# N-3 long-chain polyunsaturated fatty acids in fish physiology: From aquaculture to economic, ecological and public health challenges

Mickael Péron, Mathilde Bertrand, Elodie Baranek, Maud Martinat, Philippe Soudant, Marie Vagner, Jérôme Roy

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Mickael Péron, Mathilde Bertrand, Elodie Baranek, Maud Martinat, Philippe Soudant, et al. N-3 long-chain polyunsaturated fatty acids in fish physiology: From aquaculture to economic, ecological and public health challenges. Biochimie, 2025, 10.1016/j.biochi.2025.09.007. hal-05304245

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# N-3 long-chain polyunsaturated fatty acids in fish physiology: From aquaculture to economic, ecological and public health challenges

Mickael Péron<sup>a,1</sup>, Mathilde Bertrand<sup>a,1</sup>, Elodie Baranek<sup>b</sup>, Maud Martinat<sup>b</sup>, Philippe Soudant<sup>a</sup>, Marie Vagner<sup>a,*2</sup>, Jérôme Roy<sup>b,**2</sup>

## ARTICLE INFO

**Article history:**
Received 4 June 2025
Received in revised form
12 September 2025
Accepted 13 September 2025
Available online xxx

Handling Editor: C. Forest

**Keywords:**
n-3 LC PUFA
Fish physiology
Environmental sustainability
Fish nutritional ecology
Alternative feed sources

## ABSTRACT

N-3 long-chain polyunsaturated fatty acids (n-3 LC PUFA), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential molecules to fish physiology, influencing their development, metabolism, immunity, behavior, and reproduction. However, fish have a limited ability to synthesize these fatty acids endogenously, and must obtain them through diet. The dietary availability of these molecules is increasingly challenged by both ecological and aquaculture constraints linked to climate change and to sustainability of resources (e.g. fisheries). Currently, global changes – including ocean warming – may reduce the availability of these fatty acids in marine food webs, raising concerns for fish population dynamics and aquaculture sustainability that still largely depends on forage fish. In this review, we first summarize the metabolic pathways and tissue distribution of n-3 LC PUFA in freshwater and marine fish, highlighting differences in bioconversion capacities. We then explore the physiological and behavioral consequences of varying dietary n-3 LC PUFA levels in aquaculture feeds and natural environments, including effects on growth, locomotion, cognition, metabolic performance, oxidative status, immune response, and reproductive investment. We also review current alternatives to fish meal and fish oil, such as plant, insect, microbial, and genetically modified sources, and discuss their potential to meet fish nutritional needs. Altogether, this synthesis underscores the current challenge of n-3 LC PUFA dietary shortage for fish health, aquaculture production and nutritional security of human population, and identifies knowledge gaps that must be addressed to ensure both ecological resilience and sustainable aquaculture development in a rapidly changing world.

© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license.

(http://creativecommons.org/licenses/by/4.0/).

## Contents

<table><tr><td>1.</td><td>Introduction</td><td>00</td></tr><tr><td>2.</td><td>N-3 LC PUFA metabolism in fish</td><td>00</td></tr><tr><td>2.1.</td><td>N-3 LC PUFA bioconversion pathways in marine vs. freshwater fish</td><td>00</td></tr><tr><td>2.2.</td><td>N-3 LC PUFA composition in fish tissues</td><td>00</td></tr><tr><td>2.3.</td><td>Spatio-temporal variability in n-3 LC PUFA fish composition in the environment</td><td>00</td></tr><tr><td>2.4.</td><td>Implications in aquaculture</td><td>00</td></tr><tr><td>3.</td><td>Influence of the dietary n-3 LC PUFA availability on the fish physiology and behavior</td><td>00</td></tr><tr><td>3.1.</td><td>Growth and development</td><td>00</td></tr><tr><td>3.2.</td><td>Locomotion and behavior</td><td>00</td></tr><tr><td>3.3.</td><td>Metabolism</td><td>00</td></tr></table>

<footnote><sup>a</sup> Univ Brest, CNRS, IRD, Ifremer, UMR 6539, LEMAR, Plouzané, France</footnote>

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

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: marie.vagner@univ-brest.fr (M. Vagner), jerome.roy@inrae.fr</footnote>

<footnote>(J. Roy).</footnote>

<footnote n="1">¹ These authors share first authorship.</footnote>

<footnote n="2">² These authors have contributed equally to this work and share last authorship.</footnote>
<table><tr><td>3.4. Immunity and stress tolerance</td><td>00</td></tr><tr><td>3.5. Reproduction</td><td>00</td></tr><tr><td>4. Current alternatives to fish meal and fish oil</td><td>00</td></tr><tr><td>5. Conclusion</td><td>00</td></tr><tr><td>CRediT authorship contribution statement</td><td>00</td></tr><tr><td>Consent for publication</td><td>00</td></tr><tr><td>Funding</td><td>00</td></tr><tr><td>Declaration of competing interest</td><td>00</td></tr><tr><td>Acknowledgments</td><td>00</td></tr><tr><td>References</td><td>00</td></tr></table>

# 1. Introduction

Lipids perform biological functions that are essential to the life of organisms, as they are a source of metabolic energy (neutral or reserve lipids) and ensure the physical and chemical properties of cell membranes (polar or membrane lipids; [1,2]). Among lipids, n-3 long-chain polyunsaturated fatty acids (n-3 LC PUFA) are chains of at least 20 carbons and multiple double bonds (i.e.unsaturation) with the first one on the third carbon from the methyl terminal. The high degree of unsaturation of PUFA confers flexibility and selective permeability to cell membranes [3], and is determinant for the conformation and activity of membrane proteins [4]. Consequently, n-3 LC PUFA are crucial for the development and the maintenance of physiological functions of organisms, such as survival, growth, reproduction, cognition, or locomotion [5]. N-3 LC PUFA are also the precursors of many bioactive molecules which are of great importance in the overall metabolism and especially in inflammation and immunity [6,7].

Among n-3 LC PUFA, EPA (eicosapentaenoic acid, 20:5n-3) and DHA (docosahexaenoic acid, 22:6n-3), have been shown to play a key role in fish physiology and behavior at several ontogenetic stages [8]. DHA is present in substantial quantities in the membranes of visual and neural cells of vertebrates, including fish, enabling normal vision and neuronal function, which is essential to maintain their schooling structure as well as their feeding or escaping behavior. EPA is involved in growth and in the inflammatory response through the production of eicosanoids [5,9]. However, vertebrates, including fish, have a limited ability to efficiently bioconvert EPA and DHA from precursor fatty acids [8,10,11]. In the natural environment, these fatty acids are produced by primary producers and consequently transferred along the trophic chain to higher trophic level consumers, including humans.

Global change effects, and particularly warmer waters, are predicted to lead to a reduction in the n-3 LC PUFA production at the base of marine food webs. Some studies based on meta-analysis, prediction models, and high-resolution mass spectrometry analyses of samples have revealed that unsaturated fatty acids production is expected to decrease with increasing ocean temperature [12,13]. The availability of LC PUFA is thought to be reduced mainly in temperate zones [13,14]. These results are mainly explained by a modification in the assemblages of phytoplankton species rather than by an alteration of their physiology [12,15]. High n-3 LC PUFA-producing phytoplanktonic taxa (e.g. diatoms, dinoflagellates) are also those that are the most sensitive to global change [16]. They would be replaced by low n-3 LC PUFA-producing species (e.g., cyanobacteria), which are more tolerant to environmental changes [12,15,17,18] and also more toxic [19]. Moreover, the lack of sterols in cyanobacteria lowers zooplankton nutritional quality, with cascading effects on the entire food-web ecosystem [20,21].

The decrease in n-3 LC PUFA productivity at the base of the trophic chain would impact the dietary availability of these molecules for higher consumers. This is a cause for concern, as a reduction in dietary n-3 LC PUFA availability would be partly reflected in the fatty acid composition of the cell membranes of consumers, potentially altering membrane functions and structures [3]. These changes may lead to physiological disturbances that could ultimately impair individual fitness (e.g. survival, growth, reproductive success; [22-27]). As a result, an emerging concern is that the reduced availability of n-3 LC PUFA in the environment would have consequences for recruitment and population dynamics. In addition to being a challenge in the natural ecosystem, n-3 LC PUFA production and supply by marine ecosystems has also consequences in aquaculture as aquafeeds require to be enriched in n-3 LC PUFA to optimize the growth and health status of juveniles. n-3 LC PUFA supply still mainly rely on forage fish fished in the environment, especially in Southern countries, that are then deprived of their resources to feed the North countries. It is estimated that EPA and DHA should collectively represent around 10 % of the lipid content of dry feed in salmonids [28], between 8 and 18 % in sparids [29,30] and at least 3.5 % in European sea bass (Dicentrarchus labrax; [31]). Because of their physiological importance for development and biological functions, and their limited endogeneous origin, n-3 LC PUFA are referred to as essential fatty acids for fish.

In aquaculture, these molecules are mostly provided by fish oil (FO), that is, with fish meal (FM), considered the most nutritious aquafeed ingredients and are often referred to as the "gold standard" in fish nutrition [32]. Traditionally, commercial aquafeeds have been composed of FM/FO derived from by-products from fish processing industries or from wild caught species such as sardines (Sardina pilchardus), mackerel (Scomber sp.), anchovies (Engraulidae sp.), herring (Clupea harengus), capelin (Mallotus villosus), and menhaden (Brevoortia tyrannus). FM contains 60-72 % protein, with an optimal composition of essential amino acids (lysine and methionine) and minerals (calcium and phosphorus), which contribute to the optimal growth of farmed fish. FO, in turn, is rich in lipids (4-20 %) and essential n-3 LC PUFA (DHA and EPA), which also ensure the nutritional quality of fish flesh for human consumption [33] and fulfill the dietary needs of farmed fish [34]. However, the ecological cost of producing FM/FO for aquaculture is unsustainable as it is based on catching wild pelagic fish. Producing one ton of FM requires 4-5 tons of whole fish, while producing one ton of FO requires up to 20 tons, depending on the fish species used [33]. As a result, the continuous growth of aquaculture has contributed to the increasing anthropogenic pressure on fish stocks, which can no longer meet the demand, leading to the urgent implementation of forage fishing quotas and alternative resources. Therefore, the aquaculture and ecological challenges of n-3 LC PUFA availability are interdependent. As mentioned above, global change will affect the primary (phytoplankton) and secondary (zooplankton) productions
of these molecules and their availability for forage fish, imprinting their fatty acid composition, and impacting the stocks supporting aquaculture.

Despite extensive knowledge on the nutritional roles of n-3 LC PUFA, emerging ecological and technological shifts now call for an integrated reassessment. While the physiological importance of EPA and DHA in fish is well established, particularly within the framework of aquaculture nutrition, recent developments highlight the need to revisit this topic through a broader, dual lens. On one hand, climate-driven alterations to marine food webs are predicted to reduce the natural availability of these essential fatty acids, with potential cascading effects on wild fish populations. On the other, the progressive replacement of FM and FO in aquafeeds challenges our ability to meet the LC PUFA requirements of farmed species without compromising performance, welfare, or product quality and their related health benefits. In this review, we synthesize recent findings at the interface of fish physiology, feed innovation, and ecological stressors. We aim to (i) clarify species-specific LC PUFA synthesis and conversion abilities and nutritional requirement, (ii) assess the functional consequences of dietary n-3 LC PUFA variation on fish biological performances such as growth, behavior, metabolism, and reproduction, and (iii) explore the ecological and aquaculture implications of a declining availability of EPA and DHA, and review the current alternatives to fish meal and fish oil. Finally, we identify key knowledge gaps and propose research priorities to support fish health, ecosystem resilience, and the sustainable development of aquaculture in a rapidly changing world.

## 2. N-3 LC PUFA metabolism in fish

### 2.1. N-3 LC PUFA bioconversion pathways in marine vs. freshwater fish

Fish are not all equal when it comes to their capacity to synthesize n-3 LC PUFA, due to differences in the dietary availability of precursor fatty acids and in the functionality of their n-3 biosynthesis pathway. Most marine fish, which have evolved in a dietary environment rich in n-3 LC PUFA, are generally considered to have lost the ability to synthesize them *de novo* while, freshwater or anadromous fish, which live in an environment with lower dietary levels of n-3 LC PUFA, generally kept a greater, but still low, capacity to synthesize these molecules [8,35–38]. However, some exceptions can occur, and interestingly, some marine species do have functional desaturase pathways, particularly in the context of freshwater colonization [39].

The conversion of precursor (18:3n-3, α-linolenic acid ALA, obtained from diet) to n-3 LC PUFA is a complex process that takes place in the endoplasmic reticulum of hepatocytes, enterocytes and brain cells [36,38,40]. ALA undergoes several successive desaturation (addition of a double bond) and elongation (addition of carbon atoms) reactions catalyzed by oxygen-dependant enzymes called fatty acyl desaturases (abbreviated Fads or Δ) and fatty acyl elongases (Elovl) respectively, until EPA or DHA is obtained [36,41] Fig. 1).

In most marine teleost fish, the genes encoding desaturases and elongases are either not or very weakly expressed [36–38]. In addition, the *fads1* gene would be missing and the translated Δ5 desaturase could therefore not be produced [36,41]. In other cases, the *fads2* gene would translate in desaturase with Δ6, Δ8 and Δ5 activities [38]. DHA production can also occur from EPA, notably by involving the Sprecher pathway, which comprises two sequential elongations of EPA into 24:5n-3, desaturation of the 24:5n-3 formed into 24:6n-3 by a Δ6 and then β-oxidation to produce DHA [41] (Fig. 1). In parallel to the Sprecher pathway, a faster metabolic

pathway called the Δ4 desaturase pathway has been suggested [42]. This pathway directly desaturates 22:5n-3 to DHA, reducing the number of enzymatic reactions and avoiding translocation of 24:6n-3 from the endoplasmic reticulum to the peroxisomes [36]. The Δ4 desaturase is increasingly accepted as existing in most teleost fish [36,41,43]. For a long time, the trophic level of species was considered to be a factor influencing the different desaturase activities of the *fads2* gene, but this hypothesis has been refuted in favour of phylogeny [38,41]. Genetic expression and enzymatic activities therefore appear to be specific to each species.

Freshwater fish seem to possess a greater ability to bioconvert DHA endogenously from precursors than marine fish. At low dietary DHA availability, common carp (*Cyprinus carpio*) showed an ability to convert 20 carbons n-3 FA (particularly EPA) into DHA [44]. This conversion ability has also been shown in the Eurasian perch (*Perca fluviatilis*) fed with low levels of dietary DHA in both wild and experimental conditions [45,46]. Rainbow trout (*Onchorhynchus mykiss*) can also elongate and desaturate precursors (ALA) into EPA and DHA, as they produce the Elovl2 and Elovl5 enzymes [47]. Despite this, conversion rates remain extremely low, similar to those observed in mammals, where only about 5 % of ALA is converted into EPA and less than 0.5 % into DHA [48]. However, this higher bioconversion capacity in freshwater and anadromous species is also relevant in aquaculture, as it can reduce the need for dietary EPA and DHA supplementation, thereby decreasing dependence on marine-derived oils and improving production sustainability [44,49,50]. Numerous studies have then demonstrated the requirement of n-3 LC PUFA in the diet of farmed fish to prevent deficiency symptoms (malformation, behavior, disease susceptibility, swimming performance ...) and ensure normal growth [51]. As a result, EPA and DHA, which finalize the biosynthesis pathway of LC PUFA, must be supplied through the diet.

### 2.2. N-3 LC PUFA composition in fish tissues

N-3 LC PUFA play essential roles in fish physiology and are selectively distributed in tissues according to their specific functions. The liver, as a central organ of lipid metabolism, can accumulate high levels of EPA and DHA closely reflecting dietary input and making it a key site for transformation and redistribution/ allocation of fatty acids [52]. High DHA levels (levels depending of fish species) are consistently maintained in brain, even under dietary fluctuations, due to its vital roles in neurodevelopment, membrane structure, and cognitive function [40,53,54]. Eyes also contains elevated DHA concentrations, selectively incorporated into retinal membrane phospholipids, and essential for visual acuity and neural signaling [23,55]. The heart also contains significant levels of EPA and DHA, supporting cardiac performance [2]. Other tissues such as the head kidney and gills show high n-3 LC PUFA concentrations aligned with their immune and respiratory functions, with EPA and DHA contributing to membrane integrity and anti-inflammatory regulation [56]. The intestine, in addition to its digestive role, also participates in the biosynthesis and absorption of LC PUFA, especially when lipids from marine sources are present in the diet [2]. Muscle tissue, which constitutes the primary edible portion of fish, exhibits highly variable EPA and DHA content depending on the diet, feeding regime, and metabolic status of the fish [57]. Though often lower in n-3 LC PUFA content, the skin contributes to osmotic regulation and environmental interaction and still contains measurable levels of n-3 LC PUFA [2]. Elevated levels of EPA and DHA have been reported in fish eggs [25,26,58], revealing the role of EPA and DHA in supporting egg quality and subsequent early offspring development. N-3 LC PUFA are stored in the reserves yolk for potential use during
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Schematic representation of the biosynthesis pathways of LC PUFA of the n-9 (orange), n-6 (green) and n-3 series (blue) in teleost fish. Black and grey arrows between two FA represent reactions catalyzed by fatty acyl desaturase (Δx) and fatty acyl elongase (Elovlx) activities, respectively. ALA: α-linolenic acid, ARA: Arachidonic acid, DHA: Docosahexaenoic acid, EPA: Eicosapentaenoic acid, LA: Linoleic acid, OA: Oleic acid. Modified from Garrido et al. (2019).</figcaption></figure>

development or to be catabolized for energy after hatching [59].

The distribution of these fatty acids is not only tissue-
dependent but also influenced by environmental and biological
factors. Temperature, salinity or photoperiod can affect biosyn-
thesis and deposition of FA [60,61], while internal factors like body
size, feeding duration, and physiological state (e.g., growth or
reproduction) modulate their incorporation [62,63]. Moreover,
since the endogenous conversion of 18-carbon precursors to n-3
LC PUFA remains limited in both freshwater and marine fish, tissue
levels primarily depend on the availability of these fatty acids in
the diet [63,64].

## 2.3. Spatio-temporal variability in n-3 LC PUFA fish composition in the environment

In the environment, the fatty acid composition of fish vary ac-
cording to prey availability and composition and abiotic factors
such as temperature. In the European sardine sampled in the Bay
of Biscay (Northeast Atlantic) in spring, the muscle fatty acid
composition varied depending on their sampling location and age
[65]. These changes in fatty acid composition were linked to dif-
ferences in primary production and prey availability between the
sampling sites inducing a change in the feeding behavior [65]. The
flexible character of sardine fatty acid compositon was further
confirmed and associated with various physiological requirements
according to age class and differences in the trophic environment
[66]. The fatty acid profile of sardines from the Portugese coasts
change with seasonal variation of feeding habits and intensity as
well as with spawning season [67]. In European sea bass, fatty acid
composition can vary spatially, even at the scale of an estuary
among individuals of the same age class [68], and is further
modulated by the ontogenetic stage within an environment [69].
In general, changes in the fatty acid composition of consumers
reflect shifts in the availability of these fatty acids from primary
and secondary producers or primary consumers [70]. However, in
freshwater ecosystems, the relationship between the fatty acid
profiles of primary producers and those of fish is less obvious.
Notably, DHA consistently remains abundant in fish tissues
regardless of prey composition within their feeding habitat, sug-
gesting strong selective retention. Nonetheless, interspecific

differences in DHA content point to species-specific variations in
its metabolic utilization and physiological functions [57,71].

## 2.4. Implications in aquaculture

Aquaculture nutrition must account for species-specific and stage-dependent lipid requirements to ensure optimal growth, health, and product quality. In fish, the physiological demand for n-3 LC PUFA is influenced by dietary composition, temperature, and metabolic capacity, all of which shape the balance between energy utilization and fatty acid profile across life stages.

Lipid requirements in rainbow trout vary with age: fry need
16-18 % dietary lipids, while mature fish require 14-16 % [51].
These lipid requirement values depend on the dietary balance of
macronutrients, as proteins and carbohydrates, also sources of
energy and carbon for lipogenesis. Excess protein, which is ener-
getically costly, can be avoided by meeting lipid demands pre-
cisely. Water temperature also modulates lipid requirements:
cold-water species exploit the capacity to remodel membrane
lipid composition, especially n-3 LC PUFA, to maintain membrane
fluidity at low temperatures [72]. Species living in cold or
temperate waters require higher n-3 LC PUFA levels compared to
those living in warmer waters. Lipid source quality is critical since
their essential fatty acids, especially n-3 LC PUFA content deter-
mine the minimal lipid intake needed to meet physiological de-
mands (e.g. cholesterol, phospholipids), which vary with
developmental stage. While minimum lipid levels prevent de-
ficiencies, higher lipid inclusion can enhance growth and physio-
logical functions (e.g. reproduction, migration in salmonids). Yet,
LC PUFA dietary requirements have been established for only ~30
of the 652 aquacultured species, with most studies focusing on
marine larvae [73,74]. Despite this, data from existing species
allow extrapolations to others [2,8]. Generally, for marine fish, 1 %
of dietary dry matter as linoleic acid (LA, n-6) and α-linolenic acid
(ALA, n-3) is sufficient to meet LC PUFA requirements [75].
Although freshwater fish can usually meet their essential fatty acid
requirements through 18:3n-3 (ALA) conversion, n-3 LC PUFA (e.g.
EPA, DHA) have been shown to support growth more efficiently
[76]. Therefore, dietary LC PUFA have a high nutritional value as
conversion efficiency remains low [34].
Like total lipid needs, LC PUFA requirements are temperature-dependent, with cold-water species like rainbow trout showing a metabolic preference for n-3 over n-6 PUFA, as observed in humans. Developmental stage matters too: marine larvae have higher LC PUFA needs than adults [77], yet little data exists for freshwater larvae. Thus, for trout, recommendations do not differ across development and suggest 0.5 % each of ARA and DHA, and 1 % EPA [34]. Marine species like red seabream, Japanese flounder, and barramundi lacking Δ5-desaturase activity require n-3 LC PUFA in their diet [78,79] but the nutritional requirements are not defined. Rainbow trout, by contrast, can convert 18-carbon fatty acids into LC PUFA, making both 18-carbon fatty acids and n-3 LC PUFA essential fatty acids. Such variability in fatty acid metabolism between species could be exploited by breeding programs to identify fish strains that naturally retain more DHA or express desaturase genes more strongly on vegetable diets. This appears as an emerging field, as some breeding companies are already evaluating fillet DHA content as a trait in selection.

# 3. Influence of the dietary n-3 LC PUFA availability on the fish physiology and behavior

## 3.1. Growth and development

The effects of dietary FA on growth performance has been extensively studied in many fish species including freshwater species like the Murray cod *Maccullochella peelii* [80], Nile tilapia *Oreochromis niloticus* [81], marine species such as European sea bass [82], turbot *Scophthalmus maximus* [83,84], gilthead sea bream *Sparus aurata* [85,86] and anadromous species such as Atlantic salmon *Salmo salar* [87]. These studies were mostly involved in the context of aquaculture. Usually, freshwater and anadromous species can withstand a higher replacement of FO by vegetable oil (VO) due to their higher ability to convert the precursors included in it (e.g. 18:3n-3) into n-3 LC PUFA. In Atlantic salmon, a total replacement of FO by canola oil did not affect the growth performance over seventeen weeks [88]. The same results were observed in rainbow trout fed VO, but the substitution generated a modification of flesh FA composition, altering its nutritional quality and related health benefits for human consumption [89,90]. In European sea bass, studies have led to an estimation of the requirements for n-3 LC PUFA (EPA + DHA) in the diet of about 0.7 % of dry matter (DM) [31]. Below this threshold, the growth of the juvenile sea bass decreases, and traits like immune response or disease resistance can be affected [82].

European sea bass obtained from aquaculture farm with low dietary n-3 LC PUFA content (0.73 % n-3 LC PUFA on DM basis) led to reduced growth at both 15°C and 20°C compared to fish fed higher n-3 LC PUFA levels (1.65 %) after five months of dietary conditioning [91]. A dietary DHA limitation (0.5 % of total lipids) has also reduced the growth of wild caught European sea bass fed over six months [92]. In the same study, a negative correlation has been found between the DHA proportion in the muscle and growth rate. A tissue/diet ratio of DHA well above one in all tissues of fish fed the depleted DHA diet suggested a selective retention or biosynthesis of DHA in that group. Altogether, these results suggest an energetic trade off between selective retention or bioconversion of DHA and growth [92,93]. The same relationship has been observed in Atlantic salmon fed a diet deprived of n-3 LC PUFA (1.79 % EPA + DHA on DM basis) relative to individuals fed a control diet (8.64 % EPA + DHA on DM basis; [94]). In golden grey mullet juveniles *Chelon auratus*, a low n-3 LC PUFA content (0.2 % EPA + DHA on DM basis) also reduced growth when compared to individuals fed a standard diet (1.2 % EPA + DHA on DM basis)

during five months [27]. However, this result was not found after four months of dietary conditioning with the same low n-3 LC PUFA diet compared to a richer one (4.8 % EPA + DHA; [95,96]). The comparison between studies remain difficult since the effect of a deficiency may depend on the intensity and duration of the deficiency tested as well as on the species and ontogenetic stage considered.

## 3.2. Locomotion and behavior

### ◦ Swimming performance

Dietary n-3 LC PUFA, particularly EPA and DHA, influence cell structure and function, including that of muscle tissue, which is essential for muscle performance and, consequently, swimming [27,97]. Muscular performance depends on metabolic fuel, primarily provided by lipids in the form of triacylglycerols [98]. Swimming is the primary mode of locomotion for most fish species, and it plays a vital role in interactions with their environment, foraging [99], predator avoidance [100], reproduction [101], and migration [102]. Swimming capacity is a key determinant of survival and an important indicator of an individual's fitness [103,104]. Swimming performance is influenced by various physiological functions such as ventilation, oxygen transport, and muscle contraction, making swimming performance an indicator of overall physiological health [105].

The maximum swimming capacity of a fish, measured using the critical swimming speed ($U_{crit}$), is a standard method for assessing aerobic swimming performance. $U_{crit}$ quantifies the maximum velocity a fish can swim against a current, providing insights into its prolonged swimming abilities [106,107]. However, it has limitations and provides only an estimate of aerobic capacities, requiring careful interpretation [103]. Several studies have examined the effects of dietary fatty acids on swimming performance, with results varying according to species-specific differences and feed formulations. For example, European sea bass exhibited higher $U_{crit}$ with increased levels of monounsaturated fatty acids (MUFA), particularly oleic acid (OA, 18:1n-9), suggesting a role of this FA as an energy substrate [108]. Similarly, in Chinook salmon *Oncorhynchus tshawytscha*, triacylglycerol levels were positively correlated with swimming speed [109]. However, results regarding n-3 LC PUFA have been inconsistent. Atlantic salmon fed a n-3 LC PUFA-rich diet exhibited lower $U_{crit}$ than those fed a canola oil-enriched diet, i.e. rich in 18:3n-3 [110], while Arctic charr *Salvelinus alpinus* fed a low n-3 PUFA diet displayed reduced $U_{crit}$ compared to fish fed a n-3 LC PUFA-rich diet [111]. Wagner et al. [112] emphasized the importance of the ratio between n-3 LC PUFA and saturated fatty acids (SFA) or ARA, reporting positive correlations with $U_{crit}$ in Atlantic salmon and SFA. In juveniles golden grey mullet, $U_{crit}$ remained unchanged between fish fed a low versus standard n-3 LC PUFA diet (0.2 vs 4.8 % n-3 LC PUFA, or 0.2 % vs 1.2 % n-3 LC PUFA, on DM basis for four or five months, respectively; [27,96]). However, the low-PUFA group exhibited higher oxygen consumption per distance unit, suggesting reduced swimming efficiency [96]. All together, these results reveal the complexity of the relationship between dietary n-3 LC PUFA and swimming performance, which likely depends on species, the type and duration of the dietary deficiency.

At early developmental stages (larvae), inadequate dietary levels of n-3 LC PUFA have led to reduced swimming activity, altered feeding rhythms (delayed response to a visual stimulus; [113], impaired behavioral development (schooling), and increased abnormal behaviors (longer escape latency; [55,114]).

### ◦ Cognition and behavior
N-3 LC PUFA, particularly DHA, are crucial for maintaining optimal brain function, development, and cognition in fish [115,116]. While much of the research on DHA has focused on mammalian models due to its recognized benefits for human health [117,118], some studies have also demonstrated its importance in fish behavior and cognition, both of which are reliable indicators of health and development [54,119–123]. N-3 LC PUFA have been shown to be positively correlated with the brain volume in brown trout *Salmo trutta* which could influence the fish cognitive skills [124,125]. A recent study has shown that in both wild and experiment brown trouts, a diet lower in n-3 LC PUFA reduced n-3 LC PUFA content in the brain. This deficiency led to reduced ability to compete in a social environment, as well as a suboptimal use of habitat, leading to reduced somatic growth [126]. Recent integrative work has emphasized that DHA not only supports synaptic activity and neuronal plasticity but also enhances signal transduction through G-protein coupled receptors, such as dopamine and serotonin receptors, directly influencing cognition, sensory processing, and ecologically relevant behaviors like predator evasion and social interaction [124]. The escape response or "startle response," is a reflexive behavior exhibited by fish in response to sudden, potential threats, enabling them to quickly distance themselves from danger [127]. The underlying neural circuit for this response is conserved in fish, with the Mauthner cells in the hindbrain playing a central role in initiating rapid escape movements [128]. Research into the effects of dietary n-3 LC PUFA, especially DHA, on fish behavior has shown positive effects on escape responses in gilthead sea bream larvae [119]. For instance, DHA was found to enhance sound-induced escape behavior of larvae by influencing Mauthner cell activity, whereas a three weeks dietary deficiency in n-3 LC PUFA negatively impacted their swimming speed and response to visual stimuli [55]. Furthermore, larvae of pike perch *Sander lucioperca* fed low levels of DHA (0.4 % of total fatty acid) exhibited during three weeks delayed escape responses and developed more anxious behavior few weeks after the dietary conditioning, compared to those fed standard levels of DHA (1.7 % of total fatty acid, [129]). More recently, a study investigating the effect of alternative oils with reduced n-3 LC PUFA contents on juvenile gilthead sea bream showed no significant impairment in escape behavior and this was associated with an increased synthesis of LC PUFA, as well as a neurogenesis-related gene overexpression [130].

N-3 LC PUFA are also crucial for normal behavior and alertness in several species such as Japanese yellowtail *Seriola quinqueradiata* and gilthead seabream [55,131]. Yellowtail larvae and juveniles fed a diet enriched in n-3 LC PUFA and especially DHA (1.5–2.4 % of total fatty acid on dry matter basis vs. 0 %) during 12 days displayed a better mutual attraction, indicating a stronger scooling behavior. In gilthead seabream larvae, the reduction in dietary n-3 LC PUFA contents (from 0.1 to 3.2 vs 15.8 % of FA, on DM basis) during 16 days altered the fish response to visual stimulus. Dietary fatty acids have also been shown to influence the fish feeding behavior. For example, at juvenile stage in Senegalese sole *Solea senegalensis* and rainbow trout, the injection of fatty acids (300 µg/kg) directly into the cerebrovascular or peritoneal area have been found to reduce food intake [132,133]. Furthermore, juvenile rainbow trout have been shown to be able to distinguish between a FO-based diet and a diet containing high levels of ALA, LA, or OA [134]. In this regard, juvenile trout are able of distinguishing between different levels of n-3 LC PUFA (EPA or DHA vs ALA) in the diet and show a preference for diets with the highest DHA content (20 % vs 0 % or 5 % of total fatty acid, [53]). Therefore, dietary fatty acids depending on raw material in aquafeed composition (FM-FO vs plant based ingredient) influence feeding behavior in alevin stage from first feeding [135–137], offspring

development after nutritional programming [138] and at juvenile stage [22,139].

N-3 LC PUFA also act as regulators of neurotransmitter pathways, particularly the serotonergic and dopaminergic systems. These neurotransmitters have been shown to influence feeding behavior in both mammals and fish. In fish, tyrosine is converted into dopamine through the action of tyrosine hydroxylase, playing a crucial role in motor activity, learning, and feeding behaviors [140]. Additionally, serotonin supply in the forebrain regulates memory and learning processes. Reduced expression of certain serotonin precursors and dopamine transporter genes in the forebrain and midbrain may be linked to homeostatic and adaptive control, which is hypothesized to influence behavioral and automatic responses in the cortisol pathways, ultimately leading to decreased food intake.

In aquaculture, behavioral impairments resulting from inadequate dietary n-3 LC PUFA intake can negatively affect growth, health feed efficiency, and increase susceptibility to environmental stressors and disease. In the wild, behaviorally compromised individuals are more likely to suffer from poor predator avoidance, reduced foraging success, and impaired social interactions, which may compromise their survival and recruitment. Therefore, adequate levels of dietary n-3 LC PUFA, particularly DHA, are essential for promoting an optimal behavior and optimizing overall fitness in both aquaculture and natural environments.

### 3.3. Metabolism

N-3 LC PUFA have been shown to modulate fish aerobic metabolism at molecular, cellular and individual levels, which may have implications for organismal performance under both routine and ecologically relevant stress conditions. At individual level, some studies demonstrated that a low dietary n-3 LC PUFA content increased the standard metabolic rate (SMR), i.e. the energy necessary to maintain vital functions, such as cerebral function, osmoregulation, ventilation [96,108,141]. For example, Adriatic sturgeon *Acipenser naccarii* fed a low n-3 LC PUFA (23.8 mg of EPA and 15.4 mg of DHA per g wet weight compared to 6.1 mg EPA and 5.8 mg DHA) for a year had a SMR increased by more than 30 % compared to individuals fed a diet rich in SFA and low n-3 LC PUFA [141]. The same result was observed in European eels *Anguilla anguilla* fed the same diet for three months [141] and in juvenile golden grey mullet fed a n-3 LC PUFA deficient diet for four months [96]. In European sea bass, maximum metabolic rate (MMR), i.e. the energy required to sustain maximal aerobic activity, was lower when fish fed a high n-3 LC PUFA (2.2 % EPA and 2.3 % DHA of total fatty acid compared to 7 % and 6.3 % respectively) diet compared to fish fed a diet rich in SFA and MUFA [108]. In golden grey mullet juvenile, a low dietary n-3 LC PUFA (0.2 % of DM) content during five months induced a lower aerobic scope (AS), i.e. the amount of oxygen available to sustain non mandatory activities such as swimming, growth, reproduction, digestion [27]. However, this metabolic response was modulated according to the acclimation temperature. Fish fed n-3 LC PUFA depleted diet and acclimated at high temperature (19.9 ± 0.5 °C) displayed a higher AS, as well as a greater expenditure of energy to reach the same maximum swimming speed as other groups. This suggested a reduction of the amount of energy available to perform other physiological functions at higher temperature [96]. This result at individual level was further explained by a costly cardiac contraction strategy to sustain aerobic effort, shown by both gene expression profile and ventricular force development measurement [95], as well as by a greater mitochondrial efficiency induced by the low n-3 LC PUFA diet [142].
At cellular level, a low n-3 LC PUFA diet (1.2 % EPA + DHA of total fatty acid mass of the diets) might be beneficial for ATP production by mitochondria in response to energy-demanding cellular processes when EPA and DHA availabilities are reduced in diet [142]. However, after two months of dietary conditioning of golden grey mullet juveniles, a high ATP/O ratio was associated with a high content in EPA and DHA (17.5 % of total fatty acid mass of the diets) in biological membrane, highlighting the existence of interindividual differences in mitochondrial efficiency and its potential importance in explaining intraspecific variations in response to food web changes [142]. Another study reported a decreased mitochondrial efficiency in the muscle of juvenile wild atlantic salmon fed a n-3 LC PUFA deficient diet (1.79 % EPA + DHA on DM basis) for eight weeks [94]. Altogether, these studies highlight that further work is required to better understand the role of EPA and DHA in fish metabolism, at different biological levels, considering the interindividual variability.

### 3.4. Immunity and stress tolerance

The balance of n-3 and n-6 LC PUFA in fish diet can have a significant impact on their health, as both control and regulate cellular metabolism and, more broadly, physiological functions such as blood pressure, heart rate, immune function, and the stress response [8,24,29,85]. Indeed, ARA and EPA are precursors of eicosanoids, bioactive compounds such as prostaglandins, thromboxanes, leukotrienes, and lipoxins, which are found in all tissues and are deeply involved in inflammation and blood clotting [24,28,143]. For example, a decrease in the n-3/n-6 dietary ratio exacerbated heart and immune diseases [29,85]. A low (0.3) n-3/n-6 dietary ratio for four to five months reduced resistance of Atlantic salmon to bacterial infections, despite similar numbers of erythrocytes and leukocytes as those fed with a high (5.2) n-3/n-6 dietary ratio [144]. Similarly, in European sea bass juveniles fed for five months with low levels of FO and FM, Torrecillas et al. observed a decrease of n-3 LC PUFA in the lymphoid tissue in response to infection, delayed restoration of intestinal homeostasis post-infection, and increased rates of bacterial translocation from the intestines [82]. Furthermore, dietary LC PUFA can influence the ability of fish to tolerate environmental stressors, such as hypoxia and hypercapnia [141,145]. For example, in hypoxic conditions, Adriatic sturgeons fed optimal levels of n-3 LC PUFA (23.8 mg of EPA and 15.4 mg of DHA per g wet weight) retained oxygen better in their blood and exhibited better cardiac performance compared to individuals fed lower levels of n-3 LC PUFA (6.1 mg of EPA and 5.8 mg of DHA per g wet weight) [97,141]. Some studies have demonstrated that a diet enriched with EPA and DHA (ratio 3.88 vs 0.61) regulates genes involved in innate immunity, inflammation, and oxidative stress in various fish species [146]. A study investigating the effects of EPA and DHA on the brain of rainbow trout, showed that, similarly to what has been observed in mammals, enrichment with EPA and DHA (0 % vs 15.7 % vs 33.4 % of total fatty acids) reduces the expression of genes involved in inflammation, oxidative stress, and cortisol pathway markers [54]. Additionally, an increase in the activity of antioxidant enzymes such as glutathione reductase, glutathione peroxidase, glutathione S-transferase, superoxide dismutase, and catalase has been observed in Caspian brown trout *Salmo trutta caspius* fry fed EPA- and DHA-enriched diets (0.5 vs 2 + 1 vs 4 + 2 % of total fatty acids, DHA + EPA) [147].

### 3.5. Reproduction

N-3 LC PUFA, particularly DHA and EPA, are essential regulators of reproductive processes in both mammals and fish. In pregnant

women, DHA is preferentially transferred to the fetus via the placenta, often at the expense of the maternal stores, which decline during gestation [148]. Interestingly, a similar mechanism has been observed in rainbow trout. In trout fed plant-based diets, naturally low in DHA (0 % of total fatty acids), the levels of n-3 LC PUFA in the muscle tissue are reduced, but egg DHA levels remained comparable to those in trout fed FO-rich diets, suggesting active maternal DHA biosynthesis and preferential allocation to eggs [25,58]. Beyond this maternal investment, EPA and DHA contribute to a wide range of reproductive functions in fish. They serve as precursors for sex hormones such as estrogen and progesterone, and modulate steroidogenesis and prostaglandin pathways, as demonstrated in goldfish and European sea bass [149,150]. DHA and EPA are incorporated into gonadal tissues, eggs, and developing embryos, supporting oocyte maturation and embryogenesis, and playing critical roles in neural and retinal development of larvae, notably in rainbow trout, yellow perch, and European eel [151,152].

Sperm quality is also influenced by these fatty acids, which enhance membrane stability and motility, as shown in gilthead seabream, European eel and silver catfish [153–155]. Positive effects of n-3 LC PUFA on fertilization have been reported in Atlantic halibut, gilthead seabream and striped gourami [156,157]. Notably, in many marine species, higher EPA and DHA concentrations in eggs are positively correlated with fertilization success, hatching rate, and larval survival [158–161]. Dietary EPA and DHA supplementation improves fecundity in European sea bass (28.7 % of EPA/DHA of total fatty acids), gilthead seabream (18.8 % of EPA/DHA of total fatty acids) and common carp (22.8 % of EPA/DHA of total fatty acids) [162–164].

Moreover, egg quality, assessed by lipid content, buoyancy, and hatching rate, is strongly linked to egg EPA and DHA composition, as observed in Japanese flounder, Atlantic salmon, and crescent sweetlips [114,165,166]. During larval development, DHA enhances visual system formation and stress resistance, as demonstrated in Eurasian perch and California yellowtail [167,168]. Finally, the dietary balance among EPA, DHA and ARA is critical. DHA/EPA ratio of approximately 2:1 and ARA levels (from 0.5 to 4.8 % of total fatty acids depending of fish specie) are associated with improved reproductive outcomes, while excess n-3 LC PUFA can lead to negative effects such as yolk sac hypertrophy and increased antioxidant requirements [169]. This recent finding highlights the fact that despite the crucial necessity of having a sufficient levels of n-3 LC PUFA, consuming more is not always better and can in fact be detrimental for the health of fish.

## 4. Current alternatives to fish meal and fish oil

The aquaculture's long-term sustainability is a major challenge from both economic and ecological perspectives. It involves reducing the utilization of FM and FO from forage fish in aquafeeds, while ensuring the dietary n-3 LC PUFA requirements in farmed fish for their growth, development, immunity and normal behavior. The share of global fishery production converted into FM and FO is steadily declining, accounting for 9 % of global aquafeed production in 2020 [33]. While FM-FO inclusion remains significant in aquafeeds at certain life stages, particularly during reproduction, in finishing diets [33], and in early growth stages [170], its use in grow-out diets has significantly declined for all farmed species in recent years [171]. For example, in 1990, a commercial feed for Atlantic salmon contained 65.5 % FM and 24 % FO, but by 2020, these values had gradually decreased to 12 % and 10 %, respectively [172]. This reduction has been made possible through the development of alternative ingredients such as plant-based ingredients, including cereals (wheat, corn, and barley), oilseeds
(soybean, sunflower, rapeseed, and cottonseed), and legumes (lupin and faba beans), sourced either from crops dedicated to animal feed or from the processing of plant-based raw materials (e.g., biofuel and bioethanol production; [173]). Over the following decades (since marine ingredients were gradually replaced by plant-based ingredients representing over 60 % and reducing the total FM-FO content to 22.4 %, without compromising fish growth performance and product quality [174]). Additionally, these plant-based raw materials are available in large quantities [175], making them a viable option to meet the growing demand for aquafeed. These plant-based ingredients have become a key alternative to FM-FO as a major source of proteins and lipids in aquafeeds, although in 2020, global FM-FO production still reached 16 million metric tons with the increase in aquaculture farms worldwide [33]. However, replacing FM-FO entirely in aquafeeds with terrestrial plant-based sources is not possible as they contain less protein than FM, exhibit imbalances in essential amino acids (lysine and methionine) and are absent in n-3 LC PUFA, particularly in DHA and EPA [176,177].

The progressive reduction of FM-FO in aquafeeds over the past 20 years has led to a significant decline in EPA and DHA levels in fish flesh and, more broadly, in their nutritional quality, as observed in rainbow trout and Atlantic salmon [178,179]. For example, Sprague et al. (2016) showed that consumers eating 100 g of farmed salmon in 2015 obtained 50 % of EPA + DHA they would have obtained in 2006 with the same portion. Despite this, farmed salmon still delivers one of the highest EPA + DHA food quantity for humans. However, this reduction in EPA + DHA availability for consumers has direct public health implications, since this implies to double the portion eated to meet the current recommended intake of EPA + DHA. As global demand for aquaculture products is already very high, and supply is already struggling to keep up, doubling the proportion to meet the current requirement is not the solution that should be promoted by the guidelines. Therefore, these findings underscore why sustaining n-3 levels in farmed fish is critical. In addition to the development of novel lipid sources, feeding strategies have also been employed since several years to mitigate the decline of n-3 LC PUFA in fish fillets using finishing diets, rich in fish oil and thus n-3 LC PUFA, during the final weeks of the grow-out period. This has been shown to substantially restore tissue EPA and DHA levels relative to continuous feeding on vegetable oil-based diets, but without reaching the levels obtained with a continuous diet of FM-FO [180,181]. Beyond their inability to fully meet the nutritional requirements of carnivorous fish, plant-based ingredients also contain antinutritional factors that can hinder digestibility, nutrient absorption, and overall fish health and physiology.

This limitation has driven the search for other alternative protein sources, such as insect meal (e.g. silkworm *Bombyx mori*, black soldier fly *Hermetia illucens*, housefly *Musca domestica*, mealworm *Tenebrio molitor*, locust, and grasshopper *Zonocerus variegatus*; [180,182–184]), algae (e.g. green *Ulva rigida*, red *Gracilaria bursa pastoris*, brown algae, protists *Schizochytrium* sp.; [185–188]), and yeast (e.g. *Candida utilis*, *Kluyveromyces marxianus*, *Saccharomyces cerevisiae*, *Blastobotrys adeninivorans*, and *Wickerhamomyces anomalus*; [189–191]). Insect, microalgae, and yeast meals have gained interest due to their short life cycles and year-round production. Insects and yeasts serve as valuable macroingredients, fulfilling the nutritional requirements of farmed fish thanks to their high protein content (40–83 % for insects and 50–70 % for yeasts) and favorable amino acid profiles, relatively similar to FM [174,192]. However, insect meals include an imbalance between SFA and PUFA (high in SFA and low in PUFA), the presence of chitin that can affect protein digestibility, high production costs, and potential environmental impacts [193].

Microalgae, on the other hand, are promising alternatives to FO due to their high concentrations of essential n-3 LC PUFA such as ALA, EPA and DHA, which are crucial for fish development [194–196]. However, their incorporation in aquafeeds remains limited as they remain costly due to the challenges of large-scale production, and their current use in aquaculture is primarily limited to micronutrient supplementation [197]. Recent developments in the commercial adoption of alternative lipid sources illustrate the current transition from concept to practice. For example, DHA-rich microalgal oils have been successfully incorporated into salmonid feed, substantially reducing reliance on fish oil, while maintaining growth performance and fillet quality [188,198]. Such advances demonstrate that some emerging alternatives are already making measurable contributions to aquafeed sustainability, underscoring the importance of their use and the transition from fundamental to applied science.

Processed animal proteins, such as feather, bone, and blood meals derived from poultry and pork industry by-products, have emerged as promising alternatives to FM. These ingredients are highly digestible, palatable, protein-rich, more cost-effective than other protein sources, and free from anti-nutritional factors [199,200]. However, poultry by-product meals are low in n-3 LC PUFA (EPA and DHA) and rich in saturated fats, which can affect digestibility and quality of fish fillets for human consumption. Unlike plant-based ingredients, the inclusion of animal proteins in livestock and aquafeeds remains controversial and strictly regulated.

Transgenic *Camelina sativa* has more recently gained attention as a sustainable alternative to FO in aquaculture due to its ability to produce n-3 LC PUFA, particularly EPA and DHA. By introducing algal genes responsible for n-3 LC PUFA biosynthesis, this genetically modified oilseed can accumulate substantial levels of these fatty acids (until 26 % EPA + DHA of total fatty acids), reducing the reliance on wild-caught fish for FO production [201,202]. Feeding trials with farmed freshwater and marine species, such as Atlantic salmon, rainbow trout or European sea bass, have demonstrated that transgenic camelina oil can effectively replace FO without compromising growth, feed conversion efficiency, or tissue fatty acid composition [203–206]. For instance, salmon fed diets containing camelina oil maintained similar levels of DHA and EPA in muscle tissue, ensuring the nutritional benefits of farmed fish for human consumption [204]. The successful integration of transgenic camelina into aquafeeds presents a viable solution to the growing demand for n-3 LC PUFA in aquaculture, while alleviating pressure on marine resources and promoting more sustainable fish farming practices. However, challenges remain regarding regulatory approval, large-scale commercial adoption, and consumer acceptance of genetically modified feed ingredients.

This need for novel and sustainable sources of n-3 LC PUFA is not only critical for aquaculture production but also for global human nutrition and sustainability of the marine ecosystems. As highlighted in the comprehensive review by Tocher et al. (2019) [207], there is a widening gap between the supply and demand of EPA and DHA for human consumption. This underscores the role of aquaculture in supplying these fatty acids to human consumers and evaluates emerging solutions thereby reinforcing the urgency of diversifying and scaling up alternative n-3 LC PUFA sources to secure the long-term nutritional security of human populations.

## 5. Conclusion

N-3 LC PUFA, particularly EPA and DHA, are fundamental for fish to maintain their physiological and behavioral performance, influencing key traits such as growth, reproduction, immune competence, metabolic efficiency, cognitive and locomotor
performance (Fig. 2). Over the past few decades, a large body of research has established their nutritional requirements, tissue distribution, and physiological functions in both freshwater and marine species, primarily in the context of aquaculture. Recent concerns have raised towards ecological issues, with climate-driven disruptions in marine food webs potentially reducing n-3 LC PUFA availability, which could affect wild fish populations, the trophic networks they support, as well as the aquaculture sustainability. While significant progress has been made, several important areas still require further investigation.

One key avenue is exploring the synergy between different PUFA, such as EPA and DHA, by examining their distinct and combined effects on fish physiological functions. More research is needed to understand the impact of alternative sources of n-3 LC PUFA, such as microalgae and transgenic oilseeds, and their varying effects based on different EPA/DHA ratios. Although the current alternative lipid sources show potential, few studies rigorously assess their functional equivalence to marine oils beyond basic growth metrics, especially regarding neural, immune, and reproductive performances.

Studies should use realistic and ecologically relevant concentrations of dietary fatty acids to better understand their effects on wild fish. In addition, the long-term physiological and behavioral effects of chronic dietary LC PUFA deficiency, particularly in ecologically realistic scenarios, remain under studied. Further research should also focus on studying the effect of reduced n-3 LC PUFA in combination with other climate-change induced stressors, such as temperature and oxygen concentration in water to increase the ecological relevance of studies.

Further studies should also focus on collaboration between ecologists and physiologists to bridge the knowledge gaps in ecosystem-level research. For example, to validate the prediction of reduced availability of EPA and DHA within the trophic web, further studies should focus on long-term in situ studies

monitoring the FA profiles in different trophic levels, ranging from plankton to fish populations, as it would provide empirical evidence of how warming alters the nutritional quality of aquatic food webs. Coupling long-term physiological studies and modeling is also necessary to assess the effects observed at individual level on the population in terms of recruitment and fitness. Such results could validate current projections but also inform if declining dietary n-3 LC PUFA availability compromises fish populations or ecosystem resilience.

Using fish from wild environment instead of farmed fish in experimental study could improve the relevance of these ecological studies, as farmed fish present a genetic homogeneity that is not representative of wild fish. Our understanding of interspecific and ontogenetic variability in LC PUFA biosynthesis and retention capacities is still incomplete, especially under fluctuating environmental conditions. A more detailed assessment of the energetic trade-offs involved in n-3 LC PUFA utilization, and the combination of various environmental and biological factors influencing fish behavior and health, is crucial for both improving aquaculture management strategies, and understand the decline of some fish populations in aquatic environments (e.g. European sardine). To address these challenges, future researchs should focus on: (i) integrative studies combining lipid biochemistry with behavioral and physiological endpoints across life stages; (ii) combining modelling and physiology to better evaluate the effects at the population level, (iii) standardized testing of alternative lipid sources under both controlled and semi-natural conditions; (iv) a better understanding of ecological n-3 LC PUFA flows and bottlenecks in food web under climate change; (v) nutritional programming and selective breeding strategies to enhance n-3 LC PUFA utilization efficiency. Ultimately, ensuring a sustainable and adequate supply of n-3 LC PUFA is vital for ecological, economical and public health concerns in the context of changing climate and increasing human population.

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Schematic representation of the multifaceted physiological and behavioral roles of omega-3 long-chain polyunsaturated fatty acids (n-3 LC PUFA) in fish. Omega-3 LC PUFA (EPA and DHA) exert multiple roles in fish at different levels. At the physiological level, they contribute to growth, metabolism, reproduction, immune function, and stress tolerance. At the behavioral and neurological levels, they support cognition, sensory performance, and locomotion making them essential for robustness and aquaculture sustainability.</figcaption></figure>
CRediT authorship contribution statement

**Mickael Péron:** Writing – review & editing, Writing – original draft. **Mathilde Bertrand:** Writing – original draft. **Elodie Baranek:** Writing – review & editing. **Maud Martinat:** Writing – review & editing. **Philippe Soudant:** Writing – review & editing, Writing – original draft. **Marie Vagner:** Writing – review & editing, Writing – original draft. **Jérôme Roy:** Writing – original draft.

Consent for publication

Not applicable.

Funding

This work is a contribution to the French National Research Agency (grant no. ANR-21-CE20-0009-01 "FEEDOMEGA"), OMEGA project funded by ISblue project, Interdisciplinary graduate school for the blue planet (ANR-17-EURE-0015) and co-funded by a grant from the French government under the program "Investissements d'Avenir" embedded in France 2030, and ADJUST project funded by the French National program EC2CO (Ecosphère Continental et Côtière), the Scientific Council of the European Institute for Marine Sciences, and the Interdisciplinary graduate school for the blue planet (ANR-17-EURE-0015).

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.

Acknowledgments

NA.

For the purpose of Open Access, a CC-BY public copyright licence has been applied by the authors to the present document and will be applied to all subsequent versions up to the Author Accepted Manuscript arising from this submission.

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