Department of Zoology and Animal Cell Biology

Cell Biology in Environmental Toxicology and One Health (CBET+) Research

Group

The thicklip grey mullet Chelon labrosus (Risso, 1827), as

a candidate for intensive aquaculture diversification:
optimization of culturing conditions and development of
specific feeds based on alternative ingredients.

PhD Thesis submitted by

Markel Sanz Latorre
For the degree of
Philosophiae Doctor

Plentzia, December 2024

 Funding declaration
The present PhD Thesis has been funded with the following grants and projects, provided by

their respective public authorities:

1. Markel Sanz Latorre has received a predoctoral contract financed by Gobierno Vasco

(IKERTALENT grant programme) / Contrato predoctoral financiado por el Gobierno Vasco

(Programa de becas IKERTALENT) / Eusko Jaurlaritzak finantzatutako doktoratu aurreko

kontratua (IKERTALENT beka-programa). (00010-PIT2020-22).

2. Project AKURA I – Desarrollo de un sistema avanzado y sostenible para la cría en

cautividad del Múgil. Ayudas al sector acuícola de la Comunidad Autónoma del País Vasco.

Gobierno Vasco, Departamento de Desarrollo Económico e Infraestructuras, Dirección de Pesca

y Acuicultura. (33-2017-00250).

3. Project AKURA II – Desarrollo de un sistema sostenible para la cría en cautividad de

mugílidos. Viabilidad Técnica. Gobierno Vasco, Departamento de Desarrollo Económico e

Infraestructuras, Dirección de Pesca y Acuicultura. (00007-INA2019-33).

4. Project AKURA III – Cría en cautividad de mugílidos: viabilidad de la puesta y diseño

de piensos específicos. Gobierno Vasco, Departamento de Desarrollo Económico e

Infraestructuras, Dirección de Pesca y Acuicultura. (00003-INA2022-33).

5. Project AKURA IV – Cría en cautividad de mugílidos: viabilidad de la puesta y diseño

de piensos específicos. Gobierno Vasco, Departamento de Desarrollo Económico e

Infraestructuras, Dirección de Pesca y Acuicultura. (00005-2101022023).

6. Plan Complementario de Ciencias Marinas ThinkInAzul. European Union-

NextGenerationEU and Gobierno de Cantabria.

Index

General introduction………………………………………………………………………1

State of the art………………………………………………………………….………….63

Hypothesis and objectives…………………………………………………………....67

Results and discussion………………………………………………………...….......71

Ethical statement…………………………………………………………........73

Chapter 1................................................................................75

Chapter 2..............................................................................113

Chapter 3..............................................................................141

Chapter 4..............................................................................185

General discussion..........................................................................243

Conclusions and thesis....................................................................255

 General introduction

General introduction

1
General introduction

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 General introduction

List of abbreviations

ALA: α-linolenic acid (C18:3n-3)

ANF: Antinutritional factor

ARA: arachidonic acid (C20:4n-6)

BSG: brewer´s spent grain

DHA: docosahexaenoic acid (C22:6n-3)

dph: days post-hatching

EPA: eicosapentaenoic acid (C20:5n-3)

FAO: Food and Agriculture Organization of the United Nations

GAS: general adaptation syndrome

hCG: human chorionic gonadotropin

LA: linoleic acid (C18:2n-6)

lc-PUFA: long chain polyunsaturated fatty acids

LHRHa: luteinizing hormone-releasing hormone

P / E: protein / energy ratio

PUFA: polyunsaturated fatty acids

SBM: soybean meal

SGR: specific growth rate

TPC: thermal performance curve

3
General introduction

4
 General introduction

1. General state of aquaculture

According to the Food and Agriculture Organization of the United Nations (FAO), the world

fisheries and aquaculture production has increased dramatically since 1950, when it accounted

for 19 million tonnes, until reaching the maximum historical production in 2022, with 185.4

million tonnes of aquatic animals produced (FAO, 2024). Initially, this increase was achieved by

incrementing the volume of capture fisheries, and aquaculture only had a marginal role as a

producer of food, only producing 4 – 5 % of the aquatic animals between 1950 and 1970.

However, the landings from capture fisheries have been relatively stable at 86 – 96 million

tonnes since the late 1980s, which has instigated a rapid development of aquaculture, making it

the fastest growing animal producing industry (Tacon, 2019). In 2022, aquaculture provided 94

million tonnes of aquatic animals, 51 % of the shared production of aquaculture and fisheries

(Figure 1).

Figure 1: world fisheries and aquaculture production of aquatic animals from 1950 to 2022, excluding

aquatic mammals, crocodiles, alligators, caimans and aquatic products (corals, pearls, shells and sponges).

Adapted from FAO (2024).

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 General introduction

The projections from FAO (2024) predict an increase of aquaculture production of 10 %

by the year 2032. This will be necessary to cope with the raising demand of seafood by a global

population in continuous growth, especially in a framework where most of the world´s fish

stocks are exploited at the maximally sustainable levels (50.5 %) or overfished (37.7 %),

according to 2021 data (FAO, 2024). However, environmentally and socioeconomically

sustainable expansion of aquaculture faces several challenges (Johnson et al., 2019; Campanati

et al., 2022). One of the most relevant issues in this regard is the massive utilization of feeds and

certain feed ingredients by aquaculture, which has fuelled an intense debate in the last decades

(Naylor et al., 2000).

1.1. The aquaculture feed debate

The worldwide production of feeds for the aquaculture industry grew at an average annual rate

of 10.3 % per year from 2000 to 2015, and it is expected to reach 87.1 million tonnes by the year

2025 (Tacon & Metian, 2015). This is partially due to the overall growth of the sector, but it is

not the only driver of this trend. The total share of the non-fed aquaculture species has

decreased from 39.7 % in 2000 to 26.9 % in 2022 (FAO, 2024), evidencing a tendency towards

the intensification of aquaculture practices, and increasing the demands of feed and feed

ingredients even further (Naylor et al., 2021).

Naylor et al. (2000) raised the question of whether or not aquaculture contributes to

alleviate pressure from wild fisheries and add to the overall aquatic production. This apparent

contradiction comes from the fact that fishmeal and fish oil are important ingredients of fish

feeds due to their adequate nutritional value for fish nutrition and high digestibility (Tacon &

Metian, 2015). More importantly, they are rich in long-chain polyunsaturated fatty acids (lc-

PUFA), especially from the ω-3 group, that are highly beneficial for human health, and have to

be obtained through the diet (Calder & Yaqoob, 2009). Fishmeal and fish oil are produced from

the processing of wild fish, and in 2022 the volume of fish harvested for this purpose accounted

6
 General introduction

for 17 million tonnes (FAO, 2024). Although fishmeal and fish oil are also used for other

purposes, such as livestock and pet feeding or for manufacturing human nutritional

supplements, aquaculture is the greatest consumer of these products, using around 65 – 70 %

of the worldwide production (IFFO, 2024a). Even though the inclusion levels of fishmeal and fish

oil in aquafeeds have been substantially lowered in the last decades, the continuous increase of

feed use still makes aquaculture partially reliant on wild catches. Therefore, further reduction

of the inclusion of fishmeal and fish oil on aquaculture feeds by substituting them with

alternative ingredients will help relieve pressure from wild fisheries, and improve the

environmental sustainability of the sector (Naylor et al., 2009).

1.2. Alternative ingredients for aquaculture feeds and effects on fish nutrition

The search for alternatives to fishmeal and fish oil has spawned a great body of work in the last

decades, and several ingredient groups have been considered, such as terrestrial plants,

terrestrial animal protein (including insects), macro and microalgae, single-cell protein or food

industry by-products (Naylor et al., 2009). However, these ingredients have some key

characteristics that make them not ideal for fish nutrition or feed manufacturing. Therefore,

even though the use of these ingredients, among others, has allowed the reduction in the use

of fishmeal and fish oil significantly in aquaculture diets, the complete substitution is still a major

challenge (Naylor et al., 2009; Gasco et al., 2018). In the following sections, a brief explanation

of some of the most relevant alternative ingredients featured in the present PhD Thesis and

their effects on fish nutrition will be provided.

1.2.1. Terrestrial plants

The research for alternative aquafeed ingredients has mainly focused on land-based vegetal

ingredients. Soybean, canola or lupin meals, protein concentrates and oils are among the most

widely used (Glencross et al., 2007), for their protein content, reduced cost and stable supply

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 General introduction

(Hussain et al., 2024). However, terrestrial plants are not ideal for fish nutrition, due to their

unbalanced amino acid profiles, low contents of ω-3 PUFA and high contents of antinutritional

factors (ANFs), which can cause adverse effects on fish health (Krogdahl et al., 2010), and the

nutritional quality of the final product for the human consumer (Sprague et al., 2016). Moreover,

the production of plant based feed ingredients can compete with human food production, due

to the use of arable land and the fact that some of those products can be directly consumed by

humans, like soybean (van Riel et al., 2022).

The ω-3 PUFA, especially the eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic

(DHA; 22:6n-3) acids have well-known beneficial effects on human health, from reducing the risk

of cardiovascular disease to aiding in the correct visual and neuronal development (Calder &

Yaqoob, 2009). Currently, fish are the main ω-3 PUFA source in human nutrition. However, fish,

as well as humans, cannot produce EPA and DHA de novo, and have to obtain them through diet,

by feeding in aquatic food webs, as these fatty acids are mostly produced by algae (Sprague et

al., 2016). Therefore, an alternative fish feed ingredient having a fatty acid profile rich in ω-3

PUFA is essential from the perspective of human nutrition. Terrestrial plants, however, as

mentioned beforehand, are not a good source of ω-3 PUFA (Pickova & Mørkøre, 2007), while

these lipids are mainly produced by algae (Monroig et al., 2018). Conversely, land-based plants

are rich in ω-6 PUFA that are also essential for human nutrition (Pickova & Mørkøre, 2007).

However, ω-3 / ω-6 ratio in a human diet should ideally be between 1:1 and 1:4 (Pickova &

Mørkøre, 2007) and, consequently, complete substitution of fishmeal and fish oil by terrestrial

vegetal ingredients can skew ω-3 / ω-6 ratio excessively towards the ω-6 fraction, lowering the

nutritional quality of the final product (Pickova & Mørkøre, 2007). Atlantic salmon provides an

interesting case study for this phenomenon. Sprague et al. (2016) analysed the fatty acid profile

of more than 3000 salmons produced by the Scottish aquaculture industry from 2006 to 2015,

a period when fishmeal and fish oil were being increasingly substituted by terrestrial plant

ingredients in salmon diets. The authors reported a significant drop of EPA + DHA levels in

8
 General introduction

farmed Atlantic flesh from an average of 2.74 g per 100 g of wet weight in 2006 to 1.36 g in 2015.

Although cultured Atlantic salmon still constitutes a good source of EPA and DHA for humans,

this data implies that the salmon portions required for fulfilling the recommended weekly EPA

and DHA intakes by public health bodies, should be doubled in respect to the recommendations

based on 2006 data (Sprague et al., 2016).

However, some fish can produce lc-PUFA from the essential fatty acids linoleic (LA;

18:2n-6) and α-linolenic (ALA; 18:3n-3) acids by the processes of elongation and desaturation,

(Monroig et al., 2018). As plants are rich in LA and ALA (Pickova & Mørkøre, 2007), culturing fish

species with this ability, like Chelon labrosus (Galindo et al., 2021; Marrero et al., 2024) would

make the complete substitution of fishmeal and fish oil by terrestrial plant ingredients more

feasible.

Antinutritional factors (ANFs) or antinutrients in animal feeds are defined as

“endogenous compounds in feedstuffs that may reduce feed intake, growth, nutrient

digestibility and utilization, affect the function of internal organs or alter disease resistance”

(Krogdahl et al., 2010). The most relevant ANFs regarding terrestrial plants are fibres, phytic

acid, enzyme inhibitors, lectins, saponins, glucosinolates, phytoestrogens, phytosterols,

quinolizidine alkaloids or oligosaccharides (Krogdahl et al., 2010). The effects of these ANFs can

be manifold, from lowering protein digestibility to altering gut microbiota, but the most

common pathology associated to ANFs is enteritis (Figure 2). This condition has been widely

studied in salmonids fed soybean meal (SBM) and, thus, it is sometimes referred as SBM-induced

enteritis in the literature. However, other fish species can also be affected by this pathology, like

common carp (Urán et al., 2008a), European sea bass (Torrecillas et al., 2017) or turbot (Gu et

al., 2016). At the histological level, SBM-induced enteritis is characterized by a shortening of

intestinal mucosal folds, widening of the lamina propria, a strong infiltration of inflammatory

cells in the lamina propria and submucosa (lymphocytes, macrophages and eosinophilic granular

cells), the loss of the supranuclear vacuolation of the enterocytes and increased number of

9
 General introduction

goblet cells (Figure 2). These effects can alter the normal functioning of the intestine, lowering

nutrient absorptive capacity and the overall nutritional state of the fish (Baeverfjord & Krogdahl,

1996; Krogdahl et al., 2003; Bakke-McKellep et al., 2007; Urán et al., 2008b).

Figure 2: micrographs of intestinal villi of Atlantic salmon during the enteritis process. a: normal

epithelium, with thin lamina propria, linearly arranged supranuclear vacuolation and few goblet cells; b:

supranuclear vacuoles are small vesicles, and increased amount of goblet cells and eosinophilic

granulocytes can be seen; c: completely disturbed epithelium, without supranuclear vacuoles, numerous

goblet cells and a thick lamina propria with a high degree of infiltration of inflammatory cells, especially

eosinophilic granulocytes. SNV: supranuclear vacuoles; LP: lamina propria; GC: goblet cells; EG:

eosinophilic granulocytes. Scale bars 20 µm. Source: Urán et al. (2008b).

1.2.2. Macroalgae

Algae (both macro and microalgae) are another promising ingredient group for aquaculture feed

formulation (Øverland et al., 2019; Wan et al., 2019; Saleh, 2020; Gao et al., 2024). Macroalgae

culture, unlike terrestrial plants, does not use arable land and do not need irrigation or

fertilization and, therefore, their production does not compete with agriculture intended for

human consumption (Øverland et al., 2019; Gao et al., 2024). The suitability of algae for fish

feeding comes from their balanced amino acid profile, fatty acid profile rich in lc-ω-3 PUFA and

the content of numerous bioactive compounds that can have beneficial effects on fish health

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 General introduction

(Øverland et al., 2019; Wan et al., 2019; Saleh, 2020; Gao et al., 2024). This section will focus on

the use of macroalgae as an aquafeed ingredient.

Macroalgae are a diverse group of organisms that comprises the groups Phaeophyta

(brown algae), Chlorophyta (green) and Rhodophyta (red). Due to the early divergence of these

groups in evolutionary history, there is a great variability in terms of composition among them

(Wan et al., 2019). However, in general, they are considered a good protein and lipid source,

and are rich in simple and complex carbohydrates, as well as in bioactive compounds (Øverland

et al., 2019; Wan et al., 2019; Saleh, 2020). The protein content of macroalgae can fluctuate

between 15 % of dry matter in some brown algae species (Øverland et al., 2019) to 50 % in some

Rhodophyta species (Wan et al., 2019). Moreover, many species contain essential amino acids,

which can account for 45 % of the total amino acids in dry weight (Øverland et al., 2019; Wan et

al., 2019). Therefore, their amino acid profile can be considered well balanced. Although

macroalgae usually present low lipid contents, these are especially rich in ω-3 PUFAs, (Øverland

et al., 2019; Wan et al., 2019) and in some species, EPA in particular, can account for the 50 %

of total fatty acids (Øverland et al., 2019). Complex carbohydrates like cellulose, hemicellulose

and lignin or lignin-like compounds can constitute a significant fraction of macroalgae, but

different groups possess their own particular ones (Øverland et al., 2019; Wan et al., 2019).

Although some of these polysaccharides can be considered ANFs than can negatively affect gut

histological structure and functionality, others can have beneficial effects on fish health and

immune response, like the increase of lysozyme and complement pathway activities (Øverland

et al., 2019; Wan et al., 2019).

In general, low inclusion levels of macroalgae in fish feeds can improve growth values,

feed intake and overall health status, but high inclusion levels can carry adverse effects on said

variables (Øverland et al., 2019; Wan et al., 2019). In the case of gilthead sea bream, the brown

algae Pterocladia capillacea, at 10 % inclusion level and the green algae Ulva lactuca at 5 %

promoted growth, feed efficiency, nutrient retention and stress response, but further increases

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 General introduction

in inclusion level lead to poorer results (Wassef et a., 2005). The same algae species provided

similar results in European sea bass, with the lowest inclusion level tested (5 %) providing the

best growth and stress response results (Wassef et al., 2013). Similarly, up to 10 % of the fish

protein employed in European sea bass feed could be substituted by Gracillaria bursa-pastoris

(red) or Ulva rigida protein without compromising growth, feed utilization or body composition.

Nevertheless, protein from Gracillaria cornea could only substitute 5 % of the fish protein

without having detrimental effects on fish performance (Valente et al., 2006). In Atlantic salmon,

dietary inclusion level of 15 % of protein from the red macroalgae Palmaria palmata did not

have any adverse effect on feed conversion, growth, body composition or health status (Wan et

al., 2016). Although herbivorous and omnivorous fish could theoretically make better use of

dietary macroalgae than the aforementioned carnivorous species, there is no experimental

evidence supporting this claim (Øverland et al., 2019; Wan et al., 2019). For instance, Ulva spp.

can comprise only up to 10 % of the total protein in Nile tilapia feeds without compromising

growth (Marinho et al. 2013). Therefore, although promising as feed additives with growth, feed

utilization and health enhancing effects, the use of macroalgae as a main protein source for fish

feeds should be carefully considered.

1.2.3. Food industry by-products

Food industry by-products are interesting alternative aquafeed ingredients that have great

potential in improving the sustainability of the sector (Sandström et al., 2022). The particularity

of this group of ingredients is that they do not constitute a homogeneous array of products with

similar origins and characteristics, and the only common feature among them is that they are

by-products or residues of certain food producing industry. Therefore, some of these ingredients

might also fall into other alternative ingredient categories previously explained, with their

associated benefits and disadvantages. The main interest in the use of such ingredients comes

from the recent concept of circular economy which advocates for a more sustainable economic

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 General introduction

model, and that has already been implemented at the policies of the European Union (De

Pascale et al., 2023). It emphasizes the avoidance of waste, maintaining the value of products

and materials, and keeping resources within the economy when a product has reached the end

of its life (Geisendorf & Pietrulla, 2018).

Fishmeal and fish oil produced from fish by-products, like trimmings, are within this

category, although they are not true alternatives to traditional ingredients. However, they can

be considered sustainable alternatives to traditionally produced fishmeal and fish oil, and they

contribute to releasing pressure from wild fish stocks that are usually exploited for the

production of these ingredients. Currently, around 38 % of fishmeal and 53 % of fish oil produced

worldwide comes from this source (IFFO, 2024b). The only downside of this practice is the lower

nutritional value of the fishmeal obtained in this manner, as it generally contains lower amounts

of protein and higher amounts of minerals than its counterpart produced from whole fish (FAO,

2024).

Brewer´s spent grain (BSG) is the major by-product of the brewing industry, accounting

for about 85 % of all the waste produced, and it is gaining much attention as a potential animal

feed ingredient, including fish feeds (Mussatto et al., 2006; Aliyu & Bala, 2011; McCarthy et al.,

2013; Ikram et al., 2017). This widely and cheaply available ingredient mainly comes from barley

grains, the main cereal used for beer production. Therefore, it can also be considered a

terrestrial plant ingredient, and it shares some of the problems and benefits already mentioned

in subsection 1.2.1. Barley grains are comprised of several layers: the germ or the embryo, the

endosperm (comprised by the aleurone and the starchy endosperm) and the grain coverings,

which are divided into three parts (the seed coat, the pericarp layers and the husk). During the

brewing process, the insoluble and non-degraded part of the malted barley, mainly coming from

the last coats of the grain, rich in lignocellulose materials, forms the solid fraction of brewing

waste, which is called BSG (Mussatto et al., 2006). The proximal composition of BSG is

dominated by fibre (around 70 %) and protein (20 %), although the actual composition is highly

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 General introduction

dependent on the barley varieties used and particularities of each brewing process. The fibre

fraction can be divided into several cellulosic, non-cellulosic polysaccharides and lignin. Other

relevant components of BSG are vitamins, minerals and phenolic compounds, which can have

anti-carcinogenic, anti-inflammatory and antioxidant properties (Mussatto et al., 2006; Aliyu &

Bala, 2011; McCarthy et al., 2013; Ikram et al., 2017).

Small amounts of BSG enzymatic extracts have been successfully used as feed additives

to improve digestive functionality of European sea bass (Fernandes et al., 2021; 2022). However,

the use of raw BSG as main protein source for carnivorous fish feeds faces several difficulties,

mainly due to its composition, rich in hardly digestible carbohydrates and ANFs and poor in

essential amino acids and fatty acids (Fernandes et al., 2021; 2022). For instance, satisfactory

growth results have been obtained with diets formulated with BSG on gilthead sea bream with

inclusion levels of up to 15 %, but further increases in BSG inclusion levels rendered reduced

growth values (Estévez et al., 2021). Similar results were obtained with diets for rainbow trout

containing 15 % BSG (Estévez et al., 2022), although higher inclusion levels were not considered.

A solid-state fermentation pre-treatment of BSG by the microorganism Aspergillus ibericus

increased protein content and decreased the cellulose, hemicellulose, lignin, and lipid contents

of the ingredient. These alterations in the proximal composition of BSG allowed for increasing

its inclusion level on European sea bass diets from 10 to 20 % without causing negative effects

on digestibility (Estevão-Rodrigues et al., 2024). In the case of omnivorous and herbivorous

fishes, raw BSG generally provides better results than in carnivores. Diets containing 30 % BSG

in substitution of a portion of the rice fraction of the feed, provided better growth results than

the control devoid of BSG for the omnivorous fishes catla (Catla catla) and rohu (Labeo rohita)

(Kaur & Saxena, 2004). In the case of the stripped catfish, 50 % of the soybean of the diet can be

substituted by BSG without negatively affecting growth (Jayant et al., 2018). In diets for the

herbivorous Nile tilapia, BSG can be included in levels of up to 25 %, substituting 50 % of the

fishmeal without showing adverse effects (Zerai et al., 2008). In diets for the herbivorous Reba

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 General introduction

carp, 100 % of the fishmeal of the feed can be substituted with a combination of BSG and

microalgae meal (Chattaraj et al., 2024). Therefore, low trophic level species seem to be more

fitted for the use of BSG as a substitute for traditional ingredients than high trophic level ones.

1.3. Diversification of low-trophic level cultured species: a key step towards

sustainability

Omnivorous and herbivorous fish species generally can be fed a wider range of ingredients than

carnivorous ones and theoretically require lower levels of fishmeal and fish oil, while tolerating

higher levels of vegetal ingredients (Tacon & Metian, 2015). Therefore, a key aspect to improve

the substitution levels of fishmeal and fish oil in fish feeds and avoid some of the

aforementioned problems associated to alternative ingredients is shifting from culturing

carnivorous species (high trophic level) towards omnivorous and herbivorous ones (low trophic

level). Under the framework of the European Green Deal (EC, 2019) and the Farm to Fork

Strategy (EC, 2020), the European Commission has recommended the diversification towards

such low trophic level species in order to promote a European aquaculture that contributes to

the decarbonisation of the economy, fighting climate change and mitigate its impacts, reducing

pollution, the conservation of ecosystems and that makes a circular use of resources (EC, 2021).

2. The thicklip grey mullet Chelon labrosus Risso, 1827: a promising

alternative for sustainable aquaculture

Mugilids are interesting fish for the diversification of aquaculture mainly due to their

omnivorous feeding habits in the juvenile and adult phases, which confers them the potential

to be fed diets formulated with high inclusion levels of alternative vegetal ingredients and low

on fishmeal and fish oil (Naylor et al., 2009; Bostock et al., 2010; Tveterås & Tveterås, 2010).

Moreover, their high adaptability to fluctuating temperature and salinity conditions makes their

culture suitable for future scenarios of climate change (Martín-Montero et al., 2024). Although

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 General introduction

the majority of the research about the culture of grey mullets has been conducted on Mugil

cephalus Linnaeus, 1758, the thicklip grey mullet Chelon labrosus (Risso, 1827) is one of the most

promising mugilid for the diversification of Spanish aquaculture (García-Márquez et al., 2021).

This species is widely distributed across the coasts of the European and North African Atlantic,

the Mediterranean and the Black Sea (Turan, 2016), and it is one of the most abundant mugilids

in the coasts of the Iberian Peninsula (Cardona et al., 2008).

In the following sections, several aspects of the basic biology (systematics, anatomy,

ecology) and aquaculture (reproduction and larvae rearing, nutrition and others) of C. labrosus,

will be discussed. Finally, a brief statement will be made about the biggest gaps on the

knowledge of the culturing of this species, and some recommendations will be made for future

research.

2.1. Systematics of grey mullets

Grey mullet is the generic name with which the members of the mugilidae family are known, a

ubiquitous teleost family inhabiting most temperate, sub-tropical and tropical coastal waters of

the world (Durand et al., 2012a; Crosetti & Blaber, 2016). Stablishing the systematics and

evolutionary relationships of the different species within the group have historically been

challenging tasks due to the high similarities in external morphology across the entire family.

Therefore, a reliable taxonomy of the group did not exist until the application of modern

molecular tools based on DNA sequencing in the seminal work by Durand et al. (2012a). There,

the authors established 20 genera of grey mullets and revealed evidence pointing towards a vast

underestimation of the actual number of mugilid species. For instance, M. cephalus, classically

considered a unified species with a worldwide distribution, showed genetic variations that

allowed the species to be separated in up to 14 lineages, possibly revealing several cryptic

species (Durand et al., 2012a; Whitfield et al., 2012). Subsequent research has shed even more

light into the taxonomic understanding of this particular group of fish, and more than 91

16
 General introduction

mitochondrial lineages have been identified, from 53 morphological species and 38 putative

ones, englobed in 25 genera (Durand et al., 2012b; Durand, 2016; Xia et al., 2016).

2.2. Anatomy of C. labrosus

As previously stated, grey mullet external anatomy is highly conserved among the entire group,

although slight morphological variations between species do exist. Chelon labrosus is a small-

headed fish of fusiform appearance, grey in the dorsal area and white in the ventral, with dark-

grey lateral longitudinal stripes (Figure 3). C. labrosus has two dorsal fins, the first in the middle

area of the body with 4 or 5 spiny rays, and the second in the medial area between the first

dorsal fin and the caudal one, more or less symmetric to the anal one, with 7 to 10 soft rays.

Pectoral fins are subthoracic and have 1 spiny radius and 16 or 17 soft rays. The anal fin

possesses 3 spiny rays and 9 soft ones (García-Márquez, 2024).

Figure 3: photographs of the external anatomy of the thicklip grey mullet Chelon labrosus. A: whole body;

B: detail of the head.

17
 General introduction

Grey mullets are detritivorous fishes and a significant portion of their diet consists on

the organic matter present in the sediment. Therefore, their feeding apparatus presents highly

specialized structures that allow them to exploit said resource. The mouth is generally sub-

terminal, and in the case of C. labrosus, it has large papillated lips that grant the fish its popular

name, thicklip grey mullet. The teeth are weak and not anchored to the bone. The posterior

dorsal part of the pharynx has a filtering apparatus (Figure 4), key in the primarily sediment

filtering feeding behaviour of the family. This pharyngobranchial organ consists on several

toothed pads that, in conjunction to the gill rakers, drives the selection of ingested sediment

particles and the rejection of the coarser ones, although grey mullets can also ingest relatively

large invertebrates like mosquito larvae, amphipods, snails and polychaetes, showing that these

fish have some control over the filtering action of their feeding apparatus (Cardona, 2016).

Figure 4: position of the toothed pads of the pharyngobranchial organ (A), the branchial arches (B) within

the pharyngeal cavity and a representation of the hypothesized water flow in the pharynx of grey mullet

(C). A: PHP, pharyngeal pads; DP, denticulate part; GP, gustatory part. B: OP, Operculum; SP, secondary

18
 General introduction

processes; BA1, first branchial arch; BA5, fifth branchial arch; H, holobranchs, ARG, anterior gill raker; PGR,

posterior gill raker; IER, inner edge of the gill raker; OER, outer edge of the gill raker. C: food-laden water

(thick arrow), filtered and recirculated water (dashed arrow), and mucus (dashed/dotted arrow) around

pharyngobranchial organ. The surrounding gill arches are indicated by broken lines. Adapted from

Cardona (2016).

The stomach of mugilids is divided into two portions, the cardiac and the pyloric

stomachs (Figure 5). The oesophagus directly leads to the cardiac stomach, a thin walled pouch-

like structure with food storage function, with a blind end and a conduct in contact with the

pyloric stomach. The latter is a gizzard-like organ with a thick muscular layer that is thought to

perform mostly mechanical digestion, acting as a grinder aided by the ingested sediment. At the

ventral side of the pyloric stomach, there are a few large pyloric caeca, the number of which can

be a good tool for species identification. In the case of C. labrosus the number of caeca are six

(Sarasquete et al., 2014) and their main role seems to be related with enzyme production. The

pyloric stomach opens to the intestine which is 3.6 times longer than the body, an adaptation

for the digestion of plant material and detritus (Cardona, 2016).

Cardiac stomach
Oesophagus

Intestine
Pyloric stomach

Pyloric caeca

Figure 5: a representation of the stomach and pyloric caeca of C. labrosus. Adapted from Cardona (2016).

Liver is the main organ controlling energy metabolism in animals (Rui, 2014). In the case

of mugilids, the liver (Figure 6 A) is bilobulated, formed by hepatocytes grouped between

sinusoids (Sarasquete et al., 2014). Although the hepatocyte cytoplasm is granular, as seen in

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 General introduction

Figure 6 B (Sarasquete et al., 2014), under culturing conditions hepatocytes can accumulate high

quantities of energy reserves, in form of lipid or carbohydrates, leading to large vacuolization

and clear cytoplasm, as shown in Figure 6 C (Ojaveer et al., 1996; present PhD Thesis). The

exocrine and endocrine pancreas are other important tissues for metabolic regulation and

digestive enzyme production (Cade & Hanison, 2017). In many fish, both tissues are embedded

in adipose tissue, the exocrine pancreas forming a thin layer that envelopes the endocrine

pancreas (Morrison et al., 2004).

B C

Figure 6: micrographs of liver of C. labrosus. A: normal structure of C. labrosus liver. B: hepatocytes with

low vacuolization and granular cytoplasm. C: highly vacuolated hepatocytes. Arrows: melanomacrophage

centres; Arrow points: bile duct; Asterisks (*): blood vessels. A, C: LD group; B: T0; D: HD group.

Haematoxylin – eosin staining. Scale bars: 20 µm. Source: present PhD Thesis.

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 General introduction

2.3. Ecology of C. labrosus

Most grey mullets are euryhaline fish that preferably select coastal waters and lagoons over

freshwater (Koutrakis, 2016). In a study performed at the salt marshes of the Arcachon Bay, a

habitat with great salinity fluctuations, C. labrosus showed little ability for osmoregulation at

freshwater, evidencing that this species cannot survive for long periods at freshwater (Lasserre

& Gallis, 1975). Therefore, this mugilid selects polyhaline and mesohaline habitats (Lasserre &

Gallis, 1975), especially those with salinities between 5 and 15 ppt (Cardona, 2006). However,

other factors like interspecific competition are also relevant for its natural distribution. For

instance, in the Western Mediterranean coast, C. labrosus is the dominant fish in the grey mullet

assemblages at salinities below 13 ppt, but only when the competitor Cyprinus carpio is absent

(Cardona et al., 2008). Other research focused on C. labrosus aquaculture and nutrition

physiology seem to confirm this preference for brackish waters, as evidence points towards

protein digestive efficiency decreasing above 12 ppt (Pujante et al., 2018). Moreover, the

highest growth rate was found at 16 ppt at a nutrition experiment testing different rearing

salinities (Quirós-Pozo et al., 2024).

Despite spending most of their life in brackish waters, C. labrosus spawning happens at

sea, from December to February (Nzioka et al., 2023). After hatching, post-larvae juveniles form

large schools and migrate to coastal waters and estuaries (Koutrakis, 2016). Arguably, the most

remarkable feature of the ontogeny of grey mullets is their dietary shift from being

planktivorous carnivores at the early stages to becoming omnivorous-herbivorous detritivores

at the juvenile and adult phases. The exact moment at which this process takes place is difficult

to establish and can be variable between species, but it is known to happen very early during

development, when fry are 20 – 30 mm in length (Cardona, 2016). Adult C. labrosus feed on a

trophic level of 3, which classifies them as omnivores (Carlier et al., 2007), and this is one of the

main reasons of the interest about the intensive culture of this species. Feeding at the base of

the trophic web, their physiological versatility and adaptability confer this group of fish the

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 General introduction

ability to dominate fish assemblages in most coastal estuarine and lagoon systems (Whitfield,

2016), and they fulfil a relevant ecological role in the energy flow through the ecosystem

(Sarasquete et al., 2014).

2.4. Aquaculture of C. labrosus: past and present

Grey mullets have been cultivated in the Mediterranean region in traditional extensive systems

since the antiquity, and several written records of ancient Rome exist where mullet culture in

ponds is described, associated to other species like European sea bass or gilthead sea bream. In

fact, even Roman Emperors seem to have been very fond of this type of fish, and insisted on

keeping a captive stock at all times (Crosetti, 2016). Anecdotes about ancient history aside,

traditional mullet culture is still carried away in many countries such as Italy, Tunisia or Egypt,

mostly relying in wild caught fry for stocking the ponds (Abdel-Hady et al., 2024). The economic

importance of grey mullet fisheries and aquaculture varies greatly from country to country. In

Spain, for instance, it is not a highly valued fish, but in Egypt M. cephalus culture is an important

industry that produced 894.799 million USD in 2021 (Abdel-Hady et al., 2024). The most valuable

product obtained from mullets is the “bottarga”, the dried and salted egg roe, which can be sold

at prices higher than 200 € per kilogram (Crosetti, 2016). However, reviewing the available

information about the traditional culture of mugilids, such as the techniques used, the amount

of fish produced and its economic relevance, is outside the scope of this work and it has already

been extensively tackled in the excellent book “Biology, Ecology and Culture of Grey Mullet

(Mugilidae)”, by Blaber & Crosetti (2016). Despite the commercial importance of mugilids in

some countries and their potential for producing highly valuable delicacies like bottarga, their

intensive culture has not undergone the development other fish species had in the last decades.

Many reasons lie behind this phenomenon, the most important ones being the difficulties of

closing their life cycle in captivity, the lack of specific commercial feeds and their slow growth

(Crosetti, 2016).

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 General introduction

2.5. Reproduction and larval rearing in captivity of C. labrosus

Traditional and current aquaculture of grey mullets relies on the capture of wild individuals for

growing in captivity, as the gametogenesis is dysfunctional in captive conditions (Tang et al.,

1964; Shehadeh & Ellis, 1970; Shehadeh et al., 1973; Kuo et al., 1974; Liao et al., 1975; Nash &

Shehadeh, 1980; Lee et al., 1987; Cataudella et al., 1988; Aizen et al., 2005; de las Heras et al.,

2012; Besbes et al., 2020). In the case of female C. labrosus, oocytes can start to develop

naturally, but they get arrested at the second vitellogenic stage (Besbes et al., 2020). Males, on

the other hand, can complete gametogenesis and usually spawn in the presence of spawning

females (Cataudella et al., 1988; Besbes et al., 2020). However, artificial induction of spawning

and successful production of larvae and fry have been achieved and published by several

authors, most of the times employing hormonal treatments on females (Cataudella et al., 1988;

de las Heras et al., 2012; Besbes et al., 2020; Ortiz et al., 2020). The most complete and

comprehensive protocol about C. labrosus reproduction ever published is the one by Besbes et

al. (2020), who obtained a mean egg production of 494,655 eggs / female kg, a fertilization rate

of 83 % and a hatching success of 77 % with a two-step injection of a priming dose of 10,000 IU

human Chorionic Gonadotropin (hCG) and a resolving dose of 10,000 IU hCG + 100 µg / kg of

luteinizing hormone-releasing hormone (LHRHa). Recent research has helped refine the process,

by developing highly reliable sex determination tools based on the plasma levels of steroid

hormones, and a detailed monitoring of female and male maturity during the breeding season

(Martín-Montero et al., 2024). However, the hormone doses required for grey mullet

reproduction are remarkably high (Aizen et al., 2005) and, consequently, expensive, suggesting

a strong inhibitory factor that negatively affects the hormonal induction. Aizen et al. (2005)

found that dopamine had a great inhibitory effect over two stages of female M. cephalus

gametogenesis, at the early stages of vitellogenesis and at the final stage of oocyte maturation,

barriers that they overcame using a dopamine antagonist. This line of research is promising for

further optimization of the already successful protocol by Besbes et al. (2020), and could help

23
 General introduction

reduce the cost of the hormonal treatments, which is one of the main reasons why hatcheries

of grey mullet have never been stablished at commercial level (Crosseti, 2016). The

development of species-specific recombinant hormones for spawning induction could be

another promising direction, as has been successfully achieved on M. cephalus (Ramos-Júdez et

al., 2021; 2022).

Although reproduction in captivity is a critical aspect for the establishment of any

aquaculture species, accurate knowledge about the embryonic and larval development and

adequate rearing protocols are also essential for ensuring the viability of the hatched larvae.

Cataudella et al. (1988) described C. labrosus eggs being 1.3 – 1.4 mm in diameter and containing

2 – 13 oil droplets and established and incubation period of 72 hours. Besbes et al. (2020)

confirmed the aforementioned results with the exception of the size of the eggs, which were

reported to be of a mean diameter of 1.15 mm. Mean size of the larvae at hatching is between

3 and 4 mm. The mouth opens between 4 and 5 days post-hatching (dph), although the larvae

continue feeding partially on the yolk and oil reserves until approximately 13 dph (Cataudella et

al., 1988; Sarasquete et al., 2014; Besbes et al., 2020). With the opening of the mouth, the

digestive system starts to develop rapidly, and movable jaws, mandibular and pharyngeal teeth,

goblet cells, oesophageal and intestinal folds and taste buds appear, allowing the larvae to feed

exogenously (Sarasquete et al., 2014). At these early stages, mugilids are zooplanktivorous, and

can be reared on a microalgae-enriched rotifer diet as soon as the mouth is opened until

approximately 25 dph. Artemia larvae of increasing size can be introduced in the diet from 17

dph onwards (Ortiz et al., 2020; Besbes et al., 2020). At around 22 – 23 dph, the six pyloric caeca

of C. labrosus appear, as well as the gizzard-like cardiac stomach, which is fully formed at 78 dph

(Sarasquete et al., 2014). During weaning, from 30 to 60 dph, increasing doses of compound

feed should be introduced, in substitution of live prey (Ortiz et al., 2020; Besbes et al., 2020).

The trophic shift from carnivorous to omnivorous, characteristic to grey mullets, is expressed in

the ontogeny of expression and activity of digestive enzymes, as these fish show initially high

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 General introduction

activities of lipase and trypsin, which decrease during development, while amylase activity

increases. This change implies that the digestive system is initially well suited for protein and

lipid digestion, typical for a carnivore, but those abilities are gradually reduced in favour of a

better digestion of carbohydrates, typical of an omnivore / herbivore fish (Zouiten et al., 2008;

Gilannejad et al., 2020). Embryonic and larval development of C. labrosus is shown in Figure 7,

as described by Ortiz et al. (2020).

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 General introduction

Figure 7: embryonic and larvae development of C. labrosus. Reproduced with the permission of Ortiz et

al. (2020). Dph, days post-hatching.

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 General introduction

2.6. Artificial feeding of C. labrosus

Although the beginning of the research on mugilid artificial diets dates back to the 1970s (Vallet

et al., 1970), the first feeding experiments on juvenile C. labrosus were not made until the end

of the XXth century (Ojaveer et al., 1996; Davies et al., 1997). Unfortunately, despite the similar

feeding habits exhibited by the members of the Mugilidae family, they are different enough so

that the knowledge obtained about the formulation of a compound diet for a given species is

not completely applicable to others. For instance, M. cephalus is not capable of efficiently

utilizing diets containing protein inclusion levels much higher than 30 % (Abdel-Hakim et al.,

2001; De et al., 2012), in opposition to C. labrosus, as will be shown in this section. Therefore,

only the published feeding trials on C. labrosus have been taken into account and reviewed in

Table 1, excluding the works performed with other grey mullet species. From 1996, eight works

focusing on C. labrosus artificial feeding have been published so far (Ojaveer et al., 1996; Davies

et al., 1997; Amezcua et al., 2009; Altunok & Özden, 2017; Vílchez-Gómez et al., 2017; García-

Marquez et al., 2023a; 2024; Quirós-Pozo et al., 2024). The earliest paper on the issue, by

Ojaveer et al. (1996), had the objective of establishing the optimal protein / energy ratio (P / E)

for the species. As an imbalanced P / E will lead to inefficiencies in feed utilization, knowing the

optimum value for this parameter is crucial for the development of a specific diet for any fish.

Briefly, six feeds of varying P / E were tested, with three different protein (around 38, 49 and 59

%) and two lipid (around 12 and 24 %) inclusion levels. The best specific growth rate (SGR) was

obtained with a diet containing 38 % protein and 23 % lipid, with a P / E of 19.72 mg protein / kJ

gross energy. Surprisingly, most subsequent research on C. labrosus artificial feeding has

focused on testing the suitability of different ingredients for feeding this species, without taking

into account the results of Ojaveer et al. (1996) as baseline for establishing the proximal

composition or P / E of the feeds used. This is particularly true in the case of the lipid inclusion

levels of the experimental feeds, as only one research (Amezcua et al., 2009) has used similar

lipid levels to the ones employed by Ojaveer et al. (1996), 20 %, while the others kept a lipid

27
 General introduction

inclusion of around 10 % (Table 1). Protein inclusion, however, generally ranges between 30 and

40 %, with some notable exceptions that raise it to close to 50 % (Davies et al., 1997; Amezcua

et al., 2009). Therefore, it can be concluded that most authors agree on using feeds with more

than 30 % protein, but there is no consensus regarding the content of lipid levels for a specific

C. labrosus feed, making comparisons between studies and drawing combined conclusions very

challenging. This is not only due to the formulation of the feeds used, but also to the size of the

fish employed, particularly relevant in this species considering their characteristic trophic shift,

and to the overall experimental conditions such as water temperature or salinity (Table 1), that

can alter digestive functionality (Handeland et al., 2008; Pujante et al., 2018). Therefore, a great

variability of growth performances of C. labrosus has been reported by different authors under

different experimental conditions (Table 1). When arranging all the papers by the initial size of

the fish employed, it becomes clear that the differences in SGR between studies mostly respond

to this parameter, the highest values being obtained with the smallest fish (Davies et al., 1997)

and SGR decreasing alongside the increment of the weight studied fish. Growth being fast at the

earliest stages and slowing down during development is a common feature among fish, so the

observed pattern is not surprising. Unfortunately, it renders drawing conclusions about the

optimal proximal composition of C. labrosus diets very challenging. However, Amezcua et al.

(2009), when studying C. labrosus juveniles of approximately 15 g, reported lower SGR than

Quirós-Pozo et al. (2024) when using individuals of 26 g. The feed utilized by Amezcua et al.

(2009) contained the highest amounts of both protein and lipids of all the reviewed feeds,

causing the carbohydrate fraction to be the smallest one, although the nitrogen-free extract was

not reported. The lipase activity in C. labrosus digestive tract starts dropping at 30 dph

(Gilannejaad et al., 2020), and low lipase activity has been reported in individuals above 45 g

(Pujante et al., 2017). Therefore, most probably, lipase activity is low on juveniles of 26 g.

Considering this, it is plausible that the high lipid content in the diet used by Amezcua et al.

(2009) caused a significant reduction on the energy digestibility and availability of the feed,

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 General introduction

reflected in the slow growth obtained during the trial. In contrast, Vílchez-Gómez et al. (2017)

obtained higher SGR with an initial size of 46.45 g than Quirós-Pozo et al. (2024) with juveniles

of 26.74 g, but the lack of information about water temperature and salinity in Vílchez-Gómez

et al. (2017) make the result difficult to discuss.

However, the results regarding the suitability of C. labrosus for being fed alternative

ingredients and for the development of a diet low on fishmeal and fish oil are highly promising,

especially considering the most recent research. Vílchez-Gómez et al. (2017) designed two feeds

with high inclusion levels of brewer´s yeast (65 %), an important by-product of the brewing

industry. Brewer´s yeast is a very interesting ingredient from the perspectives of environmental

sustainability and circular economy and it is a good protein source that can have probiotic

properties (Ferreira et al., 2010). Both experimental feeds contained 20 % fishmeal, and differed

on the lipid source, as lipids were provided by fish oil in one diet, and by macroalgae in the other.

At the end of the experiment, the feed containing fish oil rendered the best SGR (1.15), but the

performance of the other group was also satisfactory (SGR of 0.89). This experiment proved the

potential of C. labrosus being fed a diet mostly comprised of a by-product of other industries

and depleted of any fish oil, although it performed better when fed that ingredient. Similarly,

microalgae are another group of interesting ingredients for aquafeeds, as they can have high

contents of protein, balanced amino acid profiles, essential fatty acids and bioactive compounds

(Gao et al., 2024). In a recent PhD Thesis, García-Márquez (2024) tested the suitability of the

microalgae Chlorella fusca as ingredient for diets of C. labrosus. In a first experiment, two diets

were formulated with fishmeal and fish oil, and one of them was supplemented with 15 %

microalgae biomass. At the end of the trial, the microalgae containing diet provided the best

growth performance, increased the muscle contents of arachidonic acid (ARA), EPA and DHA

and enhanced the intestinal absorptive capacity (García-Márquez et al., 2022), while positively

modulating the transcription of immune and stress-related genes (García-Márquez et al., 2024).

In a similar experiment, where the diet containing C. fusca was also supplemented with the

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 General introduction

probiotic Vibrio proteolyticus, it was observed that this microorganism also contributed to boost

the growth performance, muscle fatty acid profile and intestinal absorptive capacity (García-

Márquez et al., 2023a) and expression of genes related to immune and stress responses (García-

Márquez et al., 2023b). Therefore, C. fusca and V. proteolyticus were established as viable

supplements for enhancing digestive functionality and stress and immune responses in C.

labrosus. Quirós-Pozo et al. (2024) formulated two diets for C. labrosus, one of which had all of

its fish oil substituted by linseed oil, and tested them in three environmental salinities. At 16

psu, the salinity that provided the best results, the fish fed the diet with fish oil performed

slightly better than the fish fed linseed oil, but not significantly. Therefore, C. labrosus proved

its potential to be fed diets depleted of any fish oil. Another remarkable aspect of the analysed

literature is the duration of the experiments, most of them close to 90 days, with a maximum of

120 (Table 1). Although performing 90-day experiments is a standard practice in aquaculture

(Teles et al., 2020), it is surprising that the long-term viability of C. labrosus intensive culture has

never been tested, considering it is a novel species for this kind of production.

Other aspects of nutrition of C. labrosus not dealing directly with the design of specific

feeds, like digestive physiology and biochemistry have also been the focus of recent research

(Pujante, 2019). The evolution of the gene expressions and activities of different digestive

enzymes during the juvenile phase (Pujante et al., 2017) and the effects of salinity on those

variables (Pujante et al., 2018), already mentioned in subsection 2.3, are of particular interest

to optimize the diets of the species. It has already been mentioned in subsection 2.5 that during

larval development, C. labrosus experiences an alteration on the profile of its digestive enzymes,

during the trophic shift from carnivorous to omnivorous (Zouiten et al., 2008; Gilannejad et al.,

2020). However, after overcoming said process, gene expression and activity of digestive

enzymes continues to change. For example, the protease profile of the digestive tract gets

altered with age, with a clear reduction in the role of pepsin (an acid protease) and an increasing

importance of trypsin and chymotrypsin (alkaline proteases), which evidence, on the one hand,

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 General introduction

the decreasing ability of the stomach for enzymatic digestion and, on the other hand, an

adaptation for a better digestion of vegetable proteins. As C. laborosus exhibits a high activity

of alpha-amylase during the entire juvenile phase (Pujante et al., 2017) they could be fed diets

with high inclusion levels of carbohydrate independently of their weight, but the origin of the

protein and their inclusion levels remain as interesting variables to be evaluated at different size

classes.

An additional essential aspect to bear in mind to optimize the production of any cultured

fish is the feeding strategy. Quirós-Pozo et al. (2023) proved that the digestive enzyme

production pattern of C. labrosus was the typical of a continuous feeder, as the activities of

proteases and amylase did not decrease over time after feeding, a strategy suited for the

continuous presence of food inside the gut. Therefore, it was established that the daily feed

ration of this species should be split into several feedings in order to optimize its utilization. The

possibility of applying food deprivation and re-feeding strategies for this species with the aim of

triggering a compensatory growth response that might boost the overall growth is not

completely clear, as shown by Pujante et al. (2015). After being starved for 14 days and re-fed

for 7, C. labrosus did show accelerated growth, but the final weight of the starved and re-fed

fish was still lower than that of the non-starved control fish, evidencing that the growth

compensation was not complete. However, this is the only reported trial about compensatory

growth on C. labrosus, and the adjustment of the durations of the starving and re-feeding phases

could prove to be a viable way of promoting the growth of this species in intensive culture.

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 General introduction

Table 1: a review of the research on the artificial feeding of C. labrosus, arranged according to the initial weight of the fish employed, in increasing order. The data shown

corresponds to the experimental diet / trial rendering the best growth results (SGR) in each paper.

Reference IW (g) T (°C) WS (psu) Duration (days) PC (% protein/lipid/NFE) P / E (mg protein / kJ) SGR FCR
Davies et al., 1997 1 22 30 – 33 70 47.29 / 13.46 / 24.02 23.78 2.99 1.83
Altunok & Özden, 2017 6.2 ≈ 21 – 25 ≈ 36 87 30.13 / 12.08 / 45.48 15.28 2.13 1.61
Ojaveer et al., 1996 15.22 22.7 – 23.0 33 - 36 84 37.94 / 22.79 / 27.33 19.72 1.20 1.67
Amezcua et al., 2009 15.4 19.2 33.2 120 50 / 20 / NR NR 0.228 NR
Quirós-Pozo et al., 2024 26.74 25.43 16 73 41.80 / 13.27 / 27.74 NR 0.74 2.29
Vílchez-Gomez et al., 2017 46.45 NR NR 30 30.44 / 16.30 / NR NR 1.15 2.35
García-Márquez et al., 2022 84.7 17.9 – 23.8 1 – 1.2 90 37.62 / 6.8/ 41.27 NR 0.7 2.6
García-Márquez et al., 2023a 99.5 19.7 – 22.9 1 – 1.2 90 38.17 / 7.26 / 40.36 20.52 0.47 3.12
Legend: IW: mean initial weight of the fish; T: average water temperature during the experiment; WS: average water salinity during the experiment; PC: proximal composition

of the diet; NFE: nitrogen-free extract; P / E: protein / energy ratio; SGR: specific growth rate; FCR: feed conversion ratio; NR: not reported; Sea: seawater salinity.

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 General introduction

2.7. Current knowledge gaps on C. labrosus aquaculture

By far, the most studied aspects of the intensive culture of C. labrosus are reproduction and

nutrition. However, other relevant aspects like the response to stressors that are specific to

aquaculture or the effects of abiotic factors such as water temperature on fish performance

have received very little attention. These knowledge gaps will be discussed in the following

section.

2.7.1. Animal welfare and the stress response

Public interest on the issue of welfare of farmed animals, including fish, is continuously raising

(Ellis et al., 2002). Therefore, knowing the effects of intensive culturing conditions on a novel

species like C. labrosus is of upmost importance in order to develop its culture in an ethically

acceptable way. Drawing a precise definition of the concept of animal welfare is difficult, but

the “Five Freedoms of Animal Welfare” provide a good framework for its understanding and

evaluation. The Five Freedoms are the following, according to the Farm Animal Welfare Council

(2012):

1. Freedom from hunger and thirst.

2. Freedom from discomfort.

3. Freedom from pain, injury or disease.

4. Freedom to express normal behaviour.

5. Freedom from fear and distress.

Despite being a key factor influencing fish welfare, the response of C. labrosus to

stressors characteristic to intensive aquaculture (Barton & Iwama, 1991; Conte, 2004) has rarely

been studied. The definition of stress has been subjected to controversies for many years

(Barton & Iwama, 1991; Wendelaar Bonga, 1997), but it remains a relevant unifying concept for

a wide array of biological phenomena manifested at various organizational levels, from cells,

organs or organisms to populations or even ecosystems (Wendelaar Bonga, 1997). Biological

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 General introduction

stress can be defined as a condition caused by external or internal stimuli, which threaten or

disturb homeostasis. These stimuli, commonly known as stressors, elicit several coordinated

physiological or behavioural adaptive responses that help the organism recover homeostasis. If

the stressors are extremely severe or chronical, the stress response might become maladaptive,

and growth, reproduction and immune functions could become reduced or supressed

(Wendelaar Bonga, 1997), which can endanger animal welfare and cause significant economic

losses in aquaculture (Conte et al., 2004). The stress response can be summarized by the concept

of the General Adaptation Syndrome (GAS), which proposes three phases for the response: the

first phase is an alarm reaction and a concomitant physiological response. The second phase is

a resistance stage, where the animal regains homeostasis, either by returning to the pre-stress

situation or by acquiring an altered resting state. The third phase happens when the

compensation is not possible, due to the extreme severity or long duration of the stressful

situation, and the organism becomes exhausted, which could lead it to its death (Selye, 1950).

The stress response in teleost fish follows the general pattern observed in other

vertebrates, with some particularities related to their habitat, as fish are continuously in close

contact with the surrounding water through the gills and intestine. This, on the one hand, makes

them more susceptible to stressors such as chemicals or pollutants present in the water, and on

the other hand, most stressors affect branchial structure and, consequently, can disturb

hydromineral balance and respiration (Wendelaar Bonga, 1997). The stress response is mainly

coordinated by the brain via two axes, the hypothalamic-sympathetic chromaffin cell axis and

the hypothalamic-pituitary-interrenal axis. Briefly, these routes function by the activation of

brain centres, which cause the release of hormonal messengers (catecholamines and

corticosteroids) to the bloodstream, which can be called the primary stress response. The

immediate effects of those hormones at the tissue level are the secondary stress responses, like

increases in oxygen uptake, cardiac output, alterations of the hydromineral balance and energy

mobilization. The tertiary responses are the observable effects at the whole organism or even

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 General introduction

population level, like reduced growth, reproductive capacity, immune response and the

reduction of the ability to face further stressors (Wendelaar Bonga, 1997). A schematic

representation of the stress response can be found in Figure 8.

The main hormones released by the hypothalamic-sympathetic chromaffin cell axis are

catecholamines, mainly epinephrine and norepinephrine. These hormones are mostly secreted

by the chromaffin cells, which are found in small groups in the head kidneys. Although the

release of catecholamines by interrenal cells is primarily mediated by sympathetic nerves, the

control of this stress response is highly complex. The main effects of these hormones are related

to the increase of the oxygen uptake and transport capacities and the mobilization of energy

reserves. At the branchial level, catecholamines increase ventilation rate, blood flow and oxygen

diffusion capacity. The oxygen transport capacity of the blood is also raised by catecholamines,

by increasing haematocrit levels and haemoglobin’s affinity for oxygen. Moreover, high levels of

blood catecholamines can stimulate cardiac output, which also helps increase oxygen

mobilization. These hormones also stimulate glycogenolysis in the liver, causing blood

hyperglycaemia (Wendelaar Bonga, 1997).

Cortisol is the main corticosteroid hormone released by the hypothalamic-pituitary-

interrenal axis, produced by interrenal cells. These cells form layers, strands and cords around

the walls of the posterior cardinal veins and their branches through the head kidneys. The

control over cortisol secretion is mostly carried out by the pituitary gland, although, similarly to

the control of catecholamine secretion, the actual control mechanisms are highly complex.

Cortisol plays two important roles in the stress response, as a regulator of hydromineral balance

and as a metabolic modulator. Cortisol stimulates the proliferation of chloride cells, which are

the main ion transporters of gills, and increases the activity of ion-transporting enzymes in gills,

intestine and kidneys. These cells and enzymes are crucial for the compensatory mechanisms

that drive hydromineral balance within the organism, by regulating the ejection of excess ions

in hyperosmotic media (seawater) and their uptake in hypoosmotic waters (freshwater). Cortisol

35
 General introduction

also modulates carbohydrate, lipid and protein metabolism, promoting energy mobilization.

Liver glucose content can be increased by this hormone. However, the effects of cortisol on

hepatic glycogen are not completely clear and, therefore, gluconeogenesis from non-protein

substrates seems to play the main role in glucose production. Lipolysis is also stimulated by

cortisol. Cortisol secretion increases sharply and rapidly under a wide array of acute stressors,

thus, the measurement of blood cortisol levels is generally a good tool for assessing the effects

of a given stressor. However, in chronic stress situations, the response of cortisol is greatly

variable, and blood levels can remain high in the long term, or they can return to basal values.

Therefore, low blood cortisol levels do not necessarily imply an absence of stress, and the

usefulness of blood cortisol for measuring chronic stress is unclear (Wendelaar Bonga, 1997).

Figure 8: generalized diagram of main neuroendocrine elements of the integrated stress response in

teleost fish and their main physiological effects. FFA: Free fatty acids; + : stimulatory; - : inhibitory.

Adapted from Wendelaar Bonga (1997).

In intensive aquaculture, fish are routinely faced by a particular set of stressors that are

not among the ones present in their natural habitats like handling, grading, sorting, transport

and high-density stocking (Barton & Iwama, 1991). Stocking density strictly refers to the initial

density at which fish are stocked into an aquaculture system (fish biomass per unit of water

36
 General introduction

volume), but it can be used for expressing the fish biomass per unit of water at any point in time,

as the somatic growth of the animals causes this parameter to be constantly dynamic (Ellis et

al., 2002). This parameter can affect fish welfare in several ways, although the exact mechanisms

behind those effects are still unclear. In general, fish growth, feed intake and feed efficiencies

are decreased at high densities, as well as the general nutritional status, health condition and

other stress indicators (Ellis et al., 2002; Li et al., 2021). Although growth is usually negatively

affected by high stocking densities like in the cases of the gilthead sea bream (Montero et al.,

1999; Sangiao-Alvarellos et al., 2005), African catfish (Dai et al., 2011), channel catfish (Refaey

et al., 2018), Shire tilapia (M´balaka et al., 2012), turbot (Irwin et al., 1999) and the grey mullet

Mugil platanus (Sampaio et al., 2001), the contrary has also been observed on some species, like

the European sea bass (Papoutsoglou et al., 1998) or Arctic charr (Jørgensen et al., 1993). This

species-specific response might be related to the differences in social structures and

interindividual interactions between species, as social behaviour has been pointed at as one of

the main causes of stocking stress (Ellis et al., 2002). For instance, increasing stocking densities

do not only lead to a raise in damaging non-aggressive interactions, like collisions with other

individuals and with the rearing tank itself, but can also increase the aggressive ones (Ellis et al.,

2002). In intensive culturing conditions, several species like rainbow trout (Ellis et al., 2002) or

Nile tilapia (Barcellos et al., 1999) can establish strict social hierarchies, where dominant fish

might attack subordinate ones and restrict their access to food. This aggressive behaviour can

cause physical injuries in subordinate individuals like fin erosion, or elicit a stress response

evidenced by increased mobilization of energy reserves, high plasma cortisol levels, decrease

appetite and growth. Moreover, susceptibility to disease might also be increased in subordinate

fish, which is highly worrying due to disease spreading being also promoted by high stocking

densities (Ellis et al., 2002). High densities can also negatively affect several water quality

parameters like the levels of dissolved oxygen or nitrogenous waste, which are also detrimental

to fish health. Therefore, in practical culturing conditions, the negative effects of high stocking

37
 General introduction

densities themselves (caused, for instance, by increased aggressive behaviour) are hard to

separate from the effects of decreased water quality (Ellis et al., 2002).

The wide array of effects that stocking density can exert on fish welfare and aquaculture

productivity make it a particularly relevant parameter to investigate in order to establish

adequate rearing conditions for a given cultured species. In the case of C. labrosus, only one

research work has been published on this topic, on juvenile individuals of an initial weight close

to 0.5 g (de las Heras et al., 2015). In this research, the authors observed a decreased growth

rate on the animals held at the highest density (6.7 kg / m3). However, the response to stocking

density can be different depending on the life stage of the animal (Li et al., 2021). Therefore,

conducting similar studies on bigger individuals is necessary for ensuring an adequate balance

between animal welfare and farm productivity during the entire life cycle of the animal.

2.7.2. Effects of water temperature on fish

Water temperature is considered the most important abiotic factor affecting the metabolism of

poikilothermic animals such as fish (Hawkins, 1995; Nytrø et al., 2014; Sun & Chen, 2014).

Therefore, the lack of published studies related to this topic on C. labrosus is surprising.

Generally, fish performance (in terms of growth, reproductive ability or immune response)

sharply increases alongside water temperature until reaching an optimal point that is species-

specific. At that optimal temperature, performance plateaus and eventually declines if

temperature increases further, thus, creating bell-shaped thermal performance curves or TPCs

(Farrell, 2009; Schulte, 2015). The shape of the TPC answers to alterations in the metabolic rate

of the animal, and it is mainly caused by the effects of thermodynamics and protein stability.

However, the underlying mechanisms behind the effects of ambient temperature on the

metabolism of poikilothermic animals are highly complex (Clarke & Fraser, 2004; Schulte, 2015).

Therefore, a brief and simplified explanation of the processes driving the response of fish

38
 General introduction

aerobic metabolism to water temperature will be provided, focusing on the concepts of

maximum metabolic rate, basal or resting metabolic rate and the aerobic scope.

The metabolic rate is the measure of the power utilization of an organism, in this case

fish, usually expressed as the oxygen consumption rate (Clarke & Fraser, 2004). Thus, maximum

metabolic rate is the maximum oxygen and energy consumption of the fish, which is strongly

modulated by ambient temperature (Figure 8 A). At the most basic level of organization,

biochemical reactions that shape life are driven by the laws of thermodynamics, described by

the Arrhenius equation (Arrhenius, 1915). The equation expresses an exponential increase of

the metabolic rate in relation to temperature. Therefore, it provides a satisfactory explanation

to the typical initial sharp increase in metabolic rate of the first phase of TPCs. However,

maximum metabolic rates of animals exhibit a plateau at a certain temperature, and further

temperature increases do not raise metabolic rates. Although this fact defies the general

framework provided by Arrhenius (1915), several biological processes limit oxygen and energy

consumption within an animal at various organizational levels. For instance, at the molecular

level, conformational changes that lower overall catalytic activity or substrate binding of

enzymes at high temperatures might explain this phenomenon. Moreover, many of the enzymes

partaking in the aerobic metabolism are located in the mitochondrial membrane. As

temperature can alter membrane fluidity, the activity of these enzymes can be indirectly shaped

by it. However, other factors might also be involved in the limitation of the metabolic rate at the

single protein level (Schulte, 2015). However, aerobic metabolism is a complex process with

multiple steps involving complex biochemical networks. At the mitochondrial and cellular levels,

the flux through these networks might be limited by a single or multiple key steps with certain

thermal dependencies, thus, limiting metabolic rate at an optimum temperature. Another fact

that must be taken into account is that thermodynamics is not the only modulator of

metabolism. The flux through metabolic pathways is strongly controlled by the so-called

“metabolic regulation”, driven by the concentrations of substrates, products or allosteric

39
 General introduction

effectors. Therefore, the effects of temperature on such molecules should also be considered

for providing a complete explanation to metabolic regulation (Schulte, 2015). Finally, at the

organism level, oxygen supply to the cells seems to be a key limitation of the maximum

metabolic rate. To cope with the increased oxygen demand by the cells and tissues at higher

temperatures, the cardiorespiratory system increases oxygen transport capacity, mainly by

raising heart rates. However, a maximum heart rate does exist, which limits the maximum

oxygen transport within the animal (Farrel, 2009). Concomitantly, other relevant processes

might also be compromised above the optimal temperature, like neural processes at brain and

cellular levels or the maintenance of membrane potentials. However, all the explanations

provided for the limitation of the maximum metabolic rate at all organizational levels are deeply

inter-connected, and have to be understood as part of an integrated system (Schulte, 2015).

Basal metabolic rate is the minimum metabolic activity an organism has to maintain in

order to remain alive, which is also affected by ambient temperature in poikilothermic animals

(Figure 9 A). It can be defined in various ways, but a practical definition states that is the oxygen

consumption of an animal in an “inactive, postabsorptive, non-growing and non-reproducing”

state. A significant share of the basal metabolic rate corresponds to the energy expenditure

needed for protein synthesis and the maintenance of membrane energy potential gradients

(Clarke & Fraser, 2004). As membrane fluidity is increased alongside temperature, the cost of

counteracting membrane proton leak also increases. Due to this and other processes, basal

metabolic rate in fishes usually increases exponentially alongside water temperature (Farrel,

2009).

The difference between basal and maximum metabolic rates is usually called aerobic

scope (Figure 9 B), and it limits the amount of energy an animal can direct towards processes

such as growth, locomotion, immune response or reproduction. The graphical representation of

aerobic scope in relation to temperature has an initial upwards phase, when the raise of

maximum metabolic rate vastly outweighs the increase in basal metabolic rate. The second

40
 General introduction

phase is a plateau around the optimal temperature of the species, when there is a balance

between the gains of the maximum metabolic rate and the losses of the basal one. At the last

phase aerobic scope decreases, as basal metabolic rate continues to increase while the

maximum metabolic rate cannot grow above the optimum temperature. Ultimately, a point is

reached where basal metabolic rate exceeds the maximum one, and the animal dies (Farrell,

2009; Schulte, 2015). Therefore, the shape of the graphic representation of the aerobic scope in

relation to ambient temperature corresponds to the shape of the TPC.

Figure 9: representation of the relationship between basal (BMR) and maximum (MMR) metabolic rates

(A) and aerobic scope (B) in relation to water temperature in fishes. AU: arbitrary units. Adapted from

Schulte (2015).

Therefore, finding the optimal temperature where aerobic scope is at its maximum is

essential to optimize aquaculture production of a given species from the points of view of

productivity and animal welfare (Wang et al., 2019; Islam et al., 2021; Dawood et al., 2022). As

previously stated, no research on this topic has been published on C. labrosus, rendering it a

significant knowledge gap for the development of its intensive culture.

2.8. Future prospects

As previously stated, the critical bottleneck of the reproduction in captivity and larval rearing

has been surpassed with the aid of hormonal induction (Besbes et al., 2020), and great progress

41
 General introduction

has been made in regards to its nutrition (see subsection 2.6). However, the intensive

production of C. labrosus has not been developed. Most probably, this is because grey mullets

have traditionally been considered fish of secondary importance at some Mediterranean

countries (Cataudella et al., 1988), like Spain or Italy, which makes their commercial production

very economically risky. The education of the consumer through the perspective of the

environmental sustainability of C. labrosus production and the continuous research about the

optimization of its culture are of upmost importance for this industry to develop fully. In this

context, the design of specific feeds for the species, formulated with cheaply available

ingredients like food industry by-products and the determination of the effects of highly relevant

rearing parameters like water temperature and stocking density on the physiology and stress

responses of C. labrosus are key steps in order to establish this species as a viable candidate for

the diversification of intensive aquaculture.

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7345.2008.00186.x

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 State of the art

State of the art

63
State of the art

64
 State of the art

The thicklip grey mullet Chelon labrosus is an omnivorous teleost that has been extensively

cultivated in traditional systems since the antiquity. Nevertheless, its intensive culture has never

been completely developed, mainly due to the difficulties in closing its life cycle in captivity and

the lack of specific feeds for the species. Great progress has been made in recent years gaining

knowledge on reproduction and larvae rearing in captivity, and on digestive functionality and

nutrition. However, other aspects related with its intensive culture have received less attention,

like its response to aquaculture stressors like high stocking densities and the effects of abiotic

variables, such as water temperature, on its metabolism and growth. In this context, the design

of specific feeds for the species, formulated with alternative ingredients and the determination

of the effects of highly relevant rearing parameters like water temperature and stocking density

on the physiology and stress responses of C. labrosus, are key issues in order to establish this

low trophic species as a viable candidate for the diversification of intensive aquaculture.

65
State of the art

66
 Hypothesis and objectives

Hypothesis and objectives

67
Hypothesis and objectives

68
 Hypothesis and objectives

Hypothesis

The thicklip grey mullet Chelon labrosus is a suitable novel low trophic fish species for intensive

aquaculture, and the optimization of culturing conditions such as stocking density, water

temperature and the formulation of a specific feed based on alternative ingredients to fishmeal

will improve its performance, welfare status and product quality, proving its feasibility for the

development of a sustainable diversification of aquaculture.

Objectives

In order to test this hypothesis, the following objectives were established, which will be

addressed in their respective chapters:

1. To assess the growth of C. labrosus maintained in a long-term trial, as a first approach

for the understanding of the performance and nutritional needs of the species. Chapter 1.

2. To assess the effects of stocking density on C. labrosus individuals at the grow-out

phase, from the perspectives of productivity and animal welfare, focusing on secondary and

tertiary stress responses. Chapter 2.

3. To determine the effects of different water temperatures on growth, energy

metabolism, intestinal health, digestive processes and product quality (muscle fatty acid profile)

of the thicklip grey mullet C. labrosus in order to establish a range of optimal temperature

conditions for its culture. Chapter 3.

4. To determine the effects produced by diets formulated with alternative protein sources,

differing on protein inclusion levels as well as in the origin of protein, on growth, metabolism,

stress, immune status, intestinal health and nutritional quality (muscle fatty acid profile) of C.

labrosus. Chapter 4.

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Hypothesis and objectives

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Results and discussion

Results and discussion

71
Results and discussion

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Results and discussion Ethical statement

Ethical statement
All experimental procedures complied with the Guidelines of the European Union (2010/63/UE)

and the Spanish legislation (RD53/2013 and law 32/2007) for the handling and use of laboratory

animals under the supervision and acceptance of the pertinent Ethics for experimentation and

animal welfare committees and provincial authorities.

Chapter 1: the experiment was performed at Kardala LHI aquaculture school, registered

in the registry of agrarian exploitations of Gipuzkoako Foru Aldundia (20 056 003 0475), under

the supervision and acceptance of the Ethics for experimentation and animal welfare committee

of the University of the Basque Country (UPV/EHU) (CEEA M20-2018-133).

Chapter 2: the experiment was performed at Kardala LHI aquaculture school, registered

in the registry of agrarian exploitations of Gipuzkoako Foru Aldundia (20 056 003 0475), under

the supervision and acceptance of the Ethics for experimentation and animal welfare committee

of the University of the Basque Country (UPV/EHU) (CEEA M20-2018-133).

Chapter 3: the experiment was performed at The Research Centre for Experimental

Marine Biology and Biotechnology “Plentziako Itsas Estazioa” (PiE-UPV/EHU) under the

supervision and acceptance of the Ethics for experimentation and animal welfare committee of

the University of the Basque Country (UPV/EHU) (CEEA M20-2021-108).

Chapter 4: the experiment was performed at El Bocal Marine Aquaculture Plant,

Oceanographic Centre of Santander, COST-IEO (CSIC) under the supervision and acceptance of

the official ethics committee (favourable report number NTS-ES-081792).

Animal welfare is ensured in all the aforementioned centres.

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Results and discussion Ethical statement

74
Results and discussion Chapter I

Chapter I

New perspectives on the long-term culture of the

thicklip grey mullet Chelon labrosus (Risso, 1827)

fed diets of different composition.

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Results and discussion Chapter I

Congress:

Sanz-Latorre, M., Conlledo, N., Mensah, D., Goikoetxea, J., Izagirre, U., Soto, M., Sudupe, R.,

Brettes, P., de Diego, A., Lekube, X. (2022). First steps in the development of intensive

aquaculture of the thicklip grey mullet Chelon labrosus (Risso, 1827). [Oral presentation]. X

Simposio sobre el Margen Ibérico Atlántico / X Simpósio sobre a Margem Ibérica Atlântica.

Bilbao, Spain.

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Results and discussion Chapter I

List of abbreviations

ALA: α-linolenic acid (C18:3n-3)

DHA: docosahexaenoic acid (C22:6n-3)

EPA: eicosapentaenoic acid (C20:5n-3)

FA: fatty acid

FAME: fatty acid methyl esther

HP / HL: high protein / high lipid

HSI: hepatosomatic index

K: Fulton condition factor

LA: linoleic acid (C18:2n-6)

LP / HC: low protein / high carbohydrate

MUFA: monounsaturated fatty acids

ORO: oil red O

PUFA: polyunsaturated fatty acids

RAS: recirculating aquaculture system

SFA: saturated fatty acids

SGR: specific growth rate

VSI: viscerosomatic index

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Results and discussion Chapter I

Abstract

The thicklip grey mullet Chelon labrosus is a promising fish species for the diversification of

European aquaculture, as its omnivorous feeding habits provide great potential for the design

of diets with little or without inclusion of fishmeal and fish oil. In the present research, C.

labrosus juveniles (initial mean weight 26.96 ± 6.41 g) were stocked in two groups and each of

them was fed a different commercial feed for 686 days, as a preliminary approximation for the

assessment of the growth performance on the long term and a better understanding of

nutritional needs. The feeds used were anisocaloric and anisoproteic, one high on protein and

lipids (HP / HL) and the other low on protein and high on carbohydrate (LP / HC). The two groups

showed little differences in weight gain during the first year of the experiment, but after that

group HP / HL performed significantly better. This difference is probably driven by the higher

protein content of the feed, as both diets led to high energy reserve accumulation in liver,

without showing any related pathologies, indicating that energy contents were enough for

coping with the fish´s need in both groups. Similarly, final muscle lipid content was almost

identical in fish fed the two diets and interestingly, LP / HC group contained a higher level of

polyunsaturated fatty acids, including ω-3 and ω-6. This indicates that the fish has a great ability

to convert dietary carbohydrates into lipids, proving the suitability of carbohydrates as the main

energy source, and that it has a great capacity to modulate muscle fatty acid profile. Therefore,

the design of a specific feed for C. labrosus could consider diets with high amounts of protein

and low on lipids. This study provides relevant information for the development of the intensive

culture of C. labrosus, and it shows the importance of long-term feeding experiments when

studying new species for aquaculture.

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Results and discussion Chapter I

Laburpena

Lazuna (Chelon labrosus) europar akuikulturaren dibertsifikaziorako interes handiko espeziea

da, orojalea izanik, arrain irin eta olio eduki txiki edo gabeko pentsuen diseinurako aukera

eskaintzen baitu. Ikerketa honetan, C. labrosus jubenilak (hasierako bataz besteko pisua 26.96 ±

6.41 g) bi taldetan banatu eta pentsu komertzial ezberdin banarekin elikatu ziren 686 egunetan

zehar, espeziearen epe luzeko hazkuntza jardueraren atariko jakintza eta nutrizio beharren

ulermen sakonagoa lortzeko asmoz. Erabilitako pentsuak anisokalorikoak eta anisoproteikoak

izan ziren, bata proteinatan lipidotan aberatsa (HP / HL) eta bestea proteinatan xumea eta

karbohidratotan aberatsa (LP / HC). Esperimentuaren lehen urtean zehar, bi taldeen arteko

hazkuntza ezberdintasuna txikia izan zen, baina epe hori gaindituta, HP / HL taldeak emaitza

esangarriki hobeak lortu zituen. Seguruenik, ezberdintasun honen arrazoia pentsu honen

proteina eduki altua da, bi pentsuek energia erreserba metaketa handia eragin baitzuten

gibelean, honi lotutako patologiarik sortu gabe. Honek bi pentsuen energia edukia arrainaren

beharrak asetzeko bestekoa zela adierazten du. Modu berean, giharraren lipido edukia bertsua

izan zen esperimentuaren amaieran eta LP / HC taldeak gantz azido poliasegabe eduki handiagoa

zeukan, ω-3 eta ω-6 taldeetakoak barne. Hau, alde batetik, arrain honen karbohidratoak lipido

bihurtzeko ahalmenaren adierazle da, karbohidratoak energia iturri nagusitzat baliagarriak

direla ondorioztatuz eta, beste aldetik, giharreko gantz azido profila modu efizientean

kontrolatzeko ahamenarena. Beraz, C. labrosus-entzako pentsu espezifikoak proteina eduki

altua eta lipido eduki baxua eduki ahalko lituzke. Ikerketa honek C. labrosus-en akuikultura

intentsiboaren garapenerako informazio garrantzitsua dakar, eta akuikulturarako espezie

berritzaileak ikertzerakoan epe luzeko esperimentuen beharra azpimarratzen du.

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Results and discussion Chapter I

1. Introduction

Since the 1960s, worldwide fish consumption has been increasing at a rate faster than the

growth of the world´s population. This issue, reinforced by the stagnation of wild fish captures

since the late 1980s, has instigated a rapid development of the aquaculture industry; in the year

2000, aquaculture provided about 25.7 % of global fish, while in the period between 2016 and

2018 this value reached 46 % (FAO, 2020). However, further expansion is essential to satisfy the

ever-growing global fish demand. Ensuring said growth is achieved in a sustainable way is still a

challenge (Folke & Kautski, 1992; Naylor et al., 2000; Costa-Pierce, 2010; Samuel-Fitwi et al.,

2012), even though substantial improvements have been done in that regard in the last decades

(Naylor et al., 2009). Therefore, many research and policy-making efforts are being directed

towards improving said sustainability (Tezzo et al., 2021; Simmance et al., 2021; EC, 2021).

One of the main fields in the improvement of aquaculture sustainability is the research

of innovative feeds based on alternative ingredients in order to reduce the consumption of the

traditionally used fishmeal and fish oil (Naylor et al., 2000; Tveterås & Tveterås, 2010) with the

aim of exerting less pressure on wild fisheries. Omnivorous and herbivorous fish species have a

greater potential to be nurtured on a wider range of ingredients than carnivorous ones, like

land-based vegetal ones, macro and microalgae or food industry by-products (Naylor et al.,

2009; Bostock et al., 2010; Tacon & Metian, 2015). Consequently, the research of innovative

omnivorous or herbivorous alternative aquaculture species is being promoted by international

authorities to achieve a sustainable and cost-effective growth of the aquaculture production

(EC, 2021). In this context, grey mullets (family Mugilidae) are a promising group of fish for the

diversification of aquaculture (Abellán-Martínez & Arnal-Atarés, 2013; www.diversifyfish.eu).

Some of the main reasons for it are that they are cosmopolitan (Thomson, 1997), economically

relevant in many places (Heras et al., 2009; Durand et al., 2012; Whitfield et al., 2012) and, more

importantly, omnivorous in the juvenile and adult phases (trophic levels generally ranging from

2 to 3, depending on the species), despite being carnivorous in the earlier stages (Cardona,

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Results and discussion Chapter I

2016). The thicklip grey mullet Chelon labrosus (trophic level 3, according to Cardona, 2016) in

particular has been targeted as an interesting species for diversifying Spanish aquaculture

(García-Márquez et al., 2021).

Despite of being a group that has been cultivated since the antiquity, there are two main

bottlenecks for the development of the intensive culture of mugilids: the difficulties of closing

the life cycle in captivity and the lack of specific commercial feeds (Crosetti, 2016). In the past 5

decades, several attempts have been made in order to identify their specific nutritional

requirements and, by extension, an optimal proximal composition of diets for grey mullets,

although a commercial feed has never been achieved (Vallet et al., 1970; Kandasamy et al., 1987;

Benetti & Fagundes Netto, 1991; El-Sayed, 1991; Argyropoulou et al., 1992; Yoshimatsu et al.,

1992; Ojaveer et al., 1996; Abdel-Hakim et al., 2001; Amezcua et al., 2009; de Carvalho et al.,

2010; De et al., 2012; Altunok & Özden, 2017; Talukdar et al., 2020). Most of these studies,

though, do not take into account the effects of culturing conditions, especially artificial feeding,

in the long term, making them difficult to use as guidelines for the intensive aquaculture of

mugilids.

Therefore, there is a need to gain knowledge on the intensive culturing of mugilids as

alternative to the traditionally farmed carnivorous species. The aim of this work was to assess

the growth of C. labrosus maintained in a long-term trial, as a first approach for the

understanding of the performance and nutritional needs of the species. With such a purpose,

fish were fed two commercial diets (anisocaloric and anisoproteic) of different compositions.

Concomitantly, a series of supporting data (mortality, fish health, flesh proximal and biochemical

composition) was recorded.

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Results and discussion Chapter I

2. Materials and methods

2.1. Experimental design

The experimental fish (wild juvenile C. labrosus, mean fork length 12.75 ± 1.02 cm and mean

weight 26.96 ± 6.41 g) were obtained from the Fauna Marina, S. L. company (Cadiz, Spain) in

November 2018 and transported to the facilities of Kardala LHI aquaculture school (Mutriku,

Spain). The feeding experiment was performed in a recirculation aquaculture system (RAS)

comprised of two rearing tanks (1000 L), a decantation tank, mechanical, sand and biological

filters and a protein skimmer. The photoperiod was set to match the natural day/night cycle.

Water temperature ranged between 12 °C and 25.5 °C. The experiment lasted for 686 days,

starting on December 2018 and finishing in November 2020.

The fish (n = 579) were randomly separated into two well-balanced groups of similar

mean size, mean weight and stocking density (Table 1). Each group was fed a different

commercial aquaculture feed (Table 2), one designed for trout (T2-Optiline 1p; Skretting,

Stavanger, Norway), with high content of both protein and lipids (HP / HL) and the other for

tilapia (TI-3 Tilapia 3.2 mm; Dibaq, Segovia, Spain), with a low content of protein and high

content of carbohydrates (LP / HC). Due to the complex characteristics of the experimental

design and the space limitations of the RAS facility, replicates were not considered for being

impractical from a logistics standpoint. Therefore, the feed treatments were applied to the

entire aquarium, and so the experimental unit are two aquaria with 295 and 284 fish (Table 1).

The fish were fed twice a day for 5 days a week with a daily dose that accounted for 2.5 % of the

total fish biomass for each tank. Due to the high amount of uneaten feed found in the filters,

the daily dose was adjusted to 1.5 % of the total biomass after T4 (day 505). Before the

experimental period, it was noticed that the size of the feed pellets was too big for the fish´s

mouth, so the feed was ground and administered as powder for the entirety of the experiment.

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Results and discussion Chapter I

Table 1: initial characteristics of the experimental groups (HP / HL: high protein / high lipid; LP / HC: low

protein / high carbohydrate).

Group HP / HL LP / HC
Number of fish (n) 295 284
Mean length (cm) 12.70 ± 0.99 12.79 ± 1.04
Mean weight (g) 26.28 ± 5.82 27.66 ± 6.92
Stocking density (Kg / m3) 4.1 4.2

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Results and discussion Chapter I

Table 2: characteristics of the feeds used during the experimental period. Full fatty acid profiles can be

found at the Appendix (Table A1).

Group HP / HL LP / HC
Commercial name T2-Optiline 1p Dibaq Tilapia 2mm
Trout feed, 2 mm Tilapia feed, 2 mm
Proximate composition (%)
Crude protein 44.5 35
Oils and crude fats 21 6
Nitrogen free extract ~25.8 ~45
Crude fibre 2.2 6
Total ashes 6.5 8
Ca 1.4 2
P 1 1.4
Na 0.3 0.2
Energy
Digestible energy (MJ / Kg) 18.8 12.2
Protein/energy (mg / KJ) 23.67 28.69
Summary of fatty acid profile % of lipid % of feed % of lipid % of feed
Total SFA 25.57 5.11 24.48 1.47
Total MUFA 50.78 10.16 44.30 2.66
Total PUFA 23.65 4.73 31.22 1.87
Σ ω-3 PUFA 8.00 1.60 8.19 0.49
Σ ω-6 PUFA 15.56 3.11 22.90 1.37
EPA + DHA 3.31 0.66 1.93 0.12
ω-3 / ω-6 0.51 0.51 0.36 0.36
Proximate composition and digestible energy content, as provided by the manufacturers. Nitrogen free

extract: 100 - (crude protein + oils and crude fats + crude fibre + total ashes + Ca + P + Na). Fatty acid

profiles are expressed as % of fatty acids in lipid extract and in total feed. Fatty acids profile have been

analysed following the procedure described “Characterization of fatty acids profile” subsection. HP / HL:

high protein / high lipid; LP / HC: low protein / high carbohydrate; SFA: Saturated Fatty Acids; MUFA:

Monounsaturated Fatty Acids; PUFA: Polyunsaturated Fatty Acids; EPA: eicosapentaenoic acid; DHA:

docosahexaenoic acid.

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2.2. Sample collection

Six samplings were carried out, at days 0 (T0), 91 (T1), 197 (T2), 365 (T3), 505 (T4) and 686 (T5).

At each sampling time, all fish were measured (fork length) and weighed, and 10 fish from each

tank were dissected. In the dissected fish, visceral weight and liver weight were measured. At

T0 and T5, muscle samples (10 from each tank) were immediately frozen at - 40 °C after

dissection and stored for chemical analysis, and liver and proximal intestine samples were

processed for histological analysis. For fish handling (weighing and measuring), fish were

introduced in a water bath containing 100 mg / L Tricaine methanesulfonate MS 222 (Sigma-

Aldrich, Saint Louis, USA) anaesthetic. For sacrifice, the fish were introduced at a water bath of

the same compound at a concentration of 300 mg / L.

2.3. Calculations

The following parameters were measured:

Weight gain (WG) = Final weight (g) - initial weight (g)

Ln (Final body weight)− Ln (Initial body weight)

Specific growth rate % (SGR) = 100 x (% / day)
Feeding days

Visceral weight (g)

Viscerosomatic index (VSI) = 100 x (%)
Total weight (g)

Liver weight (g)

Hepatosomatic index (HSI) = 100 x (%)
Total weight (g)

Fish weight (g)

Fulton condition factor (K) = 100 x
Length3 (cm)

2.4. Measurement of total crude protein in muscle

The frozen muscle samples were lyophilized and minced. The 10 samples of each group were

mixed to obtain composite samples for further chemical analyses.

The crude protein content in each composite muscle sample was determined in

triplicate according to Kjeldahl method (Büchi Labortechnik, 2007). Approximately 0.7 g of fish

muscle sample is digested with sulphuric acid at high temperature using the Digestion Automat

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Results and discussion Chapter I

K-438; the distillation was performed with the Distillation Unit K-360 and finally the titration was

done by a Metrohm titrator. The results were calculated as percentage of nitrogen. In order to

calculate the crude protein content of the sample, the nitrogen content was multiplied with the

sample-specific protein factor 6.25.

2.5. Measurement of total lipids in muscle

The apolar lipid content in the composite muscle samples was determined (N=1) following a

modification of the Bligh and Dyer method (Navarro et al., 2010). Approximately 1 g of muscle

tissue was weighed and shaken with 15 mL of dichloromethane into an orbital shaker during 3

h. The sample was allowed to decant by itself in darkness and then was filtered using 0.45 µm

PVDF filters (Teknokroma, Spain). Finally, the extract was evaporated to dryness in a laminar

flow hood (overnight) and the resultant yellow liquid was weighed.

For the characterization of the fatty acid profile of the feeds used (Subsection 2.6), feed lipids

were extracted the same way.

2.6. Characterization of fatty acid profile

The liquid obtained after sample treatment for total lipid content analysis (Subsection 2.5) was

used in the determination of fatty acids (FA). FAs were first converted to fatty acid methyl esters

(FAME) according to ISO 12966-2:2017, using heptadecanoic acid (C17:0 ≥ 98% Sigma Aldrich,

USA) as internal standard. Briefly, 25 mg of lipid extract were transesterified with 1.5 ml of NaOH

(0.5 N in MeOH) at 100 °C for 2 min, methylated with 2 mL of BF3 (20% in MetOH) at 100 °C for

20 min and finally extracted with hexane.

The sample (1 μl) was analyzed by capillary gas chromatography on GC (Hewlett Packard

5890) equipped with a flame ionization detector and capillary DB-23 column (60 m × 0.25 mm ×

0.25 μm, Agilent Technologies; USA). The column temperature was programed as follows: initial

temperature of 100°C, hold for 2 min, rate of 5°C/min to 200°C, hold for 10 min and rate of

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2°C/min to a final temperature of 225 °C that was hold for 20 min. The injector and detector

temperatures were 250 °C and 300 °C, respectively. Helium was used as the carrier gas.

The individual FA were identified using the 37 Component FAME mix standard (Supelco,

Bellefonte, USA). Fatty acid quantification as the mass fraction, in grams of each FAME per 100

grams, was calculated using the chromatographic peak areas and the masses of the internal

standard, of the test portion and each of the FAME of the mix standard (ISO 12966-4 2015).

2.7. Histological procedures

Liver and proximal intestine samples were fixed in a 10 % formalin solution buffered with

seawater for 24 hours. Once fixed, they were dehydrated in a series of ethanol baths and

embedded in paraffin with an automated tissue processor (LEICA ASP 300S; Leica Microsystems

Nussloch GmbH, Germany) and embedded in paraffin wax blocks. Five µm thick sections were

cut with a microtome (Leica RM2125RTS; Leica Microsystems Nussloch GmbH, Germany), and

stained with a Haematoxylin-eosin staining using an Autostainer XL (Leica Microsystems

Nussloch GmbH, Germany) and mounted with cover glasses.

For the histochemical detection of neutral lipids, samples of liver and proximal intestine

frozen at - 40 °C were cryosectioned at a thickness of 5 µm with a Leica CM3050 S cryostat (Leica

Microsystems Nussloch GmbH, Germany), and stained with Oil Red O (ORO) staining (Culling,

1974).

All samples were observed under an Olympus BX61 light microscope (Olympus-

Lifescience).

2.8. Data treatment

Data are presented as means ± standard deviation. All statistical analyses were performed using

the IBM SPSS Statistics 27 software (IBM corp, 2020). After inferring that none of the data

followed a normal distribution using the Shapiro Wilk test, average weight (AW), average length

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Results and discussion Chapter I

(AL) and Fulton condition factor (K) were compared between both treatments and sampling

times using the non-parametric Kruskal-Wallis test followed by the Dunn´s post-hoc test.

Viscerosomatic index (VSI) and Hepatosomatic index (HSI) were compared among treatments

using the non-parametric Mann-Whitney U-test. The significance for every statistical test was

set at p < 0.05.

2.9. Ethical statement

All experimental procedures complied with the Guidelines of the European Union (2010/63/UE)

and the Spanish legislation (RD53/2013 and law 32/2007) for the handling and use of laboratory

animals under the supervision and acceptance of the Ethics for experimentation and animal

welfare committee of the University of the Basque Country (UPV/EHU) and provincial authorities

(CEEA M20-2018-133-Diputación Foral de Bizkaia). Kardala LHI aquaculture school is registered

in the registry of agrarian exploitations of Gipuzkoako Foru Aldundia (20 056 003 0475).

3. Results

No mortality associated to culturing conditions was recorded during the experimental period.

No differences in weight or length were observed until T3 (day 365). At that point, HP / HL group

exhibited higher weight and length than LP / HC group, as shown in Figure 1. From that moment

on, the pace of the weight and length increase was sharper in the HP / HL group. In group HP /

HL, weight gain was constant during the entire experiment, with the exception of the slow

growth recorded at the initial period (T0 – T1) and the rapid weight increase of the last one (T4

– T5). In the group LP / HC, weight increase was more irregular, starting with the same initial

slow growth period observed in group HP / HL, followed by a faster period between T1 and T2.

Then, weight gain slowed between T3 and T4 reaching the level observed in the first period.

Finally, between T4 and T5 growth increased, as seen in group HP / HL.

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Results and discussion Chapter I

A 30 310
16.46 °C 18.68 °C 21.02 °C 16.42 °C 22.04 °C
260
25
Temperature (⁰C)

210

Weight (g)
20 160
T5*

T4* 110
15
T3*
T2 60
T1
10
T0 10
0 200 Days 400 600

B 30 26
16.46 °C 18.68 °C 21.02 °C 16.42 °C 22.04 °C

T5* 24
25 22
T4*
Temperature (⁰C)

20

Length (cm)
20 T3* 18

T2 16

15 14
T1
T0 12

10 10
0 200 400 600
Days
: HP / HL : LP / HC : water temperature

Figure 1: weight gain (A) and length increase (B) of C. labrosus reared in a RAS system using two

commercial feeds during 686 days, alongside daily water temperature. HP / HL: high protein / high lipid;

LP / HC: low protein / high carbohydrate. The temperatures shown are the averages of each period

between samplings. Asterisks express significant differences between experimental groups at each

sampling time (p < 0.05).

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Results and discussion Chapter I

HP / HL feed also provided the best SGR in comparison with group LP / HC at the end of

the experiment and at every individual sampling (Table 3). VSI values between groups were not

significantly different at T5, while HSI was significantly higher in group LP / HC at T5. K values

were also significantly higher in group LP / HC at the end of the experiment.

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Results and discussion Chapter I

Table 3: growth performance and biometrical parameters of C. labrosus reared in a RAS system using two commercial feeds during 686 days.

T0 (day 0) T1 (day 91) T2 (day 197) T3 (day 365) T4 (day 505) T5 (day 686) Total (day 0 – 686)
Group HP / LP / HC HP / HL LP / HC HP / HL LP / HC HP / LP / HP / LP / HP / LP / HP / LP /
HL HL HC HL HC HL HC HL HC
26.28 ± 27.66 ± 40.38 ± 39.62 ± 71.48 ± 67.26 ± 126.77 ± 102.15 167.21 ± 119.34 ± 259.83 ± 175.84 ±
AW - -
5.82 6.92 9.37 12.21 15.01 14.44 30.43* ± 25.74 40.66* 27.60 71.64* 38.85
12.70 ± 12.79 ± 14.16 ± 14.24 ± 16.73 ± 16.45 ± 20.03 ± 18.48 ± 21.90 ± 19.39 ± 25.13 ± 21.46 ±
AL - -
0.99 1.04 1.05 2.94 1.10 1.17 1.44* 1.14 1.55* 1.44 1.89* 1.56
WG - - 14.10 11.96 31.10 27.64 55.29 34.89 40.43 17.19 92.76 56.50 233.68 148.18
LI - - 1.46 1.45 2.57 2.21 3.30 2.03 1.87 0.91 3.23 2.07 12.43 8.67
SGR - - 0.57 0.48 0.63 0.58 0.40 0.30 0.29 0.16 0.40 0.34 0.45 0.36
13.94 ± 11.88 ± 15.22 ± 13.05 ± 13.82 ± 11.89 ± 15.21 ± 12.20 ± 15.43 ± 13.02 ±
VSI - - - -
2.22 2.96 0.90* 1.83 1.51* 1.75 2.90* 2.05 0.92 3.26
2.64 ± 3.13 ± 2.44 ± 2.67 ± 1.94 ± 2.14 ± 2.06 ± 1.84 ± 1.34 ± 1.99 ±
HSI - - - -
0.92 1.34 0.38 0.55 0.34 0.52 0.51 0.46 0.28 0.25*
1.26 ± 1.30 ± 1.41 ± 1.39 ± 1.50 ± 1.49 ± 1.55 ± 1.60 ± 1.56 ± 1.61 ± 1.60 ± 1.76 ±
K - -
0.12 0.11 0.22 0.15 0.97 0.16 0.15 0.22 0.14 0.12* 0.16 0.16*
Significant differences among experimental groups at each sampling time are marked with an asterisk (*) (p < 0.05). HP / HL: high protein / high lipid; LP / HC: low protein /

high carbohydrate; AW = Average Weight (g); AL = Average length (cm); WG = Weight Gain (average g /fish); LI = Length Increase (average cm / fish); SGR = Standard Growth

Rate; VSI = Viscerosomatic Index; HSI = Hepatosomatic Index; K = Fulton condition factor.

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Results and discussion Chapter I

Muscle protein did not show any significant changes between groups or respect to T0 at

the end of the experiment (Table 4). Total lipid content was similar in both groups at T5 (22 vs

21 %), higher than at T0 (14 %). Thirty-three fatty acids where detected in total, the most

abundant ones being the C16:0 palmitic acid, the C18:1 oleic acid and C18:2 linoleic acid (LA) in

both groups and at T0. In terms of the main fatty acid groups, the content of saturated and

unsaturated fatty acids was similar in both treatments at T5, but group LP / HC was richer in

polyunsaturated fatty acids (PUFA), both relatively to the lipid extract and in absolute content

in muscle. The absolute content of ω-3 PUFA was also higher in the LP / HC group, as well as

both the eicosapentaenoic (EPA) and docosahexaenoic (DHA) essential fatty acids. The ω-3 / ω-

6 fatty acid ratio was also higher in the LP / HC group.

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Results and discussion Chapter I

Table 5: total protein and lipid contents and summary of fatty acid profiles (% of lipid extract and % in

muscle) of muscle of C. labrosus reared in a RAS system using two commercial feeds during 686 days, at

the beginning (T0) and end (day 686) of the experimental period. Full fatty acid profiles can be found in

the Appendix (Table A2).

T0 T5 (Day 686)
(Day 0) HP / HL LP / HC
Total protein (%) 71.5 ± 1.43 69.5 ± 1.39 71.3 ± 1.43
Total lipid (%) 14 22 21
Summary of fatty acid profile % of % of % of % of % of % of
lipid muscle lipid muscle lipid muscle
C16:0 37.51 5.25 24.58 5.41 27.28 5.73
C18:1 10.70 1.50 41.64 9.16 32.19 6.76
C18:2 1.17 0.16 11.45 2.52 15.29 3.21
C20:5 (EPA) 1.37 0.19 0.37 0.08 1.03 0.22
C22:6 (DHA) 0.23 0.03 0.51 0.11 1.37 0.29
Total SFA 55.21 7.73 32.15 7.07 33.61 7.06
Total MUFA 40.25 5.63 52.86 11.63 43.35 9.10
Total PUFA 4.54 0.64 14.98 3.30 23.04 4.84
Σ ω-3 PUFA 1.85 0.26 2.61 0.57 5.59 1.17
Σ ω-6 PUFA 1.43 0.20 12.15 2.67 16.47 3.46
EPA + DHA 1.60 0.22 0.88 0.19 2.40 0.50
ω-3 / ω-6 1.29 1.29 0.21 0.21 0.34 0.34
HP / HL: high protein / high lipid; LP / HC: low protein / high carbohydrate; SFA: Saturated Fatty Acids;

MUFA: Monounsaturated Fatty Acids; PUFA: Polyunsaturated Fatty Acids.

Liver samples stained with haematoxylin-eosin showed the normal structure of a healthy

liver in both experimental treatments. However, hepatocyte vacuolization was observed mainly

in the group LP / HC (Figure 2 A, B). However, no nuclei displacement towards the periphery of

the cell was noticed. The ORO staining of liver showed the same pattern, as livers of both groups

were full of neutral lipid droplets (Figure 2 C, D). Proximal intestine samples also presented

normal structure, without signs of inflammation (Figure 2 E, F). Lipid droplets were present in

enterocytes of proximal intestine stained with ORO staining (Figure 2 G, H), but no observable

differences were found. Other histopathological alterations were not observed.

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Figure 2: micrographs of liver (A, B, C, D) and proximal intestine (E, F, G, H) of C. labrosus reared in a RAS

system using two commercial feeds during 686 days. A, C, E, G: HP / HL group; B, D, F, H: LP / HC group.

A, B, E, F: haematoxylin-eosin staining; C, D, G, H: ORO staining. Asterisks indicate examples of vacuolated

hepatocytes. Arrows indicate lipid droplets.

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Results and discussion Chapter I

4. Discussion

Grey mullets are considered a promising group of fish for the diversification of aquaculture,

mainly for their omnivorous feeding habits (Abellán-Martínez & Arnal-Atarés, 2013;

www.diversifyfish.eu), which gives them a great potential for the usage of innovative feed

ingredients directed towards an aquaculture feeding less reliant on wild fisheries (Naylor et al.,

2009; Bostock et al., 2010; Tacon & Metian, 2015). Therefore, the aim of this research was to

assess the growth of juvenile C. labrosus on the long-term, fed two anisoproteic and anisocaloric

commercial feeds, to obtain information about its nutritional needs and long-term performance

in captivity.

In the present study, the best values of absolute growth parameters (weight gain, SGR)

were obtained with the HP / HL feed. Similar results have been obtained in other studies, where

diets high on both protein and lipids had provided the highest growth performance for this

species (Ojaveer et al., 1996; Amezcua et al., 2009; Altunok & Özden, 2017). In previous

nutritional research performed on C. labrosus, a wide size range of individuals has been used,

which complicates the comparisons of SGR values between studies. However, the length of the

present experiment allowed for the evaluation of C. labrosus growth at different sizes, and it

makes the results more easily comparable with those of the literature. Quirós-Pozo et al. (2024)

evaluated the growth of C. labrosus juveniles of an initial size of approximately 26 g (the initial

weight of the fish employed herein) when fed different lipid sources at different water salinities.

Due to the complexity of the cited experiment and the two variables tested, the authors

reported many different SGR values, but the ones calculated at salinities of 35 ppt (like in the

present research) ranged between 0.41 and 0.54, which are very similar to the ones obtained

herein at the period between T0 and T1 (0.48 and 0.57). However, when using fish close to a

weight of 46 g (comparable to the T1 – T2 herein), the SGR was higher (0.89 to 1.15) than the

present experiment (Vílchez-Gómez et al., 2017). At the T2 – T3 period, when fish had an initial

size close to 70 g, SGR values were 0.3 and 0.4, lower than the ones reported by García-Márquez

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Results and discussion Chapter I

et al. (2022) (0.6 to 0.7). At the next period (T3 – T4), for an initial size close to 100 g the fish

rendered SGR values of 0.16 and 0.29, which are lower than the values reported for similar fishes

(García-Márquez et al., 2023). Therefore, the overall growth performance was not very high with

any of the feeds tested herein, although it has to be taken into account that water temperature

varied greatly during the experiment, which directly affects SGR. Optimal water temperature for

C. labrosus rearing has been established at 22.8 °C (Sanz-Latorre et al., 2024), and most of the

aforementioned research kept water temperature close to that value, partially explaining the

differences in performance. Nevertheless, it is worth mentioning that differences in mean

weight between both groups started to be significant at the end of the first year of the

experiment (T3), a remarkable fact taking into account that most aquaculture feeding

experiments run for a maximum of 90 days (Teles et al., 2020). Thanks to the added value given

by the long-term design of the experiment and the high number of samplings performed

(despite of the lack of replicates), inaccurate conclusions that could have been made running

the experiment only in the short-term were avoided. For instance, taking only into account the

first 90 days no differences between groups would have been concluded.

The optimal protein and energy contents of the diet are crucial parameters when

designing any feed, and they have to be considered in conjunction in order to optimize the

growth and feed utilization (Ojaveer et al., 1996). A feed that contains a deficient amount of

energy in relation to the protein content will lead to the use of protein as an energy source

(Wilson & Halver, 1986; Vergara et al., 1999), a process that decreases the cost-effectiveness of

the feed, as protein is usually the most expensive part of it (Watanabe et al., 2002). As a result,

the feed has to contain an energy level that is enough to cope with the fish´s necessities and

allow the protein to be used for growing, a concept called the “protein sparing effect” of energy

(Watanabe et al., 2002). Ojaveer et al. (1996) concluded that the optimal protein / energy ratio

for C. labrosus should be around 19.72 mg protein / KJ, a value closer to our HP / HL diet than to

the LP / HC one. This, combined with the results presented herein, clearly state that C. labrosus

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is a fish that requires diets with high protein and energy contents. Conversely, the flathead grey

mullet Mugil cephalus is not able to make efficient use of diets with a protein content much

higher than 30 % (Abdel-Hakim et al., 2001; De et al., 2012). It seems that grey mullet feed must

be species specific, and therefore the need of considering each grey mullet species separately

when researching for aquaculture is strongly recommended, despite of having very similar

feeding habits and ecological roles in nature (Cardona, 2016; Whitfield, 2016).

Despite the unequal performances previously mentioned, no significant differences

were found in the perivisceral fat reserves between individuals of the two groups, supported by

the lack of significant differences in VSI at T5, or in the similar pattern of lipid accumulation

observed in ORO stained liver tissue sections at the end of the experiment. The higher values of

HSI observed at T5 on group LP / HC could indicate a higher carbohydrate and, especially, lipid

reserve accumulation in liver for this group (Serrano et al., 1992), and also that the energy

availability of both feeds was enough to cope with the needs of the fish, even though the main

energy source were lipids in the HP / HL feed, and carbohydrates in the LP / HC feed. For this

reason, the better growth performance of group HP / HL is most plausibly attributable to the

higher protein content of the feed rather than to a “protein sparing effect”. This high lipid

accumulation found in both groups , accompanied by the high vacuolization of the hepatic

tissue, may indicate that both feeds could not be optimal for the species, as a high accumulation

of lipids in liver can pose adverse health effects (Lu et al., 2013), although no pathologies related

to it were observed. This pattern of high lipid accumulation in liver has been previously observed

in C. labrosus under culture conditions without producing any observable damage (Ojaveer et

al., 1996).

As far as total muscle lipid contents are concerned, they were higher at the end of the

experiment than at the beginning but no differences were found between feed groups. This is

remarkable because of the huge lipid content differences between the two feeds tested. It

seems that despite of the low lipase activity found on C. labrosus digestive tract (Pujante et al.,

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2017), this animal is able to assimilate and accumulate dietary lipids successfully (as proved

herein). Stimulation of lipogenesis and fatty acid synthesis under low lipid and high carbohydrate

dietary conditions has been observed in several fish species (Fynn-Aikins et al., 1992; Dias et al.,

1998; Alvarez et al., 2000; Wang et al., 2005), including the grey mullets M. cephalus (Talukdar

et al., 2020) and Liza ramada (El Sayed, 1991). Therefore, it is conceivable that C. labrosus can

convert dietary carbohydrates into lipids via de novo lipogenesis when fed on a low lipid and

high carbohydrate diet. This hypothesis is also supported by the previously mentioned lipid

accumulation in the liver of LP / HC group. In any case, the regulation mechanisms of such an

important metabolic pathway for the product quality of the fish should be thoroughly studied in

further research. A comprehensive analysis of the muscle lipids revealed that fish at the end of

the experiment, independently of experimental group, had higher contents of monounsaturated

fatty acids (MUFA) and PUFA than at T0, which is in concordance to other findings on cultured

fish (Amoussou et al., 2022). The group fed on LP / HC diet had a higher content of PUFA than

the HP / HL group. The fish fed on a diet rich in lipids showed lower muscle contents of ω-6 and

ω-3 PUFA than fish fed on a diet rich in carbohydrates, including docosahexaenoic (DHA) and

eicosapentsaenoic (EPA) acids. In vertebrates, the biosynthesis of ω-6 and ω-3 PUFA starts from

linoleic (LA) and α-linolenic (ALA) essential fatty acids, as they lack the Δ12 and Δ15 desaturases

needed for their synthesis (Kabeya et al., 2018). In C. labrosus, the presence of desaturases with

Δ5 and Δ6 activities have previously been reported (Garrido et al., 2019; Galindo et al., 2021),

so, the synthesis of ω-3 and ω-6 PUFAs by the LP / HC group could be expected, as both LA and

ALA acids were present in the feed as starting points for their respective metabolic pathways. A

key aspect of the lipid metabolism observed in the present work was the ability of the LP / HC

group of accumulating LA and ALA to higher levels than those found on the feed and the HP / HL

group, while at the same time, using them for ω-3 and ω-6 PUFA biosynthesis. The more efficient

use of PUFA under a low lipid ingestion regime has to be considered when designing a feed for

C. labrosus to decrease the use of fish oil, which is the traditional source of ω-3 PUFA. Another

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relevant aspect to consider is the higher ω-3 / ω-6 fatty acid ratio found in the muscle of LP / HC

group, indicative of a better quality fatty acid profile for human consumption (Rabeh et al.,

2015).

As a final remark, very little research has been done about the optimal feed size for grey

mullets. To the authors´ knowledge, only one paper has been published in this area, on M.

cephalus (Ramos-Júdez & Duncan, 2022). In that paper, it was concluded that grey mullets prefer

feed pellet sizes smaller than what would be expected in other species of the same size. A similar

observation has been made in the present experiment, as fish refused to eat the administered

pellets (2 mm in diameter) at the beginning, so it had to be ground to powder in order to make

it suitable for them, bearing in mind their relatively small mouth sizes. This is a clear indicator of

the importance of considering feed size to develop a suitable commercial feed for C. labrosus.

5. Conclusions

As far as we know, this is the longest published experiment (686 days) conducted to assess C.

labrosus rearing with commercial feeds. The fish responded well and were in good health status

considering the lack of mortality or any pathologies after long periods of maintenance under

culture conditions. Significant differences in growth started after the first year of the trial,

highlighting the importance of designing long-term experiments when researching new species

for aquaculture with little available data about growth regimes, although the disadvantages such

experiments have are evident, such as the high demands of time, money and effort, and the

great logistics difficulties. The best growth results and feed utilization were obtained with the

HP / HL feed, so the design of a specific feed for C. labrosus should be focused on feeds with

higher content of protein. When fed a diet low on lipids, with a low amount of essential fatty

acids and high on carbohydrates, C. labrosus could carry out de novo lipogenesis, leading to a

muscle fatty acid profile richer in PUFA and with higher ω-3 / ω-6 fatty acid ratio than when fed

on a high amount of lipids. These results are promising under the light of the development of a

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feed high on carbohydrates and low on fish oil, but more research would be required about the

regulation of fatty acid metabolism.

6. Acknowledgements

This research was funded by the Basque Government [33-2017-00250, 00007-INA2019-33,

00010-PIT2020-22].

7. References

7th Framework Programme of the European Commission. (2013). Diversify-eu. Exploring

the biological and socio-economic potential of new/emerging candidate fish species for

expansion of the European aquaculture industry (DIVERSIFY). Available online at:

http://www.diversifyfish.eu/ (accessed November 2021).

Abdel-Hakim, N. F., Bakeer, M. N., & Soltan, M. A. (2001). Effect of dietary protein levels

on growth performance and pond productivity of Nile tilapia (Oreocromis niloticus), eel (Anguilla

anguilla) and grey mullet (Mugil cephalus) reared in polyculture system. Egyptian Journal of

Aquatic Biology and Fisheries, 5(4), 61–84. https://doi.org/10.21608/ejabf.2001.1709

Abellán- Martínez, E., Arnal-Atarés, I. (2013). Múgiles, mújoles o mugílidos. In:

Ministerio de Agricultura, Alimentación y Medio Ambiente (Ed.), Diversificación de especies en

la piscicultura marina española, pp. 506-509.

Altunok, M., & Özden, O. (2017). Effect of dietary protein on the growth of mullet,

Chelon labrosus, reared in sea cages. Archives of Polish Fisheries, 25(3), 157–164.

https://doi.org/10.1515/aopf-2017-0015

Alvarez, M. J., Diez, A., Lopez-Bote, C., Gallego, M., & Bautista, J. M. (2000). Short-term

modulation of lipogenesis by macronutrients in rainbow trout (Oncorhynchus mykiss)

hepatocytes. British Journal of Nutrition, 84(5), 619-628.

https://doi.org/10.1017/S0007114500001951

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8. Appendix
Table A1: full fatty acid profile (% of lipid extract and % in feed) of the experimental feeds.

HP / HL LP / HC
Fatty acid profile % of lipid % of feed % of lipid % of feed
C4:0 0.00 0.00 0.05 0.00
C6:0 0.04 0.01 0.05 0.00
C11:0 0.04 0.01 0.14 0.01
C13:0 0.00 0.00 0.00 0.00
C14:0 2.12 0.42 1.66 0.10
C14:1 0.07 0.01 0.07 0.00
C15:0 0.18 0.04 0.17 0.01
C15:1 0.03 0.01 0.01 0.00
C16:0 15.99 3.20 16.00 0.96
C16:1 3.87 0.77 2.65 0.16
C17:1 0.28 0.06 0.20 0.01
C18:0 4.81 0.96 4.64 0.28
C18:1 trans 0.07 0.01 0.32 0.02
C18:1 42.74 8.55 38.55 2.31
C18:2 trans 0.10 0.02 0.13 0.01
C18:2 (LA) 14.50 2.90 22.08 1.32
C18:3 (GLA) 0.11 0.02 0.09 0.01
C18:3 (ALA) 4.27 0.85 5.91 0.35
C20:0 0.51 0.10 0.50 0.03
C20:1 2.66 0.53 1.79 0.11
C20:2 0.61 0.12 0.46 0.03
C21:0 0.04 0.01 0.10 0.01
C20:3 (DGLA) 0.16 0.03 0.17 0.01
C20:3 (ETE) 0.41 0.08 0.35 0.02
C20:4 0.18 0.04 0.07 0.00
C22:0 1.71 0.34 1.02 0.06
C20:5 (EPA) 1.57 0.31 0.93 0.06
C22:1 0.05 0.01 0.13 0.01
C22:2 0.00 0.00 0.03 0.00
C23:0 0.02 0.00 0.04 0.00
C24:0 0.10 0.02 0.10 0.01
C24:1 1.02 0.20 0.58 0.03
C22:6 (DHA) 1.75 0.35 1.00 0.06

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Total SFA 25.57 5.11 24.48 1.47

Total MUFA 50.78 10.16 44.30 2.66
Total PUFA 23.65 4.73 31.22 1.87
Σ α-3 PUFA 8.00 1.60 8.19 0.49
Σ α-6 PUFA 15.56 3.11 22.90 1.37
EPA + DHA 3.31 0.66 1.93 0.12
α-3 / α-6 0.51 0.51 0.36 0.36
HP / HL: high protein / high lipid; LP / HC: low protein / high carbohydrate; SFA: Saturated Fatty Acids;
MUFA: Monounsaturated Fatty Acids; PUFA: Polyunsaturated Fatty Acids; LA: linoleic acid; GLA: γ-linolenic
acid; ALA: α-linolenic acid; DGLA: dihomo-γ-linolenic acid; ETE: eicosatrienoic acid; EPA: eicosapentaenoic
acid; DHA: docosahexaenoic acid.

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Table A2: fatty acid profiles (% of lipid extract and % in muscle) of muscle of C. labrosus reared in a RAS
system using two commercial feeds during 686 days, at the beginning (T0) and end (day 686) of the
experimental period.

T0 T5 (Day 686)
(Day 0) HP / HL LP / HC
Fatty acid profile % of % of % of % of % of % of
lipid muscle lipid muscle lipid muscle
C4:0 0.05 0.01 0.00 0.00 0.00 0.00
C6:0 0.00 0.00 0.00 0.00 0.00 0.00
C11:0 0.06 0.01 0.02 0.00 0.01 0.00
C13:0 0.10 0.01 0.00 0.00 0.00 0.00
C14:0 9.29 1.30 3.22 0.71 3.04 0.64
C14:1 0.24 0.03 0.09 0.02 0.11 0.02
C15:0 2.44 0.34 0.41 0.09 0.25 0.05
C15:1 0.17 0.02 0.16 0.03 0.11 0.02
C16:0 37.51 5.25 24.58 5.41 27.28 5.73
C16:1 15.79 2.21 7.04 1.55 8.22 1.73
C17:1 1.33 0.19 0.37 0.08 0.26 0.06
C18:0 5.14 0.72 3.20 0.70 2.61 0.55
C18:1 transa 8.99 1.26 0.35 0.08 0.19 0.04
C18:1 10.70 1.50 41.64 9.16 32.19 6.76
C18:2 trans 1.26 0.18 0.23 0.05 0.99 0.21
C18:2 (LA) 1.17 0.16 11.45 2.52 15.29 3.21
C18:3 (GLA) 0.13 0.02 0.12 0.03 0.37 0.08
C18:3 (ALA) 0.24 0.03 1.55 0.34 2.76 0.58
C20:0 0.40 0.06 0.30 0.07 0.22 0.05
C20:1 2.07 0.29 2.33 0.51 1.17 0.25
C20:2 0.05 0.01 0.40 0.09 0.44 0.09
C21:0 0.01 0.00 0.04 0.01 0.00 0.00
C20:3 (DGLA) 0.08 0.01 0.05 0.01 0.08 0.02
C20:3 (ETE) 0.00 0.00 0.17 0.04 0.43 0.09
C20:4 0.00 0.00 0.11 0.02 0.26 0.05
C22:0 0.19 0.03 0.35 0.08 0.08 0.02
C20:5 (EPA) 1.37 0.19 0.37 0.08 1.03 0.22
C22:1 0.21 0.03 0.34 0.07 0.11 0.02
C22:2 0.00 0.00 0.02 0.00 0.03 0.01
C23:0 0.00 0.00 0.00 0.00 0.08 0.02

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C24:0 0.03 0.00 0.03 0.01 0.03 0.01

C24:1 0.75 0.11 0.55 0.12 0.99 0.21
C22:6 (DHA) 0.23 0.03 0.51 0.11 1.37 0.29
Total SFA 55.21 7.73 32.15 7.07 33.61 7.06
Total MUFA 40.25 5.63 52.86 11.63 43.35 9.10
Total PUFA 4.54 0.64 14.98 3.30 23.04 4.84
Σ n-3 PUFA 1.85 0.26 2.61 0.57 5.59 1.17
Σ n-6 PUFA 1.43 0.20 12.15 2.67 16.47 3.46
EPA + DHA 1.60 0.22 0.88 0.19 2.40 0.50
n-3 / n-6 1.29 1.29 0.21 0.21 0.34 0.34
HP / HL: high protein / high lipid; LP / HC: low protein / high carbohydrate; SFA: Saturated Fatty Acids;
MUFA: Monounsaturated Fatty Acids; PUFA: Polyunsaturated Fatty Acids; LA: linoleic acid; GLA: γ-linolenic
acid; ALA: α-linolenic acid; DGLA: dihomo-γ-linolenic acid; ETE: eicosatrienoic acid; EPA: eicosapentaenoic
acid; DHA: docosahexaenoic acid.

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Results and discussion Chapter II

Chapter II

Effects of stocking density on stress and

metabolism of the thicklip grey mullet Chelon

labrosus (Risso, 1827).

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Results and discussion Chapter II

Congress:

Sanz-Latorre, M., Conlledo, N., Lekube, X., Izagirre, U., Sudupe, R., Soto, M. (2022, November
21-24). Efectos de la densidad de cultivo en niveles de estrés del múgil Chelon labrosus. [Oral
presentation]. XVIII Congreso Nacional de Acuicultura, Cádiz, Spain.

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List of abbreviations
FCR: feed conversion ratio

GAS: general adaptation syndrome

HD: High density

H/E: haematoxylin / eosin

HSI: hepatosomatic index

K: Fulton condition factor

LD: low density

MD: medium density

SGR: specific growth rate

VSI: viscerosomatic index

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Results and discussion Chapter II

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Results and discussion Chapter II

Abstract

Stocking density is one of the most important stressors specific to aquaculture, and the

identification of its effects on a novel species for intensive culture is of upmost importance for

the development of adequate rearing protocols, that optimize farm productivity while ensuring

animal welfare. Chelon labrosus is an interesting omnivorous teleost for the diversification of

European aquaculture, and the aim of this research was to assess the effects of different

stocking densities on C. labrosus individuals at the grow-out phase. With such a purpose, three

groups of fish (initial weight 219.47 ± 65.30 g) were kept at stocking densities of 5 (LD, Low

Density), 10 (MD, Medium density) and 25 (HD, High Density) kg / m3 for 100 days, and growth,

feed utilization, tissue (liver, muscle and plasma) metabolites and liver histo(patho)logical

endpoints were obtained. Growth and feed utilization were similar in the LD and MD groups,

and remarkably lower in the HD group, suggesting a stress response at this density. The

prevalence of some red areas in the skin and fins was higher in the HD group. Surprisingly, fish

maintained at high densities exhibited heavier livers, with higher triglyceride concentration and

marked hepatocyte vacuolization, which could indicate an energy accumulation strategy in

crowded situations. It can be concluded that C. labrosus starts suffering mild stress at densities

of 25 kg / m3 that, although not severe, may affect and compromise fish health in the long term.

Hence, it is not recommended to exceed this stocking density in farming conditions.

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Results and discussion Chapter II

Laburpena

Stock dentsitatea akuikulturako estresatzaile espezifikoen artean garrantzitsuenetakoa da, eta

espezie berritzaile batengan dauzkan eraginak identifikatzea lehentasuna da hazkuntza

protokolo egokiak diseinatzeko orduan, animalien ongizatea bermatuz ekoizpena optimizatzen

dutenak. Chelon labrosus europar akuikulturaren dibertsifikaziorako espezie orojale

interesgarria da. Ikerketa honen helburua stock dentsitate ezberdinek hazkuntza fasean dauden

C. labrosus banakoengan dauzkaten eraginak aztertzea zen. Helburu honekin, hiru arrain talde

(hasierako pisua 219.47 ± 65.30 g) 5 (LD, dentsitate baxua), 10 (MD, dentsitate ertaina) eta 25

(HD, dentsitate altua) kg / m3 -tan mantendu ziren 100 egunetan zehar eta hazkuntza, pentsu

erabilera, ehun (gibel, gihar eta plasma) metabolito eta gibel histo(patho)logiari lotutako

parametroak neurtu ziren. Hazkuntza eta pentsu erabilera antzekoak izan ziren LD eta MD

taldeetan, eta txikiagoak HD taldean, dentsitate honetan estres erantzuna egon zenaren seinale.

Azal eta hegatsetako gorriduren prebalentzia handiagoa izan zen HD taldean. Dentsitate

altuenean mantendutako arrainek gibel astunagoak zituzten, triazilglizerol kontzentrazio

handiagodunak eta hepatozito bakuolizazio nabarmendunak, dentsitate altuen aurreko energia

metaketa estrategia bat adieraz dezakeena. Ondoriozta daiteke C. labrosus-ek estres arina

sufritzen duela 25 kg / m3-tan, nahiz eta oso larria ez izan, epe luzera arrainen osasuna kalte

lezakeena. Beraz, ez da gomendagarria stock dentsitate hau gainditzea akuikultura baldintzetan.

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Results and discussion Chapter II

1. Introduction

The rapid expansion that the aquaculture sector is experiencing since the end of the 20th

century is essential to cope with the increasing need of aquatic products for a constantly growing

global population (FAO, 2022). The concomitant increase of feed consumption by this industry

and the continuous use of fishmeal and fish oil as feed ingredients, although their inclusion levels

in aquafeeds have been significantly lowered and substituted by vegetal ingredients, still raises

the question of whether or not aquaculture helps relieve pressure from wild fisheries (Naylor et

al., 2000; 2009). In this light, research about the culture of novel fish species feeding on low

trophic levels that could use alternative vegetal feed ingredients more efficiently than

carnivorous ones is of upmost importance in order to ensure a sustainable growth of

aquaculture production (Naylor et al., 2000).

The thicklip grey mullet Chelon labrosus is an omnivorous teleost common to the coastal

areas of the Atlantic coast of Europe and the Mediterranean basin (Turan, 2016), which is an

interesting candidate for diversifying European aquaculture (Altunok & Özden, 2017; García-

Márquez et al., 2022). Most research regarding the intensive culture of this species has dealt

with its nutrition (Ojaveer et al., 1996; Pujante et al., 2015; 2017) and the development of

specific feeds (de las Heras et al., 2012; Vílchez-Gómez et al., 2017; García-Márquez et al., 2022;

2023), but other aspects relevant for aquaculture have received less attention. One of the most

important aspects to address when targeting a novel species for intensive production is its

response to the stressors that are specific to aquaculture, in order to develop adequate rearing

protocols that not only provide the best growth results, but also ensure animal welfare (Conte,

2004). In intensive aquaculture, fish are routinely faced with stressful stimuli that would be rare

or non-existent at all in their natural habitat, like handling, transport, accumulation of

nitrogenous waste, low oxygen levels or high stocking densities (Barton & Iwama, 1991; Conte,

2004). The latter is an important parameter in aquaculture with a remarkable impact on fish

growth performance (Li et al., 2021). Density can affect the health status and welfare of the

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animals, from altering their normal social structure, to negatively influencing water quality

parameters such as the aforementioned waste accumulation and lowering oxygen availability

(Barton & Iwama, 1991; Ellis et al., 2002; Conte, 2004).

The concept of stress is controversial, and several definitions appear in the literature

(Barton & Iwama, 1991; Wendelaar Bonga, 1997). However, in the context of teleost fish it can

be broadly defined as a condition where homeostasis of the organism is threatened or altered,

caused by either internal or external stimuli (Wendelaar Bonga, 1997). These stimuli, also called

stressors, elicit a response by the animal, commonly characterized by a rapid physiological

response, a resistance phase where the animal compensates the effects of the stressor in order

to regain homeostasis, and an exhaustion phase if the stress is too severe or long lasting. This is

defined as the General Adaptation Syndrome or GAS (Selye, 1950). In this process, several

responses can be activated that affect the animal at different organizational levels, classified as

the primary, secondary and tertiary stress responses (Barton & Iwama, 1991; Wendelaar Bonga,

1997). Primary responses consist on the activation of the hypothalamic-sympathetic-chromaffin

cell axis, which releases catecholamines to the bloodstream, and the hypothalamic-pituitary-

interrenal axis, that releases corticosteroids, most importantly, cortisol. The immediate effects

of these hormones at tissue level are the secondary responses, like the mobilization of energy

reserves. The tertiary responses are the ones manifesting at the organism or even population

level, which mainly are the reduction or inhibition of growth, reproductive capacity and immune

response (Wendelaar Bonga, 1997). Many methods exist for the measurement of stress,

targeting different organization levels, and stress measurement is a useful way to assess fish

welfare.

In the case of C. labrosus, the effects of stocking density on stress, growth and

metabolism have been investigated in individuals weighing less than 1 g (de las Heras et al.,

2015), but there is a lack of information about more advanced developmental stages. Therefore,

the aim of this research was to assess the effects of stocking density on C. labrosus individuals

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at the grow-out phase, from the perspectives of productivity and animal welfare, focusing on

secondary (tissue metabolite levels and liver histopathology) and tertiary (growth, feed

utilization) stress responses. The knowledge about the effects of stocking density on this species

will be key for ensuring animal welfare in aquaculture.

2. Materials and methods

2.1. Experimental design

The experimental fish were taken from the thicklip grey mullet (C. labrosus) stock of Kardala LHI

aquaculture school (Mutriku, Spain). The stock consisted on wild individuals captured in 2018

and held in said facilities until March 2021, when the experiment started. This time allowed the

fish to acclimate to captivity and to compound feeds.

Fish from the C. labrosus stock at Kardala LHII aquaculture school (Mutriku, Spain) were

acclimated in one seawater open-flow tank, with a density of 12 kg/m3. At the beginning of the

experiment (T0) the fish (n = 187, initial length 23.22 ± 2.44 cm, initial weight 219.47 ± 65.30 g)

were separated in three experimental groups: low (LD; 5 kg/m3; n = 24), medium (MD; 10kg/m3;

n = 48) and high (HD; 25 kg/m3; n = 115) density at three 1000 L recirculating (RAS) seawater

tanks, equipped with a decantation tank, mechanical, sand and biological filters and a protein

skimmer. Water temperature was maintained at 20 °C, the photoperiod was set to match the

natural day/night cycle and the tanks were aerated to keep similar levels of oxygen in all of them,

close to 6 mg / L. The fish were fed 1% of their body weight 5 days a week using a commercial

feed designed for tilapia with 35 % protein and 6 % lipids (Dibaq, Segovia, Spain). The experiment

lasted 100 days.

2.2. Sample collection

Biometrical data (fork length and body weight) were collected at days 0 (T0), 42 (T1), 70 (T2) and

100 (T100). For handling (weighing and measuring), fish were caught with a net and introduced

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Results and discussion Chapter II

in a water bath containing 100 mg / L Tricaine methanesulfonate MS 222 (Sigma-Aldrich, Saint

Louis, USA) anaesthetic. For sacrifice, the fish were introduced at a water bath of the same

compound at a concentration of 300 mg / L.

All fish were measured to the last mm (fork length) and weighed to the last mg at the

beginning (T0) and end (T100) of the experiment. In T0 10 fish from the acclimation tank were

sacrificed and dissected, and 10 from every group at T100. Blood was collected from the caudal

vein with a heparinized 1 mL syringe with a 25 G needle. After performing glucose, lactate and

triglyceride analysis (See Section 2.4), blood was centrifuged for 15 minutes at 10000 rpm. The

plasma was collected, flash frozen in liquid nitrogen and stored at - 80 °C for metabolite analysis.

After extraction and weighing, a central section of the liver was fixed in formalin for further

histological procedures, and the remaining parts were flash frozen at -80 °C for the

measurement of metabolites, as well as a small portion of white muscle.

At the final sampling, some red areas were noticed at the ventral side of several fish

(Figure 1). These potential lesions were denominated as “light redness” when only the pelvic

fins were affected (Figure 1 A), and “severe redness” when pelvic fins and the visceral area were

noticeably red (Figure 1 B). The prevalence of such potential lesions was recorded.

Figure 1: examples of light (A) and severe (B) cutaneous redness on the visceral area and pelvic fins of C.

labrosus. Both images correspond to the fish held at the highest density (HD; 25 kg/m3).

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2.3. Calculations

The following calculations were made:

- Weight gain (WG) = Final weight (g) - initial weight (g)

𝐿𝐿𝐿𝐿 (𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡)− 𝐿𝐿𝐿𝐿 (𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡)

- Specific growth rate (SGR) = 100 x (% / day)
𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝑓𝑓𝑓𝑓𝑓𝑓

𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔/𝑓𝑓𝑓𝑓𝑓𝑓ℎ

- Feed conversion ratio (FCR) =
𝑊𝑊𝑊𝑊

𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚
- Viscerosomatic index (VSI) = 100 x 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚
(%)

𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚
- Hepatosomatic index (HSI) = 100 x 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 (%)

𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚
- Fulton condition factor (K) =
𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿ℎ3

2.4. Metabolite analyses

Blood glucose, lactate and triglycerides were measured immediately after extraction (n = 10)

using the Accutrend Plus (Roche, Basel, Switzerland) blood analyser, adapting manufacturer´s

guidelines for its use on fish: after introducing the reactive strip into the meter, a drop of blood

was placed in the reactive part using the extraction syringe. The rest of the protocol was

unchanged.

The rest of the metabolites were measured using commercial kits following the

instructions from the manufacturers. Liver glycogen was measured using ab169558-Glycogen

Assay Kit II (Colorimetric) (Abcam, Cambridge, UK), liver and muscle tryglicerides with Ab65336-

Tryglyceride Assay Kit (Abcam, Cambridge, UK), liver, muscle and plasma protein with Pierce BCA

Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA) and liver and muscle free amino acids

with Total Amino Acid (T-AA) Colorimetric Assay Kit (MyBioSource, San Diego, USA).

2.5. Histological analysis

Liver samples were fixed in a 10 % formalin solution buffered with seawater for 24 hours

(Martoja et al., 1970). Once fixed, formalin was replaced by 70 % ethanol until the processing.

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The samples (n = 10) were processed with an automated tissue processor (LEICA ASP 300S; Leica

Microsystems Nussloch GmbH, Germany) and embedded in paraffin wax blocks. Five µm thick

sections were cut with a microtome (Leica RM2125RTS; Leica Microsystems Nussloch GmbH,

Germany), stained with haematoxylin-eosin (Martoja et al., 1970) using an Autostainer XL (Leica

Microsystems Nussloch GmbH, Germany), covered by a coverslip and air-dried. The samples

were observed under an Olympus BX61 light microscope (Olympus-Lifescience, Tokyo, Japan).

2.6. Statistical analysis

Normality of the data was tested using the Shapiro-Wilk test and the homogeneity of variances

with Levene´s test. Weight, fork length, VSI, HSI, K and tissue metabolite levels (dependent

variables) were compared among groups (independent variable) using the one-way ANOVA or

Kruskal-Wallis tests (p < 0.05), depending on the normality and homogeneity of variances of the

data. All the statistical analysis were made with the IBM SPSS Statistics 27 software (IBM corp.

2020).

2.7. Ethical statement

All experimental procedures complied with the Guidelines of the European Union (2010/63/UE)

and the Spanish legislation (RD53/2013 and law 32/2007) for the handling and use of laboratory

animals under the supervision and acceptance of the Ethics for experimentation and animal

welfare committee of the University of the Basque Country (UPV/EHU) (CEEA M20-2018-133).

Kardala LHI aquaculture school is registered in the registry of agrarian exploitations of

Gipuzkoako Foru Aldundia (20 056 003 0475).

3. Results

Only one fish died during the experimental period in the HD group from unknown causes

(mortality rate= 0.9 %. At the end of the experiment, the mean weight gain was the highest at

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the LD group, closely followed by the MD group (Table 1). Concomitantly, SGR and FCR were

very similar in the LD and MD groups, and the values from HD group were very different, SGR

being almost half the value of the other groups and FCR almost double. HSI was lower than at

T0 in every group at the end of the experimental period but no significant differences were

found between groups, although group HD showed higher values. VSI and K values were not

significantly different between groups or sampling times.

Table 1: biometrical measurements, growth performance and feed utilization of C. labrosus reared at

different stocking densities (LD, MD, HD) for 100 days.

T0 T100
Acclimation tank LD MD HD
D (kg / m3) 12 5.61 10.13 27.29
W (g / fish) 219.47 ± 65.30 243.78 ± 68.54 230.25 ± 48.82 239.41 ± 80.19
WG (g / fish) - 26.20 23.35 14.30
FL (cm) 23.22 ± 2.44 23.86 ± 2.36 23.40 ± 1.68 23.80 ± 2.41
HSI 1.09 ± 0.42a 0.88 ± 0.15b 0.91 ± 0.13b 1.06 ± 0.24ab
VSI 13.06 ± 2.30 11.08 ± 2.55 12.46 ± 1.62 11.57 ± 2.43
K 1.74 ± 0.31 1.77 ± 0.20 1.77 ± 0.13 1.74 ± 0.41
SGR - 0.17 0.16 0.09
FCR - 5.00 5.44 9.02

D: stocking density; W: weight; WG: weight gain; FL: fork length; HSI: hepatosomatic index; VSI: viscero-

somatic index; K: Fulton condition factor; SGR: specific growth rate; FCR: feed conversion ratio; T0: day 0;

T100: day 100; LD: low density, 5 kg / m3; MD: medium density, 10 kg / m3; HD: high density, 25 kg / m3.

Groups with different superscript letters show statistically significant (p < 0.05) differences.

No clear pattern was found in the incidence of light redness depending on the stocking

density (Figure 2). However, there was a noticeable increase in the prevalence of severe redness

lesions alongside the increase in stocking density.

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Results and discussion Chapter II

Prevalence of skin redness (% individuals)

60

50

40

30

20

10

0
LD MD HD

Light redness Severe redness

Figure 2: prevalence of skin redness of C. labrosus reared at different stocking densities for 100 days at

the end of the experiment.

Few statistically significant differences were found in the tissue metabolite levels (Table

2). Plasma protein levels at the MD group were higher than at T0 or LD, and HD group was not

different to any other. Plasma triglycerides at T0 were higher than in every experimental group,

and among these, HD group had non-significantly higher values. In the liver, only triglyceride

levels showed significantly higher values in HD group.

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Results and discussion Chapter II

Table 2: plasma, liver and muscle metabolites of C. labrosus reared at different stocking densities for 100

days at the beginning (T0) and at the end (T100) of the experimental period.

T0 T100
Acclimation tank LD MD HD
Plasma
Lact (mmol / L) 2.77 ± 0.98 2.66 ± 0.81 2.03 ± 0.38 2.25 ± 0.59

Prot (mg / mL) 54.64 ± 8.73b 53.90 ± 10.40b 69.20 ± 6.75a 58.39 ± 11.92ab

Glu (mg / L) 115.10 ± 37.46 154.44 ± 23.13 138.20 ± 32.67 107.44 ± 56.37

TAG (mg / L) 384.50 ± 118.29a 223.00 ± 53.20b 236.90 ± 42.48b 272.67 ± 114.47ab

Liver
Prot (mg / g) 254.87 ± 134.02 328.52 ± 137.26 221.31 ± 81.68 363.25 ± 162.22
AA (mmol aa / g protein) 1.90 ± 1.04 2.05 ± 1.29 2.40 ± 0.82 1.60 ± 1.18
Gly (mg / g) 79.29 ± 35.29 50.34 ± 24.45 47.91 ± 29.76 40.70 ± 14.75

TAG (mg / g) 64.58 ± 37.44b 88.95 ± 49.95ab 69.08 ± 43.45b 135.62 ± 54.49a

Muscle
Prot (mg / g) 410.90 ± 98.10 570.54 ± 245.18 578.13 ± 188.59 598.85 ± 337.05
AA (mmol aa / g protein) 0.29 ± 0.10 0.31 ± 0.16 0.26 ± 0.06 0.36 ± 0.34
TAG (mg / g) 8.76 ± 7.58 5.76 ± 4.11 7.01 ± 5.74 14.88 ± 17.02

Lact: lactate; Prot: protein; Glu: glucose; TAG: triglycerides; AA: free amino acids; Gly: glycogen; LD: low

density, 5 kg / m3; MD: medium density, 10 kg / m3; HD: high density, 25 kg / m3. Groups with different

superscript letters show statistically significant (p < 0.05) differences.

All the analysed livers showed the normal histological structure (Figure 3 A). This is

comprised of a thin capsule of connective tissue surrounding the hepatic parenchyma, which

can be described as a compact field of hepatocytes, the main cell type present in the liver, highly

vascularised by blood vessels and sinusoidal capillaries. Melanomacrophage centres were

numerous and large in all samples. At T0, a high interindividual variability in the vacuolization

level of the hepatocytes was noticed. Highly vacuolated hepatocytes with a single large vacuole

displacing the nucleus towards the periphery were present in several samples, but this condition

was never generalized to the entire tissue (Figure 3 B). At LD and MD groups, vacuolization level

was generally low (Figure 3 C), and in group HD it was higher, but degenerative hepatocytes

were rare (Figure 3 D).

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Results and discussion Chapter II

Figure 3: micrographs of liver of C. labrosus reared at different stocking densities for 100 days. A: normal

structure of C. labrosus liver. B: liver area with highly vacuolated hepatocytes, hepatocytes with a single

large vacuole displacing the nucleus towards the periphery. C: hepatocytes with low vacuolization. D:

highly vacuolated hepatocytes. Arrows: melanomacrophage centres; Arrow points: bile duct; Asterisks

(*): blood vessels. A, C: LD group; B: T0; D: HD group. H/E. Scale bars: 20 µm.

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4. Discussion

Stocking density can have a profound impact on growth and stress of cultured fish (Barton &

Iwama, 1991), but in practical farming conditions the effects of crowding and the deterioration

of water quality parameters, which can be a direct consequence of the high stocking density,

are hard to separate (Conte et al., 2004). In fact, there is evidence pointing towards the latter

being more relevant than the stocking density itself (Ellis et al., 2002). In order to avoid said

confounding factor, water quality parameters (oxygen and ammonia concentrations) were

maintained at the same conditions during the present experiment.

Tertiary stress responses that manifest at the individual level like inhibition of growth,

reduction of food intake and assimilation, the impairment of reproduction, the reduction of

immunocompetence or increased mortalities (Barton & Iwama, 1991; Wendelaar Bonga, 1997)

are the ones directly affecting the economic viability of fish farming. Therefore, they are

arguably the most interesting for aquaculturists and, consequently, are addressed in most

studies of this field. In the present research, a clear reduction of weight gain of C. labrosus was

observed with increasing stocking density from 5 to 25 kg / m3, this reduction being especially

pronounced from 10 to 25 kg / m3. Concomitantly, SGR and FCR values of the high density group

were remarkably worse than those of the low and medium density groups. It has been previously

reported that reduced growth rates and feed efficiencies are a common effect of high stocking

densities, as seen in gilthead sea bream (Montero et al., 1999; Sangiao-Alvarellos et al., 2005),

African catfish (Dai et al., 2011), channel catfish (Refaey et al., 2018), Shire tilapia (M´balaka et

al., 2012), turbot (Irwin et al., 1999) and the grey mullet Mugil platanus (Sampaio et al., 2001).

However, the critical stocking densities are species-specific and, consequently, difficult to

compare. Likely, the flathead grey mullet Mugil cephalus has shown clear signs of stress at

crowded conditions according to Fazio et al. (2014; 2017), although the authors did not report

any growth data, and the effects of density and poor water conditions are hard to separate in

the cited studies. However, some species exhibit the opposite response to stocking density and

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perform better in crowded conditions, like European sea bass (Papoutsoglou et al., 1998) or

Arctic charr (Jørgensen et al., 1993). This seems to be related to the schooling behaviour these

species have in natural conditions (Papoutsoglou et al., 1998), but this kind of response cannot

be generalized to every species with tendency to form schools (Sampaio et al., 2001). In the only

published study about this topic on C. labrosus juveniles (initial weight around 0.5 g), similar

results to the present ones were obtained, as growth was reduced with increasing stocking

density from 0.7 to 6.7 kg / m3 (de las Heras et al., 2015). Great differences exist between that

paper and this study, both in the tested densities and fish sizes considered. In any case, the

general pattern of the response to crowding is consistent, suggesting that high stocking densities

elicit a stress response on C. labrosus and compromise growth processes. However, a density of

25 kg / m3 for individuals heavier than 200 g cannot be considered a severe stressor, as

evidenced by the fact that the fish continued growing, even if slowly, and that the mortality was

negligible during the experimental period.

The rash-like skin redness observed in several individuals can also be an indicative of

mild stress. As the incidence of light cutaneous redness lacks any pattern related to stocking

density, it is most probably due to individual variation rather to an effect of the studied stressor.

However, the incidence of severe redness is clearly affected by crowding, increasing in those

situations. Skin and fin lesions can be related to high stocking densities, although the ones

reported in the literature are usually more severe than the ones observed herein. Fazio et al.

(2014) found severe wounds and haemorrhages on M. cephalus skin when maintained at high

density conditions. The authors attributed these lesions to ammonia toxicity, as water quality

was not controlled during their experiment. Similarly, fin damage in salmon also resemble high

density conditions as a result of poor water quality, abrasion with other fish or the tank itself,

infections or aggression (Ellis et al., 2002). However, the photographs published by Fazio et al.

(2014) are very helpful examples of severe cutaneous lesions on a mugilid, and clearly suggest

that the ones found under present experimental conditions do not indicate a serious threat to

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animal welfare. Considering water quality was maintained at adequate levels, no pathologies

were found and aggressive behaviour has never been observed nor in the present experiment,

nor in all of our experience working with this species, abrasion with conspecifics or with the

rearing tank is the most probable explanation among the proposed ones. However, it is worth

mentioning that fish handling can cause lesions resembling skin ulcers, but the actual nature of

the lesion is hard to assess without a careful histopathological examination (Wolf et al., 2015).

Therefore, the present results have to be taken as preliminary data regarding skin lesions in C.

labrosus, and endpoints that are more specific should be considered in future research when

encountering similar conditions.

Despite the reduction of growth at high densities being the general trend on stressed

fish, the drivers behind that phenomenon are still unclear. A reduction of feed intake usually

accompanies high stocking densities, and it has been proposed as the direct cause of the growth

impairment (Ellis et al., 2002). This could be due to social interactions and dominance hierarchies

that would difficult feed accessibility for subordinate fish, as dominant individuals could

monopolise the feeding areas (Grand & Grant, 1994; Alanärä & Brännäs, 1996; McCarthy et al.,

1999; Boujard et al., 2002), although a general appetite reduction, independent of dominance

behaviour, has also been reported (Boujard et al., 2002). A usual effect of unequal distribution

of the feed is the increased size dispersion inside the tank (Boujard et al., 2002), which did not

happen in this experiment, as weight deviations were similar at the beginning and at the end of

the trial in all three groups. This, coupled with the lack of alterations in fish social behaviour

observed in the daily feeding, suggests that dominance relationships did not affect feed intake,

although, intake could not be measured. Future studies regarding this topic should address both

feed intake and social interactions in order to understand the mechanisms behind the observed

effects.

The aforementioned tertiary stress responses are the observable effects of the

metabolic readjustments an animal has to undergo in order to face a stressor, as energy is

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deviated from processes such as growth, reproduction or the immune response and directed

towards regaining homeostasis, threatened by the stressful situation. These readjustments are

part of the secondary stress responses, and are typically characterized by an increase in energy

and oxygen consumptions, which can result in the complete depletion of the reserves and,

ultimately, in death (Barton & Iwama, 1991; Wendelaar Bonga, 1997). Stocking density had little

effect on the measured metabolites in the present study. However, liver lipid reserves showed

a remarkable and surprising response to crowding, as triglyceride levels in the liver were the

highest in the group maintained at the highest density. This observation is backed by the non-

significantly higher HSI of this group, as well as by the large vacuolization of the tissue, showing

that livers were bigger and more lipidic. This, surprisingly, defies the expected situation under

stress (Wolf et al., 2015) and particularly crowding. Reduction of liver size and energy reserves

has been reported at several fish species held at high stocking densities and affected by low

growth rates, like gilthead sea bream (Montero et al., 1999) or brook charr (Vijayan et al., 1990).

Conversely, in the aforementioned study conducted by de las Heras et al. (2015) about stocking

density on C. labrosus juveniles, a similar response to the present one was observed. Therein,

higher HSI values were found in the fish held at the highest density, the ones with the poorest

performance. However, glycogen reserves seemed to be playing a more relevant role as energy

storage than triglycerides, driving liver size differences (de las Heras et al., 2015). In the paper

by de las Heras et al. (2015), a clear modulation of metabolism by cortisol was observed, as

specimens with higher plasma cortisol concentrations showed the highest growth rates and the

lowest liver energy reserves, indicating that fish at the lowest density had increased energy

expenditure, which optimized growth. Although primary stress responses like plasma cortisol

levels were not measured herein, a similar strategy than the one found by de las Heras et al.

(2015) can be inferred; the metabolism of C. labrosus is modulated in response to crowding

stress, lowering energy expenditure and accumulating energy reserves, and that this ability is

expressed at different life stages of the animal. This conclusion, though, could be challenged by

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different processes that could affect liver size. In a study performed with channel catfish (Refaey

et al., 2018) increasing HSI coupled with a higher catabolic activity of this organ was found at

high stocking densities and was accompanied by a higher circulating triglycerides and glucose in

plasma and lower fat content in muscle. In the present experiment, a slight raise in plasma

triglycerides was found in the HD group, but the levels of muscle triglycerides and perivisceral

fat reserves were similar between groups. These results reject the explanation of the

incremented energy mobilization, and further support the hypothesis of the energy

accumulation under crowding. The general framework of the General Adaptation Syndrome

(GAS) proposes three stages for the stress response, the alarm phase, resistance phase and the

exhaustion (Selye, 1950). In the present experiment, due to the long-term design of the trial, it

is conceivable that the initial alarm reaction and the concomitant physiological response to it

have been unnoticed. Therefore, the observed metabolic reorganization could correspond to

the resistance phase, where an altered resting state has been achieved to compensate the

negative effects of the stressor and regain homeostasis. However, this atypical response to

stress deserves further research, and this could help improve the understanding of metabolic

modulation in fish and the role of cortisol in such a process.

5. Conclusions

In conclusion, C. labrosus showed signs of mild stress in a stocking density of 25 kg / m3, with

effects only observable at the whole organism level, the tertiary stress responses: reduced

growth, minimized feed utilization and produced some skin and fin lesions, although severe

wounds or large haemorrhages were not found. Moreover, overall mortality was negligible. The

secondary stress responses, the metabolic readjustments caused by stocking density, revealed

a surprising strategy of liver energy reserve accumulation in the highest stocking density, mostly

in the form of triglycerides. This suggests a metabolic modulation towards reducing energy

expenditure under stressful situations. More research is required in order to understand the

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proper nature and the underlying mechanisms driving such response. For the aforementioned

reasons, it is recommended to keep stocking density of this species below 25 kg / m3 in the grow-

out phase. Deeper research and the improvement of the knowledge about the rearing of this

novel intensive aquaculture species, including the design of specific feeds, could help improve

its resistance to crowding stress and overall performance, allowing for higher stocking densities.

6. Acknowledgements

This research was funded by the Basque Government [00007-INA2019-33, 00003-INA2022-33,

00010-PIT2020-22].

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Results and discussion Chapter III

Chapter III

Effects of water temperature on growth, health

and digestive processes of the thicklip grey mullet

Chelon labrosus (Risso, 1827).

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Article:

Sanz-Latorre, M., Soto, M., Izagirre, U., & Lekube, X. (2024). Effects of water temperature on

growth, health and digestive processes of the thicklip grey mullet Chelon labrosus. Aquaculture,

741537.

Congress:

Sanz-Latorre, M., Lekube, X., Izagirre, U., Diaz de Cerio, O., Soto, M. (2023, September 18-21).

Effects of water temperature on growth and digestive processes of the thicklip grey mullet Chelon

labrosus. [Oral presentation]. Aquaculture Europe 2023. Vienna, Austria.

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List of abbreviations
DHA: docosahexaenoic acid (C22:6n-3)

EPA: eicosapentaenoic acid (C20:5n-3)

FA: fatty acid

FAME: fatty acid methyl esters

H / E: haematoxylin / eosin

HSI: hepatosomatic index

K: Fulton condition factor

MUFA: monounsaturated fatty acids

PUFA: polyunsaturated fatty acids

SFA: saturated fatty acids

SGR: specific growth rate

VSI: viscerosomatic index

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Results and discussion Chapter III

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Results and discussion Chapter III

Abstract

This study aimed to identify the effects of water temperature on the rearing of the omnivorous

teleost Chelon labrosus and to determine the optimal temperature for the culture of this

interesting candidate for the diversification of European aquaculture. The fish (initial weight

26.81 ± 6.2 g) were held at four water temperatures (18, 22, 26 and 30 °C) in triplicate for 90

days in 100 L recirculating tanks (n = 14 / tank). The effects of said temperatures were assessed

on growth, intestinal health and the digestive processes of the fish. At 18 °C, energy

consumption was low, leading to a high accumulation of reserves and slow growth, a typical

overwintering strategy. The best growth results were obtained at 22 °C, followed closely by 26

°C, which resulted in low energy reserves. The fact that the fish at 26 °C grew less than the ones

at 22 °C while having similar energy consumption, suggested that this temperature was close to

the end of the optimal range, but no signs of stress were detected. However, at 30 °C the fish

had lower energy reserves but grew significantly less, which can be considered indicative of

stress. Health impairment at this temperature was confirmed by the epithelial lesions found in

the intestines of this group. The growth and energy availability of the different groups is

discussed in the light of the activities of α-amylase, pepsin and alkaline proteases. As an

integration of all these results, a quadratic regression model resulting from growth values (SGR)

in relation to water temperature was performed, and it allowed predicting an actual optimal

water temperature of 22.8 °C for growth of C. labrosus. This is the first assessment of the optimal

water temperature for the culture of C. labrosus, and it will be highly valuable for the

development of its intensive culture.

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Results and discussion Chapter III

Laburpena

Ikerketa honen helburua uraren tenperaturak Chelon labrosus teleosteo orojalearen hazkuntzan

dauzkan eraginak identifikatzea izan da, eta akuikultura europarraren dibertsifikaziorako hain

interesgarria den espezie honen produkziorako tenperatura optimoa ezartzea. Arrainak

(hasierako pisua 26.81 ± 6.2 g) lau ur tenperaturatan mantendu ziren (18, 22, 26 eta 30 °C),

tratamendu bakoitza hirukoiztuta, 90 egunez 100 L-tako ur-birziklapen 12 tanketan (n = 14 /

tankeko). Tenperatura horien efektuak hazkuntzaren, hestearen osasunaren eta digestio

prozesuen ikuspegietatik ebaluatu ziren. 18 °C-tan, arrainaren energia kontsumoa baxua izan

zen, erreserba metaketa handia bultzatuz, hibernazio estrategia arrunta dena. Hazkuntza balio

onenak 22 °C-tan lortu ziren, 26 °C-tan zertxobait okerragoak izanik. 26 °C-tan mantendutako

arrainen hazkuntza 22 °C-takoena baino geldoagoa izateak, energia kontsumoa antzekoa izanda,

tenperatura tarte optimoaren amaiera 26 °C inguruan dagoela adierazten du, nahiz eta talde

honetan estres sintomarik ez zen topatu. 30 °C-tan, berriz, arrainek energia erreserba baxuak

zituzten eta haien hazkuntza esangarriki txikiagoa izan zen, estres zantzutzat har daitekeena.

Digestio epitelioan aurkitutako lesioak talde honen osasun arazoen adierazgarri dira. Talde

ezberdinen hazkuntza eta energia eskuragarritasuna α-amilasa, pepsina eta proteasa alkalinoen

testuinguruan eztabaidatu dira. Emaitza hauen guztien integraziotzat, erregresio koadratiko

modelo bat garatu zen, hazkuntza balioak (SGR) eta uraren tenperatura bilduz, eta C. labrosus-

en hazkuntzarako tenperatura optimoa 22.8 °C-tan ezartzea ahalbidetu du. Hau C. labrosus-en

hazkuntzarako ur tenperatura optimoaren lehen ebaluazioa da, eta oso erabilgarria izango da

espezie honen akuikultura intentsiboa garatzeko orduan.

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Results and discussion Chapter III

1. Introduction

Ambient temperature is considered the most important abiotic factor influencing the

metabolism and bioenergetics of poikilothermic animals, such as fish (Hawkins, 1995; Nytrø et

al., 2014; Sun & Chen, 2014). Temperature modulates the rates of biochemical reactions that

occur within an animal, increasing the oxygen and energy consumption as body temperature

increases (Schulte, 2015). This raise of energy consumption in relation to temperature, though,

plateaus and eventually declines rapidly when reaching a critical temperature. This

phenomenon is mostly explained by conformational changes in the enzymes, although other

processes are also important, as reviewed by Schulte (2015). Another factor playing a pivotal

role in the determination of the “energy budget” is the minimum amount of energy an animal

has to expend in order to maintain essential biological functions, which increases at higher

temperatures (Clarke & Fraser, 2004; Farrell, 2009; Schulte, 2015). The difference between

these two variables determines the available amount of energy to direct towards processes such

as locomotion, growth, immune response or reproduction, creating bell-shaped performance

curves in relation to water temperature in fishes (Farrell, 2009).

In the case of cultured fishes, identifying the optimal water temperature range for

maximum growth is essential to optimize production and ensure the well-being of the animals,

as suboptimal environmental conditions could eventually lead to stress, having a negative

impact on fish health (Wang et al., 2019; Islam et al., 2021; Dawood et al., 2022). Therefore,

effects of water temperature and optimal culturing conditions have been successfully identified

for commercially important aquaculture species such as Atlantic salmon (Jonsson et al., 2001),

European sea bass (Person-Le Ruyet et al., 2004), gilthead sea bream (Seginer, 2016), turbot

(Aydın et al., 2021; 2022a), Black Sea flounder (Aydın et al., 2012) or yellowtail kingfish (Bowyer

et al., 2014).

Temperature can alter digestive processes such as ingestion, gut passage time of food

(Bendiksen et al., 2002; Handeland et al., 2008), as well as the activities of digestive enzymes

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Results and discussion Chapter III

(Papoutsoglou & Lyndon, 2005; Ahmad et al., 2014; Hani et al., 2018), which directly influence

feed efficiency. The fatty acid profile of the edible part of the fish is another relevant aspect that

can be influenced by ambient temperature. Due to the homeoviscous adaptation of cell

membrane fluidity, at lower temperatures organisms tend to synthesize more unsaturated fatty

acids with lower melting points in order to compensate the loss of fluidity caused by cold

temperature (Hazel, 1995). As a result, water temperature can directly affect fish quality from

the point of view of the consumer, considering the importance unsaturated fatty acids have in

human diet.

The thicklip grey mullet Chelon labrosus has been identified as an interesting fish for the

diversification of aquaculture (Khemis et al., 2006; Zouiten et al., 2008; García-Márquez et al.,

2021; 2022). Due to its low-trophic nature, it has great potential for the usage of alternative feed

ingredients, helping in the development of the aquaculture industry without increasing its

consumption of fishmeal (Naylor et al., 2000). Grey mullets have been cultivated in the

Mediterranean region in traditional extensive systems since the antiquity and they are highly

valued for human consumption in several countries of said area (Crosetti, 2016). However, there

are several knowledge gaps about C. labrosus that pose a challenge to the development of its

intensive culture. Very little is known about the growth rates of grey mullet in the wild, but it

has been described that they alternate a slow-growing phase in autumn-winter and a faster

growing one in spring-summer, which lead to the conclusion that the species was not

competitive for aquaculture in the early 90s (Arruda et al., 1991). However, conclusions from

newer studies are more promising, although the reported data vary greatly depending on the

size of the fish considered. Specific Growth Rates (SGR) from 0.4 to 1.15 have been obtained in

nutrition researches performed with juveniles in the range of 46 to 100 g (Vílchez-Gómez et al.,

2017; García-Márquez et al., 2022; 2023), proving the potential of the species when reared

under controlled conditions. The establishment of optimal culturing conditions is of upmost

importance in order to further improve the aforementioned results. Many efforts are being

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Results and discussion Chapter III

directed towards increasing the knowledge about the nutrition of the species (Ojaveer et al.,

1996; Davies et al., 1997; Pujante et al., 2015; Pujante et al., 2017; Altunok & Özden, 2017;

García-Márquez et al., 2022, 2023; Quirós-Pozo et al., 2023). However, little research has been

conducted about optimising other aspects of culturing, apart from the effects of salinity and

stocking density (de las Heras et al., 2015; Pujante et al., 2018). Even though water temperature

is one of the most important factors for fish growth (Nytrø et al., 2014; Sun & Chen, 2014), no

research has been published on C. labrosus to our knowledge, aside from a paper focusing on

the effects of temperature on osmoregulation (Lasserre & Gallis, 1975) and the aforementioned

article by Arruda et al. (1991), where growth in wild conditions was roughly estimated.

In our previous experience working with this species, we observed the best growth

results were obtained during summer months at water temperature close to 22 °C (unpublished

data), and therefore we could expect this to be the optimal temperature for the rearing of C.

labrosus. Hence, the objective of the present research was to determine the effects of different

water temperatures on growth, energy metabolism (plasma, liver and muscle metabolites),

intestinal health (histopathological analysis), digestive processes (digestive enzyme activities)

and product quality (fatty acid profile of muscle) of the thicklip grey mullet C. labrosus in order

to establish the optimal temperature conditions for its culture, always ensuring animal welfare.

2. Materials and methods

2.1. Experimental design

The fish used in this experiment (thicklip grey mullet, C. labrosus) were wild specimens caught

at the Urola estuary (Zumaia, Basque Country, Spain) and acclimated to laboratory conditions

for eight months in three 300 L open-flow tanks, without temperature control, ensuring natural

thermal conditions for the acclimation phase. The fish (n = 168, initial fork length 13.35 ± 0.9 cm,

initial weight 26.81 ± 6.2 g) were randomly separated into 12 tanks (14 individuals per tank, n =

3) 30 days prior to the beginning of the experiment and maintained at room temperature (19

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Results and discussion Chapter III

°C). During that period and the entire experiment, fish were fed a commercial diet (Gemma

diamond 1 mm; Skretting, Stavanger, Norway) with 57 % of protein and 15 % lipid contents. The

pellet size chosen (1 mm) was smaller than the typical one for fish of this size. It was decided

based on the experience of the research team with this species, as the sediment filtering feeding

behaviour of mugilids does not allow the application of traditional aquaculture feed size tables

(Cardona, 2016). Fish were fed manually twice a day, with a total amount that accounted for 2

% of the total biomass of each tank. At the end of the acclimation period (0 D), water

temperatures were adjusted to the desired experimental treatments (± 0.5 °C), which were 18,

22, 26 and 30 °C (groups 18, 22, 26 and 30). Each temperature treatment was performed in

triplicate. The experimental period lasted 90 days. The initial average stocking density was 4.48

± 0.28 kg / m3. The stocking density of each particular tank can be found in the Appendix (Table

A1).

The experiment was performed in 12 100 L cylindrical-conical tanks. The tanks were

recirculating systems, each equipped with a Sun Sun HW - 302 canister filter (WilTec, Eschweiler,

Germany). The water flow of the filters was of 540 L / h. Each canister contained a mechanical

filter consisting on three foam layers, and a biological filter consisting of ceramic and plastic

substrate for nitrifying bacteria. Each tank was equipped with a water cooler or heater,

depending on the experimental treatment. The tanks were provided constant aeration to keep

adequate oxygen levels of around 6 mg / L. After careful examination of the feeding behaviour

of C. labrosus, it was decided to close the filters for 15 minutes at every feeding time, in order

to ensure sufficient time for an adequate feeding. After said time, a 10 L water change was

performed, and then filters were re-opened. This procedure allowed recovering much of the

leftover feed, but not all. This accounted for a daily change of 20 % of the water. At every

sampling time, all the water was renewed. Ammonia, nitrite and nitrate levels were checked

weekly using commercial kits (SERA, Heinsberg, Germany). Ammonia levels were kept at 0 – 0.5

mg / L, nitrite at 0 – 2 mg / L, and nitrate at 0 – 50 mg / L.

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Results and discussion Chapter III

2.2. Sample collection

Biometrical data (fork length and body weight) were collected at day 0 (0 D), 30 (30 D), 60 (60

D) and 90 (90 D). For handling (weighing and measuring), fish were caught with a net and

introduced in a water bath containing 100 mg / L Tricaine methanesulfonate MS 222 (Sigma-

Aldrich, Saint Louis, USA) anaesthetic. For sacrifice, the fish were introduced at a water bath of

the same compound at a concentration of 300 mg / L. Four individuals of each tank were

sacrificed and dissected at 30 D (n = 48), and the rest at 90 D (n = 120). Blood was collected from

the caudal vein with a heparinized 1 mL syringe with a 25 G needle, and centrifuged for 15

minutes at 10000 rpm. The plasma was collected, flash frozen in liquid nitrogen and stored at -

80 °C for metabolite analysis. Whole muscle of fishes were flash frozen at - 80 °C for metabolite

and fatty acid profile analyses. Liver and gastrointestinal tracts were extracted, weighed and

flash frozen at - 80 °C for the measurement of metabolites (liver) and digestive enzyme activities

(gastrointestinal tracts). A section of proximal intestine was fixed in formalin for further

histo(patho)logical analysis (See Section 2.7).

2.3. Calculations

The following calculations were made according to García-Márquez et al. (2022):

Weight gain (WG) = Final weight (g) - initial weight (g)

Ln (Final body weight)− Ln (Initial body weight)

Specific growth rate % (SGR) = 100 x (% / day)
Feeding days

Visceral weight (g)

Viscerosomatic index (VSI) = 100 x (%)
Total weight (g)

Liver weight (g)

Hepatosomatic index (HSI) = 100 x (%)
Total weight (g)

Fish weight (g)

Fulton condition factor (K) = 100 x Length3 (cm)

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Results and discussion Chapter III

2.4. Metabolite analyses

Metabolite analyses were performed in pooled samples of each tank (n = 3). Plasma glucose

(Amplex Red Glucose / Glucose Oxidase Assay Kit, Invitrogen, Carlsbad, USA), liver and muscle

glycogen (ab65620 – Glycogen Assay Kit, Abcam, Waltham, USA) and plasma, liver and muscle

triglycerides (ab65336 – Triglyceride Assay Kit, Abcam, Waltham, USA) were measured (Pujante

et al., 2015) with commercial kits, following the instructions provided by the manufacturers.

2.5. Digestive enzyme activities

The activities α-amylase (ab102523 Amylase Assay Kit, Abcam, Waltham, USA), lipase (ab102524

Lipase Activity Assay Kit, Abcam, Waltham, USA), pepsin and alkaline proteases (ab112153

Protease Activity Assay Kit, Abcam, Waltham, USA) were measured (Pujante et al., 2017, 2018)

using commercial kits. In the case of pepsin, the same kit employed for alkaline proteases was

used by changing the provided reaction buffer with 10 mM HCl (pH 2.0), as recommended by

the manufacturer.

The samples were prepared for analysis by homogenizing the stomachs and intestines

in liquid N2 conditions, using a SPEX Sample Prep 6770 freezer / mill (SPEX Sample Prep,

Metuchen, USA). Enzymes were extracted from the resulting homogenates using a Precellis 24

homogenizer (Montigny-Le-Bretonneux, France) with three 60 s passes at 6500 rpm at 4 °C,

using the buffer recommended by the manufacturer for each enzyme. The analyses were

performed in triplicate in pooled samples of each tank (n = 3). The protein content of each pool

was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA).

Each enzymatic activity was measured twice, as follows. First, all the pools were

analysed using the same incubation temperature (25 °C for α-amylase, 37 °C for lipase), as

indicated by the manufacturer. For proteases, no incubation temperature was indicated on the

protocol, so the optimum temperature was the one described by Pujante et al. (2017), that is,

50 °C for alkaline proteases and 40 °C for pepsin. This measurement was called “absolute

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Results and discussion Chapter III

activity”. Further, a second analysis was performed by incubating every group at its respective

experimental temperature. This measurement was called “actual temperature”. The enzymatic

activities were defined according to the manufacturers. 1 Unit Amylase = amount of amylase

that cleaves ethylidene-pNP-G7 to generate 1.0 μmol of nitrophenol per min at pH 7.20 at 25

°C. 1 Unit Lipase = amount of lipase that hydrolyzes triglyceride to generate 1.0 µmol of glycerol

per minute at 37°C. Proteases: alkaline proteases and pepsin activities were compared to units

of trypsin (unit not specified).

2.6. Fatty acid profile of muscle

The analysis of fatty acids was performed by ACOI Coimbra Collection of Algae. The

characterization of the fatty acid profile of muscle was performed in pooled samples of each

tank (n = 3). Muscle samples were homogenized in liquid N2, using a SPEX Sample Prep 6770

freezer / mill (SPEX Sample Prep, Metuchen, USA). The extraction of lipids from the resulting

homogenate was performed by the MTBE method (Matyash et al., 2008).

For the characterization of fatty acid profile, dried samples were resuspended in 1 mL

of hexane and 0.5 mL of methanol. After vortexing, 400 μL of sodium methoxide were added.

The top layer was filtered with a nylon membrane and 150 μL of the filtered solution was placed

in a vial, 100 μL of the internal standard methyl nonadecanoate (C19:0) (Sigma-Aldrich, Saint

Louis, USA) with a final concentration of 0.3 mg / mL were added. The gas chromatography was

performed in a NEXIS GC-2030 (Shimadzu, Kyoto, Japan) chromatograph equipped with a flame

ionization detector and a TR-CN 100 capillary column (60 m × 0.25 mm × 0.20 μm). Helium was

used as carrier gas at a pressure of 150 kPa at the top of the column. The temperature of the

injector and detector was 260°C and the split ratio was 1:25. The initial temperature of the

column was maintained at 90 °C for 7 min after the injection, increasing 5 °C / min to 220°C and

held for more 15 min. Data were acquired and analysed using Lab Solutions data analysis

software. Fatty acids (FA) were identified by comparing the relative retention times with an

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Results and discussion Chapter III

authentic external standard, Supelco 37 component FAME mix (Sigma-Aldrich, Saint Louis, USA).

The quantification of FA was based on the internal standard method (Assunção et al., 2017). The

results were expressed in percentage of the total fatty acid methyl esters (FAME) (%).

2.7. Histological analysis

Intestine samples were prepared for histological analysis following standard histological

procedures. Briefly, proximal intestine samples were fixed in a 10 % formalin solution buffered

with seawater for 24 h (Martoja & Martoja-Pierson, 1970). Once fixed, formalin was replaced by

70 % ethanol until the processing. Samples were processed with an automated tissue processor

(LEICA ASP 300S; Leica Microsystems Nussloch GmbH, Germany) and embedded in paraffin wax

blocks. Five µm thick sections were obtained with a microtome (Leica RM2125RTS; Leica

Microsystems Nussloch GmbH, Germany), stained with haematoxylin-eosin (Martoja & Martoja-

Pierson, 1970) using an Autostainer XL (Leica Microsystems Nussloch GmbH, Germany), and

mounted with coverslip. The samples were observed under an Olympus BX61 light microscope

(Olympus-Lifescience, Tokyo, Japan).

2.8. Statistical analysis

All statistical analyses were performed using the IBM SPSS Statistics 27 software (IBM corp,

2020). Normality of the data was tested using the Shapiro Wilk test. Homogeneity of variances

was tested using the Levene´s test. In the case of data with normal distributions and

homogeneous variances, Student´s T test was performed to compare the different variables

such as weight, length, SGR, HSI, VSI, K, metabolite levels, enzymatic activities, fatty acids or

histological lesions (dependent variables) across water temperature treatments or sampling

times (independent variables). When data was not normal and variances not homogeneous, the

non-parametric Shapiro-Wilk´s test was used. Different linear regression models were

performed among SGR data from 0 D to 90 D (dependent) and water temperature

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Results and discussion Chapter III

(independent), and the one with the best fit was selected. The significance for every statistical

test was set at p < 0.05. All data are presented as mean ± standard deviation.

2.9. Ethical statement

All experimental procedures complied with the Guidelines of the European Union (2010/63/UE)

and the Spanish legislation (RD53/2013 and law 32/2007) for the handling and use of laboratory

animals under the supervision and acceptance of the Ethics for experimentation and animal

welfare committee of the University of the Basque Country (UPV/EHU) and provincial authorities

(CEEA: M20_2021_108-Diputación Foral de Bizkaia).

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Results and discussion Chapter III

Results

At the end of the experiment, group 22 exhibited higher length and weight values than the

others. This difference was statistically significant (p < 0.05) between group 22 and group 30 in

both cases (Figure 1).

18

17 a
a
16 a
b
Lenght (cm)

15

14

13

12
0 30 60 90
64

59 a

54 ab
ab
49 b
Weight (g)

44
a
39 a
a
34 b

29

24
0 30 60 90
Days

: 18 °C : 22 °C : 26 °C : 30

Figure 1: length (cm) and weight (g) of C. labrosus reared at different temperatures for 90 days. Groups

with different superscript letters show statistically significant (p < 0.05) differences.

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Results and discussion Chapter III

At the end of the experiment, group 22 showed the highest SGR values, followed by

group 26, the other two having significantly lower values (Table 1). During the experimental

period, the SGR values of group 18 significantly decreased, while the opposite happened to

group 30.

Table 1: Specific Growth Rate (SGR) of C. labrosus reared at different temperatures for 90 days. Groups

with different superscript letters show statistically significant (p < 0.05) differences. Capital letter

superscripts: comparisons between temperature treatments inside a given time period (row); lower letter

superscripts: comparisons between time periods inside a given temperature treatment (column).

18 °C 22 °C 26 °C 30 °C
0 D - 30 D 0.92 ± 0.03Aa 1.02 ± 0.09Aab 1.01 ± 0.15A 0.51 ± 0.14Bb
SGR
30 D - 60 D 0.72 ± 0.14Bb 0.96 ± 0.06Ab 1.01 ± 0.16AB 0.87 ± 0.43ABab
(%/day)
60 D - 90 D 0.57 ± 0.17Bb 1.16 ± 0.10Aa 1.01 ± 0.21A 0.79 ± 0.20Ba
0 D - 90 D 0.80 ± 0.09BC 1.01 ± 0.02A 0.95 ± 0.09AB 0.79 ± 0.06C

30 D: day 30; 60 D: day 60; 90 D: day 90.

The regression model between SGR from the beginning to the end of the experiment (0

D – 90 D;dependent variable) and water temperature (independent variable) with the best fit

(R2 = 0.701) was the quadratic regression (Figure 2). The maximum of the curve was found at

22.8 °C.

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Results and discussion Chapter III

y = - 0.006 x2 + 0.274 x – 2.255

SGR (% / day)

Water temperature (°C)

Figure 2: quadratic regression between SGR (beginning to end of the experiment; 0 D – 90 D) and water

temperature of C. labrosus reared at different temperatures for 90 days.

HSI showed the same pattern at both samplings (Table 2), group 18 having the highest

values, followed by group 22, while 26 and 30 groups had the lowest. There were no significant

differences in VSI at 30 D, but at 90 D, a very similar result to HSI differences was obtained.

Regarding K value, it was also higher in group 18 than in the 26 and 30 groups at 30 D, but at 90

D, group 26 showed the lowest value.

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Results and discussion Chapter III

Table 2: weight (W), fork length (Fl), hepatosomatic (HSI) and viscerosomatic (VSI) indexes, and Fulton

condition factor (K) of C. labrosus reared at different temperatures for 90 days. Capital letter superscripts:

comparisons between temperature treatments at 30 D; lower letter superscripts: comparisons between

temperature treatments inside 90 D. Significant differences (p < 0.05) inside a given treatment over time

(30 D vs 90 D) are highlighted in bold.

18 °C 22 °C 26 °C 30 °C
30 D 90 D 30 D 90 D 30 D 90 D 30 D 90 D
40.33 ± 52.59 ± 40.25 ± 57.74 ± 38.41 ± 55.70 ± 34.86 ± 49.13 ±
W (g)
7.85a
9.56 AB
8.77 a
11.86 A
7.05 a
10.28 AB
7.36b
8.73B
14.77 ± 16.15 ± 14.96 ± 16.78 ± 14.89 ± 16.65 ± 14.49 ± 15.76 ±
Fl (cm)
0.91 0.93A 1.04 0.99A 0.88 0.97A 0.91 0.86B
1.16 ± 1.03 ± 0.80 ± 0.78 ± 0.65 ± 0.65 ± 0.63 ± 0.69 ±
HSI
0.04a 0.06A 0.12b 0.05B 0.08bc 0.03C 0.09c 0.04BC
6.79 ± 7.00 ± 6.47 ± 6.19 ± 6.10 ± 5.15 ± 6.29 ± 5.84 ±
VSI
0.47 0.33A 0.11 0.22B 0.74 0.37C 0.74 0.18B
1.24 ± 1.24 ± 1.19 ± 1.20 ± 1.15 ± 1.19 ± 1.13 ± 1.24 ±
K
0.03a
0.02A
0.02ab
0.04 AB
0.02b
0.02B
0.03b
0.02A
30 D: day 30; 90 D: day 90.

Plasma glucose (Table 3) did not show any significant difference between treatments at

any of the sampling times, but it did have an upwards trend from 30 D to 90 D in every group,

this difference being significant in group 26. Plasma triglyceride changed between groups and

sampling times. At both samplings, group 18 had the highest values, followed by group 22, while

the other groups had the lowest values. Similar to what happened with glucose, plasma

triglyceride also increased over time, but in this case the differences were always significant

between 30 D and 90 D, except at group 26. Liver glycogen only showed a significant difference

at 90 D, when group 18 had the highest values and group 22 the lowest. At 30 D, group 18

showed a significantly higher liver triglyceride content than the other groups, but at 90 D, group

30 had the highest values alongside group 18. In fact, group 30 showed the only significant

increase in liver triglycerides over time. Muscle glycogen values were the highest in group 18 at

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Results and discussion Chapter III

both samplings. Muscle triglyceride levels did not show any significant difference between

temperature treatments at any sampling, but they did increase over time at group 30.

Table 3: plasma, liver and muscle biochemical data of C. labrosus reared at different temperatures for 90

days. Groups with different superscript letters show statistically significant (p < 0.05) differences. Capital

letter superscripts: comparisons between temperature treatments at 30 D; lower letter superscripts:

comparisons between temperature treatments inside 90 D. Significant differences (p < 0.05) inside a given

treatment over time (30 D vs 90 D) are highlighted in bold.

18 °C 22 °C 26 °C 30 °C
30 D 90 D 30 D 90 D 30 D 90 D 30 D 90 D
63.56 ± 84.63 ± 58.58 ± 81.39 ± 54.10 ± 91.45 ± 64.78 ± 80.68 ±
Glu
Plasma 35.37 16.49 23.59 18.03 14.72 9.12 28.23 14.89
(mg/dL) 178. 80 243.34 ± 119.25 175.04 ± 34.97 ± 97.45 ± 38.13 ± 96.72 ±
TAG
± 20.89a 38.50A ± 12.80b 17.60B 28.71c 41.28C 23.29c 7.62C
13.23 ± 15.41 ± 7.52 ± 6.72 ± 10.67 ± 12.69 ± 8.16 ± 10.77 ±
Gly
Liver 3.37 2.03A 3.33 4.04B 2.09 3.42AB 2.91 2.24AB
(mg/g) 86.01 ± 101.35 ± 55.54 ± 61.56 ± 63.93 ± 49.41 ± 47.46 ± 86.42 ±
TAG
9.05a 25.62A 14.22b 7.18AB 26.17ab 10.16B 22.25b 15.61A
2.35 ± 1.53 ± 1.33 ± 1.30 ± 1.45 ± 0.91 ± 1.51 ± 1.12 ±
Gly
Muscle 0.31a 0.20A 0.18b 0.08AB 0.47b 0.20C 0.26ab 0.12BC
(mg/g) 5.49 ± 8.61 ± 11.79 ± 10.64 ± 9.29 ± 5.34 ± 3.45 ± 9.93 ±
TAG
1.95 5.87 5.92 3.87 6.31 2.62 0.52 1.26
30 D: day 30; 90 D: day 90; Glu: glucose; TAG: triglycerides; Gly: glycogen.

When measuring the amylase activity of every group at the same incubation

temperature (absolute activity), group 18 showed the highest value at 30 D, but at 90 D, group

22 had the highest activity (Table 4). Group 18 was the only one where absolute amylase activity

did not increase over time. When measuring the amylase activity at the experimental

temperatures (actual activity), groups 26 and 30 showed the highest values at 30 D, but at 90 D,

there were no differences between 22, 26 and 30 groups. Lipase activity was below detection

limit at every temperature tested. At 90 D group 18 had the lowest actual alkaline protease

activity, despite showing the highest absolute activity. In the case of pepsin, groups 18 and 22

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Results and discussion Chapter III

had the highest absolute activities at 90 D, while 22, 26 and 30 groups demonstrated significantly

higher actual activities than the group 18.

Table 4: α-amylase (Amy), alkaline proteases (Alk Prot) and pepsin (Pep) activities of C. labrosus digestive

tract reared at different temperatures for 90 days. Groups with different superscript letters show

statistically significant (p < 0.05) differences. Capital letter superscripts: comparisons between

temperature treatments at 30 D; lower letter superscripts: comparisons between temperature

treatments inside 90 D. Significant differences (p < 0.05) inside a given treatment over time (30 D vs 90 D)

are highlighted in bold.

18 °C 22 °C 26 °C 30 °C
30 D 90 D 30 D 90 D 30 D 90 D 30 D 90 D
100.40 ± 81.35 ± 71.20 ± 104.29 ± 73.20 ± 88.18 ± 71.46 ± 94.12 ±
Amy Abs
11.13a 5.35B 10.84b 9.50A 6.57b 3.00B 4.34b 10.89B
(U / µg
68.82 ± 53.18 ± 57.62 ± 83.98 ± 74.24 ± 85.38 ± 78.84 ± 119.49
protein) Act
10.74ab 7.16B 11.24b 6.14A 9.49a 5.62A 3.58a ± 28.39A
1.74 ± 1.78 ± 1.80 ± 1.64 ± 1.60 ± 1.21 ± 1.34 ± 1.30 ±
Alk Prot Abs
0.24 0.19A 0.16 0.22AB 0.14 0.20B 0.40 0.27AB
(mU/µg
0.93 ± 0.73 ± 1.08 ± 1.16 ± 1.29 ± 1.26 ± 0.91 ± 1.13 ±
protein) Act
0.12 0.10B 0.08 0.06A 0.25 0.19A 0.29 0.31AB
Pep 54.37 ± 55.11 ± 52.72 ± 49.56 ± 45.91 ± 11.44 ± 13.70 ± 10.57 ±
Abs
(U / g 1.52a 7.84A 3.62a 0.97A 7.81a 2.11B 7.94b 3.74B
protein) 10.63 ± 7.42 ± 14.27 ± 16.01 ± 21.37 ± 16.16 ± 11.42 ± 14.74 ±
Act
2.44b 3.31B 3.81ab 3.03A 3.38a 2.28A 4.18b 2.48A

30 D: day 30; 90 D: day 90; Abs: absolute activity measured as recommended by the manufacturer; Act:

actual activities measured at the experimental temperatures.

Fatty acid content of fish muscle (Table 5) did not differ among temperature treatments

in terms of total SFA content, but there were slight changes in MUFA and PUFA. The fish with

the lowest MUFA content at the end of the experiment were the ones held at 22 °C, while they

had the highest content of PUFA. ω-3 and ω-6 PUFA content were also equal across all

experimental treatments. Full information about fatty acids can be found at the Appendix (Table

A2).

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Results and discussion Chapter III

Table 5: summary of fatty acid contents of C. labrosus muscle reared at different temperatures for 90 days

(expressed as % of the total fatty acid methyl esters). Groups with different superscript letters show

statistically significant (p < 0.05) differences. Capital letter superscripts: comparisons between

temperature treatments at 30 D; lower letter superscripts: comparisons between temperature

treatments inside 90 D. Significant differences (p < 0.05) inside a given treatment over time (30 D vs 90 D)

are highlighted in bold. Full information about fatty acids can be found at the Appendix (Table A2).

18 °C 22 °C 26 °C 30 °C
30 D 90 D 30 D 90 D 30 D 90 D 30 D 90 D
43.00 ± 43.33 ± 41.95 ± 44.26 ± 43.12 ± 42.99 ± 40.23 ± 42.17 ±
ΣSFA
1.65 1.86AB 0.33 0.85A 2.23 0.83AB 3.32 1.07B
30.76 ± 31.33 ± 30.39 ± 27.54 ± 30.49 ± 29.31 ± 32.12 ± 31.59 ±
ΣMUFA
0.97 0.97 1.73 2.02 1.00 1.71 2.86 1.73
26.24 ± 25.34 ± 27.81 ± 28.20 ± 26.38 ± 27.70 ± 27.62 ± 26.24 ±
ΣPUFA
2.61 0.92
B
1.68 1.20A
1.23 0.99A
0.47 0.66AB
Σ ω-3 14.26 ± 15.91 ± 16.57 ± 19.63 ± 15.64 ± 18.36 ± 14.78 ± 16.12 ±
PUFA 2.63 1.29 1.99 2.45 0.24 2.33 2.53 1.64
Σ ω-6 10.61 ± 8.12 ± 9.87 ± 7.35 ± 9.40 ± 7.76 ± 11.04 ± 8.37 ±
PUFA 1.76 0.82 0.31 0.83 1.13 0.92 2.41 0.81
30 D: day 30; 90 D: day 90; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA:

polyunsaturated fatty acids; ω-3 PUFA: omega-3 polyunsaturated fatty acids; ω-6 PUFA: omega-6

polyunsaturated fatty acids.

Most of the analysed intestinal samples presented the expected structure (Figure 3A).

This consists of the serosa layer in the outermost part, muscular layer, submucosae and the

mucosa, comprised of the epithelium and lamina propria (Rašković et al., 2011). Interestingly, a

disruption of the mucosal barrier characterised by the loss of enterocytes (Figure 3B) was

detected in certain samples. Aside from the gaps in the epithelium caused by the enterocyte

loss, some debris could be seen coming out from these lesions. These anomalies were most

prevalent at the end of the experiment at group 30 (Figure 3C).

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Results and discussion Chapter III

5 4

B C 1,8
a
1,6

1,4

1,2
Lesions / sample

0,8

0,6

0,4

0,2
b b
b
0

-0,2 T3 18 T3 22 T3 26 T3 30

Figure 3: A) normal structure of intestinal villi. 1: serosa layer. 2: muscular layer. 3: submucosae. 4: lamina

propria. 5: epithelium; B) example of intestinal villi with epithelial lesions (highlighted with arrows). Note

the gap caused by enterocyte loss and the debris coming out of the lesion; C) incidence (lesions per

sample) of epithelial lesions in C. labrosus intestine reared at different temperatures at day 90. H/E

staining.

4. Discussion

In the present research, Chelon labrosus were maintained under four water temperatures (18,

22, 26 and 30 °C) to assess the one rendering the best yields in terms of growth and animal

welfare. The highest length increase, weight gain and SGR values of C. labrosus juveniles were

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achieved at 22 °C, closely followed by the fish reared at 26 °C. The other two groups (18 and 30

°C) showed significantly lower values on all the aforementioned parameters at the end of the

experiment. The SGR values obtained herein are within the range reported for C. labrosus

individuals of similar sizes in previous research, as SGRs from 0.89 to 1.15 were obtained in

juveniles weighing 46 g in a nutritional research (Vílchez-Gómez et al., 2017). These results

suggest that the optimal temperature range for a healthy growth is between 22 and 26 °C for C.

labrosus, 26 °C being close to the upper limit. The other temperatures tested are sub-optimal

for thicklip grey mullet culture. In order to summarize all these results, absolute SGR was used

as a general representation of fish performance during the experiment, and it was correlated to

water temperature. The regression model with the best fit was found to be the quadratic

regression, which accurately reflected the dome-shaped growth curves usually caused by water

temperature on fishes (Nytrø et al., 2014). The resulting model allowed predicting the actual

optimal temperature for the rearing of C. labrosus juveniles, which was 22.8 °C, a very similar

temperature to the originally expected one (22 °C). In a research focusing on fatty acids of C.

labrosus under different culturing conditions, the authors mention “good growth rates” at

temperatures from 26 to 30 °C (Rabeh et al., 2021), but further detail is lacking, as this is not the

focus of said paper. The accurate interpretation of these results, though, requires considering

the origin of the fish used. The experimental fish used herein were original from the Cantabrian

Sea, which rarely reaches temperatures above 22 °C even in summer months (Borja et al., 2019).

Moreover, it is known that optimal water temperature for fish growth is typically somewhat

higher than the temperature normally encountered at their natural habitat (Handeland et al.,

2008), which supports our observations. However, this species is widely distributed across the

North-East Atlantic Ocean from Mauritania to Norway, Mediterranean and Black Seas (Turan,

2016). Although the existence of genetically differentiated subpopulations has not been proven

(Nzioka et al., 2023), it is conceivable for populations inhabiting warmer areas, such as the

Mediterranean, to have adaptations to cope with the thermal characteristics of their habitat,

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which ultimately could affect growth. For this reason, the provenance of the fish has to be taken

into account when farming C. labrosus, and optimal temperature re-evaluated if necessary.

Other fish species of temperate waters show an optimal thermal range similar to C. labrosus,

although species-specific variations exist. Yellowtail kingfish exhibits a very narrow optimal

temperature range peaking at 24 °C, very similar to C. labrosus (Bowyer et al., 2014). The

performance curve of the European sea bass is slightly displaced towards higher temperatures,

as it performs better at temperatures close to 26 °C, and growth starts to be depressed at 29 °C

(Person-Le Ruyet et al., 2004). Gilthead sea bream also exhibits higher optimal temperatures,

close to 25 °C (Seginer, 2016). The opposite happens in the case of turbot, as its optimum

temperature is close to 18 °C (Aydın et al., 2021), which has been identified as sub-optimal for

C. labrosus. Compensatory growth is a phenomenon that could make the interpretation of these

results more difficult. It consists of accelerated growth appearing when recovering optimal

conditions after a period of adversity like food deprivation or sub-optimal temperature (Py et

al., 2022). Water temperature during the acclimation phase was around 19 °C, close to the 18

°C identified as sub-optimal for C. labrosus rearing. Therefore, increasing water temperature to

22 and 26 °C, in the optimal range of the species, could trigger compensatory growth, and the

effects observed would be a product of the temperature change, not of the experimental

temperature itself. However, compensatory growth is known to be stronger after periods of

severe adverse conditions, when energy reserves of the fish have been strongly exploited (Py et

al., 2022). As seen in the present study, fish at 18 °C had the highest energy reserves, so the sub-

optimal conditions of the acclimation period probably were not severe enough to induce a

compensatory growth response.

It is worth noting that while the performance of groups 22 and 26 was constant across

the entire experimental period, the other two groups showed remarkable alterations over time,

as evidenced by the changes in SGR across the different sampling times. In the case of group 30,

the performance improvement observed over time might be a result of acclimation after a sharp

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increase in water temperature. A wide range of mechanisms for thermal acclimation exist in

teleost fishes, some of them being almost instantaneous while others can take several weeks to

activate (Johnston & Dunn, 1987). However, despite the ability showed by C. labrosus to

acclimate to temperatures up to 30 °C and improve their performance over time, their

compensatory mechanisms are not enough to overcome the challenge posed by this extreme

ambient temperature completely. Feed intake data could allow for a better understanding of

the decrease of growth observed at group 18 from the beginning to the end of the experiment,

but unfortunately, it was not recorded. It is known that feed intake can vary depending on

several factors, including water temperature, although the understanding of appetite regulation

on fish is still a “work in progress” (Volkoff & Rønnestad, 2020). In general, food intake in fishes

increases alongside water temperature until reaching a maximum, usually at a temperature a

bit higher than the optimal one for growth (Burel et al., 1996). A decrease in feed intake when

lowering water temperature has been observed in fishes such as the yellowtail kingfish Seriola

lalandi (Miegel et al., 2010), Asian catfish Clarias batracus (Ahmad et al., 2014), turbot

Scophtalmus maximus (Guerreiro et al., 2016), cobia Rachycentron canadum (Sun & Chen, 2014)

or Atlantic salmon Salmo salar (Bendiksen et al., 2002). This hypothetical low feed intake of the

fish at 18 °C does not completely explain the decrease in the growth rate observed after the first

30 days. Excessive fat reserve accumulation can hinder feed intake by providing negative

feedback on the food intake regulation centres (Jobling & Johansen, 1999) and cause retardation

of growth (Jobling et al., 2002). In this experiment, perivisceral fat was extracted and weighed

alongside the digestive tract, so VSI could be used as an approximation of perivisceral fat

reserves in this particular case. This index was higher on group 18, and this high fat accumulation

could lead to lower feed intake and consequently, a decrease in the performance of this group

over time. However, there is not enough evidence in order to support this hypothesis.

As expected, water temperature also had an impact on the bioenergetics of the fish. The

fish held at 18 °C had the highest levels of triglycerides, in both plasma and liver, as well as

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glycogen in liver and muscle. Therefore, the slow growth observed on this group cannot be

attributed to low energy availability. High energy storage was also reflected on HSI, as this group

had the highest values of said index, and on VSI, as previously mentioned. In winter, food

availability might be compromised, and some fishes do exhibit a reserve accumulation behaviour

at cold temperatures (Schultz & Conover, 1997), coupled with a depressed metabolic activity

(Reeve et al., 2022). On a research conducted on juvenile roach Rutilus rutilus it was seen that

growth was completely stopped at 12 °C, and that the energy reserves were higher in winter

than in summer, especially in fish acclimated at 4 °C (Van Dijk et al., 2005). The extent of this

behaviour and the temperature at which it takes place is variable among species and responds

to the environmental factors they encounter in their natural habitat (Schultz & Conover, 1997).

In the case of our experimental fish, 18 °C seems to be too high to trigger an overwintering

behaviour, because this temperature is high for winter in their natural habitat at the Cantabrian

Sea (Borja et al., 2019). In a research about wild grey mullet populations of the Atlantic coast of

Portugal, it was seen that most of the growth experienced by C. labrosus happened during spring

and summer, when water temperature was above 14 °C (Arruda et al., 1991). Perhaps, in natural

conditions, 18 °C could be close to the threshold at which this fish starts to reorganize energy

partitioning, withdrawing energy from growth towards reserve accumulation, as a preparation

for winter dormancy. The results of plasma, liver and muscle metabolites, as well as HSI and VSI

suggest that fish of group 26 had a higher energy expenditure than the fish of group 22, which

was not reflected as a growth increase. This could imply that at this temperature, the fish are

starting to deviate energy from growth in order to fuel the mechanisms for thermal stress

compensation (Alfonso et al., 2021), a strategy that could be considered successful at this

temperature, as it did not compromise growth. A similar but more pronounced response was

seen at 30 °C, were higher energy expenditure did not cause growth improvement, this being

the group with the lowest SGR. Interestingly, after 30 D, an increase in lipid reserves was noted,

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as seen by the high liver triglyceride levels and VSI, coinciding with the moment where the

growth in this group started to raise.

In a previous study about the activity of digestive enzymes of fish reared at different

water temperatures, the enzymatic extracts of every experimental group at the same reaction

temperature were tested to estimate enzyme secretion levels (Navarro-Guillén et al., 2022),

called “absolute activity” herein. However, the enzymatic activities measured at the

experimental temperatures are more relevant to elucidate the actual digestive capabilities of

the fish, as they are poikilotherms and strict temperature conformers (Volkoff & Rønnestad,

2020), with the exception of some species with big body sizes that can produce and keep enough

heat so as to increase their temperature above said threshold (Carey et al., 1971). The overall

low lipase activity found in the present research is in concordance with the results obtained by

Pujante et al. (2017) on the same species, which was almost negligible. This is in agreement with

the expected lipase activity of herbivorous fish, which theoretically would have a low intake of

dietary lipids in the wild (Opuszynski & Shireman, 2019), and it suggests that C. labrosus has a

limited ability to digest lipids (Pujante et al., 2017), independently of water temperature. As a

result, these species would rely more on carbohydrates as energy source. One of the most

important carbohydrases in fishes is α-amylase (Kaushik et al., 2022). When measuring absolute

activity, all groups except group 18 showed an increase in α-amylase activity in the

gastrointestinal tract over time. It has been reported that α-amylase activity of C. labrosus

increases alongside the size of the fish (Pujante et al., 2017), and the raise of activity over time

found in the present experiment could respond to the same principle. Even though not

statistically significant, the α-amylase activity of the 18 °C group decreased over time. This could

potentially lower carbohydrate digestibility, and it could partially explain the decrease of SGR

experienced over time. Although feed digestibility was not measured, the alteration of the

pattern observed on the rest of the groups seems relevant, and the possibility of a decrease on

energy digestibility cannot be ruled out. When measured at their respective physiological

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temperatures, groups 22, 26 and 30 showed similar α-amylase activities, while group 22 showed

the highest absolute activity of said enzyme. Enzymatic activities increase with reaction

temperatures (Schulte, 2015), so it is plausible for group 22 to be producing a higher amount of

α-amylase in order to compensate for the lower activity at said temperature. Even though this

mechanism has been observed at some cases (Savoie et al., 2008), it does not seem to be a

widespread strategy. Despite being a general trend, the increase of the α-amylase activity at 30

°C from 30 D to 90 D was the most pronounced and may explain the improvement of

performance during the experimental period, as well as the increase of lipid reserves. Protease

activities showed similar patterns to α-amylase, but there were not any remarkable differences

over time. At 18 and 22 °C, higher absolute pepsin and alkaline protease activities were found,

suggesting a higher concentration of said enzymes, but when measuring at the physiological

temperatures, actual activities were similar at groups 22, 26 and 30, and lower at 18. The higher

production of proteases at 18 and 22 °C could respond to the same compensation mechanism

previously hypothesized for α-amylase, but with different effectiveness. At 22 °C, the increased

enzyme production would be enough to reach the same physiological activity than at warmer

temperatures, whereas at 18 °C would not. The pepsin activity measured herein is remarkably

low when compared to alkaline protease activity, contrary to the findings by Pujante et al.

(2017), who found that these fish do have a relatively high pepsin activity at early life stages,

which is almost lost during development. Therefore, our results align better with the expected

protease profile for older fish, as the experimental fish of the present research were even

younger/smaller than the fish analysed by Pujante et al. (2017). This discrepancy deserves more

attention in future research, and the understanding of the different experimental strategies,

protocols, feeding, origin of the fish and sampling would make the comparison between

researches easier.

Even though group 30 was not the only one showing epithelial damage in the intestines,

the prevalence of such lesions was significantly higher than at other temperatures. It is known

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that stressful conditions can alter fish digestive epithelial integrity by damaging enterocyte

junctions (Olsen et al., 2002). The observed enterocyte damage could be a result of such

weakening of the epithelia, and it could lead to a disruption of its barrier function, endangering

the osmotic balance and facilitating pathogen infection (Del-Pozo et al., 2010). This condition

strongly suggests that 30 °C are stressful for C. labrosus, and the health and well-being of the

fish could be compromised at this temperature.

Water temperature did not have a profound impact on C. labrosus muscle fatty acid

profile. Even though cell membranes show the ability to alter their fatty acid composition

depending on ambient temperature (Hazel, 1995), other biotic factors such as phylogeny, diet,

age, reproductive status or ploidy have been found to be more relevant when explaining the

fatty acid profile of a given fish species (Kaushik et al., 2006; Skalli et al., 2006; Senso et al., 2007;

Sushchik et al., 2018; Aydın et al., 2022b). Among abiotic factors, water salinity or pH can also

have a profound impact on fish fillet fatty acid profile (Rabeh et al., 2022). This would support

the lack of differences found among groups in the present research when it comes to muscle

fatty acids. In any case, groups 22 and 26 did show a higher content of PUFA, especially of the

ω-3 group. Conversely, Rabeh et al. (2022) reported significant changes in C. labrosus fatty acid

profile depending on water temperature and salinity, but the interaction between those two

abiotic variables make comparisons with our results difficult.

5. Conclusions

In conclusion, optimal water temperature for rearing C. labrosus juveniles was found to be 22.8

°C. Colder temperatures of around 18 °C caused growth delay and high energy reserve

accumulation. At 30 °C, fish exhibited a high energy expenditure that did not lead to fast growth.

This, coupled with the epithelial lesions observed in intestines of these fish, evidences that 30

°C are stressful for C. labrosus juveniles. Even though the exact water temperature were said

stress started could not be elucidated, at 26 °C the fish showed similar growth values to the ones

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reared at 22 °C, but they spent more energy for that, therefore, it is not recommended to

increase water temperature above 26 °C. However, fish performance in relation to water

temperature and, concomitantly, optimal temperature for growth, is highly variable not only

across different species, but also across different life stages of a given one (McCauley & Huggins,

1979). Consequently, the results obtained in this research cannot be extrapolated to the entire

life cycle, and conducting similar experiments to the present one is recommended with

individuals at different development stages in order to obtain more accurate information about

the optimal water temperature for C. labrosus across its entire life cycle. The results obtained

herein are particularly interesting for the extensive culture of the thicklip grey mullet, as the lack

of temperature control mandates a deep understanding of the response of the fish to said

variable, in order to adjust farming conditions, feeding protocols or harvesting strategies

accordingly.

6. Acknowledgements

This research was funded by the Basque Government [00007-INA2019-33, 00003-INA2022-33,

00005-2101022023, 00010-PIT2020-22].

The authors are thankful for the technical and human support from the ACOI Coimbra

Collection of Algae at the fatty acid analysis, and from Kardala LHII at fish capture and delivery.

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8. Appendix

Table A1: initial stocking density of every experimental tank at the beginning of the experiment. T:

temperature treatment (°C); SD: stocking density (kg / m3).

T (°C) 18 22 26 30

Tank 18.1 18.2 18.3 22.1 22.2 22.3 26.1 26.2 26.3 30.1 30.2 30.3

SD (kg / m3) 4.73 4.85 4.38 4.85 4.35 4.42 4.74 4.10 4.15 4.45 4.58 4.10

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Table A2: fatty acid contents of C. labrosus muscle reared at different temperatures for 90 days (expressed

as % of the total fatty acid methyl esters). 30 D: day 30; 90 D: day 90. SFA: saturated fatty acids; MUFA:

monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; ω-3 PUFA: omega-3 polyunsaturated

fatty acids; ω-6 PUFA: omega-6 polyunsaturated fatty acids. Groups with different superscript letters

show statistically significant (p < 0.05) differences. Capital letter superscripts: comparisons between

temperature treatments at 30 D; lower letter superscripts: comparisons between temperature

treatments inside 90 D. Significant differences (p < 0.05) inside a given treatment over time (30 D vs 90 D)

are highlighted in bold.

18 °C 22 °C 26 °C 30 °C
30 D 90 D 30 D 90 D 30 D 90 D 30 D 90 D
3.77 ± 3.52 ± 3.42 ± 3.18 ± 3.48 ± 3.67 ± 3.63 ± 3.80 ±
C14:0
0.28 0.24 0.12 0.38 0.05 0.55 0.48 0.44
0.40 ± 0.33 ± 0.38 ± 0.35 ± 0.39 ± 0.40 ± 0.41 ± 0.41 ±
C15:0
0.03 0.01 0.02 0.04 0.02 0.03 0.04 0.02
28.72 ± 26.64 ± 25.98 ± 26.88 ± 26.39 ± 26.10 ± 24.65 ± 25.61 ±
C16:0
2.69 1.58 0.44 0.85 1.78 0.57 2.03 0.95
0.00 ± 1.10 ± 0.80 ± 0.75 ± 1.16 ± 0.92 ± 1.26 ± 0.83 ±
C17:0
0.00 0.14 0.70 0.66 0.09 0.38 0.12 0.72
6.43 ± 5.84 ± 6.07 ± 6.97 ± 6.70 ± 6.10 ± 5.68 ± 6.13 ±
C18:0
0.59 0.59 0.24 0.80 0.98 1.01 1.60 1.21
0.10 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ±
C20:0
0.17 0.00 0.00 0.00 0.00 0.00 0.00 0.00
3.57 ± 5.90 ± 5.30 ± 6.14 ± 5.00 ± 5.79 ± 4.62 ± 5.40 ±
C24:0
3.13 0.18 0.15 0.28 0.00 0.30 0.35 0.07
0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.02 ± 0.00 ±
C14:1
0.00 0.00 0.00 0.00 0.00 0.00 0.03 0.00
6.96 ± 6.96 ± 6.45 ± 5.24 ± 6.36 ± 5.68 ± 6.77 ± 6.17 ±
C16:1
0.34 0.28 0.54 1.30 0.14 1.30 1.06 1.67
21.17 ± 21.13 ± 20.43 ± 19.33 ± 20.76 ± 20.34 ± 21.47 ± 22.07 ±
C18:1n9c
1.09 0.90 1.15 0.57 0.46 0.62 0.97 0.52
2.63 ± 3.21 ± 3.51 ± 2.96 ± 3.38 ± 3.29 ± 3.89 ± 3.36 ±
C20:1n9
0.64 0.46 0.25 0.24 0.44 0.43 0.88 0.29
8.87 ± 7.92 ± 9.58 ± 7.15 ± 9.21 ± 7.53 ± 10.86 ± 8.14 ±
C18:2n6c
0.84 0.74 0.31 0.70 1.02 0.75 2.48 0.62

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1.07 ± 1.09 ± 1.09 ± 1.04 ± 1.07 ± 1.15 ± 1.30 ± 1.19 ±

C20:2
0.12 0.20 0.03 0.11 0.14 0.16 0.34 0.18
0.33 ± 0.22 ± 0.28 ± 0.19 ± 0.28 ± 0.43 ± 0.50 ± 0.56 ±
C20:3n5
0.02 0.21 0.03 0.33 0.01 0.39 0.25 0.04
0.92 ± 1.12 ± 1.04 ± 1.46 ± 1.09 ± 1.37 ± 1.21 ± 1.52 ±
C20:3n3
0.14 0.06 0.15 0.12 0.08 0.10 0.30 0.30
1.74 ± 0.20 ± 0.30 ± 0.20 ± 0.19 ± 0.22 ± 0.18 ± 0.23 ±
C22:2n6
2.55 0.17 0.02 0.17 0.16 0.20 0.16 0.20
C20:5n3 0.33 ± 0.34 ± 0.24 ± 0.49 ± 0.42 ± 0.57 ± 0.28 ± 0.53 ±
EPA 0.11 0.04 0.04 0.14 0.17 0.06 0.17 0.08
C22:6n3 13.01 ± 14.45 ± 15.29 ± 17.68 ± 14.12 ± 16.42 ± 13.29 ± 14.07 ±
DHA 2.55 1.24 1.82 2.34 0.36 2.18 2.33 1.27
43.00 ± 43.33 ± 41.95 ± 44.26 ± 43.12 ± 42.99 ± 40.23 ± 42.17 ±
ΣSFA
1.65 1.86AB
0.33 0.85 A
2.23 0.83AB
3.32 1.07B
30.76 ± 31.33 ± 30.39 ± 27.54 ± 30.49 ± 29.31 ± 32.12 ± 31.59 ±
ΣMUFA
0.97 0.97 1.73 2.02 1.00 1.71 2.86 1.73
26.24 ± 25.34 ± 27.81 ± 28.20 ± 26.38 ± 27.70 ± 27.62 ± 26.24 ±
ΣPUFA
2.61 0.92B 1.68 1.20A 1.23 0.99A 0.47 0.66AB
Σ ω-3 14.26 ± 15.91 ± 16.57 ± 19.63 ± 15.64 ± 18.36 ± 14.78 ± 16.12 ±
PUFA 2.63 1.29 1.99 2.45 0.24 2.33 2.53 1.64
Σ ω-6 10.61 ± 8.12 ± 9.87 ± 7.35 ± 9.40 ± 7.76 ± 11.04 ± 8.37 ±
PUFA 1.76 0.82 0.31 0.83 1.13 0.92 2.41 0.81

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Chapter IV

Alternative protein sources and different inclusion

levels in diets for the thicklip grey mullet Chelon

labrosus (Risso, 1827): effects on growth,

metabolism, animal welfare and product quality.

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Congress:

Sanz-Latorre, M., Salazar-Moreno, D., Sánchez-Ruiz, D., Martín, I., León-San Emeterio, J.,Martos-

Sitcha, J. A., Soto, M., Martínez-Llorens, S., Lekube, X., Aguado-Giménez, F. (2024, June 17 – 20).

Fuentes de proteína alternativas y diferentes niveles de inclusión en dietas para Chelon labrosus:

efectos sobre el crecimiento, metabolismo energético y bienestar animal. [Oral presentation].

XIX Congreso Nacional de Acuicultura, Las Palmas de Gran Canaria, Spain.

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List of abbreviations

35 V: diet containing 35 % protein, of 100 % vegetal origin

35 VA: diet containing 35 % protein, of animal and vegetal origin

40 V: diet containing 40 % protein, of 100 % vegetal origin

40 VA: diet containing 40 % protein, of animal and vegetal origin

AB-PAS: Alcian blue – periodic acid Schiff staining

ANF: antinutritional factors

BSG: brewer´s spent grain

DHA: docosahexaenoic acid (C22:6n-3)

EPA: eicosapentaenoic acid (C20:5n-3)

FAME: fatty acid methyl esters

FM: diet containing 40 % protein, of animal and vegetal origin, including fishmeal

H-E: haematoxylin / eosin

HSI: hepatosomatic index

K: Fulton condition factor

MSI: mesenteric index

MUFA: monounsaturated fatty acids

P / E: protein / energy ratio

PUFA: polyunsaturated fatty acids

SFA: saturated fatty acids

SGR: specific growth rate

SSI: spleen somatic index

TGC: thermal growth coefficient

VSI: viscerosomatic index

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Results and discussion Chapter IV

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Results and discussion Chapter IV

Abstract

Omnivorous fish like Chelon labrosus are ideal candidates to be fed diets with vegetal protein,

favouring a more sustainable aquaculture, less reliant on fishmeal. Five (isolipidic, isoenergetic)

diets were formulated with the macroalgae Ulva lacinulata and brewer´s spent grain as

alternative ingredients. Two diets contained 100 % vegetal protein, at 35 and 40 % inclusion

(35V and 40V), other two diets had a mix of vegetal and animal (swine) protein at 35 and 40 %

(35VA and 40VA), and the last one contained 40 % protein, with a mixture of animal (fishmeal)

and vegetal protein (FM). Chelon labrosus (72.3 ± 18.5 g) were fed those diets in triplicate for

153 days and biometrical data, feed intake, liver, plasma and muscle metabolites, liver

antioxidant capacity, plasma cortisol levels, muscle fatty acid profile and liver and intestine

histological status were analysed. V diets provided the best growth values, especially 35V,

followed by FM diet, and the VA feeds rendered negligible growth. This mirrored feed intake,

and the low acceptance of the VA feeds may suggest low palatability of the animal ingredients,

most probably haemoglobin meal. Metabolite and cortisol levels and muscle fatty acids revealed

no relevant metabolic alterations. Liver morphology and antioxidant capacity were unchanged.

Some signs of intestinal inflammation were found in all treatments, suggesting adverse effects

of the diets. This species has proven its potential to be fed diets containing 100 % vegetal

protein, and further research is recommended to improve the feed formulation, avoiding the

aforementioned problems and achieve better growth rates.

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Results and discussion Chapter IV

Laburpena

Chelon labrosus bezalako arrain orojaleak proteina begetalekin elikatzeko aukera ona dira,

arrain irin behar txikiagodun akuikultura jasangarriagoa bultzatuz. Bost dieta (isolipidiko,

isoenergetiko) formulatu ziren Ulva lacinulata makroalga eta garagardo ahotz osagai

alternatibodunak. Dietetako bik % 100 proteina begetala zeukaten, % 35 eta 40 inklusio mailatan

(35 V eta 40 V), beste bik proteina begetal eta animal (txerria) nahasketa % 35 eta 40 inklusio

mailatan (35 VA eta 40 VA), eta azkenak % 40 proteina, begetal eta animal (arrain irina)

nahasketa (FM). Chelon labrosus (72.3 ± 18.5 g) jubenilak dieta horiekin elikatu ziren 153

egunetan zehar eta datu biometrikoak, pentsu ingestioa, gibel, plasma eta gihar metabolitoak,

gibelaren gaitasun antioxidatzailea, plasma kortisol maila, gihar gantz azido profila eta gibel eta

hestearen egoera histologikoa analizatu ziren. Hazkuntza balio onenak V dietekin lortu ziren,

batez ere 35 V dietarekin. VA pentsuekin hazkuntza mesprezagarria lortu zen, eta FM dietarekin

tarteko balioak lortu ziren. Hau pentsu ingestioaren isla da, eta VA pentsuen onarpen maila

baxuak animalia jatorriko osagaiek, seguru asko hemoglobinak, sortutako palatabilitate

arazoren bat adieraz dezake. Metabolito eta kortisol mailek eta gihar gantz azido profilak ez

zuten aldaketa metaboliko esangarririk azaleratu. Gibelaren gaitasun antioxidatzailea eta

egitura histologikoa ez ziren aldatu. Heste hantura sintoma arinak aurkitu ziren tratamendu

guztietan, dietek eragin negatiboren bat eduki dezaketela adieraziz. Espezie honek % 100

proteina begetaldun dieten bidez elikatua izateko gaitasuna erakutsi du, eta ikerketa

sakonagoak beharrezkoak dira pentsu formulazioa hobetzeko, ikusitako arazoak ekidin eta

hazkuntza balio hobeak lortzeko.

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1. Introduction

Aquaculture is the fastest growing animal production industry (Tacon, 2019), but it has to

experience further development in order to satisfy the ever-growing demand for high quality

aquatic products, as capture fisheries have been stagnated since the 1990s (FAO, 2022). The

concomitant increase of feed consumption by this industry makes the use of fishmeal as a feed

ingredient still a worrying issue, despite the vast improvements achieved in lowering its inclusion

levels in aquafeeds. Therefore, finding alternatives to fishmeal is important to help aquaculture

rely less on the exploitation of wild fisheries (Naylor et al., 2000; 2009), and omnivorous and

herbivorous fish species have the ability to utilize a wider range of ingredients than carnivorous

ones, including protein of vegetal origin (Tacon & Metian, 2015). Therefore, diversifying cultured

species towards lower trophic level ones is interesting for the development of a more

sustainable aquaculture industry, less dependent on animal protein (Naylor et al., 2000).

As previously mentioned, the inclusion levels of fishmeal have decreased substantially

in aquaculture feeds in the last decades, and it has been partially substituted by land-based plant

protein (Naylor et al., 2009). Nevertheless, the complete substitution faces several issues caused

by these ingredients having unbalanced amino acid profiles, low essential fatty acids and high

contents of antinutritional factors (ANF), among others (Fjelldal et al., 2010). Those ANFs include

compounds like protease inhibitors, lectins, saponins or oligosaccharides, which can cause

inflammatory disorders on fish digestive tract, hindering digestion and endangering the health

of the animals (Krogdahl et al., 2010). Such pathologies have been found in several carnivorous

species of high market value, like salmonids (Baeverfjord & Krogdahl, 1996), European sea bass

(Torrecillas et al., 2017) or turbot (Gu et al., 2016).

Even though terrestrial based plant and animal products have been the main focus of

the research for alternative feed ingredients (Glencross et al., 2007), other candidates like

macroalgae are also worth considering (Øverland et al., 2017; Wan et al., 2019; Saleh, 2020).

Macroalgae do not show most of the aforementioned problems associated with terrestrial

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vegetal ingredients, as their amino acid profiles can be more balanced and some species have

high contents of polyunsaturated fatty acids (PUFA), most interestingly, those from the ω-3

group. On top of that, they are rich in bioactive compounds like polysaccharides, pigments or

phenolic compounds, which have beneficial effects for fish, acting as probiotics,

immunostimulants and improving antioxidant defence mechanisms. However, macroalgae

inclusion in aquafeeds carries its own problems that should be treated carefully, as they are not

completely devoid of ANFs and they can accumulate high levels of toxic metals like arsenic, lead

and mercury. All these benefits and problems, among others, have been extensively reviewed

by some authors, like Øverland et al. (2017), Wan et al. (2019) or Saleh (2020).

Food industry by-products are another interesting group of novel feed ingredients that

fit into the framework of circular economy (Geisendorf & Pietrulla, 2018). Brewer´s spent grain

(BSG) or beer bagasse is the main by-product of the brewing industry (Fernandes et al., 2022b).

This by product has already been successfully tested both as a main feed ingredient and as an

additive for many aquaculture species like gilthead sea bream (Estévez et al., 2021), European

sea bass (Fernandes et al., 2022a; Fernandes et al., 2022b), rainbow trout (Estévez et al., 2022),

stripped catfish (Jayant et al., 2018) or Pacific white shrimp (He et al., 2020).

The thicklip grey mullet Chelon labrosus is an omnivorous teleost fish common to the

coastal areas of many European, Middle Eastern and North African countries (Turan, 2016). The

omnivorous nature of the species and the ease to cultivate it make it a good candidate for

intensive culture (Altunok & Özden, 2017; García-Márquez et al., 2022), and there is great

potential for the use of alternative vegetal ingredients in its diets (de las Heras et al., 2012;

Vílchez-Gómez et al., 2017; García-Márquez et al., 2022; García-Márquez et al., 2023).

Given the potential of C. labrosus for diversifying aquaculture systems, the present study

aimed to determine the effects produced by diets formulated with alternative protein sources,

differing on protein inclusion levels as well as in the origin of protein on growth, metabolism

(plasma, liver and muscle metabolites), stress (plasma cortisol), immune status (liver antioxidant

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capacity), intestinal health (histological analysis) and nutritional quality (fatty acid profile) in this

low trophic level fish.

2. Materials and methods

2.1 Experimental fish

The experimental fish (thicklip grey mullet C. labrosus, n = 300, initial weight 72.3 ± 18.5 g) were

obtained from the grey mullet stock at the facilities of the Research Centre for Experimental

Marine Biology & Biotechnology (PiE-UPV/EHU). One month before the beginning of the

experiment, fish were acclimated to the experimental tanks and they were fed a commercial

diet intended for gilthead sea bream, containing 44 % protein and 20 % lipids (D-2 Optibream

AE 1P, Skretting, Stavanger, Norway).

2.2. Experimental design

The trials were carried out at the facilities of the Marine Aquaculture Plant ‘El Bocal’ (COST-

IEO/CSIC) located in Santander (Cantabria, N Spain). The fish were distributed into five

experimental groups in triplicate (20 fish / tank). Fifteen 360 L cylindrical tanks arranged in an

open-flow seawater system were used. Each one was provided with constant aeration to keep

adequate oxygen levels. Each treatment group was fed a different diet (Table 1) ad libitum twice

a day seven days a week and the uneaten feed was siphoned out for the measurement of the

real intake. The experimental period lasted 153 days (June – November 2023).

2.3. Experimental diets

The experimental diets were manufactured by the cooking-extrusion process at the Feed

Manufacture of the Institute of Animal Science and Technology at the Universitat Politècnica de

València (Valencia, Spain). A semi-industrial extruder from Clextral model BC45 (Saint Etienne,

France) was used for this purpose. The processing conditions included 100 rpm screw speed,

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110 °C temperature, 20 atm pressure, and 2 mm diameter pellets. Five feeds were formulated

based on alternative protein sources (BSG and the macroalgae Ulva lacinulata), differing on

protein inclusion level (35 and 40 %) and the origin of protein (100 % vegetal and mixture of

vegetal + animal). The experimental diets were denominated 35 V (35 % protein of 100 % vegetal

origin), 40 V (40 % protein of 100 % vegetal origin), 35 VA (35 % protein, 65 % of which being

vegetal protein + 35% animal protein), 40 VA (40 % protein, 65% vegetal + 35% animal protein)

and FM (fishmeal diet, 40 % protein, 65% vegetal + 35% animal protein, with 20 % fishmeal). The

complete formulations and proximal compositions are shown in Table 1.

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Table 1: composition of the experimental diets used for the experiment. Ingredient composition is

expressed as g ingredient / kg of feed, proximate composition as % of feed wet weight and fatty acid

profile as % of lipid extract. Full fatty acid profiles can be found in the appendix (Table A1).

35 V 40 V 35 VA 40 VA FM
Ingredients (g / kg)
Fishmeal 0 0 0 0 200
Soybean meal 262 302 173 198 168
Wheat gluten 150 205 60 100 0
Swine haemoglobin meal 0 0 64 75 47
Pig meat meal 0 0 95 110 75
BSG 90 100 80 90 70
Ulva lacinulata 115 125 90 100 80
Wheat 185 72 256 147 206
Fish oil 50 50 50 50 30
Soy oil 38 36 32 30 54
Soy lecithin 20 20 20 20 0
Maltodextrin 50 50 50 50 50
Monocalcium Phosphate 30 30 20 20 10
Vitamins and mineral
10 10 10 10 10
premix1
Proximate composition (% wet weight)
Crude protein 34.96 40.01 34.95 39.96 39.99
Crude lipids 12.04 12.00 11.97 12.03 11.98
Carbohydrates 33.82 29.20 32.47 25.53 26.23
Moisture 10.87 10.21 10.56 11.68 10.47
Ash 8.31 8.58 10.05 10.80 11.33
Energy content
Energy (MJ / kg)2 16.04 16.10 15.79 15.49 15.59
Protein / energy (g
21.79 24.85 22.13 25.80 25.64
protein / MJ)
Fatty acid profile (% of lipid extract)
Σ SFA 24.39 24.87 45.40 40.37 28.82
Σ MUFA 24.79 24.46 40.35 35.96 29.85
Σ PUFA 50.81 50.67 14.25 23.66 41.33
Σ ω-3 FA 12.99 13.60 1.09 4.13 8.53
Σ ω-6 FA 36.50 35.66 12.91 19.01 31.95
EPA + DHA 7.36 7.94 0.35 1.98 4.21
ω3 / ω-6 FA 0.36 0.38 0.08 0.22 0.27
35 V: 35 % protein of vegetal origin; 40 V: 40 % protein of vegetal origin; 35 VA: 35 % protein, vegetal +

animal protein; 40 VA: 40 % protein, vegetal + animal protein; FM: Fishmeal diet; BSG, Brewer’s spent

grain; NFE, nitrogen-free extract; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; SFA, saturated

fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; FA, fatty acids.

Vitamin-and-mineral mix (per kg): colloidal silica, 176.7 g; sepiolitic clay, 357.3 g; Butylhydroxytoluene, 20 g; Vitamin B12, 0.010 g;
1

niacinamide, 20 g; Folic Acid, 1.5 g; Vitamin D3, 2 x 105 UI; Vitamin A, 2 x 106 UI; Vitamin E, 10 g; Vitamin K3, 2.5 g; Vitamin B1, 3 g;

Vitamin B2, 3 g; Vitamin B6, 2 g; Calcium d-pantothenate, 10 g; Biotin, 0.3 g; Inositol, 50 g; Betaine Anhydrous, 50 g; Iron (II) sulphate,

monohydrate, 0.6 g; potassium iodide 0.05 g; Copper (II) Sulphate Pentahydrate, 0.9 g; Manganese (II) oxide, 0.96 g; Zinc sulphate

monohydrate, 0.75 g; sodium selenite, 0.001 g; Medium: calcium carbonate, sodium chloride, potassium chloride.

2Calculated considering 16.7 kJ / g for protein and carbohydrates and 37.7 kJ / g for lipids (Fellows, 2022).

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2.4. Sample collection

Biometrical data (fork length and body weight) were collected at day 0 (T0), 30 (T1), 60 (T2), 90

(T3), 120 (T4) and 153 (T5). For that, all specimens from each tank were previously sedated with

clove oil (eugenol) dissolved in 96% ethanol (20 ppm) in the holding tanks, and then, they were

anaesthetized with the same anaesthetic (40 ppm) in a 100 L tank. At T5, four fish of each tank

(n = 60) were anaesthetized for blood sample collection, and then sacrificed for inner organs

sampling. Blood was collected from the caudal vein with a heparinized 1 mL syringe with a 25 G

needle and centrifuged for 15 minutes at 10000 rpm. The plasma was collected and frozen at -

80 °C for metabolite and cortisol analyses. Liver, gastrointestinal tract, perivisceral fat, spleen

and eviscerated body weights were recorded. A central piece of each liver and three pieces of

every intestine (proximal, medial and distal) were taken for further histological procedures. The

remaining liver parts were preserved frozen at - 80 °C until metabolite and total antioxidant

capacity analyses. Three more fish were slaughtered by anaesthetic overdose, grinded and

preserved frozen at - 80 °C until analysis of proximate composition and fatty acid profile.

2.5. Calculations

The following calculations were made:

Ln (Final body weight)− Ln (Initial body weight)

Specific growth rate (SGR) = 100 x (% / day)
Feeding days

3√ (Final body weight)− 3√ (Initial body weight)

Thermal-unit growth coefficient (TGC) =
𝛴𝛴 °𝐶𝐶 𝑥𝑥 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑

Liver weight (g)

Hepatosomatic index (HSI) = 100 x (%)
Total weight (g)

Visceral weight (g)

Viscerosomatic index (VSI) = 100 x (%)
Total weight (g)

Spleen weight (g)

Spleen somatic index (SSI) = 100 x (%)
Total weight (g)

Perivisceral fat weight (g)

Mesenteric index (MSI) = 100 x (%)
Total weight (g)

Fish weight (g)

Fulton condition factor (K) = 100 x
Length3 (cm)

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2.6. Metabolite analyses

Glucose, lactate, triglycerides, cholesterol and proteins were measured in plasma. Glucose,

glycogen, lactate, triglycerides and cholesterol were measured in liver and muscle. Liver and

muscle samples were mechanically homogenized in an acid medium. Briefly, 7.5 mL of 0.6 N

Perchloric acid (HClO4) was added to 1 g of tissue and it was homogenized using an Ultra Turrax

homogenizer T 25 basic (IKA-Werke; Staufen, Germany) with a single pass of approximately 6

seconds. After that, the reaction was stopped adding 7.5 mL of 1 M potassium bicarbonate

(KHCO3) per 1g of tissue. The samples were thoroughly vortexed in order to avoid the lipid phase

to separate from the rest, and an aliquot was immediately collected for cholesterol and

triglyceride analyses. The rest of the sample was centrifuged at 4000 rpm at 4 °C for 30 minutes,

and the aqueous phase was recovered for lactate, glucose and glycogen analyses. Plasma was

directly analysed. The metabolites (except protein) were analysed using commercial kits form

Spinreact (Barcelona, Spain): Spinreact Glucose-HK (cod. 1001200) was used for glucose and

glycogen, Spinreact Lactate (cod. 1001330) for lactate, Spinreact Triglycerides (cod. 1001311)

for triglycerides and Spinreact Cholesterol-LQ (cod. 41021) for cholesterol. Plasma protein was

measured using the Pierce BCA Protein Assay Kit (cod. 23225) from Thermo Fisher Scientific

(Waltham, USA).

2.7. Stress and antioxidant capacity

Plasma cortisol was directly measured using the DetectX Cortisol Enzyme Immunoassay Kit (cod.

K003-H1/H5) from Arbor Assays (Ann Arbor, USA). Total antioxidant capacity of liver was

measured using the Total Antioxidant Capacity Assay Kit (Colorimetric) (cod. Ab65329) from

Abcam (Waltham, USA).

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2.8. Fatty acid profile of muscle

The analysis of the fatty acid profile of muscle were performed at the “Analysis Service Unit

facilities of ICTAN-CSIC for the analysis of Chromatography”. Fatty acid methyl esters (FAME)

were obtained following the internal procedure from the aforementioned laboratory, using

sodium methoxide 0.5 M in methanol and acetyl chloride in methanol (1:10 v / v). FAMEs were

extracted by hexane. Samples were analysed in triplicate by gas chromatography, using a gas

chromatograph with a flame ionization detector (FID) Agilent 7820A (Agilent; Santa Clara, USA),

with the control software EZChrom Elite. The carrier gas used was helium (1 mL / minute). The

oven temperature was initially set at 50 °C for 1 minute and it was raised to 175 °C at a rate of

25 °C / minute. After that, temperature was increased to 230 °C at a rate of 4 °C / minute. The

injector and column were set at 250 °C. The split ratio was 40:1. Fatty acids were identified by

comparing retention times of the peaks with the FAME standards FAME 37, PUFA Nº2 Animal

Source and PUFA Nº3 Menhaden oil (Supelco; Bellefonte, Pennsylvania).

2.9. Histological and morphometric analyses

Sections of liver and proximal, middle and distal intestine were fixed in a 10 % formalin solution

buffered with seawater for 24 hours. Once fixed, formalin was replaced by 70 % ethanol until

processing. Samples were processed with an automated tissue processor (LEICA ASP 300S; Leica

Microsystems Nussloch GmbH, Germany) and embedded in paraffin wax blocks. Five µm thick

sections were obtained with the aid of a microtome (Leica RM2125RTS; Leica Microsystems

Nussloch GmbH, Germany). For the intestine samples, three slices with a distance of 20 µm

between them were placed in each slide, one slide per sample. For performing the

morphometric analysis and the assessment of the histopathological status of the intestine and

liver, Haematoxilin-Eosin (H-E) staining was performed. The histochemical staining Alcian Blue-

PAS (AB-PAS) (Suvarna et al., 2019) was applied for the identification of intestinal goblet cells.

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Both staining techniques were carried out using an Autostainer XL (Leica Microsystems Nussloch

GmbH, Germany) and slides were mounted in a permanent medium (DPX) with cover slides.

For the morphometric analysis of the intestine, the following structures were measured

in every section (3 section / sample): diameter (2 measurements / section), longitudinal muscle

thickness (8 measurements / section), circular muscle (8 measurements / section), submucosa

(8 measurements / section), lamina propia (4 measurements / section), villi length (4

measurements / section) and epithelium height (3 measurements / villi). All measurements are

illustrated in Figure 1 A, B. In order to ensure an accurate analysis, samples that were not

sectioned perfectly perpendicular were excluded (Figure 1 C). Similarly, only villi longitudinally

sectioned through their medial area across their entire length were considered for measuring

(Figure 1B, D). Therefore, up to four villi in each section were selected for villi length, submucosa,

lamina propria and epithelium height measurements. That quantity was lower if not enough

suitable villi were found.

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A B
7

7
1
7
6

2
3 5

Figure 1: A, B; representation of the morphometric measurements of the intestine. 1: diameter; 2:

longitudinal muscle; 3: circular muscle; 4 submucosa; 5: lamina propria; 6: villi length; 7: epithelium

height. C: example of an intestine cross-section not perfectly transversal. D: villi not suited for analysis

because the entire length is not sectioned. H-E staining.

For the histopathological analysis of liver and intestine, samples were observed under

an Olympus BX61 light microscope (Olympus-Lifescience) looking for possible pathological

alterations in the tissue.

A semi quantitative scale was designed for the quantification of goblet cells in the

intestine, ranging from 1 to 7 (Figure 2). 1: almost no goblet cells; 2: some of the villi have few

goblet cells, mostly in the base area; 3: some villi do not have any goblet cells; 4: every villi have

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goblet cells; 5: many goblet cells, individualised; 6: groups of goblet cells with scarce space

between them; 7: almost no space between goblet cells.

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Figure 2: examples of the subjective scale used for the semi-quantification of goblet cells in the intestine

epithelium after AB-PAS staining. A: score 1. Almost no goblet cells; B: score 2. Some of the villi have few

goblet cells, mostly in the base area; C: Score 3. Some villi do not have any goblet cells. D: score 4. Similar

to 3, but every villi have goblet cells; E: score 5. Many goblet cells, individualised; F: score 6. Similar to 5,

groups of goblet cells with scarce space between them. G: score 7. Almost no space between goblet cells.

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2. 10. Statistical analysis

All statistical analyses were carried out with the aid of the IBM SPSS Statistics 27 software (IBM

corp. 2020). Normality of the data was checked with the Shapiro-Wilk test and the homogeneity

of variances with Levene´s test. Body weight, feed intake and SGR were tested across treatments

using the repeated measures ANOVA test, said variables being the dependent ones, the diets

independent and the samplings the within-subject factor, with five levels. Every other variable

was tested using one-way ANOVA followed by Tukey´s post-hoc test for variables with normal

distributions and homogenous variances, or Kruskal-Wallis test followed by Dunn-Bonferroni´s

post-hoc test, when said criteria was not met. In every case, the tested variable was dependent

and diets independent. The significance of every statistical test was set at p < 0.05. Data are

shown as means ± standard deviation.

Most relevant muscle fatty acids were selected as the ones explaining the highest

amount of variance in the first two axes of PCA. Differences in composition of muscle fatty acid

profiles across dietary treatments were analysed by PERMANOVA, based on S17 Bray Curtis

similarity (4999 permutations).

2.11 Ethical statement

The fish were always handled (routine management and experimentation) according to the

Guidelines of the European Union (2010/63/UE) and the Spanish legislation (RD 53/2013) for

the use of laboratory animals. Moreover, all the people involved in the experiments had the

required FELASA accreditations for each procedure (ECC556/2015). The project was evaluated

by the official ethics committee with favourable report number NTS-ES-081792.

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3. Results

Mean water temperature was 21.67 °C between T0 – T1 samplings, 23.85 °C between T1 – T2,

22.21 °C between T2 – T3, 20.59 °C between T3 – T4, 17.48 °C between T4 – T5 and 21.21 °C

between T0 – T5.

Mortality was not observed during the experimental period in any of the experimental

groups. After 153 days of experimentation, repeated measures ANOVA showed that there were

significant differences between dietary treatments with regards to weight increase (p < 0.001)

and feed intake (p < 0.001). Tukey test (p < 0.05) revealed that fish fed the 35 V and 40 V diets

had significantly higher final body weights, followed by the fish fed the FM diet, while the lowest

ones were achieved by the 35 VA and 40 VA groups. In the last period of the experiment (T4 –

T5) weight increase slowed down in the groups fed the V and FM diets, and the VA groups

showed a decrease in weight (Figure 3 A). Feed intake was also higher in the V groups (the

highest in 35 V group), followed by the fish fed FM, and lower in the VA groups (Tukey test, p <

0.05). Between T4 and T5, there was a drop in feed intake, generalized to every treatment,

evidenced by the flattening tendency of the accumulated feed intake data shown in Figure 3 B).

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Figure 3: weight gain (A) and accumulated feed intake (B) of C. labrosus fed with experimental diets for

153 days. 35 V: 35 % protein, 100% vegetal protein; 40 V: 40% protein, 100 % vegetal protein; 35 VA: 35

% protein, vegetal + animal protein; 40 VA: 40 % protein, vegetal + animal protein; CTR: Control diet.

Groups with different letters at the last sampling point (T5) show statistically significant differences (p <

0.05). X axis: days; Y axis: A) mean fish body weight (g); B) accumulated feed intake (g feed / tank).

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At every sampling, SGR results mirrored the results observed in weight gain (Table 2).

The highest SGR values were achieved by the fish fed the V diets and the lowest by the groups

fed the VA diets, which were close to zero or even negative throughout the trial. Considering

the entire experimental period (T0 – T5), the tested diets were separated into three statistically

differentiated groups (Tukey, p < 0.05), V diets forming the group with the highest SGR, followed

by the FM diet with intermediate values and VA diets rendered the lowest growth results

(repeated measures ANOVA, p < 0.001).

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Figure 4: Specific Growth Rate (SGR) and Thermal-unit Growth Coefficient (TGC) of C. labrosus fed

experimental diets for 153 days. Groups with different superscripts show statistically significant

differences (p < 0.05). Statistics were only calculated considering the entire period (T0 – T5). Data are

shown as means ± standard deviation. 35 V: 35 % protein of vegetal origin; 40 V: 40 % protein of vegetal

origin; 35 VA: 35 % protein, vegetal + animal protein; 40 VA: 40 % protein, vegetal + animal protein; FM:

Fishmeal diet.

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One-way ANOVA revealed significant differences (p < 0.05) in HSI, being the highest in

the FM group, followed by the V groups (Table 2). The VA diets provided the lowest amounts of

perivisceral fat, as shown by the MSI (one-way ANOVA, p < 0.05), and those fish had a leaner

morphology, as their lower K values show (one-way ANOVA, p < 0.05). No differences were

found in the relative size of the spleen, SSI (one-way ANOVA, p > 0.05).

Table 2: somatic indexes of C. labrosus fed experimental diets for 153 days. Groups with different

superscripts show statistically significant differences (p < 0.05). Data are shown as means ± standard

deviation.

35 V 40 V 35 VA 40 VA FM
EvisW (g) 97.66 ± 14.46ab 105.45 ± 5.53a 72.17 ± 15.14b 73.25 ± 9.05b 87.11 ± 9.59ab
DigestL/ForkL 2.85 ± 0.09 2.88 ± 0.13 2.78 ± 0.06 2.93 ± 0.09 2.79 ± 0.29
HSI 0.92 ± 0.05ab 1.03 ± 0.33ab 0.69 ± 0.09bc 0.64 ± 0.11c 1.00 ± 0.17a
VSI 12.27 ± 0.78a 12.40 ± 0.35a 10.17 ± 0.25bc 9.37 ± 0.49c 11.40 ± 1.15ab
SSI 0.21 ± 0.02 0.23 ± 0.02 0.17 ± 0.02 0.17 ± 0.04 0.20 ± 0.02
MSI 3.72 ± 0.82a 4.37 ± 0.73a 1.67 ± 0.57b 1.53 ± 0.32b 3.31 ± 0.01a
K 1.44 ± 0.06ab 1.50 ± 0.02a 1.31 ± 0.07bc 1.29 ± 0.05c 1.47 ± 0.03a

35 V: 35 % protein of vegetal origin; 40 V: 40 % protein of vegetal origin; 35 VA: 35 % protein, vegetal +

animal protein; 40 VA: 40 % protein, vegetal + animal protein; FM: Fishmeal diet; EvisW: eviscerated

weight; DigestL / ForkL: digestive tract length / fork length; HSi: Hepatosomatic Index; VSI: Viscerosomatic

Index; SSI Spleen somatic index; MSI: Mesenteric index; K: Fulton condition factor.

Levels of liver glucose, glycogen, lactate, triglycerides and cholesterol were not

significantly different across any of the experimental treatments (Table 3), as shown by the one-

way ANOVA or Kruskal-Wallis tests, depending on the normality and the homogeneity of

variances of the data (p < 0.05). In muscle, cholesterol levels were higher in the 35 V group, but

the rest of metabolites were constant (Kruskal-Wallis + Dunn-Bonferroni, p < 0.05). In the case

of plasma, the levels of most of the metabolites were significantly higher in the V groups than in

the VA ones (one-way ANOVA / Kruskal-Wallis, p < 0.05), glucose being an exception, as it was

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similar in all of them (one-way ANOVA, p > 0.05). No differences in plasma cortisol Kruskal-

Wallis, p > 0.05) or liver antioxidant capacity (one-way ANOVA, p > 0.05) were found.

Table 3: liver, muscle and plasma metabolite levels of C. labrosus fed experimental diets for 153 days, as

well as plasma cortisol and liver antioxidant capacity. Groups with different superscripts show statistically

significant differences (p < 0.05). Data are shown as means ± standard deviation.

35 V 40 V 35 VA 40 VA FM
Liver
Glu (mM / gww) 13.20 ± 0.86 12.68 ± 2.42 14.78 ± 2.59 13.60 ± 2.57 12.47 ± 1.56
Gly (mM / gww) 59.34 ± 5.90 52.64 ± 6.69 63.45 ± 4.04 58.04 ± 15.39 59.37 ± 8.08
Lact (mM / gww) 1.48 ± 0.43 1.05 ± 0.14 1.25 ± 0.41 0.89 ± 0.04 1.05 ± 0.12
TAG (mM / gww) 26.24 ± 13.15 18.14 ± 4.37 22.91 ± 1.59 19.42 ± 9.23 16.21 ± 9.55
Chol (mM / gww) 14.03 ± 10.95 9.46 ± 4.90 12.82 ± 1.46 9.65 ± 7.68 6.96 ± 5.98
Ant (nmol Trolox / mg 151.30 ±
147.96 ± 5.79 157.92 ± 15.41 156.36 ± 5.37 157.14 ± 5.02
protein) 11.88
Muscle
Gly (mM / gww) 26.52 ± 2.92 25.19 ± 5.03 20.82 ± 2.20 17.72 ± 1.66 28.38 ± 8.59
Lact (mM / gww) 65.23 ± 10.21 71.07 ± 7.94 53.57 ± 8.95 53.17 ± 4.59 70.88 ± 4.98
TAG (mM / gww) 32.40 ± 6.91 24.46 ± 5.21 23.47 ± 3.28 26.33 ± 8.26 25.54 ± 1.59
Chol (mM / gww) 36.84 ± 3.87a 26.33 ± 4.43b 28.37 ± 2.64b 24.51 ± 1.22b 26.34 ± 2.08b
Plasma
Glu (mM) 36.00 ± 8.62 31.78 ± 7.40 30.99 ± 3.27 32.32 ± 3.54 31.98 ± 4.21
Lact (mM) 0.86 ± 0.12a 0.74 ± 0.12ab 0.60 ± 0.11bc 0.45 ± 0.07c 0.64 ± 0.11b
TAG (mM) 3.86 ± 2.07ab 6.03 ± 2.56a 2.56 ± 0.87b 2.52 ± 0.75b 3.19 ± 1.40ab
Chol (mM) 9.15 ± 1.37a 8.50 ± 0.64ab 5.87 ± 0.71b 5.88 ± 1.52b 8.67 ± 0.26a
Prot (mg / mL) 37.11 ± 5.0a 36.85 ± 4.57a 28.25 ± 3.38b 30.43 ± 4.52b 34.08 ± 3.45ab
Cort (ng / mL) 12.96 ± 6.29 12.81 ± 7.25 7.87 ± 2.58 10.95 ± 5.09 11.42 ± 4.59

35 V: 35 % protein of vegetal origin; 40 V: 40 % protein of vegetal origin; 35 VA: 35 % protein, vegetal +

animal protein; 40 VA: 40 % protein, vegetal + animal protein; FM: Fishmeal diet; Glu: glucose; Gly:

glycogen; Lact: lactate; TAG: triglycerides; Chol: colesterol; Prot: proteins; Ant: liver antioxidant capacity;

Cort: cortisol.

The histological structure of the liver was similar in all the dietary groups, and resembled

that of a healthy individual, with regularly shaped hepatocytes with a central nucleus (Figure 5

A). There was a high individual variability in the vacuolization level of the hepatocytes, but cells

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containing a single large vacuole causing the displacement of the nucleus were rare, and such

condition was never generalized to the entire sample (Figure 5 A). However, increased lipid-like

vacuolation was common at the fish sampled at the beginning of the experiment (Figure 5 B).

Figure 5: histological structure of C. labrosus liver fed experimental diets for 153 days. A: normal structure

of liver, comprised of slightly vacuolated hepatocytes with a central nucleus (35 V group). Insert: some

highly vacuolated hepatocytes surrounded by healthy tissue. B: liver comprised of highly vacuolated

hepatocytes, with a single large vacuole displacing the nucleus to the periphery (T0 group). H-E staining.

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All the intestinal samples presented the usual structure. This consists of the serosa layer

in the outermost part, muscular layer (longitudinal and circular layers), submucosae and the

mucosa, comprised of the epithelium and lamina propria (Rašković et al., 2011). However,

serosa layer was very thin and fragile, and, consequently, it was lost during processing in several

samples, and therefore, it was not measured. The lamina propria was relatively thick in general,

irrespective of the treatment, sampling time or intestinal section. At the beginning of the

experiment, in the proximal, medial and distal sections, enterocyte nuclei were aligned at the

basal region of the epithelia, and few leukocyte infiltrations could be found, possibly

lymphocytes (Figure 6 A). In the V groups, this kind of infiltration was higher than at the

beggining (Figure 6 B, C). However, this infiltration was lower in the distal section of the 40 V

group, similar to the observations carried out at the initial (T0) samples. In the VA groups cell

infiltrations were scarce in every intestinal section with the exception of the distal intestine of

35 VA group, for which cell infiltrations were higher, similar to the V feeds. In the FM group,

epithelial infiltration was low in the medial and distal intestine sections, but higher in the

proximal one. The morphometric analysis of the intestines did not reveal any significant

differences between diet groups (one-way ANOVA / Kruskal-Wallis, p > 0.05), with the exception

of the length of the villi at the medial section of the intestine, being the highest in group 35 V,

as shown by the one-way ANOVA + Tukey test, with a significance of p < 0.05 (appendix; Table

A2).

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Figure 6: histological structure of C. labrosus intestines fed experimental diets for 153 days. A: normal

structure of intestinal villi (T0 group). B (35 VA group), C (35 V group): intestinal villi with leukocyte

infiltration in the epithelia (arrows). H-E staining.

The semi quantitative goblet cell count (Figure 7) revealed that in every treatment,

goblet cells were more numerous in the distal section than in the proximal or medial sections

(Kruskal-Wallis + Dunn-Bonferroni, p < 0.05). No significant differences were found between

treatments in any of the intestinal sections (Kruskal-Wallis, p > 0.05).

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Figure 7: semi quantitative goblet cell count of C. labrosus intestines fed experimental diets for 153 days.

T0: beginning of the experiment; 35 V: 35 % protein, 100% vegetal protein; 40 V: 40 % protein, 100%

vegetal protein; 35 VA: 35 % protein, vegetal + animal protein; 40 VA: 40 % protein, vegetal + animal

protein; FM: Fishmeal diet; AU: arbitrary units. Groups with different superscripts show statistically

significant differences (p < 0.05) between intestinal sections inside a given treatment. Error bars represent

standard deviation.

Thirty-three fatty acids were identified in total in all the experimental groups (Table 4).

Seven of them were selected for statistical analysis, as they accumulated 95.1 % of the variance

on the axes 1 and 2 of the PCA. These fatty acids were C16:0 (palmitic acid), C16:1n7 (palmitoleic

acid), C18:1n9 (oleic acid), C18:2n6c (linoleic acid), C18:3n3 (linolenic acid), C20:5n3

(eicosapentaenoic acid, EPA) and C22:6n3 (docosahexaenoic acid, DHA). PERMANOVA analysis

only revealed significant differences (p < 0.05) in the contents of those seven fatty acids between

the beginning of the experiment (T0) and the 35 V, 40 V, 40 VA and FM groups. In all cases,

linoleic and oleic acids accounted for most of the differences, from 56.98 to 81.8 % of the

accumulated variance (Simper test). EPA and DHA did not significantly drive differences in fatty

acid profiles. In terms of the main fatty acid groups, saturated fatty acid (SFA) levels were

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constant across all the dietary treatments (Kruskal-Wallis, p > 0.05), and monounsaturated fatty

acid (MUFA) levels were the lowest in the V groups (one-way ANOVA + Tukey, p < 0.05), opposite

to the PUFA content, as it was the highest in the V groups (Kruskal-Wallis + Dunn-Bonferroni, p

< 0.05). The difference in PUFA levels was mainly driven by the fatty acids of the ω-6 group

(Kruskal-Wallis, p < 0.05), as no differences were found in the ω-3 fatty acids, as well as in the

sum of EPA and DHA and in the ω-3 / ω-6 fatty acid ratio (Kruskal-Wallis, p > 0.05).

Table 4: summary of the fatty acid profile (% of total lipids) of C. labrosus muscle fed experimental diets

for 153 days. Groups with different superscripts show statistically significant differences (p < 0.05). Full

fatty acid profiles can be found in the appendix (Table A3). Data are shown as means ± standard deviation.

T0 35 V 40 V 35 VA 40 VA FM
Fatty acid profile (% of lipid extract)
C16:0 20.78 ± 2.78 18.94 ± 0.42 18.72 ± 1.16 19.07 ± 0.97 20.10 ± 1.69 20.66 ± 1.86
C16:1n7 6.63 ± 1.55 5.51 ± 0.30 5.38 ± 0.80 5.74 ± 0.57 6.48 ± 0.82 6.28 ± 0.94
C18:1n9c 29.95 ± 5.13 24.46 ± 1.36 24.82 ± 2.03 29.56 ± 1.76 27.47 ± 2.90 27.79 ± 1.65
C18:2n6c 13.34 ± 3.40c 23.30 ± 0.74a 22.48 ± 3.15ab 15.51 ± 1.09bc 14.26 ± 1.98c 16.16 ± 1.75bc
C18:3n3 2.42 ± 0.74 3.35 ± 0.17 3.35 ± 0.46 2.76 ± 0.25 2.45 ± 0.49 2.55 ± 0.44
C20:5n3
2.79 ± 1.23 2.76 ± 0.15 2.85 ± 0.55 2.66 ± 0.38 3.08 ± 0.53 2.66 ± 0.41
EPA
C22:6n3
4.67 ± 2.18 5.17 ± 1.17 5.23 ± 1.62 6.21 ± 1.17 7.11 ± 1.31 5.23 ± 0.48
DHA
Σ SFA 28.79 ± 4.12 25.53 ± 0.61 25.51 ± 1.72 26.05 ± 1.20 27.64 ± 2.41 28.32 ± 2.57
Σ MUFA 43.52 ± 4.07a 35.37 ± 1.03b 35.77 ± 1.14b 41.93 ± 1.34ab 40.56 ± 1.86ab 40.53 ± 0.73ab
Σ PUFA 27.70 ± 5.02b 39.10 ± 0.56a 38.73 ± 1.50a 32.03 ± 0.81ab 31.80 ± 0.62ab 31.16 ± 2.35ab
Σ ω-3 FA 12.20 ± 4.14 13.68 ± 1.25 14.05 ± 1.91 14.26 ± 1.37 15.29 ± 1.43 12.85 ± 1.23
Σ ω-6 FA 14.61 ± 3.51 c 24.65 ± 0.71a 23.89 ± 3.12ab 17.05 ± 1.13bc 15.73 ± 2.03c 17.54 ± 1.84abc
EPA +
7.47 ± 3.40 7.92 ± 1.32 8.08 ± 2.17 8.87 ± 1.54 10.19 ± 1.82 7.89 ± 0.85
DHA
ω-3 / ω-6
0.92 ± 0.56 0.56 ± 0.07 0.60 ± 0.16 0.84 ± 0.13 0.99 ± 0.23 0.74 ± 0.10
FA
T0: beginning of the experiment; 35 V: 35 % protein of vegetal origin; 40 V: 40 % protein of vegetal origin;

35 VA: 35 % protein, vegetal + animal protein; 40 VA: 40 % protein, vegetal + animal protein; FM: Fishmeal

diet; BSG, Brewer’s spent grain; NFE, nitrogen-free extract; DE, digestible energy; EPA, eicosapentaenoic

acid; DHA, docosahexaenoic acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA,

polyunsaturated fatty acids; FA, fatty acids.

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4. Discussion

The suitability of an ingredient for feeding any given aquaculture species is a complex issue,

driven by the interactions of multiple nutritional factors (Glencross et al., 2007; Glencross,

2020). When the target organism of the nutritional study is a novel species for intensive

aquaculture, like C. labrosus, another layer of complexity is added to the equation, arising from

the general lack of knowledge about nutritional needs and adequate rearing or feeding

strategies. In the present research, remarkable differences in growth were observed among the

experimental groups. Growth performance of the fish fed the diets based on vegetable protein

(35 V and 40 V) was the highest since the beginning of the experiment, a difference that

increased over time until the end of the experimental period. In fact, the fish of the groups fed

with mixtures of protein, vegetable and animal (35 VA and 40 VA) showed negligible growth,

rendering final SGR values barely above zero. The growth values of the group fed with both

protein origins plus conventional fishmeal (FM) were intermediate between the ones fed with

only vegetable protein and the mixture.

Highly variable growth data of C. labrosus juveniles fed experimental diets can be found

in the literature. As usual, the highest published SGR results, to our knowledge, have been

obtained with very small individuals of approximately 1 g, in an experiment evaluating the

potential of the seaweed Porphyra purpurea as an alternative feed ingredient, were SGR values

of up to 2.99 were reported (Davies et al., 1997). Similarly, high SGR values ranging from 1.77 to

2.13 were obtained on fish of 6 g fed traditional ingredients (Altunok & Özden, 2017). When

considering bigger fish (around 15 g), Ojaveer et al. (1996) obtained SGR values ranging from

1.05 to 1.20, only using traditional feed ingredients, much greater values than the ones reported

by Amezcua et al. (2009) with juveniles of similar size, which ranged from 0.110 to 0.228. Quirós-

Pozo et al. (2024), reported SGR values between 0.36 and 0.74 in juveniles around 26 g, when

testing different water salinities and lipid sources. SGR ranging from 0.89 to 1.15 were observed

in C. labrosus of 46 g when brewer´s yeast was tested as an alternative protein source (Vílchez-

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Gómez et al., 2017). García-Márquez et al. (2022) tested the suitability of the microalgae

Chlorella fusca as a feed ingredient for C. labrosus juveniles around 85 g, and SGR values of 0.6

and 0.7 were obtained. When the same authors used slightly bigger fish, close to 100 g, and fed

diets formulated with C. fusca and Vibrio proteolyticus, lower SGR values between 0.42 and 0.47

were reported, (García-Márquez et al., 2023). Although comparisons between results obtained

by different methodologies are difficult to make, the SGR values obtained herein fall within the

wide range of SGR previously reported for C. labrosus juveniles, although they are not

particularly high.

Thermal-unit growth coefficient (TGC) is a useful coefficient to make the comparisons

more accurate, as it represents the growth values according to the ambient temperature.

However, the interpretation has to be made carefully, as TGC can be calculated either with the

absolute temperature or only with the effective temperature (Jauralde et al., 2013), that is the

temperature above the thermal threshold at which the fish stops feeding. As the lower

temperature at which C. labrosus stops feeding has never been published, only absolute TGC

can be calculated, and considered for comparisons between species. The highest TGC values

obtained herein, in the groups fed 100 % vegetable protein (V), are lower than ones reported

for already stablished aquaculture species like Atlantic salmon of 148 g (Thodesen et al., 2001).

These results prove the potential of this species for being fed diets based on alternative

ingredients and devoid of animal protein sources, while also emphasizing the need for further

research in order to optimize its diet and rearing conditions, which would help to boost the

obtained growth values.

Growth results mostly mirror the feed intake data, as the highest intake was recorded

with the V diets, followed by the FM one and the VA feeds were the least ingested. Feed intake

can be affected by multiple biotic and abiotic factors, like water temperature, nutrient and

energy balance, palatability, stress or the life stage of the animal (Burel et al., 1996; Bendiksen

et al., 2002; Glencross et al., 2007; Handeland et al., 2008; Eriegha & Ekokotu, 2017; Glencross,

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2020). Great variations in weight gain and feed intake were observed in relation to experimental

time independently of the treatment. This was especially remarkable in the case of the period

comprised between the last two sampling times, when growth slowed down in every group, and

feed intake dramatically dropped. Changes in water temperature can account for most of these

variations. Water temperature is considered the most important abiotic factor affecting fish

metabolism (Nytrø et al., 2014), and growth and feed intake are closely linked to it. Optimal

water temperature for rearing C. labrosus juveniles (32 – 54 g) has been reported to be close to

23 °C (Sanz-Latorre et al., 2024) and in the present research the best growth values were

obtained when water temperature was the closest to that value (T1 – T3). In the same paper,

the authors also state that growth slowed down significantly at temperatures around 18 °C

(Sanz-Latorre et al., 2024), temperatures encountered in the last period of the experiment (T4 –

T5).

Presently, the inclusion of animal protein, the ingredients of swine origin in particular,

seemed to have a greater negative effect than any other variable in feed acceptability and

ingestion. This is in opposition to the results obtained in other fish species when fed pork and

other terrestrial animal industry by-products. For instance, promising results have been

obtained in carnivorous species like Atlantic salmon (Hatlen et al., 2013; Liland et al., 2015),

rainbow trout (Bureau et al., 1999) or Japanese sea bass (Hu et al., 2012; Wang et al., 2012),

herbivores like Nile tilapia (Hernández et al., 2008) and omnivores like sharp snout sea bream

(Nogales-Mérida et al., 2010), channel catfish (Li et al., 2019) or flathead grey mullet Mugil

cephalus (Luzzana et al., 2005). However, haemoglobin meal at an inclusion level of 10 % has

been reported to reduce growth rates of the carnivorous gilthead seabream (Martínez-Llorens

et al., 2008). Moreover, in the cited paper, Martínez-Llorens et al. (2008) also observed that the

inclusion of haemoglobin meal caused the fish to reject the feed, although this apparent

reduction of feed intake could not be recorded due to complexities of the experimental setup

(personal communication). Therefore, haemoglobin meal could be causing a similar effect

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herein, reducing the palatability of the diet and, concomitantly, feed intake. However, feed

pellet characteristics could also be a factor to consider. All the experimental diets were

manufactured following the same methodology, but the different ingredients used for each of

them could have altered some key physical property of the pellet, like floatability or stability,

which are also important for feed intake and utilization (Glencross et al., 2007). However, no

remarkable difference in such physical characteristics was observed in the daily feeding of the

fish, so palatability is the most plausible explanation for the differences in ingestion. It is worth

mentioning that the FM feed contained the same haemoglobin meal as the VA diets, but its

ingestion was significantly higher. This could be because it also had fishmeal, an ingredient that

usually enhances palatability in fish diets (Dong et al., 2016), and it could partially compensate

for the negative effect of the other animal protein, although this feed did not reach the

acceptability of the diets formulated only with vegetal protein sources. Several strategies can be

implemented in order to improve the palatability of a given ingredient, like applying certain pre-

treatments to it or adding other additives to the feed, but it is generally more advisable to avoid

ingredients with such a negative impact on feed palatability and ingestion (Glencross et al.,

2007). Therefore, it would be appropriate to perform palatability tests before committing to a

long-term feeding experiment when using ingredients of swine origin in C. labrosus diets.

Among the diets that provided the best results, 35 V and 40 V, the highest final weight

and SGR values were obtained with the 35 V feed, although the differences were not statistically

significant. In terms of ingestion, fish fed the 35 V had a significantly higher feed intake than the

ones fed the 40 V diet. Although 35 V and 40 V feeds did not have exactly equal composition,

the same ingredients at similar inclusion rates were used for both, therefore, the difference on

feed intake is not likely to be driven by palatability issues in this case. Regulation of food intake

by fish is still a source of debate among researchers (Volkoff & Rønnestad 2020), and aside from

the factors previously discussed, energy and nutrient content of the feed seems to be a key

modulator of it. The main hypothesis is that fish eat in order to satisfy their energy requirement,

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and they stop feeding once energy need is met (Wilson & Halver, 1986). However, our results

seem to challenge this principle, as the experimental feeds were isoenergetic in terms of crude

energy. Therefore, energy intake was not the same between the two treatments, although

actual digestible energy content was not measured. Recent research has improved the

understanding of appetite regulation in fish, and glucose and fatty acid sensing mechanisms that

activate several signalling pathways that ultimately lead to food intake regulation have been

described, as reviewed by Conde-Sieira & Soengas (2017). These mechanisms give a satisfactory

explanation to the classic hypothesis of the regulation of energy intake, and are very similar to

the ones known for mammals. Aside from carbohydrates and lipids, the other main nutrient are

amino acids, and appetite regulation driven by amino acid sensing in mammals is well known.

There is a surprising knowledge gap related to this on fish, but it is reasonable to think that these

mechanisms could also exist in this group of animals (Conde-Sieira & Soengas, 2017). In the

present experiment, a decrease of ingestion without a significant impact on growth was

observed at increased protein inclusion levels, and further research is needed in order to confirm

if protein intake can be a modulator of overall food ingestion in this species. The non-

significantly higher growth obtained with the 35 V diet cannot be overlooked, and it could be

related to the lower energy intake of the 40 V group, that could cause some of the protein to be

catabolized in order to obtain energy (Wilson & Halver, 1986), a slight inefficiency of feed usage.

In a previous research about the effects of the protein / energy ratio (P / E) on C. labrosus

growth, similar results were obtained (Ojaveer et al., 1996). There, varying P / E were tested in

two different lipid inclusion levels. When comparing increasing protein contents inside each lipid

inclusion level, a decrease of feed intake was observed alongside the increment of protein level.

Lower P / E also resulted in higher growth and feed efficiency. However, it has to be taken into

account that the P / E tested herein are higher than the optimal one found by Ojaveer et al.

(1996), and that the actual digestibility of the feeds was not measured in either of the two

researches. Therefore, it can be concluded that C. labrosus, when fed diets of similar

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compositions, shows the best growth results with a P / E between 21.79 (present research) and

19.72 mg protein / MJ (Ojaveer et al., 1996) at the cost of increasing feed intake in respect to

feeds with higher P / E.

The liver, muscle and the perivisceral fat are the main tissues for energy storage in fish

(Weil et al., 2013). Alterations on the relative weights of liver and perivisceral fat were observed

between treatments, fish fed the VA diets showing the lowest values for both of them at the

end of the experiment. This is not surprising on fish that have been with such a low nutrient and

energy intake for 153 days, which would force them to consume their energy reserves in order

to survive. However, the relative abundances of liver metabolites did not show any alteration in

the patterns of consumption or accumulation of any given tested metabolite. In fact, among the

main reserve tissues, muscle cholesterol was the only metabolite that showed a statistically

significant difference, being the highest in the 35 V group. This could be attributable to this

group being the one with the highest energy intake. The relative stability in reserve tissue

metabolites when nutrient and energy intakes were so dramatically different among dietary

groups could be due to an adaptation to adverse conditions generalized in fish (Foster & Moon,

1991). In their natural habitats, fish can routinely face long fasting periods caused by several

factors like spawning migrations or seasonal variations in temperature or food availability

(Costas et al., 2011; Pujante et al., 2015). These factors can induce a hypometabolic state

characterized by low activity and energy expenditure that allow some animals to survive with

little to no food intake without endangering their survival (Storey & Storey, 2004). This state

seemingly happened in fish fed with the mixture of animal and vegetal protein (VA). Lower

energy mobilization in these groups was evidenced by the lower concentrations of plasma

lactate, triglycerides, cholesterol and proteins, whereas plasma glucose levels were unaltered

by the dietary regime. While in some fish species the ability to maintain the glycaemia when

facing starvation has been found (Polakof et al., 2006; Costas et al., 2011), this is in disagreement

with what has been reported in the case of C. labrosus. In a previous research about fasting and

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re-feeding of C. labrosus, plasma glucose levels decreased under food deprivation (Pujante et

al., 2015), but great differences exist between that research and the present one in terms of

objectives and methodology. In our experiment, unlike the one conducted by Pujante et al.

(2015), there was no real food deprivation, and the time period considered was much longer,

153 days, in contrast to the 21 days of the cited paper. It is plausible that the fish in the present

trial suffered an initial decrease in plasma glucose, but compensatory mechanisms for the

maintenance of glycaemia might have been successfully activated by the time of the final

sampling, probably by redirecting perivisceral fat reserves for gluconeogenesis, considering the

stable liver glycogen reserves and the reduction of MSI. In the same paper by Pujante et al.

(2015), a food deprivation period of 21 days triggered an increase in plasma cortisol levels, in

opposition to the lack of plasma cortisol differences observed herein. In fact, fish fed the VA

diets had non-significantly lower plasma cortisol levels than any other group. A similar principle

applied for plasma glucose can be used to explain such disparity between studies.

In the presence of a stressor, like starvation, one of the main primary stress responses

in fish is the activation of the hypothalamic-pituitary-interrenal axis, which ultimately releases

cortisol to the bloodstream, leading towards the mobilization of energy in order to face the

stressor (Wendelaar Bonga, 1997). However, it is known that under situations of chronic stress,

plasma cortisol concentration can return to basal levels. Therefore, low cortisol concentrations

do not necessarily imply an absence of stressors (Wendelaar Bonga, 1997), and that might be

the case in the present research. Cortisol is a modulator of metabolism and its secretion can

enhance the energy mobilization of the fish, supporting growth, as seen in C. labrosus held at

low stocking density by de las Heras et al. (2015). Thus, lower plasma cortisol levels could play a

part on the induction of the hypometabolic state previously suggested for the VA groups.

Despite fish being able to survive for long periods with very little food intake, like in the present

experiment, in the long term the consumption of energy reserves of liver and perivisceral fat

could eventually lead to their complete depletion and, consequently, death.

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The lack of significant alterations in liver metabolism is also reflected in the similarity of

the structure of this organ and its vacuolization level across all the experimental groups. The

increased lipid-like vacuolation (Wolf et al., 2015) could be related to the high lipid content (20

%) of the commercial feed provided during the acclimation period. High lipid intake can cause

the accumulation of excessively large lipid droplets in the hepatocytes, leading to the

displacement of the nucleus and the impairment of the normal functioning of the cell (Rueda-

Jasso et al., 2004), although there was no evidence to support that the observed vacuolation

level was a pathological alteration (Wolf et al., 2015). Similarly, no significant differences in liver

antioxidant capacity were found, which indicates that the experimental diets did not affect this

relevant mechanism for the maintenance of homeostasis (Martínez-Álvarez et al., 2005), despite

inclusion of macroalgae from the Ulva genera in fish diets has been reported to improve

antioxidant defences in other species like zebrafish (Rouhani et al., 2022). The similar relative

sizes of the spleen found herein in every group point towards a lack of differential effect of the

diet on this aspect of the immune system of the fish. It has been observed that an alteration in

spleen size might be due to pathogen infections or stress situations in general, due to its roles

in blood filtering and as blood cell reservoir (Bjørgen & Koppang, 2022).

The evaluation of the histological status of the intestine is an important tool to assess

the possible beneficial or adverse effects a novel diet could have in the digestive system of a fish

(Rašković et al., 2011). Ingredients of terrestrial plant origin can cause intestinal inflammation

in several aquaculture species like Atlantic salmon (Baeverfjord & Krogdahl, 1996; Knudsen et

al., 2008; Urán et al., 2008) European sea bass (Torrecillas et al., 2017) or turbot (Gu et al., 2016),

induced by antinutriotional factors (ANFs) abundant in this type of ingredients. Soybean meal

induced enteritis in Atlantic salmon is a classic example of such pathologies and is characterized

by a thickening of the lamina propria with high infiltration of inflammatory cells, shortening of

intestinal villi and loss of the normal vacuolization of enterocytes (Baeverfjord & Krogdahl,

1996). The morphometric analysis of the intestine did not detect any significant alteration in the

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structure of this organ, with the exception of an increase of villi length in the middle section of

the intestine in the 35 V group. Being the only parameter showing a difference and the lack of

any consistent pattern dependant of protein inclusion level or its origin imply that this is most

probably a result of individual variation rather than an effect of the diet. However, symptoms of

minor intestinal inflammation were found in the experimental fish, with some differences

between treatments. As a general observation, leukocyte infiltrations in the epithelia were

higher at the V groups and enterocyte nuclei displacement and disruption of their basal

alignment was more common than at the VA or FM groups, although these were not completely

devoid of these anomalies. However, these symptoms of inflammation were light, and in no case

reached the levels of severity typically reported on fish affected by enteritis (Baeverfjord &

Krogdahl, 1996; Urán et al., 2008). Interestingly, these signs of slight inflammation were scarce

in the intestines of the fish at the beginning of the experiment (T0). This suggests all the

experimental diets produced some kind of adverse effect on the digestive tract of C. labrosus.

Goblet cells are mostly mucine secreting cells that produce a protective mucus layer,

and are located alongside the enterocytes or absorptive cells in the digestive epithelia of the

intestine (Marchetti et al., 2006). In the present study, the relative abundance of goblet cells

was always higher in the distal intestine than in any other intestinal section. However, this value

was not significantly different between treatments. The distal section of the intestine is usually

the most affected by enteritis-like pathologies (Urán et al., 2008; Martínez-Llorens et al., 2012),

and high abundances of goblet cells have been previously linked to the need of protecting the

distal intestine in the presence of vegetal ingredients in fish diets (Monge-Ortiz et al., 2016).

However, goblet cells being more abundant at the distal intestine is a common feature across

several fish species, like the omnivorous banded tilapia (Okuthe & Bhomela, 2021), and it cannot

be considered a definitive sign of intestinal inflammation, but coupled with the aforementioned

epithelial infiltrations, it supports the conclusion that all the feeds tested herein might have a

slight negative effect for C. labrosus intestinal health.

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Considering all the experimental groups showed minor signs of intestinal inflammation,

the causative agent is most likely an ingredient common to all. Soy is a typical pro-inflammatory

ingredient for fish (Krogdahl et al., 2010), but the feed used before the experimental period also

contained it without causing evident signs of enteritis, as shown by the scarce amount of cell

infiltration in the digestive epithelia and goblet cells found in the distal intestine. Moreover, it

has been successfully used as a feed ingredient for this species without compromising intestinal

health (García-Márquez et al., 2022, 2023). Macroalgae, like U. lacinulata, can also contain ANFs

that could potentially cause a pathological alteration in the gastrointestinal tract of fish (Wan et

al., 2019). Research about the inclusion of macroalgae in C. labrosus diets is scarce. Davies et al.

(1997) found a reduction in growth when substituting a significant fraction of fishmeal by the

seaweed Porphyra purpurea in diets for C. labrosus, and the authors attributed this effect to the

different nutritional value of the algae and fishmeal. However, no histopathological analysis was

conducted. BSG is also rich in ANFs like cellulose, hemicellulose and lignin, and it is generally

considered of little use for feeding monogastric animals such as fish in its raw form (Fernandes

et al., 2022a; Fernandes et al., 2022b). However, raw BSG has been successfully used as a feed

ingredient for different fish species at inclusion levels higher than the tested herein. Kaur &

Saxena (2004) found that diets containing 30 % BSG were suitable for the freshwater

omnivorous fishes rohu (Labeo rohita) and catla (Labeo catla). Similarly, on the striped catfish

(Plotosus lineatus), another freshwater omnivorous species, BSG inclusion levels of 36.4 %, in

substitution of 50 % of the soybean meal of the fishmeal diet, provided the same growth values

as the fishmeal feed, devoid of BSG (Jayant et al., 2018). In an experiment conducted on the

herbivorous freshwater fish reba carp (Cirrhinus reba), the best growth results were obtained at

BSG inclusion levels of 13 %, when substituting 100 % of the fishmeal and soybean meal of the

reference diet (Chattaraj et al., 2024). In the case of Nile tilapia, a freshwater herbivorous, BSG

inclusion of 25 % did not negatively affect growth, when substituting 50 % of the fishmeal

protein of the fishmeal (Zerai et al., 2008). On the carnivorous fishes gilthead seabream and

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rainbow trout, BSG has been found to be an adequate feed ingredient at inclusion levels of up

to 15 % (Estévez et al., 2021; 2022). Disappointingly, none of the aforementioned papers

evaluates the effects of raw BSG on the intestinal histopathology of the fish. Cellulose can up-

regulate the expression of several inflammatory genes in a dose-dependent manner in fish

(Zhang et al., 2021). Therefore, it is plausible that the cellulose and other ANFs present in U.

lacinulata and BSG caused an inflammatory response in C. labrosus intestine, although, algae

having a negative impact on the digestive tract of C. labrosus seems unlikely, as both macro and

micro algae are relevant components of the natural diet of mullets (Cardona, 2016). However,

the present experimental design does not allow separating the effects of both ingredients. In

conclusion, U. lacinulata and BSG have been proven promising for their use in C. labrosus diets,

given the SGR values obtained, in line with other papers focusing on this species, but further

research is needed in order to alleviate the potential mild pathologies found in the intestines of

the fish. One possible solution for that would be the pre-processing of BSG to produce a

concentrate rich in protein and devoid of ANFs, which can be made using a combination of

biological, chemical and enzymatic treatments (Karlsen & Skov, 2022). In particular, solid-state

fermentation of BSG by the fungus Aspergilus ibericus has already been successfully used with

that purpose, and used for European sea bass with positive results in terms of growth and

intestinal health (Estevão-Rodrigues et al., 2024).

The muscle fatty acid profile of C. labrosus found herein, dominated by palmitic, oleic

and linoleic acids, is similar to previously reported ones for the same species (Rabeh et al., 2015;

García-Márquez et al., 2022; Rabeh et al., 2022; García-Márquez et al., 2023; Marrero et al.,

2024; Quirós-Pozo et al., 2024; Sanz-Latorre et al., 2024), as well as others, like turbot (Peng et

al., 2014), European sea bass (Tibaldi et al., 2015) or meagre (Fountoulaki et al., 2017). Although

the experimental feeds differed greatly in terms of the main fatty acid groups, V diets containing

more than 50 % PUFA and around 25 % of SFA and MUFA, while VA feeds had less than half

PUFA and more than 40 % SFA, the final fatty acid profiles of muscle were very similar, with only

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slight differences. This shows the ability of C. labrosus to regulate their muscle fatty acid profile

with a great independence of the fatty acid content of the diet, which is not surprising due to its

strong capacity for the modulation of fatty acid metabolism (Marrero et al., 2024). This ability is

especially remarkable when considering the dramatic differences in ingestion between the V

and VA groups. For instance, the VA diets contained very small amounts of ω-3 fatty acids, but

the low acceptance of these feeds implies that the ingestion of this type of fatty acid was almost

negligible by the fish treated with these diets. However, no statistical difference was found in

the levels of ω-3 fatty acids of muscle. The sum of EPA and DHA contents and the ω-3 / ω-6 fatty

acid ratio are usually used for the evaluation of the fatty acid quality of fish (Rabeh et al., 2015),

and the lack of significant differences in both parameters evidence a very similar nutritional

quality of the edible part for human consumption, which is fish muscle.

5. Conclusions

In conclusion, C. labrosus has proven its suitability to be fed diets formulated solely with vegetal

protein sources, as these feeds were the ones providing the best growth values, especially at

the 35 % protein inclusion level. The potential for the design of fishmeal-free diets for this

species presents a promising opportunity for advancing in the development of sustainable

aquaculture practices, reducing its reliance on marine resources.

6. Acknowledgements

This research was funded by the Basque Government [00003-INA2022-33, 00005-2101022023,

00010-PIT2020-22] and by the European Union-NextGenerationEU and Governement of

Cantabria through “Plan Complementario de Ciencias Marinas ThinkInAzul”.

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8. Appendix

Table A1: complete fatty acid profile of the experimental diets used for the experiment.

Fatty acid profile

35 V 40 V 35 VA 40 VA FM
(% of lipid extract)
C14:0 2.66 2.84 4.82 4.22 2.89
C15:0 0.24 0.25 0.44 0.39 0.29
C16:0 16.42 16.72 29.79 26.62 18.59
C17:0 0.62 0.64 0.60 0.59 0.62
C18:0 3.54 3.53 8.19 7.20 5.36
C20:0 0.35 0.34 0.64 0.56 0.46
C22:0 0.36 0.35 0.57 0.50 0.40
C24:0 0.20 0.19 0.34 0.29 0.21
C16:1n7 3.15 3.35 5.01 4.48 3.35
C18:1n7c 2.25 2.30 3.58 3.23 2.60
C18:1n9c 18.33 17.70 30.08 26.69 22.69
C20:1n9 0.74 0.77 1.14 1.04 0.84
C22:1n9 0.13 0.14 0.34 0.32 0.19
C24:1n9 0.19 0.19 0.20 0.22 0.18
C16:2n4 0.53 0.56 0.25 0.27 0.39
C18:2n6c 36.08 35.21 12.50 18.59 31.57
C20:2n6 0.09 0.10 0.00 0.00 0.00
C22:2n6 0.00 0.00 0.42 0.30 0.11
C16:3n4 0.80 0.86 0.00 0.24 0.46
C18:3n3 3.85 3.76 0.74 1.68 3.29
C18:4n3 0.89 0.95 0.00 0.28 0.50
C20:4n3 0.22 0.23 0.00 0.00 0.15
C20:4n6 0.33 0.35 0.00 0.13 0.27
C20:5n3 EPA 4.09 4.41 0.22 1.20 2.48
C22:5n3 0.67 0.72 0.00 0.20 0.39
C22:6n3 DHA 3.28 3.53 0.13 0.78 1.73
Σ SFA 24.39 24.87 45.40 40.37 28.82
Σ MUFA 24.79 24.46 40.35 35.96 29.85
Σ PUFA 50.81 50.67 14.25 23.66 41.33
Σ ω-3 FA 12.99 13.60 1.09 4.13 8.53
Σ ω-6 FA 36.50 35.66 12.91 19.01 31.95
EPA + DHA 7.36 7.94 0.35 1.98 4.21
ω3 / ω-6 FA 0.36 0.38 0.08 0.22 0.27
35 V: 35 % protein of vegetal origin; 40 V: 40 % protein of vegetal origin; 35 VA: 35 % protein, vegetal +

animal protein; 40 VA: 40 % protein, vegetal + animal protein; FM: Fishmeal diet; EPA, eicosapentaenoic

acid; DHA, docosahexaenoic acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA,

polyunsaturated fatty acids; FA, fatty acids.

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Table A2: morphometric parameters of C. labrosus intestines fed experimental diets for 153 days. Groups

with different superscripts show statistically significant differences (p < 0.05). Data are shown as means ±

standard deviation.

35 V 40 V 35 VA 40 VA FM
Proximal intestine
Diam (mm) 2.09 ± 0.57 1.58 ± 0.01 1.52 ± 0.16 1.59 ± 0.18 1.88 ± 0.20
Area (mm ) 2 3.48 ± 1.81 1.85 ± 0.01 1.80 ± 0.37 1.89 ± 0.42 2.70 ± 0.52
Lmusc (µm) 18.73 ± 1.55 14.16 ± 1.68 15.78 ± 3.09 18.54 ± 0.16 22.06 ± 10.32
Cmusc (µm) 22.00 ± 2.12 18.17 ± 2.12 20.66 ± 3.58 24.57 ± 1.55 24.92 ± 10.45
Mext (µm) 40.73 ± 0.68 32.33 ± 3.80 36.44 ± 6.67 43.10 ± 1.71 46.99 ± 20.55
Sub (µm) 32.79 ± 11.78 21.85 ± 0.09 20.31 ± 3.30 32.17 ± 9.84 24.52 ± 7.80
Lpropr (µm) 202.10 ± 52.16 160.19 ± 1.00 155.62 ± 44.89 147.98 ± 28.04 180.53 ± 18.43
Vheig (µm) 554.61 ± 136.81 364.14 ± 38.08 350.82 ± 77.40 346.25 ± 25.96 433.67 ± 59.57
Eheig (µm) 43.78 ± 4.14 40.97 ± 2.78 38.53 ± 4.82 40.86 ± 4.60 44.40 ± 1.71
Medial intestine
Diam (mm) 1.56 ± 0.08 1.47 ± 0.18 1.32 ± 0.11 1.32 ± 0.08 1.52 ± 0.24
Area (mm2) 1.89 ± 0.20 1.62 ± 0.37 1.37 ± 0.23 1.33 ± 0.22 1.75 ± 0.57
Lmusc (µm) 17.09 ± 1.50 19.74 ± 2.70 18.38 ± 0.81 17.92 ± 0.11 18.59 ± 3.57
Cmusc (µm) 23.22 ± 1.93 27.43 ± 2.89 23.30 ± 4.27 24.96 ± 1.41 26.16 ± 6.17
Mext (µm) 40.31 ± 3.42 47.17 ± 5.59 41.68 ± 4.42 42.88 ± 1.53 44.74 ± 9.74
Sub (µm) 33.44 ± 4.81 30.31 ± 0.99 24.35 ± 5.76 33.23 ± 5.15 36.76 ± 11.94
Lpropr (µm) 135.51 ± 18.94 160.65 ± 1.86 135.23 ± 18.68 124.87 ± 20.10 146.35 ± 6.34
Vheig (µm) 380.76 ± 31.24a 307.77 ± 14.87ab 293.02 ± 43.11b 300.76 ± 14.47ab 361.85 ± 8.53ab
Eheig (µm) 37.71 ± 2.66 35.67 ± 0.74 34.76 ± 3.25 33.33 ± 1.00 38.37 ± 2.93
Distal intestine
Diam (mm) 1.60 ± 0.27 1.70 ± 0.13 1.70 ± 0.13 1.85 ± 0.33 1.71 ± 0.03
Area (mm2) 2.03 ± 0.65 2.20 ± 0.36 2.29 ± 0.31 2.63 ± 0.97 2.23 ± 0.07
Lmusc (µm) 38.11 ± 13.03 43.00 ± 10.59 50.86 ± 34.96 37.92 ± 6.61 36.12 ± 10.33
Cmusc (µm) 26.06 ± 1.56 27.89 ± 3.68 24.40 ± 2.77 26.91 ± 5.84 24.25 ± 5.82
Mext (µm) 64.17 ± 13.88 70.88 ± 14.27 75.26 ± 37.73 64.82 ± 12.42 60.37 ± 16.10
Sub (µm) 35.12 ± 7.36 38.17 ± 5.45 39.01 ± 5.42 52.70 ± 18.70 36.44 ± 7.48
Lpropr (µm) 173.91 ± 15.82 212.20 ± 6.05 176.62 ± 27.17 216.11 ± 35.11 200.08 ± 41.13
Vheig (µm) 433.79 ± 88.04 464.57 ± 84.90 463.97 ± 95.93 435.43 ± 47.08 441.06 ± 22.85
Eheig (µm) 40.72 ± 5.25 43.85 ± 4.03 42.03 ± 2.74 45.22 ± 4.35 40.41 ± 5.39
35 V: 35 % protein of vegetal origin; 40 V: 40 % protein of vegetal origin; 35 VA: 35 % protein, vegetal +

animal protein; 40 VA: 40 % protein, vegetal + animal protein; FM: Fishmeal diet; Diam: transversal section

diameter; Area: transversal section area; Lmusc: longitudinal muscle layer thickness; Cmusc: circular

muscle layer thickness; Mext: muscularis externa thickness; Sub: submucosa layer thickness; Lpropr:

lamina propria thickness; Vheig: villus height; Eheig: enterocyte height.

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Table A3: full fatty acid profile (% of total lipids) of C. labrosus muscle fed experimental diets for 153 days.

Groups with different superscripts show statistically significant differences (p < 0.05). Data are shown as

means ± standard deviation.

T0 35 V 40 V 35 VA 40 VA FM
Fatty acid profile (% of lipid extract)
C12:0 0.05 ± 0.01 0.03 ± 0.01 0.03 ± 0.01 0.03 ± 0.01 0.04 ± 0.00 0.04 ± 0.01
C14:0 3.78 ± 0.98 2.91 ± 0.16 2.96 ± 0.42 3.08 ± 0.19 3.48 ± 0.48 3.43 ± 0.45
C15:0 0.39 ± 0.10 0.31 ± 0.02 0.32 ± 0.03 0.33 ± 0.03 0.38 ± 0.06 0.37 ± 0.03
C16:0 20.78 ± 2.78 18.94 ± 0.42 18.72 ± 1.16 19.07 ± 0.97 20.10 ± 1.69 20.66 ± 1.86
C17:0 0.55 ± 0.12 0.48 ± 0.01 0.50 ± 0.04 0.47 ± 0.02 0.51 ± 0.07 0.52 ± 0.05
C18:0 2.82 ± 0.27 2.44 ± 0.06 2.56 ± 0.08 2.61 ± 0.04 2.68 ± 0.11 2.86 ± 0.19
C20:0 0.24 ± 0.02 0.20 ± 0.02 0.21 ± 0.00 0.24 ± 0.01 0.23 ± 0.00 0.23 ± 0.01
C21:0 0.04 ± 0.01 0.04 ± 0.00 0.04 ± 0.01 0.04 ± 0.01 0.05 ± 0.01 0.04 ± 0.01
C22:0 0.07 ± 0.01b 0.09 ± 0.01a 0.09 ± 0.01a 0.08 ± 0.01ab 0.09 ± 0.01a 0.09 ± 0.00a
C24:0 0.07 ± 0.01 0.07 ± 0.01 0.07 ± 0.00 0.08 ± 0.01 0.09 ± 0.01 0.08 ± 0.00
C14:1n5 0.06 ± 0.02 0.05 ± 0.01 0.05 ± 0.01 0.05 ± 0.01 0.06 ± 0.01 0.05 ± 0.01
C16:1n7 6.63 ± 1.55 5.51 ± 0.30 5.38 ± 0.80 5.74 ± 0.57 6.48 ± 0.82 6.28 ± 0.94
C18:1n7c 4.14 ± 0.45a 3.51 ± 0.02c 3.53 ± 0.25bc 3.92 ± 0.10abc 3.99 ± 0.28abc 4.06 ± 0.29ab
C18:1n9c 29.95 ± 5.13 24.46 ± 1.36 24.82 ± 2.03 29.56 ± 1.76 27.47 ± 2.90 27.79 ± 1.65
C20:1n9 2.27 ± 0.20a 1.51 ± 0.00d 1.65 ± 0.06cd 2.20 ± 0.14ab 2.12 ± 0.07ab 1.94 ± 0.04bc
C22:1n9 0.25 ± 0.04 a 0.17 ± 0.01c 0.17 ± 0.01bc 0.23 ± 0.01ab 0.24 ± 0.01a 0.21 ± 0.01abc
C24:1n9 0.21 ± 0.04 0.16 ± 0.02 0.16 ± 0.02 0.22 ± 0.01 0.21 ± 0.03 0.19 ± 0.01
C16:2n4 0.56 ± 0.14 0.48 ± 0.02 0.48 ± 0.07 0.47 ± 0.04 0.53 ± 0.08 0.51 ± 0.06
C18:2n6c 13.34 ± 3.40c 23.30 ± 0.74a 22.48 ± 3.15ab 15.51 ± 1.09bc 14.26 ± 1.98c 16.16 ± 1.75bc
C20:2n6 0.50 ± 0.11 0.53 ± 0.02 0.56 ± 0.05 0.58 ± 0.07 0.50 ± 0.08 0.52 ± 0.05
C16:3n4 0.26 ± 0.08 0.25 ± 0.01 0.25 ± 0.02 0.18 ± 0.02 0.20 ± 0.03 0.20 ± 0.04
C18:3n3 2.42 ± 0.74 3.35 ± 0.17 3.35 ± 0.46 2.76 ± 0.25 2.45 ± 0.49 2.55 ± 0.44
C18:3n4 0.07 ± 0.01 0.05 ± 0.00 0.06 ± 0.00 0.06 ± 0.01 0.06 ± 0.01 0.05 ± 0.01
C18:3n6 0.16 ± 0.04b 0.24 ± 0.02a 0.26 ± 0.02a 0.23 ± 0.01a 0.19 ± 0.02ab 0.24 ± 0.03a
C20:3n3 0.19 ± 0.05 0.17 ± 0.01 0.19 ± 0.02 0.23 ± 0.03 0.20 ± 0.04 0.19 ± 0.04
C20:3n6 0.10 ± 0.02 0.09 ± 0.01 0.09 ± 0.01 0.11 ± 0.01 0.10 ± 0.02 0.10 ± 0.02
C18:4n3 0.62 ± 0.26 0.69 ± 0.02 0.77 ± 0.13 0.57 ± 0.06 0.66 ± 0.11 0.61 ± 0.08
C20:4n3 0.36 ± 0.10 0.32 ± 0.01 0.34 ± 0.02 0.40 ± 0.02 0.38 ± 0.01 0.35 ± 0.04
C20:4n6 0.45 ± 0.14 0.42 ± 0.05 0.43 ± 0.07 0.53 ± 0.07 0.58 ± 0.05 0.46 ± 0.04
C22:4n6 0.07 ± 0.02 0.07 ± 0.01 0.07 ± 0.01 0.09 ± 0.01 0.09 ± 0.01 0.07 ± 0.01
C20:5n3
2.79 ± 1.23 2.76 ± 0.15 2.85 ± 0.55 2.66 ± 0.38 3.08 ± 0.53 2.66 ± 0.41
EPA
C22:5n3 1.14 ± 0.39 1.22 ± 0.06 1.31 ± 0.18 1.44 ± 0.08 1.42 ± 0.03 1.26 ± 0.13
C22:6n3
4.67 ± 2.18 5.17 ± 1.17 5.23 ± 1.62 6.21 ± 1.17 7.11 ± 1.31 5.23 ± 0.48
DHA
Σ SFA 28.79 ± 4.12 25.53 ± 0.61 25.51 ± 1.72 26.05 ± 1.20 27.64 ± 2.41 28.32 ± 2.57
Σ MUFA 43.52 ± 4.07a 35.37 ± 1.03b 35.77 ± 1.14b 41.93 ± 1.34ab 40.56 ± 1.86ab 40.53 ± 0.73ab
Σ PUFA 27.70 ± 5.02b 39.10 ± 0.56a 38.73 ± 1.50a 32.03 ± 0.81ab 31.80 ± 0.62ab 31.16 ± 2.35ab
Σ ω-3 FA 12.20 ± 4.14 13.68 ± 1.25 14.05 ± 1.91 14.26 ± 1.37 15.29 ± 1.43 12.85 ± 1.23
Σ ω-6 FA 14.61 ± 3.51c 24.65 ± 0.71a 23.89 ± 3.12ab 17.05 ± 1.13bc 15.73 ± 2.03c 17.54 ± 1.84abc
EPA +
7.47 ± 3.40 7.92 ± 1.32 8.08 ± 2.17 8.87 ± 1.54 10.19 ± 1.82 7.89 ± 0.85
DHA
ω-3 / ω-6
0.92 ± 0.56 0.56 ± 0.07 0.60 ± 0.16 0.84 ± 0.13 0.99 ± 0.23 0.74 ± 0.10
FA
T0: beginning of the experiment; 35 V: 35 % protein of vegetal origin; 40 V: 40 % protein of vegetal origin;

35 VA: 35 % protein, vegetal + animal protein; 40 VA: 40 % protein, vegetal + animal protein; FM: Fishmeal

diet; BSG, Brewer’s spent grain; NFE, nitrogen-free extract; DE, digestible energy; EPA, eicosapentaenoic

acid; DHA, docosahexaenoic acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA,

polyunsaturated fatty acids; FA, fatty acids.

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General discussion

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General discussion

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 General discussion

List of abbreviations

35 V: diet containing 35 % protein, of 100 % vegetal origin

40 V: diet containing 40 % protein, of 100 % vegetal origin

ALA: α-linolenic acid

LA: linoleic acid

lc-PUFA: long-chain polyunsaturated fatty acid

P / E: protein / energy ratio

VA: diet containing both animal and vegetal protein

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 General discussion

Diversifying cultured species towards low trophic level ones is a key aspect to improve the

sustainability of aquaculture, as recommended by the European Commission (EC, 2021) under

the framework of the European Green Deal (EC, 2019). Low trophic level fish (omnivorous /

herbivorous) can be fed a wider range of feed ingredients than high trophic level ones

(carnivorous) and generally tolerate higher inclusion levels of vegetal ingredients in their diet.

Therefore, feeds for low trophic level fish can be formulated with little amounts of fishmeal and

fish oil, or even completely devoid of such traditional ingredients (Tacon & Metian, 2015).

The thicklip grey mullet Chelon labrosus, due to its omnivorous feeding habits during the

juvenile and adult phases, is an interesting fish for the diversification of aquaculture (García-

Márquez et al., 2021). Despite being a popular species in some Mediterranean countries and

cultivated in extensive traditional systems for millennia, its intensive culture has never been

developed (Crosetti, 2016). In recent years, great advancements have been achieved about its

nutritional needs (Ojaveer et al., 1996), digestive physiology (Pujante et al., 2017, 2018) and the

formulation of diets based on alternative ingredients (Davies et al., 1997; Vílchez-Gómez et al.,

2017; García-Marquez et al., 2022; 2023; Quirós-Pozo et al., 2024), but the development of a

specific diet for the thicklip grey mullet C. labrosus remains elusive.

The lack of any published data about long-term culturing of C. labrosus is surprising,

considering it is widely considered an interesting species for the diversification of European

aquaculture. Relevant information regarding this topic has been reported in the present PhD

Thesis for the first time (Chapter 1). After 686 days under intensive culturing conditions, C.

labrosus did not show any pathology or other signs of stress, and there was no mortality during

the trial. Moreover, the growth rates obtained during the experiment were in line with other

published growth rates of C. labrosus. However, besides proving the suitability of the species for

intensive aquaculture, the most relevant observation carried out in the long-term culturing

experiment was that differences in weight between the two vastly different dietary treatments

(45 % protein + 21 % lipid vs 35 % protein + 6 % lipid) started to be statistically significant at the

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 General discussion

end of the first year of the trial. The implication of this observation is that an average

aquaculture nutritional experiment, running for approximately 90 days (Teles et al., 2020),

would have missed such difference, rendering a false negative. This is not an argument indicating

that most aquaculture experiments are poorly designed or that their conclusions are inaccurate,

as 90 days are enough to fulfil the objectives of most research of this type. However, when the

aim is to gain knowledge about the nutritional needs of a novel aquaculture species, the

aforementioned false negatives could lead future research in an incorrect direction, and to the

failure of the establishment of such species as a viable aquaculture alternative.

As feeding involves the highest cost associated to aquaculture production (Naylor et al.,

2000), the design of a cost-effective specific feed that provides satisfactory growth results is

essential for the development of intensive aquaculture of C. labrosus. In both nutritional

experiments performed herein, the feed with the lowest protein to energy ratio (P / E) provided

the best growth results, 23.67 mg protein / kJ in Chapter 1 and 21.79 mg protein / kJ in Chapter

4. Although comparisons between different experiments are always challenging, it is remarkable

that Ojaveer et al. (1996), when conducting a research about the optimal P / E for diets of C.

labrosus, also obtained the best results with the lowest tested P / E (19.72 mg protein / kJ),

similar to the results obtained herein. These comparatively low P / E maximize growth rates, but

also feed intake (Ojaveer et al., 1996; Chapter 4). Therefore, it seems that P / E is the key

parameter for optimizing diets for C. labrosus, as concluded in Chapters 1 and 4. In Chapter 1,

the fish fed the diet containing 44.5 % of protein performed better than the ones fed 35 %

protein, indicating that increasing protein inclusion above 35 % is beneficial for promoting the

growth of this species. In contrast, in Chapter 4, the diet including 35 % protein (35 V) provided

a better growth performance than the one containing 40 % (40 V). However, the high protein

content of the feed from Chapter 1 was energetically balanced by a high lipid content (21 %),

while in Chapter 4 lipid levels were around 12 %, keeping P / E close to the optimal one in both

cases (Ojaveer et al., 1996).

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 General discussion

Chelon labrosus has proven its potential for being fed diets with a protein fraction of 100

% vegetal origin, as the feeds formulated this way provided better growth results than the ones

containing vegetal and animal protein, even fishmeal (Chapter 4). However, it is worth

mentioning that some of the animal protein employed, probably haemoglobin meal, caused a

significant reduction in feed intake. The inclusion of the macroalgae Ulva lacinulata and the food

industry by-product brewer´s spent grain proved suitable for C. labrosus nutrition, as the

obtained growth rates were in line with other published results (Ojaveer et al., 1996; Davies et

al., 1997; Vílchez-Gómez et al., 2017; García-Marquez et al., 2022; 2023; Quirós-Pozo et al.,

2024), although they were not particularly high. Some histological observations in the intestine

of the fish showed mild signs of inflammation that could partially explain the slow growth rates,

but those effects at intestinal level cannot be confidently attributed to any particular ingredient,

due to the experimental design. This evidences the need for further nutritional research on C.

labrosus and a careful evaluation of its response to alternative feed ingredients.

It is well known that C. labrosus has fatty acid desaturases with Δ5 and Δ6 activities,

which confer it the ability for the production of long chain polyunsaturated fatty acids (lc-PUFA)

from the essential precursors linoleic (LA) and α-linolenic (ALA) acids (Galindo et al., 2021).

However, currently, C. labrosus has shown a remarkable ability for the regulation of the fatty

acid profile of its muscle, independently of water temperature (Chapter 3) and fatty acid intake

(Chapters 1 and 4). The results regarding the fatty acid intake are especially relevant, as they

lead to the conclusion that feeds containing very low amounts of dietary fatty acids, including

lc-PUFA and essential fatty acids LA and ALA are enough for C. labrosus without compromising

the nutritional quality of the muscle, which is the edible part for the human consumer. As fish

oil is the main source of such fatty acids for aquaculture feeds, C. labrosus has great potential to

be fed diets formulated with low levels of fish oil, or even without any (Quirós-Pozo et al., 2024).

Moreover, diets low on lipids and high on carbohydrates lead to high lipid accumulation in liver

(Chapter 1), suggesting a successful lipogenesis starting from carbohydrates. This indicates that

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 General discussion

feeds with low lipid inclusion levels could also be suitable for C. labrosus, but P / E should always

be considered and balanced for the nutritional needs of the species.

High stocking densities of 25 kg / m3 caused a retardation of growth when compared to

densities of 10 kg / m3 and especially 5 kg / m3 (Chapter 2). Although this is a common stress

response (Wendelaar Bonga, 1997), the only sign of stress when fish being held at the highest

stocking density was a higher incidence of a rash-like skin condition on the ventral side of the

animal. However, no pathologies or significant mortality occurred during the experiment, and

fish behaviour was not altered in any observable way. The usual energy mobilization associated

to stressful situations was not observed at muscle, liver or plasma metabolite levels or biometry

of the main energy reserve tissues, liver and perivisceral fat. In fact, liver size and triglyceride

levels increased at the highest stocking density, which is an atypical response under chronic

stress. However, the retardation of growth and increased liver energy reserves under high

stocking densities are in agreement with previous reports of C. labrosus (de las Heras et al.,

2015).

The optimal temperature for rearing C. labrosus juveniles has been established at 22.8

°C, thanks to the quadratic regression model developed in Chapter 3. Empirically, growth was

maximized at 22 °C. Even though temperatures of up to 30 °C did not increase the mortality rate,

some evidence indicates that the end of the optimal temperature range is around 26 °C. At this

temperature, growth was not significantly lower than at 22 °C, but tissue metabolite levels

showed diminished energy reserve accumulation, which is a sign of higher energy expenditure.

This might suggest that energy is being deviated for the compensation of thermal stress, but

such stressor was not severe enough to affect growth. Nevertheless, at 30 °C energy reserves

were even scarcer and growth was significantly lower than at 26 °C. This effect is a sign that the

thermal stress at 30 °C was severe enough to cause an energy reorganization that impaired

biological functions such as growth. Furthermore, some lesions in the epithelia of the intestine

of the fish reared at 30 °C confirmed that such temperature was affecting somehow the health

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 General discussion

status of C. labrosus. At colder temperatures (18 °C), growth was slowed down when compared

to 22 °C, but the high energy reserves observed in this group of fish did not point towards a

growth impairment caused by thermal stress. This could be due to a diminished metabolic rate

caused by cold temperatures, general to poikilothermic animals (Schulte, 2015). Therefore, the

slow growth observed at 18 °C in Chapter 3 could be attributed to a general effect of

thermodynamics rather to a stress response. However, similar responses were observed in

Chapters 2 and 4 that cannot be explained the same way.

Those responses are the slow growth accompanied by high lipid reserve accumulation

observed at the highest stocking density in Chapter 2, and the metabolic modulation observed

in the fish fed the VA diets in Chapter 4. The response observed at high stocking densities

(Chapter 2), is atypical, as some of the most common effects of chronic stress are the reduction

of growth and the depletion of energy reserves, not their accumulation (Wendelaar Bonga,

1997). A similar observation was carried out by de las Heras et al. (2015) when conducting

another experiment to assess the effects of stocking density on C. labrosus, as they obtained the

lowest growth rates at the highest density accompanied by increased reserve accumulation.

These results suggest a modulation of metabolism under crowding stress that would lead to

diminishing energy consumption, allowing the fish to accumulate reserves. This modulation

could be mediated by a reduction of cortisol secretion at the highest stocking density (de las

Heras et al., 2015), but the plasma levels of this hormone were not measured when assessing

the effects of high stocking densities herein (Chapter 2). However, plasma cortisol was measured

when assessing the effects of different protein inclusion rates of varying sources (Chapter 4). In

this chapter, some of the fish (VA groups) ingested very low amounts of feed, which was

reflected in negligible growth rates. In this case, energy reserves of these potentially stressed

fish were lower than the reserves of the well-fed fish, as they were being consumed for survival.

However, plasma cortisol levels were the lowest at the VA fish. This could be interpreted as a

strategy for reducing energy consumption when food ingestion is low, as cortisol secretion

251
 General discussion

promotes energy mobilization. The combination of the observations from chapters 2 and 4 and

from de las Heras et al. (2015) suggest the ability of C. labrosus for metabolic modulation under

chronic stress, mediated by the reduction of cortisol secretion, reducing energy consumption

and favouring reserve accumulation. If the stressor is too severe or long lasting, reserves might

eventually become depleted, as seen in Chapter 4. This could be a very useful survival strategy

for inhabiting hostile environments like highly polluted estuaries, where C. labrosus thrive.

However, this hypothesis cannot be proven herein and would be an interesting topic for future

research.

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J. M., ... & Abdala-Díaz, R. T. (2022). Dietary use of the microalga Chlorella fusca Improves

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Conclusions and thesis

255
Conclusions and thesis

256
 Conclusions and thesis

Conclusions

1. Chelon labrosus is a suitable species for intensive culture, evidenced by the lack of

mortality or pathologies and the overall good health status of the individuals reared in captivity

for 686 days.

2. The effects of certain culturing conditions, like growth rates under different diet

compositions, are only observable in the long term, and the average 90-day aquaculture

experiments might fail to detect them. These false negatives are especially important when

considering novel species for aquaculture diversification, as they could condition the direction

of subsequent research and limit its success. Therefore, it can be highlighted that long-term

rearing trials are essential to avoid drawing inaccurate conclusions regarding highly relevant

parameters such as optimal diet composition for novel aquaculture species.

3. Protein to energy ratio (P / E) is a more relevant parameter to evaluate than absolute

protein or energy content in C. labrosus diets. P / E should be close to 22 – 24 mg protein / kJ.

There is no benefit in raising protein inclusion levels of the feed if the energy content is not

increased accordingly.

4. Diets formulated with 100 % vegetal protein and significant inclusion levels of the

alternative ingredients Ulva lacinulata and brewer´s spent grain are suitable for C. labrosus.

These feeds can provide better results than diets containing animal protein, including fishmeal.

However, the selection of alternative ingredients for C. labrosus has to be cautious, evidenced

by some slight intestinal health impairments.

5. Chelon labrosus has a great ability for the regulation of its muscle fatty acid profile,

independently of water temperature and the amount of ingested lipids, including the essential

fatty acids linoleic (LA) and α-linolenic (ALA) acids. Therefore, a diet low on lipids, including LA

and ALA would not affect lipid quality of the edible part of C. labrosus, thus, potentially allowing

for feed formulations with low fish oil content, or even completely devoid of such ingredient.

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 Conclusions and thesis

6. Stocking densities of 25 kg / m3 elicit a mild stress response by C. labrosus. This stress

response is evidenced by lower growth rates than at 10 kg / m3 and, especially, 5 kg / m3, but

the lack of mortality or pathologies and the high energy reserves found in the fish reared at 25

kg / m3 indicate the low severity of the stressor.

7. Optimal water temperature for culturing juvenile C. labrosus is around 22.8 °C. At

lower temperatures (around 18 °C), the fish show high energy reserve accumulation and slow

growth. At 26 °C, signs of thermal stress start to arise, as fish have higher energy expenditure

than at 22 °C, and non-significantly lower growth rates. At 30 °C, growth was significantly

impaired despite the higher energy consumption than at 26 °C, a definitive sign of thermal stress,

further evidenced by the epithelial lesions found in the intestines of the fish reared et 30 °C.

8. Under suboptimal conditions, like high stocking densities or low feed intake, C.

labrosus can modulate its metabolism and enter a hypometabolic state that lowers energy

expenditure and favours reserve accumulation. This metabolic modulation could be driven by

lowering cortisol secretion under chronic stress. However, if the suboptimal condition is too

severe or long lasting, reserves might become depleted.

Thesis

The optimization of long-term intensive culturing conditions for the thicklip grey mullet, the

setting up stocking densities and optimal water temperature (22.8 °C), together with the design

of specific feeds without animal protein and high inclusion levels of alternative vegetal

ingredients, make this fish suitable novel low trophic species for a sustainable diversification of

intensive aquaculture.

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Conclusions and thesis

259