<figure><img src="image_8.png" /></figure>

E Average profile - Plant-based

<figure><img src="image_9.png" /></figure>

F Average profile - Tuna-based

<figure><img src="image_10.png" /><figcaption>Fig. 2. Mean of normalized value (Z-score) through time of all the 68 parameters measured in plant- (A) and tuna-based diets (B). Clusters of temporal response in plant- (C) and tuna-based diets (D), using the k-means clustering approach. Average profile of identified clusters in plant- (E) and tuna-based diets (F).</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Fig. 3.** PCA biplots of all parameters measured in fish fed a based diet not supplemented or supplemented with MeHg or in combination with SeMet (control, MeHg supplemented or MeHg and SeMet supplemented) during 21, 84 or 168 days, in plant (A) and tuna-based diets (B). Arrows represent the 5 most contributing variables for the representation of the dataset: 18:3.n-4, fatty acid 18:3 ω-4; 22:4.n-6, fatty acid 22:4 ω-6; 15:0, fatty acid 15:0; 22:0, fatty acid 22:0; 22:5.n-3, fatty acid 22:5 ω-3 in plant-based diets (A), 16:4.n-3, fatty acid 16:4 ω-3; 14:1, fatty acid 14:1; 22:2.n-6, fatty acid 22:2 ω-6; 17:1, fatty acid 17:1; GSH/GSSG, ratio GSH/GSSG in tuna-based diets (B). Ellipses represent a 95 % confidence intervals around 4 individuals in plant and tuna-diets at 21 and 84 days. Except for PHO group at 84 days with *n* = 5. For PHO group at 21 days too individuals were used to display an ellipse (*n* = 3) only the barycenter for this group was shown. For groups in plant- and tuna-based diets at 168 days *n* = 6.</figcaption></figure>

Fish on the PHO diet had even lower δ-tocopherol concentrations than those on PH and PC diets. In tuna-based diets, δ-tocopherol increased in fish fed the TH diet compared to the control, but decreased in the THO dietary group. No significant dietary effects were observed on GSH concentrations in fish fed either plant- or tuna-based diets. However, GSSG levels were significantly influenced by diet in fish fed plant-based diets. Fish fed, PH and PHO diets had lower GSSG concentrations compared to the control (PC). A significant diet and time interaction was also detected for GSSG in both plant- and tuna-based diet groups. After 21 days, fish fed PH and PHO diets showed reduced GSSG levels. At day 84, GSSG remained lower in fish fed the PHO diet compared to PC, and at day 168, it was lower in the PH group versus PC. In tuna-based diets, no significant GSSG differences were found between groups at individual time points. However, temporal patterns were different depending on the diet. In the TC group, GSSG levels remained stable. In contrast, TH-fed fish showed an increase at day 84, with no further change at day 168. Fish on the THO diet exhibited increased GSSG only at day 168, compared to both earlier time points. The GSH:GSSG ratio was influenced by the diet in both plant- and tuna-based groups. Fish fed PH and PHO diets showed higher GSH:GSSG ratio than the PC. Although a dietary effect was also observed in tuna-based diets, Tukey's *post-hoc* test revealed no significant differences. A significant diet and time

interaction was detected in both diet types. At day 21, PHO-fed fish showed a higher GSH:GSSG ratio than PC, while TH-fed fish had a higher ratio than both TC and THO groups. At day 168, PH-fed fish showed a higher ratio compared to both PC and PHO diets (Table 5). No significant differences were found among tuna-based groups at this time point (Tables 5 and 6).

# 4. Discussion

## 4.1. Influence of based-diets on lipid composition, oxylipin generation and antioxidant activity

The differences in muscle fatty acid profiles between fish fed plant- and tuna-based diets closely reflected the fatty acid composition of the respective diets, consistent with the well-established role of diet as the primary determinant of muscle fatty acid composition [38]. Tuna-based diets contained higher levels of ALA, ARA and DHA, which may have contributed to the elevated formation of their respective oxylipins, found in higher amount in muscle of tuna fed fish. A result that suggests a potential enhancement of oxidative stress in these animals. This interpretation is supported by reduced levels of all tocopherol isoforms and a lower GSH/GSSG ratio in the muscle of tuna-fed fish, suggesting
<table><caption>**Table 5**
Timing and supplementation effects on selected parameters in muscle of fish fed plant-based diets not supplemented (PC), Supplemented with MeHg (PH) or with MeHg and SeMet (PHO).</caption><thead><tr><th rowspan="2">Timing</th><th colspan="3">Day 21</th><th colspan="3">Day 84</th><th colspan="3">Day 168</th><th colspan="3">p-value</th></tr><tr><th>PC</th><th>PH</th><th>PHO</th><th>PC</th><th>PH</th><th>PHO</th><th>PC</th><th>PH</th><th>PHO</th><th>PTiming</th><th>PDiet</th><th>PTiming x Diet</th></tr></thead><tbody><tr><td colspan="14"><strong>Lipid peroxides levels</strong></td></tr><tr><td colspan="14">Peroxides derived from ALA (pg.mg<sup>-1</sup>)</td></tr><tr><td>ent-16A-9-epi-ST-D<sup>14</sup>-10-PhytoF</td><td>2.14 ± 0.2</td><td>2.42 ± 0.3</td><td>1.98 ± 0.1</td><td>2.47 ± 0.3</td><td>3.44 ± 0.5</td><td>3.16 ± 0.3</td><td>2.27 ± 0.2</td><td>2.11 ± 0.2</td><td>2.05 ± 0.1</td><td><strong>&lt;0.01</strong></td><td>0.23</td><td>0.21</td></tr><tr><td>ent-16B-9-epi-ST-D<sup>14</sup>-10-PhytoF</td><td>4.18 ± 0.2</td><td>4.52 ± 0.8</td><td>3.77 ± 0.4</td><td>5.50 ± 0.7</td><td>7.23 ± 0.8</td><td>6.39 ± 0.8</td><td>4.89 ± 0.4</td><td>4.00 ± 0.5</td><td>3.58 ± 0.3</td><td><strong>&lt;0.01</strong></td><td>0.43</td><td>0.25</td></tr><tr><td>ent-16-epi-16-F<sub>1</sub><sub>1</sub>-PhytoP</td><td>1.22 ± 0.3</td><td>1.01 ± 0.2</td><td>0.92 ± 0.2</td><td>1.25 ± 0.2</td><td>1.70 ± 0.3</td><td>1.89 ± 0.3</td><td>1.20 ± 0.2</td><td>1.35 ± 0.2</td><td>1.10 ± 0.1</td><td><strong>0.01</strong></td><td>0.75</td><td>0.25</td></tr><tr><td>9-F<sub>1</sub><sub>1</sub>-PhytoP</td><td>1.89 ± 0.3</td><td>1.43 ± 0.3</td><td>1.41 ± 0.2</td><td>2.02 ± 0.3</td><td>2.71 ± 0.5</td><td>2.94 ± 0.4</td><td>1.60 ± 0.2</td><td>2.18 ± 0.2</td><td>1.83 ± 0.2</td><td><strong>&lt;0.01</strong></td><td>0.53</td><td>0.21</td></tr><tr><td>ent-16-F<sub>1</sub><sub>1</sub>-PhytoP</td><td>1.01 ± 0.2</td><td>0.73 ± 0.1</td><td>0.79 ± 0.2</td><td>1.17 ± 0.2</td><td>1.46 ± 0.3</td><td>1.54 ± 0.2</td><td>1.03 ± 0.1</td><td>1.24 ± 0.2</td><td>1.07 ± 0.2</td><td><strong>&lt;0.01</strong></td><td>0.86</td><td>0.45</td></tr><tr><td>9-epi-9-F<sub>1</sub><sub>1</sub>-PhytoP</td><td>1.93 ± 0.3</td><td>1.53 ± 0.3</td><td>1.55 ± 0.2</td><td>2.36 ± 0.2</td><td>2.78 ± 0.4</td><td>2.87 ± 0.3</td><td>1.88 ± 0.2</td><td>2.29 ± 0.2</td><td>2.13 ± 0.3</td><td><strong>&lt;0.01</strong></td><td>0.76</td><td>0.43</td></tr><tr><td>16-B<sub>1</sub><sub>1</sub>-PhytoP</td><td>4.15 ± 0.6</td><td>4.05 ± 0.4</td><td>4.31 ± 0.5</td><td>6.92 ± 0.7</td><td>7.13 ± 0.9</td><td>7.97 ± 0.5</td><td>6.92 ± 0.4</td><td>7.80 ± 1.2</td><td>7.62 ± 0.6</td><td><strong>&lt;0.01</strong></td><td>0.52</td><td>0.91</td></tr><tr><td>9-L<sub>1</sub>-PhytoP</td><td>3.19 ± 0.6</td><td>3.51 ± 0.4</td><td>3.49 ± 0.5</td><td>6.00 ± 0.8</td><td>6.41 ± 0.8</td><td>7.35 ± 0.6</td><td>6.28 ± 0.5</td><td>7.09 ± 1.1</td><td>7.11 ± 0.7</td><td><strong>&lt;0.01</strong></td><td>0.33</td><td>0.94</td></tr><tr><td colspan="14">Peroxides derived from ARA (pg.mg<sup>-1</sup>)</td></tr><tr><td>5-F<sub>2</sub><sub>2</sub>-IsoP</td><td>0.68 ± 0.1</td><td>1.03 ± 0.1</td><td>0.87 ± 0.0</td><td>1.48 ± 0.2</td><td>1.47 ± 0.1</td><td>1.41 ± 0.1</td><td>1.76 ± 0.1</td><td>1.50 ± 0.1</td><td>1.64 ± 0.1</td><td><strong>&lt;0.01</strong></td><td>0.95</td><td>0.17</td></tr><tr><td>PGF<sub>2</sub></td><td>0.19 ± 0.0</td><td>0.24 ± 0.0</td><td>0.20 ± 0.0</td><td>0.28 ± 0.0</td><td>0.29 ± 0.0</td><td>0.32 ± 0.0</td><td>0.23 ± 0.0</td><td>0.24 ± 0.0</td><td>0.31 ± 0.0</td><td><strong>&lt;0.01</strong></td><td>0.09</td><td>0.29</td></tr><tr><td colspan="14">Peroxides derived from DHA (pg.mg<sup>-1</sup>)</td></tr><tr><td>4(RS)-4-F<sub>4</sub><sub>1</sub>-NeuroP</td><td>1.12 ± 0.1</td><td>1.28 ± 0.2</td><td>1.14 ± 0.1</td><td>1.40 ± 0.2</td><td>1.38 ± 0.1</td><td>1.68 ± 0.2</td><td>1.23 ± 0.0</td><td>1.26 ± 0.1</td><td>1.40 ± 0.2</td><td><strong>0.02</strong></td><td>0.33</td><td>0.59</td></tr><tr><td>10(S)-10-F<sub>4</sub><sub>1</sub>-NeuroP</td><td>0.50 ± 0.0</td><td>0.60 ± 0.1</td><td>0.53 ± 0.1</td><td>0.56 ± 0.0</td><td>0.60 ± 0.0</td><td>0.66 ± 0.1</td><td>0.45 ± 0.0</td><td>0.46 ± 0.0</td><td>0.54 ± 0.1</td><td><strong>0.06</strong></td><td>0.37</td><td>0.77</td></tr><tr><td>10(R)-10-F<sub>4</sub><sub>1</sub>-NeuroP</td><td>0.91 ± 0.0</td><td>1.00 ± 0.1</td><td>0.90 ± 0.1</td><td>0.98 ± 0.1</td><td>1.05 ± 0.0</td><td>1.21 ± 0.1</td><td>0.84 ± 0.0</td><td>0.85 ± 0.1</td><td>0.96 ± 0.1</td><td><strong>&lt;0.01</strong></td><td>0.17</td><td>0.35</td></tr><tr><td colspan="14"><strong>Antioxidant molecules</strong></td></tr><tr><td colspan="14">Vitamin E metabolites (nmol.g<sup>-1</sup>)</td></tr><tr><td>α-tocopherol</td><td>18.07 ± 0.97<sup>bc</sup></td><td>19.47 ± 0.99<sup>bc</sup></td><td>21.48 ± 0.77<sup>b</sup></td><td>34.14 ± 2.51<sup>a</sup></td><td>31.48 ± 1.44<sup>a</sup></td><td>35.95 ± 0.66<sup>a</sup></td><td>14.25 ± 0.77<sup>c</sup></td><td>19.44 ± 0.91<sup>bc</sup></td><td>15.58 ± 0.88<sup>bc</sup></td><td><strong>&lt;0.01</strong></td><td>0.17</td><td><strong>0.03</strong></td></tr><tr><td>γ-tocopherol</td><td>0.76 ± 0.10<sup>d</sup></td><td>0.53 ± 0.04<sup>d</sup></td><td>0.26 ± 0.02<sup>d</sup></td><td>14.85 ± 0.84<sup>b</sup></td><td>11.86 ± 0.49<sup>c</sup></td><td>25.27 ± 1.37<sup>a</sup></td><td>23.69 ± 0.75<sup>a</sup></td><td>22.38 ± 0.38<sup>a</sup></td><td>24.53 ± 0.71<sup>a</sup></td><td><strong>&lt;0.01</strong></td><td><strong>&lt;0.01</strong></td><td><strong>&lt;0.01</strong></td></tr><tr><td>δ-tocopherol</td><td>0.24 ± 0.01<sup>e</sup></td><td>0.25 ± 0.04<sup>e</sup></td><td>0.23 ± 0.02<sup>e</sup></td><td>1.44 ± 0.07<sup>a</sup></td><td>1.13 ± 0.08<sup>b</sup></td><td>0.56 ± 0.04<sup>d</sup></td><td>0.97 ± 0.03<sup>bc</sup></td><td>0.83 ± 0.05<sup>c</sup></td><td>0.87 ± 0.03<sup>c</sup></td><td><strong>&lt;0.01</strong></td><td><strong>&lt;0.01</strong></td><td><strong>&lt;0.01</strong></td></tr><tr><td colspan="14"><strong>Glutathione metabolites</strong></td></tr><tr><td>GSH (pg/g of proteins<sup>-1</sup>)</td><td>41.65 ± 2.28</td><td>47.53 ± 1.75</td><td>42.61 ± 1.62</td><td>17.97 ± 2.24</td><td>23.73 ± 4.93</td><td>31.74 ± 3.95</td><td>45.45 ± 5.64</td><td>41.62 ± 4.20</td><td>43.73 ± 2.13</td><td><strong>&lt;0.01</strong></td><td>0.73</td><td>0.79</td></tr><tr><td>GSSG (pg/g of proteins<sup>-1</sup>)</td><td>1.77 ± 0.26<sup>ab</sup></td><td>0.79 ± 0.25<sup>ab</sup></td><td>0.31 ± 0.09<sup>d</sup></td><td>1.81 ± 0.24<sup>a</sup></td><td>0.99 ± 0.19<sup>ad</sup></td><td>0.95 ± 0.08<sup>bd</sup></td><td>1.75 ± 0.17<sup>ab</sup></td><td>0.43 ± 0.11<sup>d</sup></td><td>1.37 ± 0.15<sup>abc</sup></td><td><strong>0.15</strong></td><td><strong>&lt;0.01</strong></td><td><strong>&lt;0.01</strong></td></tr><tr><td>Ratio GSH/GSSG</td><td>26 ± 4<sup>b</sup></td><td>97 ± 31<sup>ab</sup></td><td>186 ± 45<sup>a</sup></td><td>10 ± 1<sup>b</sup></td><td>27 ± 5<sup>b</sup></td><td>35 ± 7<sup>b</sup></td><td>27 ± 4<sup>b</sup></td><td>125 ± 27<sup>a</sup></td><td>33 ± 3<sup>b</sup></td><td><strong>&lt;0.01</strong></td><td><strong>&lt;0.01</strong></td><td><strong>&lt;0.01</strong></td></tr></tbody></table>

Values are represented as mean ± SEM. For parameters related to fatty acid profile, oxylipins and GSH metabolites (n = 6), for tocopherol at 21 and 84 days n = 9, at 168 n = 6. Means not sharing a common superscript letter are significantly different according to two-way ANOVA (significance *p-value* < 0.05) to test the effect of Timing (P<sub>Timing</sub>), Diet (P<sub>Diet</sub>) and the interaction (P<sub>Timing×Diet</sub>) followed by a Tukey's post-hoc test.
<table><caption>**Table 6**
Timing and supplementation effects on selected parameters in muscle of fish fed tuna-based diets not supplemented (TC), Supplemented with MeHg (TH) or with MeHg and SeMet (THO).</caption><thead><tr><th rowspan="2">Timing</th><th colspan="3">Day 21</th><th colspan="3">Day 84</th><th colspan="3">Day 168</th><th colspan="3">p-value</th></tr><tr><th>TC</th><th>TH</th><th>THO</th><th>TC</th><th>TH</th><th>THO</th><th>TC</th><th>TH</th><th>THO</th><th>PTiming</th><th>PDiet</th><th>PTiming x Diet</th></tr></thead><tbody><tr><td colspan="14"><strong>Lipid peroxides levels</strong></td></tr><tr><td colspan="14">Peroxides derived from ALA (pg.mg<sup>-1</sup>)</td></tr><tr><td>ent-16A-9-epi-ST-D<sup>14</sup>-10-PhytoF</td><td>2.31 ± 0.2</td><td>1.58 ± 0.2</td><td>2.36 ± 0.3</td><td>2.37 ± 0.2</td><td>2.59 ± 0.2</td><td>2.52 ± 0.4</td><td>2.45 ± 0.4</td><td>2.92 ± 0.2</td><td>2.99 ± 0.3</td><td><strong>0.01</strong></td><td><strong>0.43</strong></td><td><strong>0.21</strong></td></tr><tr><td>ent-16B-9-epi-ST-D<sup>14</sup>-10-PhytoF</td><td>4.34 ± 0.3</td><td>3.35 ± 0.3</td><td>4.48 ± 0.5</td><td>5.29 ± 0.5</td><td>5.64 ± 0.4</td><td>5.16 ± 0.5</td><td>4.98 ± 0.8</td><td>5.86 ± 0.5</td><td>6.55 ± 0.7</td><td><strong>&lt;0.01</strong></td><td><strong>0.44</strong></td><td><strong>0.22</strong></td></tr><tr><td>ent-16-epi-16-F<sub>1</sub><sub>1</sub>-PhytoP</td><td>0.92 ± 0.1</td><td>1.16 ± 0.3</td><td>0.91 ± 0.1</td><td>1.38 ± 0.1</td><td>1.58 ± 0.3</td><td>1.70 ± 0.2</td><td>1.62 ± 0.3</td><td>2.18 ± 0.3</td><td>2.38 ± 0.2</td><td><strong>&lt;0.01</strong></td><td><strong>0.11</strong></td><td><strong>0.53</strong></td></tr><tr><td>9-F<sub>1</sub><sub>1</sub>-PhytoP</td><td>1.47 ± 0.2</td><td>1.75 ± 0.5</td><td>1.43 ± 0.2</td><td>2.25 ± 0.1</td><td>2.43 ± 0.3</td><td>2.73 ± 0.3</td><td>2.42 ± 0.4</td><td>3.44 ± 0.5</td><td>3.87 ± 0.4</td><td><strong>&lt;0.01</strong></td><td><strong>0.07</strong></td><td><strong>0.26</strong></td></tr><tr><td>ent-16-F<sub>1</sub><sub>1</sub>-PhytoP</td><td>0.86 ± 0.1</td><td>1.04 ± 0.3</td><td>0.76 ± 0.1</td><td>1.27 ± 0.1</td><td>1.37 ± 0.2</td><td>1.57 ± 0.2</td><td>1.51 ± 0.3</td><td>1.93 ± 0.3</td><td>2.18 ± 0.2</td><td><strong>&lt;0.01</strong></td><td><strong>0.21</strong></td><td><strong>0.42</strong></td></tr><tr><td>9-epi-9-F<sub>1</sub><sub>1</sub>-PhytoP</td><td>1.60 ± 0.1</td><td>1.89 ± 0.4</td><td>1.48 ± 0.1</td><td>2.52 ± 0.2</td><td>2.63 ± 0.3</td><td>3.05 ± 0.2</td><td>2.40 ± 0.3</td><td>3.41 ± 0.5</td><td>3.83 ± 0.3</td><td><strong>&lt;0.01</strong></td><td><strong>0.04</strong></td><td><strong>0.07</strong></td></tr><tr><td>16-B<sub>1</sub><sub>1</sub>-PhytoP</td><td>3.15 ± 0.2<sup>e</sup></td><td>4.35 ± 0.3<sup>de</sup></td><td>3.48 ± 0.3<sup>e</sup></td><td>7.57 ± 0.7<sup>bc</sup></td><td>7.76 ± 0.9<sup>bc</sup></td><td>7.77 ± 0.8<sup>bc</sup></td><td>6.96 ± 0.8<sup>cd</sup></td><td>10.77 ± 0.6<sup>a</sup></td><td>10.13 ± 0.6<sup>ab</sup></td><td><strong>&lt;0.01</strong></td><td><strong>&lt;0.01</strong></td><td><strong>0.04</strong></td></tr><tr><td>9-L<sub>1</sub><sub>1</sub>-PhytoP</td><td>2.90 ± 0.2</td><td>4.05 ± 0.3</td><td>3.14 ± 0.3</td><td>7.76 ± 0.8</td><td>7.92 ± 1.0</td><td>7.85 ± 0.9</td><td>7.27 ± 1.0</td><td>11.23 ± 0.8</td><td>10.65 ± 1.0</td><td><strong>&lt;0.01</strong></td><td><strong>0.03</strong></td><td><strong>0.09</strong></td></tr><tr><td colspan="14">Peroxides derived from ARA (pg.mg<sup>-1</sup>)</td></tr><tr><td>5-F<sub>2</sub><sub>2</sub>-IsoP</td><td>0.93 ± 0.1</td><td>0.79 ± 0.1</td><td>0.89 ± 0.2</td><td>2.13 ± 0.3</td><td>1.77 ± 0.1</td><td>1.80 ± 0.1</td><td>2.11 ± 0.3</td><td>2.33 ± 0.3</td><td>2.77 ± 0.2</td><td><strong>&lt;0.01</strong></td><td><strong>0.55</strong></td><td><strong>0.19</strong></td></tr><tr><td>PGF<sub>2</sub></td><td>0.29 ± 0.0</td><td>0.32 ± 0.0</td><td>0.28 ± 0.0</td><td>0.42 ± 0.0</td><td>0.37 ± 0.0</td><td>0.45 ± 0.0</td><td>0.39 ± 0.1</td><td>0.46 ± 0.0</td><td>0.43 ± 0.0</td><td><strong>&lt;0.01</strong></td><td><strong>0.82</strong></td><td><strong>0.34</strong></td></tr><tr><td colspan="14">Peroxides derived from DHA (pg.mg<sup>-1</sup>)</td></tr><tr><td>4(RS)-4-F<sub>4</sub><sub>1</sub>-NeuroP</td><td>1.24 ± 0.1</td><td>1.34 ± 0.1</td><td>1.35 ± 0.1</td><td>1.96 ± 0.1</td><td>1.95 ± 0.1</td><td>1.88 ± 0.1</td><td>1.90 ± 0.1</td><td>2.08 ± 0.2</td><td>2.11 ± 0.2</td><td><strong>&lt;0.01</strong></td><td><strong>0.62</strong></td><td><strong>0.84</strong></td></tr><tr><td>10(S)-10-F<sub>4</sub><sub>1</sub>-NeuroP</td><td>0.57 ± 0.0</td><td>0.54 ± 0.0</td><td>0.54 ± 0.0</td><td>0.73 ± 0.0</td><td>0.72 ± 0.1</td><td>0.75 ± 0.0</td><td>0.71 ± 0.1</td><td>0.81 ± 0.1</td><td>0.90 ± 0.1</td><td><strong>&lt;0.01</strong></td><td><strong>0.43</strong></td><td><strong>0.42</strong></td></tr><tr><td>10(R)-10-F<sub>4</sub><sub>1</sub>-NeuroP</td><td>1.09 ± 0.1</td><td>1.04 ± 0.1</td><td>1.04 ± 0.1</td><td>1.34 ± 0.1</td><td>1.32 ± 0.1</td><td>1.40 ± 0.0</td><td>1.16 ± 0.1</td><td>1.44 ± 0.1</td><td>1.36 ± 0.1</td><td><strong>&lt;0.01</strong></td><td><strong>0.56</strong></td><td><strong>0.33</strong></td></tr><tr><td colspan="14"><strong>Antioxidant molecules</strong></td></tr><tr><td colspan="14">Vitamin E metabolites (nmol.g<sup>-1</sup>)</td></tr><tr><td>α-tocopherol</td><td>11.62 ± 0.57<sup>c</sup></td><td>10.12 ± 0.46<sup>c</sup></td><td>10.28 ± 0.32<sup>c</sup></td><td>19.96 ± 0.96<sup>b</sup></td><td>22.61 ± 1.56<sup>b</sup></td><td>28.37 ± 1.27<sup>a</sup></td><td>9.79 ± 0.58<sup>c</sup></td><td>13.07 ± 0.45<sup>c</sup></td><td>13.38 ± 0.26<sup>c</sup></td><td><strong>&lt;0.01</strong></td><td><strong>&lt;0.01</strong></td><td><strong>&lt;0.01</strong></td></tr><tr><td>γ-tocopherol</td><td>0.76 ± 0.05<sup>e</sup></td><td>0.85 ± 0.02<sup>e</sup></td><td>0.36 ± 0.02<sup>e</sup></td><td>3.15 ± 0.18<sup>d</sup></td><td>3.71 ± 0.28<sup>d</sup></td><td>9.35 ± 0.16<sup>a</sup></td><td>5.03 ± 0.37<sup>c</sup></td><td>6.24 ± 0.26<sup>b</sup></td><td>6.92 ± 0.35<sup>b</sup></td><td><strong>&lt;0.01</strong></td><td><strong>&lt;0.01</strong></td><td><strong>&lt;0.01</strong></td></tr><tr><td>δ-tocopherol</td><td>0.23 ± 0.01<sup>d</sup></td><td>0.22 ± 0.02<sup>d</sup></td><td>0.23 ± 0.03<sup>d</sup></td><td>0.87 ± 0.04<sup>b</sup></td><td>1.04 ± 0.05<sup>a</sup></td><td>0.30 ± 0.01<sup>d</sup></td><td>0.58 ± 0.03<sup>c</sup></td><td>0.53 ± 0.01<sup>c</sup></td><td>0.67 ± 0.02<sup>c</sup></td><td><strong>&lt;0.01</strong></td><td><strong>&lt;0.01</strong></td><td><strong>&lt;0.01</strong></td></tr><tr><td colspan="14"><strong>Glutathione metabolites</strong></td></tr><tr><td>GSH (pg/g of proteins<sup>-1</sup>)</td><td>50.81 ± 3.15</td><td>48.31 ± 2.99</td><td>53.38 ± 4.57</td><td>29.37 ± 3.46</td><td>23.42 ± 2.24</td><td>32.78 ± 6.28</td><td>49.59 ± 4.69</td><td>48.04 ± 2.96</td><td>54.53 ± 4.23</td><td><strong>&lt;0.01</strong></td><td><strong>0.12</strong></td><td><strong>0.98</strong></td></tr><tr><td>GSSG (pg/g of proteins<sup>-1</sup>)</td><td>1.44 ± 0.20<sup>bd</sup></td><td>0.62 ± 0.09<sup>d</sup></td><td>1.27 ± 0.28<sup>cd</sup></td><td>1.77 ± 0.34<sup>bd</sup></td><td>2.68 ± 0.27<sup>abc</sup></td><td>1.73 ± 0.17<sup>bd</sup></td><td>3.06 ± 0.31<sup>ab</sup></td><td>2.29 ± 0.23<sup>ad</sup></td><td>3.88 ± 0.72<sup>a</sup></td><td><strong>&lt;0.01</strong></td><td><strong>0.36</strong></td><td><strong>0.01</strong></td></tr><tr><td>Ratio GSH/GSSG</td><td>36 ± 8<sup>b</sup></td><td>76 ± 15<sup>a</sup></td><td>34 ± 9<sup>b</sup></td><td>16 ± 2<sup>b</sup></td><td>9 ± 1<sup>b</sup></td><td>16 ± 1<sup>b</sup></td><td>17 ± 1<sup>b</sup></td><td>20 ± 2<sup>b</sup></td><td>14 ± 2<sup>b</sup></td><td><strong>&lt;0.01</strong></td><td><strong>0.03</strong></td><td><strong>&lt;0.01</strong></td></tr></tbody></table>

Values are represented as mean ± SEM. For parameters related to fatty acid profile, oxylipins and GSH metabolites (n = 6), for tocopherol at 21 and 84 days n = 9, at 168 n = 6. Means not sharing a common superscript letter are significantly different according to two-way ANOVA (significance *p-value* < 0.05) to test the effect of Timing (P<sub>Timing</sub>), Diet (P<sub>Diet</sub>) and the interaction (P<sub>Timing×Diet</sub>) followed by a Tukey's post-hoc test.
an increased utilization of antioxidant defenses in response to elevated pro-oxidant lipid mediators. A comparable decline in α-tocopherol has previously been observed in trout muscle following a 12 week exposure to oxidized dietary lipids [39]. The apparent oxidative imbalance in fish fed the tuna-based diet may be partially attributable to the diet itself, in which no tocopherol isoforms were detected. Quantitative analysis revealed that α-tocopherol was either absent or near detection limits, while γ- and δ-tocopherol levels were 2- and 1.7-fold lower, respectively, compared to the plant-based diet. As tocopherols are not synthesized in *situ*, both diets were supplemented with 50 IU of tocopherol acetate (see Ref. [32]). However, this esterified form lacks inherent antioxidant activity [40] and requires *in vivo* hydrolysis to release biologically active α-tocopherol [41]. The absence of detectable α-tocopherol in tuna-fed fish may therefore reflect increased oxidative consumption, reinforcing the hypothesis of ongoing oxidative stress. It should be noted that only the free, biologically active forms of tocopherols were quantified in the diets, tocopherol acetate was not measured. Moreover, oxylipins were not measured in the diets, previous research has highlighted the critical role of oxidized dietary lipids in modulating antioxidant responses in fish muscle [39]. Future investigations should quantify both dietary oxylipin and tocopherol acetate content to clarify their contribution to the observed redox disturbances. Interestingly, the oxidative imbalance observed in tuna-fed fish contrasts with our prior findings on Hg and Se accumulation. Fish exposed to tuna-based diets accumulated 40 % less Hg and 57 % more Se than those on plant-based diets [21]. Given narrow window of Se between essentiality and toxicity in fish [42], particularly in its inorganic forms [43], the initial Se concentration of ~7 mg/kg in the tuna-based diet may have contributed to redox dysregulation. Inorganic Se has been shown to increase oxidative stress markers at dietary concentrations as low as 5.4 mg/kg [31]. Therefore, the evaluation of dietary Se speciation in the diets is essential for accurately evaluating their pro-oxidant potential.

## 4.2. Influence of diets on the temporal shifts in lipid composition, oxylipin generation, and antioxidant activity

The stabilization of muscle fatty acid (FA) profiles in response to dietary changes is influenced by multiple factors, including fish size and age [44], complicating predictions regarding the time required for equilibration. In our PCA, we observed a strong overlap in data from fish sampled at 84 and 168 days, indicating stabilization of several parameters, including muscle FA composition. This was further supported by the absence of significant differences in most FA-related variables between these two time points. A similar stabilization was observed in oxylipin levels, which increased by day 84 and remained elevated at day 168. This temporal pattern mirrors previous findings in rainbow trout muscle where lipid peroxidation, measured by TBARS, increased over a three-year period of dietary exposure [13]. Interestingly, the trends observed for oxylipins were not directly aligned with changes in precursor FA levels. Of the three key precursors: ALA, ARA, and DHA, only DHA declined over time, and this decrease was detected only at day 168, potentially reflecting its oxidative conversion to DHA-derived oxylipins. In contrast, ALA exhibited the same temporal pattern as the oxylipins, while ARA remained stable throughout the study. The greater abundance of ALA in diets compared to DHA may have compensate its disappearance through oxidation and explain why ALA levels were not correlated to oxylipins dynamics. Methodological differences in measurement units may also contribute to the lack of direct correlation between oxylipins and FA levels: fatty acids were expressed as a percentage of total fatty acids and not as a percentage of total lipid or muscle mass, whereas oxylipins were quantified in pg per mg muscle. Notably, oxylipin concentrations correlated more closely with antioxidant parameters. The decline in GSH/GSSG ratio at 84 and 168 days likely reflects increased GSH oxidation to counteract elevated oxylipin levels. This suggests a transient reduction in antioxidant capacity, consistent with similar observations in brown trout (*Salmo trutta*) under oxidative stress

[14]. Interestingly, while GSH and the GSH/GSSG ratio responded by 84 days, reductions in tocopherol isoforms were only evident by day 168, suggesting delayed antioxidant depletion. GSH and vitamin E are key molecules for lipid peroxide reduction [10–12]. It is noteworthy that between the two antioxidant systems analyzed, the glutathione system responded immediately to the increase in oxylipins, whereas tocopherols depletion occurred later. The glutathione system plays a central role in maintaining cellular redox homeostasis. GSH is abundant, can be synthesized directly within cells, and serve as substrate for enzymatic reactions that reduce oxidative compounds such as oxylipins. These characteristics enable a finely regulated system that responds rapidly to even minor shifts in cellular redox potential [45]. In contrast, tocopherol cannot be synthesized *in situ* and must be obtained from the diet. Additionally, its reduction does not involve enzymatic processes [10]. These fundamental differences likely explain the distinct response kinetics in our study.

Diet type influenced FA dynamics. Saturated FAs, remained stable in plant-fed fish but gradually increased in tuna-fed fish, reflecting their higher dietary content. Conversely, ω-6 FA, rose in plant-fed fish but declined in tuna-fed fish after 84 and 168 days. In contrast, ω-3 FA showed similar increases across both diet groups at 84 days, with no further change at 168 days, consistent with prior studies on tilapia fed tuna by-product diets [20]. Among key FA, ALA increased similarly in both groups. ARA increased over time only in tuna-fed fish, peaking at 84 days. DHA declined in both groups, though earlier in plant-fed fish (84 days) and later in tuna-fed fish (168 days). These differences may underlie the divergent oxylipin dynamics observed between diet groups. In tuna-fed fish, all oxylipins clustered together, displaying a gradual increase as main trend. This pattern was further supported by statistical analyses, with most oxylipins either continuously increasing or rising after 84 days and remaining elevated at 168 days. In contrast, most oxylipins in plant-fed fish peaked at 84 days, and returned to baseline at 168 days. This decreasing trend, was already evident after day 21 by the clustering approach. These trends were mirrored in antioxidant responses. In plant-fed fish, the GSH/GSSG ratio declined at 84 days and recovered by 168 days, matching the oxylipin curve. This suggests that GSH was mobilized to counteract oxylipin accumulation. A comparable pattern was reported in white sea bream (*Diplodus sargus*) brains under oxidative stress [23]. In tuna-fed fish, the GSH/GSSG ratio remained low at 84 and 168 days. A similar trend was observed for α-tocopherol and GSH levels, as shown by our clustering approach. These results are in line with the continued rise in oxylipins. This may reflect sustained GSH consumption and insufficient replenishment, leading to decreased antioxidant capacity. Overall, the GSH system in both dietary groups may represent an adaptive mechanism in trout to meet increased antioxidant demands, as previously suggested in juvenile rainbow trout exposed to oxidized lipids [39]. Regarding tocopherols, all three isoforms declined similarly across diets, with the exception of α-tocopherol. In plant-fed fish, α-tocopherol initially increased at 84 days but fell below day 21 levels by day 168. In tuna-fed fish, α-tocopherol returned to baseline by day 168. Whereas, γ- and δ-tocopherols increased in both groups at 84 and 168 days. Vitamin E plays a key role in reducing lipid peroxides [10], with α-tocopherol being the most potent antioxidant, whereas γ- and δ-tocopherols have lower biological activity [46,47]. This suggests that α-tocopherol is preferentially utilized, as proposed by Fontagné-Dicharry et al. [39]. Their study on juvenile rainbow trout exposed to oxidized fish oil for 12 weeks showed a reduction in α-tocopherol alongside with an increase in γ-tocopherol, leading the authors to suggest that γ-tocopherol may not effectively prevent free radical proliferation in rainbow trout. Altogether, these data suggest that fish fed tuna-based diets experienced progressive oxidative stress, beginning at 84 days and persisting throughout the experiment. Surprisingly, this occurred despite previously reported lower Hg and higher Se accumulation in tuna-fed fish [21]. While Hg accumulation may partly explain the oxylipin rise, this hypothesis is weakened by the oxylipin dynamics in plant-fed fish, despite their higher and gradual Hg
bioaccumulation. Interestingly, oxylipin and antioxidant levels aligned more closely with Se dynamics: Se increased at 84 days and decreased thereafter. However, Se concentrations at 168 days were still 52 % higher in tuna-fed fish compared to plant-fed counterparts. Given the narrow margin of Se between essentiality and toxicity in fish [42], even this reduction may not have been sufficient to mitigate pro-oxidant effects in tuna fed fish and lead to the long-lasting pro-oxidant effects.

Oxidative stress is considered one of the main toxic mechanisms of Se in fish [48]. In salmon, exposure to diets containing approximately 15 mg per kg of total Se for 3 months led to increased hepatic oxidative damage, characterized by elevated TBARS and reduced levels of anti-oxidant molecules such as vitamin C, α-, γ-, δ-tocopherols, and GSH [43]. Based on these biomarkers, the authors established a safe dietary limit for total Se between 1 and 2.8 mg per kg in salmon [31]. In our study, fish fed the tuna-based diet received, on average, 2.8 times more Se than the 2.8 mg per kg established upper limit. This observation strengthens the hypothesis that excessive dietary Se from tuna by-products may lead to adverse effects, particularly oxidative stress. Selenium speciation plays a crucial role in its toxicity. Inorganic Se has a lower toxicity threshold than organic forms, and therefore a lower safe dietary limit in salmon [31,43]. In the tuna-based diets used in this study, approximately 80 % of the total selenium originated from the natural background of tuna by-products and likely accounted for the major biological effects observed. To better interpret the observed effects, Se speciation analysis of the tuna-based diets would be highly informative. Although such analysis was not conducted directly on the diets, Se speciation was performed on the same set of rainbow trout examined in this study, revealing a specific selenocompound profile. Notably, selenoneine was the only Se species detected in the muscle of tuna-fed fish [22]. This finding is consistent with previous studies identifying selenoneine as the predominant Se form in tuna [49], and supports the hypothesis that tuna by-products are a major source of selenoneine. However, to date, no toxicological data are available regarding the effects of selenoneine in aquatic organisms, and further research is needed to assess its biological significance.

### 4.3. Influence of dietary MeHg and SeMet supplementations on lipid profile, oxylipins and antioxidant metabolites dynamics

Dietary supplementation with MeHg (2 mg kg⁻¹) and SeMet (1.5 mg kg⁻¹) had minimal impact on lipid profile evolution, oxylipin formation, and antioxidant metabolites. Consistent with previous findings in salmon fed MeHg-supplemented diets (5 mg kg⁻¹) for three months [50], the muscle fatty acid profile remained largely unaffected. This limited responsiveness may be attributed to the high abundance of structural proteins such as actin and myosin, which can bind Hg without inducing oxidative damage [50]. In contrast, organs such as the brain in salmon [50] and the liver in rainbow trout [51] have shown lipid profile alteration under MeHg exposure, highlighting the need for further investigation of lipid metabolism in more sensitive tissues, such as the liver and brain [52]. Similarly, the limited effects on fatty acid composition were mirrored in oxylipin formation, likely due to the absence of significant oxidative stress induced by dietary MeHg supplementation. In flounder (*Paralichthys olivaceus*) larvae exposed to waterborne MeHg, muscle exhibited the lowest sensitivity to pro-oxidative effects compared to the liver and gills [53]. Additionally, in salmonids, lipid peroxidation in the brain was only induced at much higher MeHg concentrations (10 mg kg⁻¹) after four months of exposure [24]. Dietary MeHg (2 mg kg⁻¹) had negligible effects on the GSH system and did not significantly alter vitamin E isoform levels. In contrast, a transient decrease in brain GSH levels was reported in white sea bream after acute MeHg exposure (8.7 mg kg⁻¹), followed by a compensatory increase after 14 days [23]. Similarly, in salmon exposed to dietary MeHg (5 mg kg⁻¹) for three months, α-tocopherol levels remained unchanged, while γ-tocopherol was reduced in the liver [54]. These findings suggest that higher MeHg concentrations may be required to elicit significant

oxidative stress response in muscle, a relatively insensitive tissue. Previous work indicated that dietary SeMet supplementation in tuna-based diets could slow down Hg bioaccumulation in fish muscle [21]. Organic Se compounds, such as diphenyl selenide, have been shown to counteract Hg-induced oxidative stress by reducing reactive oxygen species production and lipid peroxidation in fish [25]. However, given the limited oxidative effects of dietary MeHg in our study, no such antagonism was observed.

SeMet can influence antioxidant status and lipid metabolism in Atlantic salmon exposed to 15 ppm of SeMet over a three-month feeding period [31,43], thus higher SeMet dietary supplementation, than the one used in this study, is expected to detect such effects.

The MeHg concentrations used in our study are environmentally relevant, reflecting potential contamination scenarios in fishmeal and providing insights into realistic exposure levels. Moreover, previous research on our fish model confirmed that those fed a control tuna-based diet did accumulate Hg in muscle beyond EU safety limits for human consumption [21,55]. However, the elevated Se content observed in tuna by-products raises important questions about their safe inclusion in aquafeeds, warranting further investigation.

## 5. Conclusion

Our findings demonstrate that fish fed tuna-based diets exhibited a higher oxidative stress, as evidenced by sustained elevations in oxylipin concentrations and concomitant reductions in muscle antioxidant metabolites. On the dynamics of the parameters measured, oxylipin levels were shown to persist longer in fish fed tuna-based diets. Antioxidant metabolites, which counteract oxylipin formation, exhibited dynamic fluctuations over time. Notably, the GSH system responded promptly after oxylipins increased, while α-tocopherol was the only vitamin E isoform to decline and this response appeared latter. These dynamics suggest a prompt and adaptive response of the GSH against the increase of oxylipins while for α-tocopherol the answer was delayed. Moreover, neither MeHg supplementation nor dietary SeMet influenced the parameters measured in this study. The presented results were suprising based on our previous results on Hg and Se bioaccumulation dynamics in fish muscle fed plant- or tuna-based diets. It appears that the dynamics of the parameters observed in our study were less related to the bioaccumulation of the pro-oxidant Hg than the deposition of Se, an antioxidant oligo-element. Thus, despite Hg levels in rainbow trout muscle remaining below the European regulatory limit (0.3 mg kg⁻¹ wet weight) [55], which underscore the viability of tuna by-products as sustainable aquafeed ingredients, levels of Se might deserve more attention. In fact too high dietary Se levels might have caused the prolonged increase in oxylipin levels observed in tuna-fed fish and warrants further investigation into potential long-term implications for fish health and product quality.

### CRediT authorship contribution statement

**Marius Bidon:** Writing – original draft, Investigation, Formal analysis, Data curation, Conceptualization. **Cécile Heraud:** Formal analysis, Data curation. **Claudia Marchán-Moreno:** Formal analysis, Data curation. **Laurence Larroquet:** Formal analysis, Data curation. **Guillaume Reversat:** Formal analysis. **Claire Vigor:** Formal analysis, Data curation. **Camille Oger:** Formal analysis, Data curation. **Jean-Marie Galano:** Formal analysis, Data curation. **Thierry Durand:** Funding acquisition. **Zoyne Pedrero-Zayas:** Writing – original draft, Funding acquisition, Conceptualization. **Stéphanie Fontagné-Dicharry:** Writing – original draft, Supervision, Investigation, Formal analysis, Data curation, Conceptualization. **Jérôme Roy:** Writing – review & editing, Writing – original draft, Supervision, Data curation, Conceptualization.
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.

Acknowledgements

This study was supported by I-site E2S: Energy and Environment Solutions from the University of Pau and Pays de l'Adour, UPPA contract 2020-32 (M B., PhD fellowship). The authors thank the Agence Nationale de la Recherche (MERSEL Project no. ANR-18-CE34-0004-01) for their financial support. This project has also received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement no. 101007962. The authors wish to thank Marine Biotechnology Products (Port-Louis, Mauritius) and Orffa (Breda, The Netherlands) for the kind supply of tuna meal and selenomethionine, respectively. They are also very grateful to F. Terrier and A. Lanuque for the preparation of diets and for the care of fish and A. Surget, K. Dias, V. Véron, E. Baranek, M. Marchand, T. Callet, F. Vallée, E. Plagnes-Juan, M. Delaygues, A. Soisson, A. Braun for their help during samplings.

Abbreviations

ALA, alpha-linolenic acid; ARA, arachidonic acid; BCA, bixinchninic acid; DHA, docosahexaenoic acid; FAO, Food and Agriculture Organization of the United Nations; GC-FID, gas chromatography coupled with flame ionization detector; GSH, glutathione; GSSG, oxidized glutathione; Hg, mercury; HPLC, high-performance liquid chromatography; HPLC, high performance liquid chromatography; ICP-MS, inductively coupled plasma mass spectrometry; MeHg, methylmercury; microLC MS/MS, liquid chromatography with tandem mass spectroscopy; PC, control plant-based diet; PH, plant-based diet supplemented with methylmercury; PHO, plant-based diet supplemented with methylmercury and selenomethionine; PUFA, polyunsaturated fatty acid; Se, selenium; SeMet, selenomethionine; TBARS, thio-barbituric acid reactive substances; TC, control tuna-based diet; TH, tuna-based diet supplemented with methylmercury; THO, tuna-based diet supplemented with methylmercury and selenomethioine; UV, ultraviolet.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.freeradbiomed.2025.07.043.

Data availability

Data will be made available on request.

References

[1] Mladenka Malenica Staver, Igor Jerković, Jasminka Giacometti, Ante Malenica, Zvonimir Marijanović, « fatty-acid profile of total and polar lipids in cultured rainbow trout (Oncorhynchus mykiss) raised in freshwater and seawater (Croatia) determined by transmethylation method, Chem. Biodivers. 9 (8) (2012) 1591–1598, https://doi.org/10.1002/cbdv.201200011.

[2] Fereidoon Shahidi, Ambigaipalan Priyatharini, Omega-3 polyunsaturated Fatty acids and their health benefits, Annu. Rev. Food Sci. Technol. 9 (1) (2018) 345–381, https://doi.org/10.1146/annurev-food-111317-095850.

[3] Anna Abramova, Jamie Bride, Camille Oger, Marie Demion, Jean-Marie Galano, Thierry Durand, Jérôme Roy, Metabolites derived from radical oxidation of PUFA: neo-pufas, promising molecules for health? Atherosclerosis (2024) 118600 https://doi.org/10.1016/j.atherosclerosis.2024.118600 septembre.

[4] Camille Oger, Tereza Pavlíčková, Valérie Bultel-Poncé, Alexandre Guy, Jean-Marie Galano, Ullrich Jahn, Thierry Durand, « An update of isoprostanoid nomenclature, Prog. Lipid Res. 96 (novembre) (2024) 101301, https://doi.org/10.1016/j.plipres.2024.101301.

[5] Clara Boglione, Enric Gisbert, Paulo Gavaia, Paul E. Witten, Mori Moren, Stéphanie Fontagné, Giorgos Koumoundouros, Skeletal anomalies in reared E uropean fish Larvae and juveniles. Part 2: main typologies, occurrences and causative factors, Rev. Aquacult. 5 (s1) (2013), https://doi.org/10.1111/raq.12016.

[6] A.A. Horton, Steven Fairhurst, S. Bus James, Lipid peroxidation and mechanisms of toxicity, CRC Crit. Rev. Toxicol. 18 (1) (1987) 27–79, https://doi.org/10.3109/10408448709089856.

[7] G. Hölmer, Mechanisms of oxidation – autooxidation and enzymic oxidation, in: J. M. Diplock, C. Gutteridge, V.K.S. Shukla (Eds.), Antioxidants, Free Radicals and Polyunsaturated Fatty Acids in Biology and Medicine, International Food Science Centre A/S, Lystrup, Denmark, 1993, pp. 15–30.

[8] E.N. Frankel, Lipid Oxidation, The Oily Press, Dundee, Scotland, 1998, p. 303.

[9] Shashank Kumar, Rapalli Krishna Chaitanya, Victor R. Preedy, Assessment of antioxidant potential of dietary components, in: HIV/AIDS, Elsevier, 2018, pp. 239–253, https://doi.org/10.1016/B978-0-12-809853-0.00020-1.

[10] K. Hamre, Metabolism, interactions, requirements and functions of vitamin E in fish: vitamin E in fish, Aquac. Nutr. 17 (1) (2011) 98–115, https://doi.org/10.1111/j.1365-2095.2010.00806.x.

[11] Aalt Bast, Guido R.M. M. Haenen, Regulation of lipid peroxidation by glutathione and lipoic acid: involvement of liver microsomal vitamin E free radical reductase, in: *Antioxidants in Therapy and Preventive Medicine*, 264:111-16. Advances in Experimental Medicine and Biology, Springer US, Boston, MA, 1990, https://doi.org/10.1007/978-1-4684-5730-8_15.

[12] O. Ritola, L.D. Peters, D.R. Livingstone, P. Lindström-Seppä, Effects of in Vitro exposure to ozone and/or hyperoxia on superoxide dismutase, catalase, glutathione and lipid peroxidation in red blood cells and plasma of rainbow Trout, *Oncorhynchus mykiss* (Walbaum): in Vitro exposure to ozone and/or Hyperoxia, Aquac. Res. 33 (3) (2002) 165–175, https://doi.org/10.1046/j.1365-2109.2002.00649.x.

[13] Siro Passi, Riccardo Ricci, Stefano Cataudila, Irene Ferrante, Francesco De Simone, et Luca Rastrelli, « fatty acid pattern, oxidation product development, and antioxidant loss in muscle tissue of rainbow trout and *Dicentrarchus labrax* during growth, J. Agric. Food Chem. 52 (9) (2004) 2587–2592, https://doi.org/10.1021/jf030559t.

[14] Carney Almroth, Bethanie, Angelica Johansson, Lars Förlin, Sturve Joachim, Early-Age changes in oxidative stress in brown trout, Salmo Trutta, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 155 (4) (2010) 442–448, https://doi.org/10.1016/j.cbbp.2010.01.012.

[15] M.J. Alvarez, C.J. Lopez-Bote, A. Diez, G. Corraze, J. Arzel, J. Dias, S.J. Kaushik, J. M. Bautista, Dietary fish oil and digestible protein modify susceptibility to lipid peroxidation in the muscle of rainbow trout (*Oncorhynchus mykiss*) and Sea bass (*Dicentrarchus labrax*), Br. J. Nutr. 80 (3) (1998) 281–289.

[16] Melissa K. Gregory, Robert O. Collins, Douglas R. Tocher, Michael J. James, Giovanni M. Turchini, Nutritional regulation of long-chain PUFA biosynthetic genes in rainbow trout (*Oncorhynchus mykiss*), Br. J. Nutr. 115 (10) (2016) 1721–1729, https://doi.org/10.1017/S0007114516000830.

[17] Stéphanie Fontagné-Dicharry, Emilie Lataillade, Anne Surget, Laurence Larroquet, Marianne Cluzeaud, Sadasivam Kaushik, Antioxidant defense System is altered by dietary oxidized lipid in first-feeding rainbow trout (*Oncorhynchus mykiss*), Aquaculture (2014) 220–227, https://doi.org/10.1016/j.aquaculture.2014.01.009, 424–425 (mars).

[18] Rohana Subasinghe, Doris Soto, Jiansan Jia, « global aquaculture and its role in sustainable development, Rev. Aquacult. 1 (1) (2009) 2–9, https://doi.org/10.1111/j.1753-5131.2008.01002.x.

[19] FAO 2024, Fao. https://doi.org/10.4060/cd0683en, 2024.

[20] Kyochan Kim, Youngjin Park, Hyeong-Woo Je, Minji Seong, Jim Hyacinth Damusaru, Soohwan Kim, Joo-Young Jung, Sungchul C. Bai, Tuna byproducts as a fish-meal in tilapia aquaculture, Ectotoxicol. Environ. Saf. 172 (mai) (2019) 364–372, https://doi.org/10.1016/j.ecoenv.2019.01.107.

[21] Claudia Marchán-Moreno, Silvia Queipo-Abad, Warren T. Corns, Maite Bueno, Florence Pannier, David Amouroux, Stéphanie Fontagné-Dicharry, Zoyne Pedrero, Assessment of dietary selenium and its role in Mercury fate in cultured fish rainbow trout with two sustainable aquafeeds, Food Chem. 447 (juillet) (2024) 138865, https://doi.org/10.1016/j.foodchem.2024.138865.

[22] El Hanafi, Khouloud, Tamara Fernández-Bautista, Laurent Ouerdane, Warren T. Corns, Maite Bueno, Stéphanie Fontagné-Dicharry, David Amouroux, Pedrero Zoyne, Exploring Mercury detoxification in fish: the role of selenium from Tuna byproduct diets for sustainable aquaculture, J. Hazard Mater. 480 (décembre) (2024) 135779, https://doi.org/10.1016/j.jhazmat.2024.135779.

[23] Olívia Cardoso, Sónia Puga, Fátima Brandão, João Canário, Nelson J. O'Driscoll, Maria Ana Santos, Mário Pacheco, Patrícia Pereira, Oxidative stress profiles in brain point out a higher susceptibility of fish to waterborne Divalent Mercury compared to dietary organic Mercury, Mar. Pollut. Bull. 122 (1-2) (2017) 110–121, https://doi.org/10.1016/j.marpolbul.2017.06.029.

[24] Marc H.G. Berntssen, Aase Aatland, Richard D. Handy, Chronic dietary Mercury exposure causes oxidative stress, brain lesions, and altered behaviour in Atlantic salmon (*Salmo salar*) Parr, Aquat. Toxicol. 65 (1) (2003) 55–72, https://doi.org/10.1016/S0166-445X(03)00104-8.

[25] Matheus D. Baldissera, Carine F. Souza, Aleksandro S. da Silva, Alessandra S. Henn, Erico M.M. Flores, Baldisserotto Bernardo, Diphenyl diselenide dietary supplementation alleviates behavior impairment and brain damage in grass carp (*Ctenopharyngodon idella*) exposed to Methylmercury Chloride, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 229 (mars) (2020) 108674, https://doi.org/10.1016/j.cbpc.2019.108674.
[26] Nicholas V.C. Ralston, Laura J. Raymond, Mercury's neurotoxicity is characterized by its disruption of selenium biochemistry, *Biochim. Biophys. Acta Gen. Subj.* 1862 (11) (2018) 2405–2416, https://doi.org/10.1016/j.bbagen.2018.05.009.

[27] Nicholas V.C. Ralston, Carla R. Ralston, Laura J. Raymond, Selenium health benefit values: updated criteria for Mercury risk assessments, *Biol. Trace Elem.* 171 (2) (2016) 262–269, https://doi.org/10.1007/s12011-015-0516-z.

[28] Chloé J. Zampetti, Jessica E. Brandt, Co-Considering Selenium concentrations alters mercury-based fish and seafood consumption advice: a data compilation and critical assessment, *Environ. Sci. Technol. Lett.* 10 (2) (2023) 179–185, https://doi.org/10.1021/acs.estlett.2c00925.

[29] Tamara Fernández-Bautista, Beatriz Gómez-Gómez, Emma Gracia-Lor, Teresa Pérez-Corona, et Yolanda Madrid, «Selenium health benefit values and Hg and Se speciation studies for elucidating the quality and safety of highly consumed fish species and fish-derived products, *Food Chem.* 435 (mars) (2024) 137544, https://doi.org/10.1016/j.foodchem.2023.137544.

[30] Romina V. Barbosa, David Point, Anais Médieu, Valérie Allain, David P. Gillikin, Lydie I.E. Couturier, Jean-Marie Munaron, François Roupsard, Lorrain Anne, Mercury concentrations in tuna blood and muscle mirror seawater methylmercury in the Western and central Pacific Ocean, *Mar. Pollut. Bull.* 180 (juillet) (2022) 113801, https://doi.org/10.1016/j.marpolbul.2022.113801.

[31] M.H.G. Berntssen, M. Betancor, M.J. Caballero, M. Hillestad, J. Rasinger, K. Hamre, V. Sele, H. Amlund, R. Ørnsrud, Safe limits of selenomethionine and selenite supplementation to plant-based Atlantic salmon feeds, *Aquaculture* 495 (octobre) (2018) 617–630, https://doi.org/10.1016/j.aquaculture.2018.06.041.

[32] M. Bidon, A.J.P. Philip, A. Braun, A. Herman, J. Roy, Z. Pedrero-Zayas, S. Fontagné-Dicharry, Interaction between dietary selenium and methylmercury on growth performance, deposition and health parameters in rainbow trout Fed selenium-rich tuna-based diets or selenium-poor plant-based diets, *Aquaculture* 572 (juillet) (2023) 739550, https://doi.org/10.1016/j.aquaculture.2023.739550.

[33] Cécile Heraud, Théo Hirschinger, Elodie Baranek, Laurence Larroquet, Anne Surget, Franck Sandres, Anthony Lanuque, Frederic Terrier, Jérôme Roy, « detection and modulation of olfactory sensing receptors in carnivorous rainbow trout (*Oncorhynchus mykiss*) Fed from first feeding with plant-based diet, *Int. J. Mol. Sci.* 23 (4) (2022) 2123, https://doi.org/10.3390/ijms23042123.

[34] J. Folch, M. Lees, G.H. Sloane Stanley, A simple method for the isolation and purification of total lipides from animal tissues, *J. Biol. Chem.* 226 (1) (1957) 497–509.

[35] N.C. Shantha, R.G. Ackman, «advantages of total lipid hydrogenation prior to lipid class determination on chromoars-SIII, Lipids 25 (9) (1990) 570–574, https://doi.org/10.1007/BF02537167.

[36] Pauline Wischhusen, Laurence Larroquet, Thierry Durand, Camille Oger, Jean-Marie Galano, Amandine Rocher, Claire Vigor, et al., Oxidative stress and antioxidant response in rainbow trout Fry exposed to acute hypoxia is affected by selenium nutrition of parents and during first exogenous feeding, *Free Radic. Biol. Med.* 155 (août) (2020) 99–113, https://doi.org/10.1016/j.freeradbiomed.2020.05.006.

[37] Pauline Wischhusen, Takaya Saito, Cécile Heraud, Sadasivam J. Kaushik, Benoit Fauconneau, Philip Antony Jesu Prabhu, Stéphanie Fontagné-Dicharry, Kaja H. Skjærven, Parental Selenium nutrition affects the one-carbon metabolism and the hepatic DNA methylation pattern of rainbow trout (*Oncorhynchus mykiss*) in the progeny, *Life* 10 (8) (2020) 121, https://doi.org/10.3390/life10080121.

[38] J.H. Robin, C. Regost, J. Arzel, S.J. Kaushik, «Fatty acid profile of fish following a change in dietary fatty acid source: model of Fatty acid composition with a dilution Hypothesis, *Aquaculture* 225 (1–4) (2003) 283–293, https://doi.org/10.1016/S0044-8486(03)00296-5.

[39] Stéphanie Fontagné-Dicharry, Laurence Larroquet, Karine Dias, Marianne Cluzeaud, Cécile Heraud, Corlay Dominique, Effects of dietary oxidized fish oil supplementation on oxidative stress and antioxidant defense System in juvenile rainbow trout (*Oncorhynchus mykiss*), *Fish Shellfish Immunol.* 74 (mars) (2018) 43–51, https://doi.org/10.1016/j.fsi.2017.12.039.

[40] V. Mambro, A.E.C.S. Azzolini, Y.M.L. Valim, M.J.V. Fonseca, Comparison of antioxidant activities of tocopherols alone and in pharmaceutical formulations, *Int. J. Pharm.* 262 (1–2) (2003) 93–99, https://doi.org/10.1016/S0378-5173(03)00333-8.

[41] P.M. Mathias, J.T. Harries, T.J. Peters, D.P. Muller, « studies on the in vivo absorption of micellar solutions of tocopherol and Tocopheryl acetate in the rat: demonstration and partial characterization of a mucosal esterase localized to the endoplasmic reticulum of the enterocyte, *JLJ* (J. Lipid Res.) 22 (5) (1981) 829–837, https://doi.org/10.1016/S0022-2275(20)37355-7.

[42] Seunghan Lee, Rebecca Walugembe Nambi, Seonghun Won, Katya Kumar, Sungchul C. Bai, Dietary Selenium requirement and toxicity levels in Juvenile Nile Tilapia, *Oreochromis niloticus*, *Aquaculture* 464 (novembre) (2016) 153–158, https://doi.org/10.1016/j.aquaculture.2016.06.027.

[43] M.H.G. Berntssen, T.K. Sundal, P.A. Olsvik, H. Amlund, J.D. Rasinger, V. Sele, K. Hamre, M. Hillestad, I. Buttle, R. Ørnsrud, Sensitivity and toxic mode of action of dietary organic and inorganic selenium in Atlantic salmon (*Salmo salar*), *Aquat. Toxicol.* 192 (novembre) (2017) 116–126, https://doi.org/10.1016/j.aquatox.2017.09.014.

[44] James H. Tidwell, H. Randall Robinette, Changes in proximate and fatty acid composition of fillets from channel catfish during a two-year growth period, *Trans. Am. Fish. Soc.* 119 (1) (1990) 31–40, https://doi.org/10.1577/1548-8659(1990)119<0031:CIPAPA>2.3.CO;2.

[45] Andreas J. Meyer, The integration of glutathione homeostasis and redox signaling, *J. Plant Physiol.* 165 (13) (2008) 1390–1403, https://doi.org/10.1016/j.jplph.2007.10.015.

[46] Graham W. Burton, Maret G. Traber, Vitamin E: Antioxidant activity, *biokinetics*, and bioavailability, *Annu. Rev. Nutr.* 10 (1) (1990) 357–382, https://doi.org/10.1146/annurev.nu.10.070190.002041.

[47] X. Wang, Vitamin E and its function in membranes, *Prog. Lipid Res.* 38 (4) (1999) 309–336, https://doi.org/10.1016/S0163-7827(99)00008-9.

[48] Md Helal Uddin, Jinnath Rehana Ritu, Sravan Kumar Putnala, Mahesh Rachamalla, Douglas P. Chivers, Niyogi Som, Selenium toxicity in fishes: a Current perspective, *Chemosphere* 364 (septembre) (2024) 143214, https://doi.org/10.1016/j.chemosphere.2024.143214.

[49] Michiaki Yamashita, Yumiko Yamashita, Tamami Suzuki, Yoko Kani, Nanami Mizusawa, Shintaro Imamura, Kenji Takemoto, et al., « selenoneine, a novel selenium-containing compound, mediates detoxification mechanisms against methylmercury accumulation and toxicity in zebrafish embryo, *Mar. Biotechnol.* 15 (5) (2013) 559–570, https://doi.org/10.1007/s10126-013-9508-1.

[50] Heidi Amlund, Lise Andreasen, Bente E. Torstensen, Dietary methylmercury and vegetable oil affects brain lipid composition in Atlantic salmon (*Salmo salar*), *Food Chem. Toxicol.* 50 (3–4) (2012) 518–525, https://doi.org/10.1016/j.fct.2011.12.041.

[51] Gilles Tinant, Mélusine Van Larebeke, Lemaire Benjamin, Courteille Marine, Cécile Gardin, Ineke Neefs, Das Krishna, et al., Dietary methylmercury and fatty acids affect the lipid metabolism of adipose tissue and liver in rainbow trout, *Aquat. Toxicol.* 263 (octobre) (2023) 106673, https://doi.org/10.1016/j.aquatox.2023.106673.

[52] James A. Hamilton, Cecilia J. Hillard, Arthur A. Spector, Paul A. Watkins, Brain uptake and utilization of fatty acids, lipids and lipoproteins: application to neurological disorders, *J. Mol. Neurosci.* 33 (1) (2007) 2–11, https://doi.org/10.1007/s12031-007-0060-1.

[53] Zhonghua Ren, Jinhu Liu, Wei Huang, Liang Cao, Wenting Cui, Shuozeng Dou, «antioxidant defenses and immune responses of flounder *Paralichthys olivaceus* Larvae under methylmercury exposure, *Comp. Biochem. Physiol. C Toxicol. Pharmacol.* 225 (novembre) (2019) 108589, https://doi.org/10.1016/j.cbpc.2019.108589.

[54] Pål A. Olsvik, Heidi Amlund, Bente E. Torstensen, Dietary lipids modulate methylmercury toxicity in Atlantic salmon, *Food Chem. Toxicol.* 49 (12) (2011) 3258–3271, https://doi.org/10.1016/j.fct.2011.09.025.

[55] EU, Commission regulation (EU) 2022/617 of 12 April 2022 amending regulation (EC) no 1881/2006 as regards maximum levels of mercury in fish and salt, *Official Journal of the European Union* L115 65 (2022) 60–63. http://eur-lex.europa.eu/l egal-content/EN/TXT/PDF/?uri=OJ:L:2014:015:FULL&from=EN.