# Discarded blue mussel (Mytilus edulis): a feed ingredient that maintains growth performance of juvenile gilthead seabream (Sparus aurata) while fishmeal and fish oil are removed

Christophe Jaeger, Geneviève Corraze, Vincent Gayet, Laurence Larroquet, Anne Surget, Frederic Terrier, Joël Aubin

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Christophe Jaeger, Geneviève Corraze, Vincent Gayet, Laurence Larroquet, Anne Surget, et al. Dis- carded blue mussel (Mytilus edulis): a feed ingredient that maintains growth performance of juvenile gilthead seabream (Sparus aurata) while fishmeal and fish oil are removed. Journal of Applied Aqua- culture, 2024, pp.1-17. 10.1080/10454438.2024.2407122. hal-04705390

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# Discarded blue mussel (*Mytilus edulis*): a feed ingredient that maintains growth performance of juvenile gilthead seabream (*Sparus aurata*) while fishmeal and fish oil are removed

Christophe Jaeger, Geneviève Corraze, Vincent Gayet, Laurence Larroquet, Anne Surget, Frédéric Terrier & Joël Aubin

**To cite this article:** Christophe Jaeger, Geneviève Corraze, Vincent Gayet, Laurence Larroquet, Anne Surget, Frédéric Terrier & Joël Aubin (22 Sep 2024): Discarded blue mussel (*Mytilus edulis*): a feed ingredient that maintains growth performance of juvenile gilthead seabream (*Sparus aurata*) while fishmeal and fish oil are removed, Journal of Applied Aquaculture, DOI: 10.1080/10454438.2024.2407122

**To link to this article:** https://doi.org/10.1080/10454438.2024.2407122

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Published online: 22 Sep 2024.

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# Discarded blue mussel (*Mytilus edulis*): a feed ingredient that maintains growth performance of juvenile gilthead seabream (*Sparus aurata*) while fishmeal and fish oil are removed

Christophe Jaeger<sup>a</sup>, Geneviève Corraze<sup>b</sup>, Vincent Gayet<sup>c</sup>, Laurence Larroquet<sup>b</sup>, Anne Surget<sup>b</sup>, Frédéric Terrier<sup>b</sup>, and Joël Aubin<sup>a</sup>

<sup>a</sup>INRAE, Institut Agro, SAS, Rennes, France; <sup>b</sup>INRAE, Université de Pau et des Pays de l'Adour, E2S UPPA, Saint Pée-sur-Nivelle, France; <sup>c</sup>Lycée de la Mer et du Littoral, Bourcefranc-le-Chapus, France

## ABSTRACT

The total substitution of fishmeal (FM) and fish oil (FO) with sustainable feed ingredients, while maintaining acceptable growth performances and fish composition, is a challenge. Mussels have a high protein content, with essential amino acid and fatty acid (FA) profiles similar to those of FM. This study aimed to assess the potential to rear gilthead seabream (*Sparus aurata*) on a plant-based feed (without FM and FO) to which mussels were added. Subsequent effects on growth performance, protein content, and FA concentrations were studied. Juvenile gilthead seabream were reared for 46 days in water temperature of 21°C, in a recirculated aquaculture system composed of three fish tanks, each corresponding to one treatment: in the FMFO treatment, 342 fish were fed a commercial feed that contained FM and FO; in the plant-based feed (PBF) treatment, 345 fish were fed an experimental feed that contained only plant-based ingredients; in the mussel feed (MF) treatment, 342 fish were fed the feed from PBF supplemented with fresh mussel flesh from which the shells had been removed. Fish weighed a mean of 6.7 ± 1.3 g at stocking in each treatment. At harvest, the mean weight of fish from FMFO and MF were significantly different (23.1 ± 4.3 and 23.1 ± 5.2 g, respectively) than that from PBF (15.5 ± 3.6 g) (p < .05). Compared to the feed from FMFO, the feed from PBF decreased the percentages of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and saturated FA (14:0, 16:0) in fish, but increased those of 18:1, 18:2 *n*-6 and 18:3 *n*-3 FA. Compared to the feed from PBF, the feed from MF increased the EPA+DHA concentration in fish by 21%. Thus, adding fresh mussels to a plant-based feed yielded similar growth performances and survival rate as a commercial feed. It also improved the FA composition of fish compared to that of fish fed a plant-based feed.

## KEYWORDS

Fish feed; eicosapentaenoic acid (EPA); docosahexaenoic acid (DHA); plant-based feed

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Introduction

Aquaculture is expected to develop sustainably. Several strategies are possible like using environmentally friendly production systems, such as integrated aquaculture (i.e. combining multiple species). To this end, domesticating new species and promoting native species that grow more efficiently and are more resilient to climate change might be considered. Adopting responsible feed-management practices that consider the need to reuse water and minimize nutrient discharge are also of importance. Another strategy stands in using novel feed ingredients to partially or completely replace unsustainable ingredients like fishmeal (FM) and fish oil (FO) (Boyd et al. 2020). At the same time, it is necessary to use alternative ingredients that do not compete with those used by terrestrial animal production systems (Boyd et al. 2020; Saleh, Wassef, and Abdel-Mohsen 2022). Replacing FM is also crucial because its production is decreasing and its prices are increasing (Jannathulla et al. 2019). Several feed ingredients are used to replace FM in fish feed, such as plant-based ingredients (e.g. soy protein concentrate, wheat gluten, rapeseed meal), single-cell protein sources, fishery and aquaculture byproducts, and insect meal (Qian et al. 2024). FO is usually replaced with vegetable oils (e.g. rapeseed, linseed, soybean) (Aas, Asgard, and Ytrestoyl 2022; Albrektsen et al. 2022; Hua et al. 2019; Richard et al. 2021; Tacon, Metian, and Hasan 2009; Teves and Ragaza 2016; Woodgate et al. 2022). Nonetheless, attention must be paid to essential amino acids and long-chain polyunsaturated fatty acids (PUFA) supplied in fish feed (Tocher 2015) to maintain growth performances and fish quality (Naylor et al. 2009, Matos et al. 2016). It is of importance more specifically for marine finfish, which cannot synthesize eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Tocher 2010).

Currently, the main strategy is to partially replace FM and FO with a blend of plant-based ingredients, with potential negative consequences on filet quality (Matos et al. 2016), for instance in decreasing *n*-3 PUFA:*n*-6 PUFA ratio in the filet (Castro et al. 2016; Matos et al. 2016; Richard et al. 2021). Then, it can negatively influence potential health benefits provided to consumers (Matos et al. 2016). In feed for carnivorous species, the total replacement of FM and FO negatively impacts growth performances, as well as metabolism and flesh characteristics (Richard et al. 2021). For gilthead seabream (*Sparus aurata*), growth or feed and protein use efficiency are not influenced when FO is replaced (up to 70%) with soybean or rapeseed oil (Castro et al. 2016; Fountoulaki et al. 2009; Wassef, Saleh, and El-Abd El-Hady 2009).

Mussels have high protein (50–70% dry matter (DM)) and lipid (5–16% DM) contents, with essential amino acid and fatty acid (FA) profiles (Albrektsen et al. 2022; Berge and Austreng 1989) similar to those of FM (Sauvant et al. 2004). Thus, mussels may be able to replace FM as a feed ingredient (Jusadi et al. 2021). However, few studies have examined this
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practice, and most only partially replaced FM. Berge and Austreng (1989) observed lower growth, lower protein digestibility, and a negative effect on flesh pigmentation when increasing the quantity of blue mussel (*Mytilus edulis*) in the diet of rainbow trout (*Oncorhynchus mykiss*). Similar trends (i.e. a decrease in specific growth rate (SGR) and feed intake) were observed when increasing the quantity of mussel added to the diets of other species. Hence, it is suggested to limit the replacement to 25% in the diet of turbot (*Scophthalmus maximus*) (Weiß and Buck 2017) and to 50% in the diet of juvenile Ussuri catfish (*Pseudobagrus ussuriensis*) (Luo et al. 2019). For juvenile common sole (*Solea solea*), the SGR, feed conversion ratio (FCR), protein efficiency ratio, and viscero-somatic index were significantly improved when 25-75% of FM were replaced with mussel meal (MM) compared to those of a diet without MM (Mongile et al. 2015). Metabolically, MM is considered a suitable feed ingredient, without adverse effects, when it is introduced in the diet of Arctic char (*Salvelinus alpinus*) (Wagner et al. 2019) or Ussuri catfish (Luo et al. 2019). To be definitive, no work considered the gilthead seabream as the target species in the replacement of FM with MM.

However, gilthead seabream prey on shellfish by crushing their shells (Richard et al. 2020; Wassef and Eisawy 1985). Since shellfish can become a large part of the diet of wild seabream (Avignon et al. 2017), it makes sense to use mussels as an ingredient in seabream feed. Mussels that do not compete with human food can come from several sources, such as artificial substrates (e.g. wind turbines) or farms (e.g. undersized mussels (i.e. too small to sell)) (Weiß and Buck 2017). The Charente-Maritime department in France is known for its production of oysters and mussels. Once harvested, some of them are discarded because they are undersized. However, they could be a local and suitable source of nutrients for gilthead seabream. Hence, from the perspective of potentially giving up FM and FO in the future, there is an interest to establish if blue mussel is a suitable feed ingredient, for carnivorous species, like gilthead seabream. The experiment aimed to assess the influence of rearing gilthead seabream on a plant-based feed (without FM and FO), with or without the addition of fresh blue mussels, on growth performances and body composition (i.e. protein content and FA concentrations).

# Materials and methods

## Experimental design

The experiment was conducted at the facilities of the Lycée de la Mer in Boursefranc-le-Chapus (France). Juvenile gilthead seabream were purchased two weeks before the beginning of the experiment, from a commercial hatchery. They were stocked at a density of 1 kg/m³ in tanks similar to that used for the experiment. Then, at the beginning of the experiment, fish were stocked in
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1.6 m³ tanks at a density of 1.5 kg/m³. They were reared for 46 days in a single recirculating aquaculture system composed of three square rearing tanks (1.6 m³ each, composed of glass fiber reinforced, polyester resin), a mechanical filter (Japan matting, 0.5 m³) to remove suspended matter, and a biological filter (0.5 m³) on a fluidized bed to transform ammonia into nitrate. The water was circulated using an airlift pump and maintained at a constant temperature of 21 ± 1°C. Water was renewed at a rate of 2 m³/day.

Three treatments were tested, one per tank. In the control treatment (FMFO), 342 fish were fed a commercial feed (feed FMFO) that contained FM and FO (Neo Start, Le Gouessant, France) used for juvenile marine fish. In the “plant-based” treatment (PBF), 345 fish were fed an experimental feed (feed PBF) that contained only plant-based ingredients. This experimental feed was formulated without marine ingredients and synthetic amino acids. The ingredients used were produced locally (<100 km), or if necessary regionally (i.e. European). And at last, few processed products were used, but an attractant mixture was incorporated to encourage consumption (Actipal, Symrise Aqua Feed), as performed by Kikuchi and Furuta (2009) for the diet of tiger puffers (*Takifugu rubripes*). The feed PBF was formulated to be

<table><caption>**Table 1.** Formulation and composition of the feeds and mussel flesh used in the experiment.</caption><thead><tr><th></th><th>Control feed<sup>b</sup></th><th>Plant-based feed</th><th>Mussel flesh</th></tr></thead><tbody><tr><td><strong>Ingredient (%)</strong></td><td></td><td></td><td></td></tr><tr><td>Fish meal</td><td>X</td><td></td><td></td></tr><tr><td>Fish oil</td><td>X</td><td></td><td></td></tr><tr><td>Pea protein</td><td>X</td><td></td><td></td></tr><tr><td>Faba bean</td><td>X</td><td></td><td></td></tr><tr><td>Wheat flour</td><td>X</td><td></td><td></td></tr><tr><td>Yeast</td><td>X</td><td></td><td></td></tr><tr><td>Wheat</td><td>X</td><td></td><td></td></tr><tr><td>Wheat gluten</td><td>X</td><td>18.32</td><td></td></tr><tr><td>Vitamin and mineral premix</td><td>X</td><td>3.00</td><td></td></tr><tr><td>Faba bean protein concentrate</td><td></td><td>20.00</td><td></td></tr><tr><td>Corn gluten</td><td></td><td>20.00</td><td></td></tr><tr><td>Pea protein concentrate</td><td></td><td>18.00</td><td></td></tr><tr><td>Linseed oil</td><td></td><td>10.00</td><td></td></tr><tr><td>Rapeseed oil</td><td></td><td>4.11</td><td></td></tr><tr><td>Di-calcium phosphate</td><td></td><td>3.00</td><td></td></tr><tr><td>Soy lecithin</td><td></td><td>2.00</td><td></td></tr><tr><td>Rapeseed meal</td><td></td><td>1.07</td><td></td></tr><tr><td>Attractant mixture<sup>c</sup></td><td></td><td>0.50</td><td></td></tr><tr><td><strong>Composition<sup>a</sup></strong></td><td></td><td></td><td></td></tr><tr><td>Dry matter (%)</td><td>91.52</td><td>97.32</td><td>18.61</td></tr><tr><td>Crude protein (%)</td><td>51.09</td><td>53.49</td><td>11.09</td></tr><tr><td>Crude fat (%)</td><td>15.08</td><td>19.50</td><td>3.70</td></tr><tr><td>Crude energy (kJ/g)</td><td>22.00</td><td>22.76</td><td>4.35</td></tr><tr><td>Ash (%)</td><td>10.33</td><td>6.75</td><td>3.23</td></tr></tbody></table>

<footnote><sup>a</sup>Values were determined from analyses, except for the crude energy which was calculated for the control and plant-based feeds, and, for the flesh of mussel, is reported from: Projet « Composition nutritionnelle des produits aquatiques » (https://www.nutraqua.com/).</footnote>

<footnote><sup>b</sup>Because the feed is a private commercial formula (Neo Start, Le Gouessant, France), the manufacturer specified whether an ingredient was included (X) but not its percentage.</footnote>

<footnote><sup>c</sup>Actipal, Symrise Aqua Feed.</footnote>
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isoenergetic with feed FMFO. In the “mussel” treatment (MF), 342 fish were fed the feed PBF supplemented with fresh mussel flesh from which the shells had been removed (Table 1). The mussels, purchased locally from a nearby aquaculture farm, were undersized.

## Feeding management

For each treatment, the feed was weighed daily, prior to the first manual distribution and monitoring of dead fish. Feed FMFO and feed PBF were distributed for treatments FMFO and PBF, respectively, twice a day and six days per week. For the treatment MF, feed PBF was distributed twice a day, five days per week and only mussels were distributed on the 6th day, once a day. In order to determine the quantity of feed to deliver and to follow the appropriate growth of fish, once a week throughout the experiment, the mean weight of fish was measured. A batch of 50 fish from each treatment were weighed in a group and then restocked. The quantity of feed distributed in each treatment was based on the same table, which considered fish weight (measured weekly) and corresponded to an overall mean of 3% of live weight per day. Since it was not possible to collect unfed feed, the feed delivered was considered as the feed intake. Nevertheless, the quantity of feed distributed was daily adjusted according to the feed consumption observed the day before to avoid unfed feed. The quantity of mussel distributed in treatment MF was based on a daily ratio that was isoenergetic with the quantity of feed PBF distributed the previous day.

## Determination of growth performance, survival, and feed conversion ratio

Prior to Measurements, fish were fasted for 16–18 h. At stocking (i.e. the beginning of the experiment), 200 of the fish were individually weighed (W, g), and their length at the caudal fork (L, mm) was measured to calculate Fulton's (1904) condition factor (K):

$$K = \frac{W}{L^3} \times 100$$

At stocking, for each treatment, the total number and weight of fish was also determined by weighing groups of 30 individuals. At harvest (i.e. the end of the experiment), W and L were determined for each treatment from a representative sample of fish (n = 114, 120, and 104 for FMFO, PBF, and MF, respectively), and the remaining fish were counted and weighed in groups. Survival, specific growth rate (SGR), and feed conversion ratio (FCR) were calculated based on group measurements:
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$$Survival(\%) = \left( \frac{N_s}{N_h} \right) \times 100$$

where Ns and Nh are the number of fish at stocking and harvest, respectively.

$$SGR(\%/day) = \left( \frac{\ln(Wh) - \ln(Ws)}{duration(days)} \right) \times 100$$

where Wh and Ws are the mean weight at harvest and stocking (g), respectively.

The quantity of feed distributed throughout the experiment was recorded to calculate the FCR:

$$FCR = \frac{feed \ intake(g)}{fish \ net \ weight \ gain(g)}$$

Since feeds differed in composition and presentation, a standardized FCR was calculated in which the quantity of feed was standardized to a common crude energy content (i.e. that of feed FMFO) and DM (94%).

## Body and feed proximate compositions

Proximate composition (i.e. DM, crude protein, lipids, and ash contents) and FA profiles were determined based on a single sample of (i) each feed, (ii) mussels composed of samples of flesh collected each time they were distributed to fish, (iii) 10 fish pooled at stocking, and (iv) 10 fish pooled at harvest for each treatment. The fish were deeply anesthetized by immersing them in a water bath containing an excess of 10% isoeugenol (0.4 ml/l), prior to exsanguination by cutting the brachial artery while in a cold water bath (Neiffer and Stamper 2009). The flesh of mussels was removed from the shell after heating whole mussels for 2 min in a 1 300 W microwave oven (Besse and Mazurie 2003). Immediately after sampling, the feed, mussel flesh, and fish (whole body) sampled were kept at -20°C until analysis.

Proximate composition was determined according to the standard methodology of AOAC (2000) as follows: DM content was measured by drying the samples to a constant weight at 105°C for 24 h. Crude protein content was determined using the Kjeldahl method after acid digestion and estimated by multiplying the nitrogen content by 6.25. Gross energy content was measured in an adiabatic bomb calorimeter (IKA, Staufen, Germany).

## Fatty acid analysis

Total lipid content of feed, mussel flesh, and whole fish was quantified gravimetrically after extraction by dichloromethane/methanol (2:1, v/v) containing 0.01% butylated hydroxytoluene as an antioxidant, according
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to Folch, Lees, and Stanley (1957). FA methyl esters were prepared by acid-catalyzed transmethylation of the lipid extract using boron trifluoride, according to Shantha and Ackman (1990), and then analyzed in a Varian 3900 gas chromatograph equipped with a fused silica DB-Wax capillary column (30 m × 0.25 mm internal diameter, film thickness 0.25 µm; JW Alltech, France). The injection volume was 1 µl, using helium as a carrier gas (1 mL/min). The temperatures of the injector and the flame ionization detector were 260°C and 250°C, respectively. The thermal gradient was as follows: 100–180°C at 8°C/min, 180–220°C at 4°C/min, and then a constant temperature of 220°C for 20 min. FA were identified with reference to a known standard mixture (Sigma, St. Louis, Missouri, USA), and peaks were integrated using Varian Star Chromatography Software (Star Software, version 5). Results for each FA were expressed as the percentage of total FA identified.

## Nutritional retention indicators

The retention of protein and lipids was determined for each treatment according to proximate and FA analyses performed on body and feed, according to the formula:

$$Re(\%) = \frac{Qhe(g) - Qse(g)}{Qfe(g)} \times 100$$

where e is protein or lipids, Qhe is the total quantity of e in all fish harvested, Qse is the total quantity of e in all fish stocked, and Qfe is the total quantity of e in all feed distributed.

The protein efficiency ratio (PER) (Mongile et al. 2015) was also calculated according to:

$$PER = \frac{\text{fish weight gain(g)}}{\text{total protein intake(g)}}$$

## Statistical analyses

Since the samples included more than 50 individuals, data obtained from individual measurements (i.e. W, L, and K), were assessed visually for normality in using the quantile-quantile plot (package ggpubr) in addition to the Shapiro-Wilk test (Ghasemi and Zahediasl 2012). For W, L, and K, the distribution was normal and equality of variance assumed. Thus, W, L, and K were compared between treatments using type III analysis of variance (ANOVA). When significant differences were detected, multiple comparison test was performed using Tukey's HSD (package multcomp) to identify significant differences among treatments. Results are presented as means ± s.e
<figure><img src="image_20.png" /><figcaption>**Table 2.** Growth performance, survival, and feed efficiency parameters of gilthead seabream at harvest for fish from the control (FMFO), plant-based (PBF), and plant-based+mussel (MF) treatments. Means ± s.e. On a line with different letters differ significantly (*p* < .05) and were calculated from individual measurements, whereas the other values were obtained from measurements in group.</figcaption></figure>

<table><thead><tr><th rowspan="2">Performance</th><th colspan="3">Treatments</th><th rowspan="2">RSE<sup>a</sup></th><th rowspan="2">Significance<sup>b</sup></th></tr><tr><th>FMFO</th><th>PBF</th><th>MF</th></tr></thead><tbody><tr><td>Weight (g)</td><td>23.1 ± 4.3<sup>a</sup></td><td>15.5 ± 3.6<sup>b</sup></td><td>23.1 ± 5.2<sup>a</sup></td><td>4.4</td><td>***</td></tr><tr><td>Length<sup>c</sup> (mm)</td><td>109.9 ± 5.9<sup>a</sup></td><td>95.4 ± 7.0<sup>b</sup></td><td>107.9 ± 7.4<sup>a</sup></td><td>6.8</td><td>***</td></tr><tr><td>Fulton's condition factor - K</td><td>1.72 ± 0.13<sup>a</sup></td><td>1.75 ± 0.09<sup>a</sup></td><td>1.81 ± 0.11<sup>b</sup></td><td>0.1</td><td>***</td></tr><tr><td>Survival (%)</td><td>99.7</td><td>98.0</td><td>99.4</td><td></td><td></td></tr><tr><td>Total feed delivered (g fresh weight)</td><td>5663</td><td>5197</td><td>7696</td><td></td><td></td></tr><tr><td>Specific growth rate (%/day)</td><td>2.69</td><td>1.82</td><td>2.69</td><td></td><td></td></tr><tr><td>Feed conversion ratio (FCR)</td><td>1.03</td><td>1.77</td><td>1.49</td><td></td><td></td></tr><tr><td>Standardized FCR<sup>d</sup></td><td>1.00</td><td>2.35</td><td>1.26</td><td></td><td></td></tr></tbody></table>

and the significance level of 5% was applied in all statistical analyses. All the statistical analyses were performed using R software (R Core Team 2021).

## Results

### Growth performance, survival, and feed efficiency parameters

At harvest, the mean W for treatments FMFO and MF were significantly higher than that for treatment PBF. The mean L for FMFO and MF were significantly longer than that for PBF. The mean K for FMFO and PBF were significantly lower than that for MF. Nevertheless, the survival was similar between treatments (Table 2). Fish growth was similar for FMFO and MF, while that for PBF slowed after the first week of the experiment (Figure 1), as illustrated by the SGR from stocking to harvest (Table 2).

A total of 5 182 g DM of feed were distributed in FMFO, 5 057 g DM in PBF and 5 240 g DM in MF (i.e. 4 708.5 g DM of feed PBF +531.5 g DM of fresh flesh of mussel). The total net weight gain of fish was 5 501 g for FMFO, 2 928 g for PBF, and 5 159 g for MF. The standardized FCR decreased the value of the FCR of 0.03 and 0.23 for FMFO and MF, respectively, whereas the value for PBF increased by 0.58. The total quantity of protein distributed in each treatment was 2 891 g for FMFO, 2 781 g for PBF, and 2 907 g for MF, and the total quantity of gross energy distributed was 98 MJ for FMFO, 122 MJ for PBF, and 116 MJ for MF.

### Body and feed proximate compositions

Whole fish DM was 24.32% at stocking and 27.80, 28.90 and, 30.68% for FMFO, PBF, and MF, respectively, at harvest. Whole fish crude protein

<footnote><sup>a</sup>Residual standard error.</footnote>

<footnote><sup>b</sup>***: *p* < .001.</footnote>

<footnote><sup>c</sup>Length at the caudal fork.</footnote>

<footnote><sup>d</sup>FCR for which the feed was standardized to 94% dry matter (DM) and 18.9 kJ/g DM.</footnote>
<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /><figcaption>**Figure 1.** Mean weight of gilthead seabream from the beginning (day 0) to the end of the experiment (day 46) for the control (FMFO), plant-based (PBF), and plant-based+mussel (MF) treatments. Mean weight was obtained from individual weights at day 0 (n = 200) and day 46 (n = 114, 120, and 104 for FMFO, PBF and MF, respectively), and from bulk weights for the intermediate points (group of 50 fish for each treatment).</figcaption></figure>

content was 15.52% at stocking and 15.81, 15.35, and 15.98% for FMFO, PBF, and MF, respectively, at harvest. And whole fish lipids content was 6.07% at stocking and was 9.38, 11.87, and 12.15% for FMFO, PBF, and MF, respectively, at harvest.

Feed proximate composition is reported in Table 1.

## Fatty acid analysis

Compared to feed FMFO, feed PBF was about two times more concentrated in 18:1 monounsaturated FA, five times more in n6 PUFA 18:2, 30 times more in n-3 PUFA 18:3 and 4 times less in saturated 16:0 FA. In contrast to feed FMFO and mussel flesh, EPA (n-3 PUFA 20:5) concentration in the feed PBF was nearly null (i.e. 0.04% of the total FA) and DHA (n-3 PUFA 22:6) concentration was null. Thus, in treating MF, flesh of mussel was nearly the only source of EPA (98% of the total EPA content) and the only source of DHA (Table 3).

Regarding the FA profiles at harvest, fish content from PBF and MF were around 1.7 times more concentrated in monounsaturated (18:1) FA, 3 times more in n6 PUFA (18:2 and 18:3 n-6), and 17 times more in n-3 PUFA 18:3 n-3 than those from FMFO. In contrast, the content in 16:0, EPA and DHA was around 0.6, 0.2, and 0.2 times, respectively,
<figure><img src="image_23.png" /><figcaption>**Table 3.** Fatty acids profile (percentage of total fatty acids) of the commercial feed (feed FMFO), the experimental feed (feed PBF), and mussel flesh used in the experiment.</figcaption></figure>

<table><thead><tr><th>Category</th><th>Fatty acid</th><th>Feed FMFO</th><th>Feed PBF</th><th>Mussel flesh</th></tr></thead><tbody><tr><td rowspan="10">Saturated</td><td>12:0</td><td>0.074</td><td>0</td><td>0.266</td></tr><tr><td>14:0</td><td>5.483</td><td>0.082</td><td>6.476</td></tr><tr><td>15:0</td><td>0.390</td><td>0.045</td><td>0.363</td></tr><tr><td>16:0</td><td>18.687</td><td>7.963</td><td>12.984</td></tr><tr><td>17:0</td><td>0.358</td><td>0.069</td><td>0.515</td></tr><tr><td>18:0</td><td>3.126</td><td>3.024</td><td>2.854</td></tr><tr><td>20:0</td><td>0.218</td><td>0.290</td><td>0.144</td></tr><tr><td>22:0</td><td>0.082</td><td>0.199</td><td>0.030</td></tr><tr><td>24:0</td><td>0.101</td><td>0.131</td><td>0.013</td></tr><tr><td>Total saturated MUFA<sup>a</sup></td><td>14:1</td><td>28.520</td><td>11.803</td><td>23.645</td></tr><tr><td rowspan="7">Total MUFA<sup>b</sup></td><td>15:1</td><td>0.076</td><td>0</td><td>0.025</td></tr><tr><td>16:1</td><td>7.607</td><td>0.216</td><td>8.579</td></tr><tr><td>17:1</td><td>0.063</td><td>0.030</td><td>0.127</td></tr><tr><td>18:1</td><td>13.326</td><td>29.907</td><td>3.176</td></tr><tr><td>20:1</td><td>3.346</td><td>0.490</td><td>4.992</td></tr><tr><td>22:1</td><td>4.256</td><td>0.221</td><td>2.507</td></tr><tr><td>n-3 PUFA<sup>b</sup></td><td>16:4 n-3</td><td>0.091</td><td>0.042</td><td>4.537</td></tr><tr><td rowspan="10">Total PUFA<sup>b</sup></td><td>18:3 n-3</td><td>1.202</td><td>32.503</td><td>0.954</td></tr><tr><td>18:4 n-3</td><td>2.338</td><td>0</td><td>2.260</td></tr><tr><td>20:3 n-3</td><td>0.086</td><td>0.025</td><td>0.108</td></tr><tr><td>20:4 n-3</td><td>0.659</td><td>0</td><td>0.287</td></tr><tr><td>20:5 n-3 (EPA<sup>c</sup>)</td><td>13.187</td><td>0.044</td><td>22.767</td></tr><tr><td>21:5 n-3</td><td>0.568</td><td>0.032</td><td>0.843</td></tr><tr><td>22:4 n-3</td><td>0.045</td><td>0</td><td>0.000</td></tr><tr><td>22:5 n-3</td><td>1.267</td><td>0</td><td>1.058</td></tr><tr><td>22:6 n-3 (DHA<sup>d</sup>)</td><td>9.950</td><td>0.000</td><td>9.663</td></tr><tr><td rowspan="10">n-6 PUFA<sup>b</sup></td><td>18:2 n-6</td><td>5.305</td><td>24.018</td><td>0.842</td></tr><tr><td>18:3 n-6</td><td>0.224</td><td>0</td><td>0.199</td></tr><tr><td>20:2 n-6</td><td>0.157</td><td>0.043</td><td>0.353</td></tr><tr><td>20:3 n-6</td><td>0.126</td><td>0.018</td><td>0.163</td></tr><tr><td>20:4 n-6</td><td>0.669</td><td>0</td><td>3.386</td></tr><tr><td>22:2 n-6</td><td>0.125</td><td>0.049</td><td>0.180</td></tr><tr><td>22:4 n-6</td><td>0.045</td><td>0</td><td>0.339</td></tr><tr><td>22:5 n-6</td><td>0.177</td><td>0</td><td>0.282</td></tr><tr><td>Other PUFA<sup>b</sup></td><td>4.868</td><td>0.262</td><td>4.527</td></tr><tr><td>Total PUFA<sup>b</sup></td><td>41.089</td><td>57.036</td><td>52.748</td></tr><tr><td>Total n-3 PUFA<sup>b</sup></td><td>29.393</td><td>32.646</td><td>42.477</td></tr><tr><td>Total n-6 PUFA<sup>b</sup></td><td>6.828</td><td>24.128</td><td>5.744</td></tr><tr><td>n-3:n-6 ratio</td><td>4.305</td><td>1.353</td><td>7.395</td></tr></tbody></table>

less concentrated in fish from PBF and MF than that from FMFO. Thus, from stocking to harvest, the *n*-3:*n*-6 PUFA ratio decreased in fish from PBF (−54%) and MF (50%) but increased in fish from FMFO (+36%) (Table 4). The sum of EPA+DHA was 1.05 g/100 g of fish at stocking and 1.74, 0.38, and 0.46 g/100 g of fish at harvest for FMFO, PBF, and MF, respectively. The EPA+DHA concentration at harvest was 21% higher in fish from MF than that from PBF.

<footnote><sup>a</sup>Monounsaturated fatty acids.</footnote>

<footnote><sup>b</sup>Polyunsaturated fatty acids.</footnote>

<footnote><sup>c</sup>Eicosapentaenoic acid.</footnote>

<footnote><sup>d</sup>Docosahexaenoic acid.</footnote>
<figure><img src="image_24.png" /><figcaption>**Table 4.** Fatty acid profile (percentage of total fatty acids) of gilthead seabream at stocking and harvest from the control (FMFO), plant-based (PBF), and mussel (MF) treatments.</figcaption></figure>

<table><thead><tr><th rowspan="2">Category</th><th rowspan="2">Fatty acid</th><th rowspan="2">Stocking</th><th colspan="3">Harvest</th></tr><tr><th>FMFO</th><th>PBF</th><th>MF</th></tr></thead><tbody><tr><td rowspan="10">Saturated</td><td>12:0</td><td>0.055</td><td>0.051</td><td>0.024</td><td>0.026</td></tr><tr><td>14:0</td><td>4.567</td><td>4.613</td><td>1.415</td><td>1.396</td></tr><tr><td>15:0</td><td>0.380</td><td>0.373</td><td>0.150</td><td>0.158</td></tr><tr><td>16:0</td><td>16.648</td><td>17.964</td><td>11.177</td><td>11.430</td></tr><tr><td>17:0</td><td>0.346</td><td>0.344</td><td>0.175</td><td>0.189</td></tr><tr><td>18:0</td><td>3.675</td><td>3.304</td><td>3.733</td><td>3.837</td></tr><tr><td>20:0</td><td>0.293</td><td>0.220</td><td>0.209</td><td>0.203</td></tr><tr><td>22:0</td><td>0.091</td><td>0.084</td><td>0.140</td><td>0.130</td></tr><tr><td>24:0</td><td>0.085</td><td>0.077</td><td>0.104</td><td>0.088</td></tr><tr><td>Total saturated MUFA<sup>a</sup></td><td>14:1</td><td>0.055</td><td>0.066</td><td>0.025</td><td>0.029</td></tr><tr><td rowspan="7">Total MUFA<sup>a</sup></td><td>15:1</td><td>0.060</td><td>0.072</td><td>0.034</td><td>0.025</td></tr><tr><td>16:1</td><td>6.912</td><td>8.105</td><td>2.717</td><td>2.928</td></tr><tr><td>17:1</td><td>0.047</td><td>0.052</td><td>0.120</td><td>0.092</td></tr><tr><td>18:1</td><td>17.502</td><td>17.228</td><td>29.398</td><td>28.788</td></tr><tr><td>20:1</td><td>4.248</td><td>3.358</td><td>1.214</td><td>1.352</td></tr><tr><td>22:1</td><td>4.240</td><td>3.529</td><td>0.813</td><td>0.664</td></tr><tr><td>n-3 PUFA<sup>b</sup></td><td>16:4 n-3</td><td>0.269</td><td>0.199</td><td>0.093</td><td>0.098</td></tr><tr><td rowspan="10">Total PUFA<sup>b</sup></td><td>18:3 n-3</td><td>1.264</td><td>1.101</td><td>19.031</td><td>19.453</td></tr><tr><td>18:4 n-3</td><td>1.653</td><td>1.847</td><td>1.199</td><td>1.107</td></tr><tr><td>20:3 n-3</td><td>0.139</td><td>0.106</td><td>0.380</td><td>0.450</td></tr><tr><td>20:4 n-3</td><td>0.675</td><td>0.776</td><td>0.333</td><td>0.343</td></tr><tr><td>20:5 n-3 (EPA<sup>c</sup>)</td><td>7.905</td><td>9.204</td><td>1.314</td><td>1.818</td></tr><tr><td>21:5 n-3</td><td>0.441</td><td>0.526</td><td>0.108</td><td>0.123</td></tr><tr><td>22:4 n-3</td><td>0.068</td><td>0.072</td><td>0.000</td><td>0.000</td></tr><tr><td>22:5 n-3</td><td>2.107</td><td>2.235</td><td>0.394</td><td>0.453</td></tr><tr><td>22:6 n-3 (DHA<sup>d</sup>)</td><td>10.736</td><td>10.777</td><td>2.099</td><td>2.210</td></tr><tr><td rowspan="7">n-6 PUFA<sup>b</sup></td><td>18:2 n-6</td><td>7.706</td><td>5.993</td><td>19.983</td><td>18.973</td></tr><tr><td>18:3 n-6</td><td>0.484</td><td>0.292</td><td>0.898</td><td>0.751</td></tr><tr><td>20:2 n-6</td><td>0.215</td><td>0.177</td><td>0.248</td><td>0.272</td></tr><tr><td>20:3 n-6</td><td>0.278</td><td>0.187</td><td>0.186</td><td>0.200</td></tr><tr><td>20:4 n-6</td><td>0.861</td><td>0.669</td><td>0.151</td><td>0.292</td></tr><tr><td>22:2 n-6</td><td>0.098</td><td>0.168</td><td>0.088</td><td>0.091</td></tr><tr><td>22:4 n-6</td><td>0.126</td><td>0.101</td><td>0.032</td><td>0.039</td></tr><tr><td>22:5 n-6</td><td>0.228</td><td>0.194</td><td>0.028</td><td>0.046</td></tr><tr><td>Other PUFA<sup>b</sup></td><td></td><td>3.114</td><td>3.873</td><td>0.693</td><td>0.571</td></tr><tr><td>Total PUFA<sup>b</sup></td><td></td><td>38.366</td><td>38.135</td><td>47.256</td><td>47.289</td></tr><tr><td>Total n-3</td><td></td><td>25.256</td><td>26.841</td><td>24.949</td><td>26.054</td></tr><tr><td>Total n-6</td><td></td><td>9.996</td><td>7.781</td><td>21.614</td><td>20.664</td></tr><tr><td>n-3:n-6 ratio</td><td></td><td>2.527</td><td>3.450</td><td>1.154</td><td>1.261</td></tr></tbody></table>

## Nutritional retention indicators

According to results from proximate and FA analyses, it was determined that the protein retention was 30, 16, and 29% for FMFO, PBF, and MF, respectively. The protein efficiency ratio was 1.90, 1.05, and 1.99 for FMFO, PBF, and MF, respectively. And the lipid retention was 69, 45, and 70 for FMFO, PBF, and MF, respectively.

<footnote><sup>a</sup>Monounsaturated fatty acids.</footnote>

<footnote><sup>b</sup>Polyunsaturated fatty acids.</footnote>

<footnote><sup>c</sup>Eicosapentaenoic acid.</footnote>

<footnote><sup>d</sup>Docosahexaenoic acid.</footnote>
<figure><img src="image_25.png" /></figure>

Discussion

In the present work, with isoenergetic diets, the lack of significant difference between the individual mean weight of fish from treatments FMFO and MF at harvest indicates that feeding seabream mussels in addition to a plant-based feed maintained growth performance, unlike feeding the plant-based feed alone (treatment PBF). This is supported by the results for SGR, which were similar for FMFO and MF, both of which were 48% higher than that for PBF. In previous works, a decrease in specific growth rate and in feed intake were observed when FM was substituted with blue mussel. Whatever the experimental design, taking into account the fact that the concentrations of protein and lipids were less important in the flesh of mussel (Luo et al. 2019) or not (Berge and Austreng 1989; Weiß and Buck 2017), they concluded that FM can be replaced with mussel up to 50%. According to the results from Mongile et al. (2015), it was observed an improvement of the SGR, the protein efficiency ratio, and the FCR, for isoproteic and isolipidic diets containing up to 75% of mussel meal, in substitution of FM.

The FCR for PBF and MF were 72% and 45% lower, respectively, than that for FMFO. For carnivorous species, feed intake and the resulting growth performances of fish fed diets without FM or FO have been observed to decrease (Benedito-Palos et al. 2008; Lazzarotto et al. 2018; Torrecillas et al. 2017). In the present study, the quantities of feed distributed were checked visually each day to correspond to the quantities consumed. The quantity of feed distributed did not differ among treatments, likely because the attractant mixture was added to feed PBF. Since similar quantities of feed, energy, and protein were distributed in the treatments, the difference in FCR could be due to differences in how the fish used the nutrients. The low energy content of mussel, compared to those of FMFO and PBF, can be also an explanation. Thus, standardizing the quantities of feed based on energy and DM contents balanced the FCRs, which decreased the difference in FCR between FMFO and MF from 45% to 26% but increased the difference in FCR between MF and PBF from 19% to 87%.

As observed in previous studies, gilthead seabream fed a plant-based diet had a higher lipid content than those fed an FO-based diet (Benedito-Palos et al. 2008; Castro et al. 2016; Matos et al. 2016). While the ingredients may explain this difference, it could also be explained by the higher lipid content of feed PBF than that of feed FMFO (19.50% and 15.09%, respectively), while their crude energy content was nearly the same (22.76 and 22.00 MJ/kg, respectively). The FA composition of feed modified the FA composition of the fish, especially when fed feed PBF. Due to the composition of feed PBF, the percentages of EPA, DHA, and saturated FA (14:0, 16:0) of fish decreased from stocking to harvest, while those of 18:1, 18:2 *n*-6, and
<figure><img src="image_26.png" /></figure>

18:3 *n*-3 increased. The added mussel increased the EPA+DHA concentration of fish from MF by 21% compared to that of fish from PBF. This result agrees with previous observations of the influence of feed composition on fish body composition (Benedito-Palos et al. 2008; Fountoulaki et al. 2009; Matos et al. 2016; Wassef, Saleh, and El-Abd El-Hady 2009). Overall, regarding the experimental design, the present study indicates that mussel and vegetable oil ingredients cannot yield the same EPA+DHA concentrations as FM and FO ingredients can. However, positive results were obtained for SGR and survival rate. Nevertheless, the concentrations of EPA and DHA of a feed are also considered to meet the requirements of consumers. Assuming that the FA content of the whole body is similar to that of the filet, the recommended daily intake of EPA+DHA (500 mg, (Tocher 2015)) could be met by eating only 29 g of fish from FMFO *vs* 132 or 109 g of fish from PBF or MF, respectively. This indicates that a meal containing a serving size of 150 g of gilthead seabream from FMFO would provide five days of the recommended daily intake, while wild gilthead seabream would provide only three days of it (Grigorakis 2007), and those from MF would provide slightly more than one day of it.

As a result, mussel remains an interesting feed ingredient, with high percentages of EPA (23% of the total FA) and DHA (10% of the total FA), which increases their percentages in fish. With a lipid content of 3.7%, the quantity of mussel added (once every 6 days) to the plant-based feed (5 out of 6 days) was not enough to result in EPA+DHA concentrations as high as those in fish fed feed FMFO. Consequently, increasing the quantity and frequency of mussels in the diet should be considered (e.g. once every 2 days) while reconsidering the plant-based feed formulation to promote the use of new ingredients such as oil from microalgae (Albrektsen et al. 2022; Santigosa, Brambilla, and Milanese 2021; Teves and Ragaza 2016). This is especially important since re-feeding fish a finishing diet that contains FO does not guarantee that EPA and DHA concentrations will become as high as those in fish fed FO from the start (Fountoulaki et al. 2009). Thus, using environmentally friendly feed ingredients, such as discarded mussels, to meet requirements for human health highlights the existence of nutrition-environment tradeoffs and the need to consider them when developing sustainable aquaculture (Shepon et al. 2021).

The protein content is also to consider. Thus, the protein retention rate for FMFO and MF (30 and 29%, respectively) were close to that already observed (i.e. 34%) on growing gilthead seabreams fed with a feed formulated with low concentrations in FM and FO (Aragao et al. 2022). The protein retention rate for PBF was 45% lower than those of FMFO and MF, but the protein content in fish at harvest was similar among the three treatments, which indicates that mussel intake increased the protein retention rate.
<figure><img src="image_27.png" /></figure>

**Conclusion**

Formulating a sustainable diet in which fishmeal and fish oil are totally substituted with a plant-based feed and fresh flesh of mussels provides depletion in EPA and DHA but similar growth performance, survival and nutritional retention performances to those of commercial fish feed. So, in the future, feeding juvenile in such way should be considered to improve the environmental footprint of production. It would be useful to investigate feeding of such diet to growing fish, as well as its effects on growth performances, survival, and body composition, and the feeding strategy to adopt until a marketable size. In order to ensure supply, it would be interesting to investigate the possibility to set up mussel cultures around fish cages to exclusively feed gilthead seabream. Another way to explore is to find other potential marine ingredients from low trophic level. Discarded oysters could be another sustainable alternative to ensure a steady supply of marine protein.

**Acknowledgments**

The authors warmly thank M. Chalon, the head of the Lycée de la Mer et du Littoral de Bourcefranc-le-Chapus, who allowed us to work at the facilities of the teaching establishment and to share this work with students. The authors thank Michelle and Michael Corson for the proofreading and the improvement of the text.

**Disclosure statement**

No potential conflict of interest was reported by the author(s).

**Funding**

This experiment was financially supported by the SIMTAP project (Self-sufficient Integrated Multi-Trophic Aquaponic systems for improving food production sustainability and brackish water use and recycling, https://www.simtap.eu/index.php/) supported by the PRIMA program (Partnership for Research and Innovation in the Mediterranean Area) under grant (18110-2).

**Availability of data and materials**

All raw data are available in open access (Jaeger, Gayet, and Aubin 2023).
<figure><img src="image_28.png" /></figure>

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