Marine and Fishery sciences 40 (1): xxx-xxx (2027)

https://doi.org/10.47193/mas.4012027010101

Marine and

Fishery Sciences

MAFIS

*Correspondence:

fgimenez@mdp.edu.ar

yrodriguez@mdp.edu.ar

Received: 9 June 2026

Accepted: 17 September 2026

ISSN 2683-7595 (print)

ISSN 2683-7951 (online)

https://ojs.inidep.edu.ar

Journal of the Instituto Nacional de

Investigación y Desarrollo Pesquero

(INIDEP)

This work is licensed under a Creative

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International License

ORIGINAL RESEARCH

Baseline physiological data and enzymatic valorization of shery waste for

female Odontesthes argentinensis nutrition

yaMila e. rodriguez1, 2, *, Juana c. del Valle1, iVana s. FriedMan1, María Victoria laitano1,

clara liébana3, aldo n. zanazzi2 and analia V. Fernández-giMenez1, *

1Instituto de Investigaciones Marinas y Costeras (IIMyC), Facultad de Ciencias Exactas y Naturales (FCEyN),Universidad Nacional de

Mar del Plata (UNMdP), Consejo Nacional de Investigaciones Cientícas y Técnicas (CONICET), Juan B. Justo 2550, B7608FBY -

Mar del Plata, Argentina. 2Universidad Tecnológica Nacional (UTN), Facultad Regional Mar del Plata, Av. Dorrego 281, B7600CLF -

Mar del Plata, Argentina. 3Instituto Nacional de Investigación y Desarrollo Pesquero (INIDEP), Paseo Victoria Ocampo Nº 1, Escollera

Norte, B7602HSA - Mar del Plata, Argentina. ORCID Yamila E. Rodriguez https://orcid.org/0000-0002-4596-3586,

Juana C. del Valle https://orcid.org/0000-0003-1095-8098, Ivana S. Friedman https://orcid.org/0000-0002-9075-0539,

María Victoria Laitano https://orcid.org/0000-0003-2436-0839, Clara Liébana https://orcid.org/0000-0002-7062-8675,

Aldo N. Zanazzi https://orcid.org/0009-0008-7736-2567, Analia V. Fernández Gimenez https://orcid.org/0000-0001-9232-4560

ABSTRACT. This study investigated the energy storage patterns in wild female Odontesthes

argentinensis collected during autumn in the coastal waters of Mar del Plata (Argentina) to assess

their suitability as breeders and explored innovative enzyme-based feeding strategies to improve

food quality, nutrient utilization, and nutritional management of breeders in this species. Biochemical

analyses showed that the liver was the primary storage organ for lipids and proteins, while the fatty

acid proles were consistent with a capital breeding strategy associated with seasonal reproduction.

However, because this study was based on a single seasonal sampling and lacked ovarian stage

classication, gonadosomatic or hepatosomatic indices, and a temporal series, these ndings should

be interpreted as preliminary evidence and should not be taken as sufcient to denitively classify

the reproductive strategy. To improve the nutritional value of soybean meal, enzymatic treatment

with peptidases from Urophycis brasiliensis under acidic conditions (pH 2) reduced antinutritional

compounds while preserving bioactive molecules, thereby enhancing protein quality. In addition, in

vitro combination of exogenous peptidase extracts from sheries waste with digestive enzymes of O.

argentinensis increased proteolytic activity, suggesting synergistic interactions that warrant testing in

future feeding trials. Together, these ndings provide physiological and nutritional insights that can

contribute to the development of feeding strategies for O. argentinensis females. Furthermore, the use

of shery waste as sources of digestive enzymes represents a promising biotechnological approach

to improve feed digestibility while promoting more sustainable aquaculture practices. Nevertheless,

further studies assessing digestibility, sh performance, extraction yields, safety, economic feasibility,

and environmental impacts are needed to substantiate these potential benets.

Key words: Peptidases, lipases, antinutritional factors, sh, soybean meal, sh wastes.

Datos siológicos basales y valorización enzimática de residuos pesqueros para la nutrición de

hembras de Odontesthes argentinensis

RESUMEN. Este estudio investigó los patrones de almacenamiento de energía en hembras sil-

vestres de Odontesthes argentinensis capturadas durante el otoño en aguas costeras de Mar del Plata

(Argentina) para evaluar su idoneidad como reproductoras, y exploró estrategias de alimentación

innovadoras basadas en enzimas para mejorar la calidad del alimento, la utilización de nutrientes y

el manejo nutricional de los reproductores de esta especie. Los análisis bioquímicos mostraron que

Marine and Fishery sciences 40 (1): xxx-xxx (2027)

INTRODUCTION

Odontesthes argentinensis (Valenciennes, 1835)

(Atherinopsidae, Atheriniformes), commonly

known as the Southwest American marine silver-

side, inhabits temperate and cold-temperate coast-

al waters of the southern Atlantic Ocean, ranging

from southern Brazil to northeastern Argentine Pa-

tagonia (Llompart et al. 2013). Owing to its com-

mercial value, desirable esh quality, and promis-

ing aquaculture potential, this species has attracted

increasing interest as a candidate for marine aqua-

culture (Guidi et al. 2021). However, important as-

pects of its reproductive biology, energy allocation,

digestive physiology, and nutritional requirements

remain poorly understood, limiting the develop-

ment of species-specic culture technologies.

Reproduction is one of the most energetically

demanding phases of the sh life cycle, requiring

the mobilization of stored energy to support go-

nadal development, spawning, and embryo produc-

tion. In O. argentinensis the reproductive cycle is

characterized by marked changes in the hepatoso-

matic index and body condition, suggesting shifts

in energy allocation throughout the reproductive

season (Llompart et al. 2013). Consequently, char-

acterizing the quantity and distribution of energy

reserves in storage tissues is essential for under-

standing the reproductive physiology of the species

and for designing appropriate nutritional strategies

for broodstock management (Rahman et al. 2020).

Recent physiological studies of the population

of O. argentinensis from Mar Chiquita Coastal La-

goon (Argentina) have revealed marked seasonal

and ontogenetic plasticity in digestive and meta-

bolic traits associated with reproduction. Méndez

et al. (2024) showed that females maintain body

condition throughout ovarian development by ad-

justing digestive capacity and energy metabolism;

in particular, during the spawning-capable phase,

increased intestinal aminopeptidase-N activity

was accompanied by the selective mobilization of

hepatic and muscle glycogen, triglycerides, and

proteins to support gonadal maturation. In addi-

tion, Méndez et al. (2026) demonstrated marked

seasonal changes in digestive capacity, reected

by changes in the intestinal alkaline phosphatase

activity, as well as in the mobilization of energy re-

serves. Collectively, these ndings indicate that O.

argentinensis relies on endogenous energy reserves

that are mobilized in a coordinated and tissue-spe-

cic manner to meet the energetic demands of re-

production. However, these studies were conduct-

ed on an estuarine population and did not include

el hígado fue el principal órgano de almacenamiento de lípidos y proteínas, mientras que los perles de ácidos grasos fueron coherentes

con una estrategia reproductiva basada en reservas corporales (capital breeding) asociada a la reproducción estacional. Sin embargo,

dado que este estudio se basó en un único muestreo estacional y careció de clasicación de estadios ováricos, índices gonadosomáticos

o hepatosomáticos y series temporales, estos hallazgos deben interpretarse como evidencia preliminar y no considerarse sucientes para

clasicar denitivamente la estrategia reproductiva. Para mejorar el valor nutricional de la harina de soja, el tratamiento enzimático con

peptidasas de Urophycis brasiliensis en condiciones ácidas (pH 2) redujo los compuestos antinutricionales y preservó las moléculas

bioactivas, mejorando así la calidad proteica. Además, la combinación in vitro de extractos de peptidasas exógenas provenientes de

residuos pesqueros con enzimas digestivas de O. argentinensis aumentó la actividad proteolítica, lo que sugiere interacciones sinér-

gicas que justican su evaluación en futuros ensayos de alimentación. En conjunto, estos hallazgos aportan información siológica y

nutricional que puede contribuir al desarrollo de estrategias de alimentación para hembras de O. argentinensis. Asimismo, el uso de

residuos pesqueros como fuente de enzimas digestivas representa un enfoque biotecnológico prometedor para mejorar la digestibilidad

del alimento y promover prácticas acuícolas más sostenibles. No obstante, se requieren estudios adicionales que evalúen la digestibili-

dad, el desempeño de los peces, los rendimientos de extracción, la seguridad, la viabilidad económica y los impactos ambientales para

corroborar estos benecios potenciales.

Palabras clave: Peptidasas, lipasas, factores antinutricionales, peces, harina de soja, residuos pesqueros.

RodRiguez et al.: FRom waste to Feed oF OdOntesthes argentinensis 3

fatty acid proling or a direct comparison of the

biochemical composition of liver and ovary. The

present study therefore extends this knowledge by

providing the rst fatty acid prole and a compre-

hensive biochemical characterization of liver and

ovary in wild females from the marine population

off Mar del Plata, together with baseline digestive

peptidase and lipase activities that have not previ-

ously been reported for this species.

Digestive physiology is another key aspect in-

uencing feed utilization and reproductive perfor-

mance in aquaculture. Unlike many teleosts, O. ar-

gentinensis lacks both a stomach and pyloric caeca.

As in other agastric sh species, the intestine is

relatively long, increasing the surface area availa-

ble for nutrient digestion and absorption (Le et al.

2019). Under these conditions, alkaline digestive

enzymes, particularly peptidases and lipases, play

a central role in nutrient hydrolysis. Nevertheless,

the digestive enzyme prole of O. argentinensis

remains poorly characterized, representing an im-

portant knowledge gap for the development of nu-

tritionally adequate formulated diets.

Beyond its reproductive physiology, O. argentin-

ensis is a euryhaline species that inhabits both ma-

rine and estuarine environments, and its digestive

and metabolic traits reect this ecological plasticity.

In this sense, Albanesi et al. (2025) showed that

estuarine individuals from Mar Chiquita Coastal

Lagoon display higher intestinal carbohydrase ac-

tivities (amylase, maltase, sucrase) and greater li-

pid and glycogen reserves than marine conspecics,

with no differences in protein content or trypsin

activity, highlighting the species’ capacity for hab-

itat-related digestive/metabolic adjustment. At the

interspecic level, Albanesi et al. (2026) compared

juveniles of O. argentinensis with the detritivorous

Mugil liza and the carnivorous Oligosarcus jenynsii

within the same lagoon system and found that O.

argentinensis exhibits an intermediate enzymatic

prole (combining carbohydrase and peptidase ac-

tivities) together with a marked reliance on hepatic

triglycerides as its principal energy reserve, con-

sistent with its omnivorous feeding habits.

Adequate broodstock nutrition is essential to en-

sure successful ovarian development, spawning per-

formance, and offspring quality. Fishmeal remains

the primary protein source in aquafeeds, whereas

soybean meal is widely used as sustainable alter-

native because of its availability and favorable nu-

tritional composition (Soltan et al. 2023). However,

soybean meal contains several antinutritional fac-

tors, including trypsin inhibitors, which can impair

digestive enzyme activity, reduce nutrient utiliza-

tion, and negatively affect sh performance (Weng

et al. 2023). Enzymatic treatment has emerged as

an effective strategy to reduce these antinutritional

compounds while improving protein digestibility and

generating bioactive peptides that may benet intesti-

nal microbiota (Deng et al. 2023; Soltan et al. 2023).

Marine enzymes recovered from shery by-products

represent an alternative to commercial enzymes for

feed processing. Previous studies have demonstrated

the biotechnological potential of digestive enzyme

extracts obtained from discarded marine organisms,

including Percophis brasiliensis (Quoy and Gai-

mard, 1824), Urophycis brasiliensis (Kaup, 1858),

and the crab Ovalipes trimaculatus (de Haan, 1833).

In addition, dietary supplementation with exogenous

digestive enzymes has been shown to improve di-

gestive efciency and nutrient utilization in several

cultured sh species (Bonadero et al. 2022; Friedman

et al. 2022). Nevertheless, the effectiveness of this

approach depends on species-specic digestive phys-

iology, and the interactions between endogenous and

exogenous enzymes remain insufciently understood

(Liang et al. 2022). In vitro studies have demonstrat-

ed that these interactions may be synergistic or inhib-

itory, highlighting the importance of evaluating their

compatibility before application in formulated diets

(Rodriguez et al. 2017, 2021; del Valle et al. 2022).

Considering the limited knowledge of the re-

productive physiology and digestive biology of O.

argentinensis, this study aimed to generate infor-

mation supporting the development of nutritional

strategies for broodstock culture. Specically, the

objectives were: (1) to characterize the energy re-

serves in the muscle, liver, and ovaries of wild fe-

Marine and Fishery sciences 40 (1): xxx-xxx (2027)

male collected during autumn as a partial indicator

of nutritional and physiological status during the

reproductive period, and (2) to assess the effects of

two functional ingredients for diet by evaluating

their effects in vitro (i) soybean meal treated with

marine enzymes as an improved protein source,

and (ii) exogenous marine enzymes as dietary

supplements.

MATERIALS AND METHODS

Fish sampling

Twenty wild adult females Odontesthes argentin-

ensis (body weight: 186.70 g ± 7.17; total length:

30.02 cm ± 0.42) were collected during autumn

(April-May) by artisanal shers off the coast of Mar

del Plata, Argentina (38° 04′ S, 57° 30′ W) (supple-

mentary material, Table S1). Fish were transported

on ice to the laboratory and immediately dissected.

The digestive tract, liver, ovaries, and muscle were

individually excised for subsequent analyses. Only

females with ovaries at a resting stage and no food

contents in the digestive tract were included in the

study; individuals that did not meet either of these

criteria were excluded.

Energy reserve characterization

Crude protein contents in muscle, liver, and

ovary tissues (n = 20) were determined using the

Kjeldahl method (AOAC Method No. 2001.11)

with a nitrogen-to-protein conversion factor of 6.25

(Thiex et al. 2002). Crude lipid content was deter-

mined using the Soxhlet extraction method (AOAC

Method 920.39C) with petroleum ether as solvent

(AOAC 1995). Glycogen concentration was de-

termined according to the method of Dubois et al.

(1956). Fatty acid proles were analyzed by gas

chromatography with ame ionization detection

(GC-FID) following COI-T.20; DOC. 24-2001. All

biochemical analyses were performed by CEDEAC

Group Laboratories (Mar del Plata, Argentina).

Digestive enzyme extraction and characteriza-

tion

Digestive tracts were individually homogenized

in ice-cold 50 mM Tris-HCl buffer (pH 7.5) at a

ratio of 1:2 (w/v) using a Teon-glass homogenizer.

Homogenates were centrifuged at 10,000 × g for

30 min at 4 °C (KIMADI 3H12RI, China), and the

resulting supernatants were stored at -20 °C as the

O. argentinensis digestive enzyme extract (Oa).

Alkaline peptidase activity was determined ac-

cording to García-Carreño (1992) using 0.5% (w/v)

azocasein (Sigma A2765) prepared in 50 mM Tris-

HCl buffer (pH 7.5) as substrate. Absorbance was

measured at 366 nm using a SPECTROstar Nano

microplate spectrophotometer (BMG LABTECH,

Germany) following González-Zamorano et al.

(2013). Lipase activity was determined according

to the micro-method described by Nolasco-Soria

et al. (2018) using 20 mM β-naphthyl caprylate

(Goldbio N-100) as substrate. β-Naphthol release

was measured at 540 nm.

One unit (U) of enzyme activity was dened as

the change in absorbance per minute. Total activity

(TA) was expressed as U ml-1. Protein concentra-

tion was determined following Bradford (1976),

and specic activity (SA) was calculated as U mg

protein-1. All enzymatic assays were performed in

triplicate.

Marine enzymes for functional feed applications

Digestive enzyme extracts were obtained from

the viscera of Percophis brasiliensis (Pb), Uroph-

ycis brasiliensis (Ub), and Ovalipes trimaculatus

(Ot). Fish specimens were collected by commercial

shing vessels operating off the coast of Mar del

Plata, Argentina. Fish stomachs and intestine-py-

loric caeca, as well as crab midgut glands, were

excised immediately after capture and transported

on ice until processing. Cephalothorax by-prod-

ucts of the Argentine red shrimp Pleoticus mu-

elleri (Pm) were obtained from a local seafood

processing plant (Moscuzza SACI, Mar del Plata,

RodRiguez et al.: FRom waste to Feed oF OdOntesthes argentinensis 5

Argentina). Enzyme extracts were prepared as pre-

viously described. Ot extracts were used both for

soybean meal treatment and as exogenous enzyme

supplements, whereas Pb and Ub extracts were

used exclusively for soybean meal treatment and

Pm extracts only as dietary enzyme supplements.

Soybean meal treatment with marine enzymes

Acid peptidase activity from sh stomach ex-

tracts (Pb and Ub) was determined at pH 2 us-

ing 100 mM glycine-HCl buffer containing 0.5%

hemoglobin (Sigma H2625) as substrate (Anson

1938). Absorbance was measured at 280 nm, and

total activity was calculated as described above.

Alkaline peptidase activity from intestine-pyloric

caeca (Pb and Ub) and crab midgut gland (Ot) ex-

tracts was measured at pH 8 following the method

described above.

Soybean meal (5% w/v) was hydrolyzed accord-

ing to Wu and Muir (2010), with modications.

Each enzyme preparation was added at a concen-

tration of 5 U g-1 soybean meal, and reactions were

carried out under the optimal pH conditions previ-

ously established for each extract (Bonadero et al.

2022; Friedman et al. 2022). Hydrolysis was per-

formed at 30 °C for 150 min under gentle stirring

using stomach extracts at pH 2 (Pb2 and Ub2) and

intestinal or crab extracts at pH 8 (Pb8, Ub8, and

Ot8). Reaction pH was maintained by continuous

adjustment with 0.5 M NaOH or HCl. Following

incubation, samples were centrifuged (6,000 g,

20 min), washed with distilled water, resuspended

in 10 mL distilled water, centrifuged again, dried

at 40 °C, and stored at room temperature until anal-

ysis. All treatments were performed in triplicate.

Evaluation of antinutritional and antioxidant

properties

Antinutritional activity was evaluated by pepti-

dase inhibition assays adapted from Alarcón et al.

(1999), with modications. Aqueous extracts of

untreated soybean meal (SM) and enzyme-treated

soybean meals (Pb2, Pb8, Ub2, Ub8, and Ot8) were

prepared at 100 mg ml-1 by shaking for 60 min.

Following centrifugation (6,000 g, 10 min), 10 µl

of supernatant was mixed with 10 µl of Oa di-

gestive extract and incubated at 25 °C for 60 min.

Control reactions contained digestive extract and

Tris-HCl buffer only. Residual peptidase activity

was determined as described above and expressed

as U ml-1. Antioxidant activity was evaluated by

electron paramagnetic resonance (EPR) spectros-

copy using the DPPH radical scavenging assay ac-

cording to Díaz et al. (2014). Soybean meal sam-

ples (20 mg) were suspended in 900 µL absolute

ethanol, sonicated for 30 min, mixed with 100 µL

DPPH solution (0.8 mmol l-1), and incubated in

darkness for 1 h. Spectra were recorded at 293 K

using a Bruker ELEXSYS E500T spectrometer

equipped with an ER 4102ST rectangular resonator

operating in the TE102 mode at X-band frequency

(9.8 GHz). Total radical scavenging activity was

quantied from the area under the rst integral of

each EPR spectrum, which is proportional to the

remaining DPPH concentration.

In vitro compatibility of exogenous enzymes with

digestive enzymes of O. argentinensis

Potential synergistic interactions between endog-

enous digestive enzymes of O. argentinensis (Oa)

and exogenous crustacean enzyme extracts from

Pleoticus muelleri (Pm) and Ovalipes trimaculatus

(Ot) were evaluated in vitro. Equal volumes (5 µl)

of Oa extract, and each exogenous enzyme prepa-

ration were mixed prior to determining alkaline

peptidase and lipase activities. Individual enzyme

extracts were assayed separately under identical

conditions and served as controls.

Statistical analysis

Differences among soybean meal treatments

were evaluated by one-way analysis of variance

(ANOVA) followed by Tukey’s multiple com-

parison test. Comparisons between endogenous

Marine and Fishery sciences 40 (1): xxx-xxx (2027)

enzyme activity and endogenous + exogenous

enzyme activity were performed separately for

proteases and lipases and for Pm and Ot, using

Student’s t-tests with individual sh considered as

the experimental unit (n = 10). Data were tested

for normality and homogeneity of variance before

statistical analyses. All analyses were performed

using R software (R Core Team 2023).

RESULTS AND DISCUSSION

Although recent studies on O. argentinensis aqua-

culture have addressed larval nutrition and feeding

ecology (Führ et al. 2016; Thompson and Volpe-

do 2018), broodstock reproductive physiology and

digestive enzyme characterization remain poorly

understood, representing key knowledge gaps for

the development of culture technologies. Nutrition

is essential during reproduction because it directly

inuences maturation, fecundity, and larval survival.

Successful reproduction requires sufcient energy

reserves to meet the high energetic demands of gam-

ete production and reproductive behavior. Females

invest substantial amounts of energy in maintaining

egg reserves (Volkoff and London 2018; Hernandez

de Dios et al. 2022). Therefore, maternal nutrition

strongly inuences egg quality and larval devel-

opment during endogenous feeding (Izquierdo et

al. 2001). In sh, the liver and ovary are the main

organs involved in nutrient storage and provision-

ing for reproduction (Volkoff and London 2018). In

the present study, the liver and ovary of wild adult

females exhibited similar glycogen concentrations.

In contrast, the liver contained higher concentrations

of fatty acids and proteins than the ovary (Table 1).

The predominance of the liver as a major storage

site for lipids and proteins is consistent with pre-

vious ndings for this species. In O. argentinensis

from Mar Chiquita Coastal Lagoon, hepatic gly-

cogen, triglycerides, and proteins are mobilized in

relation to reproductive stage and season, whereas

muscle appears to play a comparatively minor role

in energy storage (Méndez et al. 2024, 2026; Al-

banesi et al. 2025). Notably, hepatic triglycerides

have been identied as the main lipid reserve in

this species, in contrast to the muscle glycogen or

protein-based energy storage strategies reported for

co-occurring species (Albanesi et al. 2026). These

ndings further support the importance of hepatic

lipid reserves in meeting the energetic demands of

gonadal development in O. argentinensis.

In sh, glucose serves as a primary energy

source for gonadal maturation. Additionally, gly-

cogen may serve as the main energy source for

oocytes during the rst days after ovulation (So-

engas et al. 1995). Before vitellogenesis, proteins

accumulate in the female liver to support vitello-

genin synthesis (Yilmaz et al. 2024). Protein mo-

bilization from the liver to the ovary is regulated

by estrogens. The higher protein content observed

in the liver of O. argentinensis compared with the

ovary suggests that this organ constitutes an im-

portant source of amino acids for the synthesis of

the yolk precursor protein vitellogenin (Table 1).

Adequate protein reserves in females facilitate the

synthesis of lipoproteins, hormones, and enzymes

Table 1. Biochemical composition of liver and ovary in female Odontesthes argentinensis. Values are means ± SE (n = 20).

Liver Ovary Muscle

Glycogen (mg glucose/g tissue) 5.7 ± 0.02 5.8 ± 0.07 2.0 ± 0.02

Protein (%) 15.4 ± 0.20 15.0 ± 0.17 22.3 ± 0.25

Total fatty acids (%) 18.4 ± 0.24 4.4 ± 0.03 1.43 ± 0.01

RodRiguez et al.: FRom waste to Feed oF OdOntesthes argentinensis 7

required for oocyte development, thereby inu-

encing reproductive performance in aquaculture.

Proteins are the major nutrient in sh eggs and play

a pivotal role throughout the reproductive cycle, af-

fecting spawning success, fertilization, embryonic

development, hatching rate, and larval survival and

growth (Hernandez de Dios et al. 2022).

Fish obtain lipids primarily from their diet, par-

ticularly essential fatty acids that cannot be synthe-

sized de novo. Seasonal capital breeders, such as

the marine silverside, rely on energy reserves ac-

cumulated before the reproductive period (Fuiman

and Perez 2015). In these species, lipids are mainly

stored in the liver and muscle and are subsequent-

ly mobilized to the ovary during gametogenesis

for incorporation into the egg yolk (Volkoff and

London 2018). Accordingly, the liver of female O.

argentinensis contained the highest lipid content

(18.4%), followed by the ovary (4.4%), whereas

muscle did not represent a major lipid storage tis-

sue (Table 1). Previous studies in red drum have

shown that maternal nutrition can induce long-

term metabolic effects in the offspring (Fuiman

and Perez 2015). Maternal provisioning of essen-

tial fatty acids enhances offspring tness. In this

context, palmitic acid and unsaturated fatty acids

stored in the liver of O. argentinensis females may

serve as energy reserves that are transferred to the

ovaries during the reproductive season to support

follicular development and egg provisioning (Table

2). Palmitic acid (16:0) was the predominant satu-

rated fatty acid in both the liver and ovary (Table

2), and may provide an important substrate for ATP

production during vitellogenesis (Zhao et al. 2023).

Likewise, the presence of the polyunsaturated fatty

acids linoleic acid (18:2 n-6), alpha linolenic acid

(18:3 n-3), and arachidonic acid (20:4 n-6) (Table

2) may contribute to eicosanoid (e.g. prostaglandin)

synthesis, which is required for steroid hormone

production (Izquierdo et al. 2001), whereas vitello-

genin synthesized in the liver serves as the primary

precursor of egg yolk (Hara et al. 2016).

The nding of arachidonic acid (20:4n-6) as the

only HUFA in the present work, together with the

absence of detectable EPA and DHA, may reect

differences in fatty acid metabolism and tissue-spe-

cic lipid allocation rather than an analytical in-

consistency. Marine sh have a limited capacity to

synthesize long-chain polyunsaturated fatty acids

from C18 precursors and therefore rely largely on

dietary sources to meet their requirements for these

fatty acids (Xu et al. 2022). Under conditions of

low dietary n-3-PUFA availability, changes in tis-

sue fatty acid composition may support the incor-

poration of n-6 fatty acids, including 20:4n-6 and

20:3n-6, into membrane phospholipids, helping to

preserve membrane structure and function (Bou

et al. 2017). This reciprocal relationship between

n-6 and n-3 fatty acids has been reported in marine

sh, including Atlantic salmon, in which increased

dietary 18:2n-6 resulted in higher tissue ARA (20:

4n-6) levels accompanied by a reduction in EPA

(Katan et al. 2020; Sissener et al. 2020).

Tissue fatty acid proles are also inuenced by

lipid class composition and by the physiological

role of each tissue. Dietary effects, for example,

can be particularly evident in liver polar lipids,

whereas tissues such as the brain and retina tend

to maintain relatively conserved fatty acid proles

(Sissener et al. 2020). Reproductive tissues may

show an additional degree of regulation, as fatty

acids can be selectively retained or mobilized ac-

cording to reproductive demands rather than simply

reecting dietary intake (Rodríguez-Barreto et al.

2012). In this context, the presence of 20:4n-6 as

the only detectable HUFA in the present samples

could be related to preferential retention of ARA,

reduced availability of dietary n-3-PUFA, or se-

lective utilization of EPA and DHA. Nevertheless,

these mechanisms cannot be distinguished from the

present data alone, and the absence of detectable

EPA and DHA should therefore be interpreted with

caution.

By comparison, the proximate composition of O.

argentinensis muscle showed a protein content of

22.3%, glycogen content of 2.0%, and a low lipid

content (1.43%). The low glycogen concentration

suggests limited carbohydrate storage in skeletal

Marine and Fishery sciences 40 (1): xxx-xxx (2027)

muscle, consistent with the metabolic character-

istics of marine teleost, which rely primarily on

hepatic glycogen reserves. Furthermore, the low

lipid content (< 2%) indicates that O. argentinensis

can be classied as a lean sh with high nutritional

value due to its elevated protein content.

The biochemical composition observed in fe-

male O. argentinensis collected during autumn sug-

gests that the liver represents the principal energy

reserve, particularly for proteins and lipids, which

are likely to support vitellogenesis. This pattern

is consistent with the central role of the liver in

Table 2. Fatty acid prole (% total fatty acid) of liver and ovary of Odontesthes argentinensis female (n = 20) collected from the

coastal waters of Mar del Plata during autumn.

Fatty acid Liver Ovary

14:0 5.2 8.2

16:0 31.1 37.1

17:0 1.2 1.3

18:0 8.3 9.1

22:0 0.2 1.5

24:0 0.1 1.1

∑SFA 46.1 58.3

16:1 n-7 11.7 7.8

17: 1: n-7 0.9 0.7

18:1 n-9 33.5 27.2

20:1 n-11 1.0 0.6

∑MUFA 47.1 36.3

18:2 n-6 4.3 4.0

18:3 n-3 2.0 1.2

∑PUFA 6.3 5.2

20:4 n-6 0.5 0.3

∑HUFA 0.5 0.3

Ratio ∑ω6/∑ω3 2.4 3.6

∑SFA: sum of saturated fatty acids. ∑MUFA: sum of monounsaturated fatty acids. ∑PUFA: sum of polyunsaturated fatty acids

(18:2n-6, 18:3n-3). ∑HUFA: sum of highly unsaturated fatty acids (≥ 20 carbons, ≥ 3 double bonds); in this study, represented

only by arachidonic acid (20:4n-6), as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) were

below the quantication limit of the GC-FID method used and are therefore not reported. Values represent percentage of total

fatty acids identied.

RodRiguez et al.: FRom waste to Feed oF OdOntesthes argentinensis 9

nutrient storage and mobilization described for

many marine teleosts. However, because the pres-

ent study was based on wild adult females’ samples

at a single seasonal point, these ndings should be

interpreted as a physiological snapshot rather than

denitive evidence of the species’ overall repro-

ductive energy allocation strategy. This limitation

is consistent with previous evidence from the same

species in Mar Chiquita Coastal Lagoon, where

digestive and metabolic traits vary substantially

across both seasons and stages of ovarian matura-

tion (Méndez et al. 2024, 2026). In this population,

hepatic glycogen content and intestinal alkaline

phosphatase activity decline during winter-spring,

coinciding with increased investment in gonadal

development. Conversely, lipid reserves accumu-

lated during summer-autumn are subsequently

mobilized to support spawning, while muscle pro-

tein reserves decrease during active reproduction.

These patterns highlight the extent of physiological

remodeling that occurs throughout the reproductive

cycle and underscore the need for future studies

covering multiple seasons to fully characterize

the energy allocation strategy of O. argentinensis

from Mar del Plata. While our results provide a

detailed biochemical baseline for females in au-

tumn, the absence of a temporal series prevents a

direct quantication of energy mobilization or a

denitive classication of the reproductive strategy.

These ndings represent a physiological state prior

to the peak of spawning, which aligns with, but

does not independently prove, the seasonal trajec-

tories described for other populations of the species.

Comparisons with other marine sh indicate that

O. argentinensis shares some characteristics with

species that rely, at least partially, on stored endog-

enous reserves for reproduction. For example, the

Patagonian grouper (Acanthistius patachonicus)

exhibits mobilization of hepatic lipid reserves to

the developing gonads, a pattern consistent with

a capital breeding strategy, although concurrent

feeding during the reproductive period suggests a

certain degree of energy income (López et al. 2025).

Likewise, the Patagonian redsh (Sebastes ocula-

tus) accumulates proteins and lipids in muscle and

gonadal tissues before reproduction, also display-

ing characteristics associated with capital breeding

(Rodrigues et al. 2025). In contrast, reproductive

energy allocation in the European sardine (Sardina

pilchardus) varies geographically, behaving as a

typical capital breeder in the northern Mediterra-

nean through seasonal lipid accumulation prior to

spawning, whereas populations from the northern

Adriatic show greater dependence on recently ac-

quired resources (Schismenou et al. 2024). Similarly,

the snapper (Chrysophrys auratus) exhibits a mixed

strategy in which hepatic fatty acid reserves coexist

with ovarian enrichment in polyunsaturated fatty

acids, reecting contributions from both stored and

recently assimilated nutrients (Sabetian et al. 2025).

In the present study, the relatively high protein

content and low lipid concentration in muscle in-

dicate that O. argentinensis is a lean species, while

the low muscle glycogen content is consistent with

the limited carbohydrate storage generally reported

for marine teleosts. Together with the high pro-

tein and lipid contents observed in the liver, these

ndings suggest that hepatic reserves may play

an important role in supporting ovarian develop-

ment. Nevertheless, because the present work was

restricted to autumn-collected females, further

studies covering the complete reproductive cycle,

together with assessments of seasonal changes in

tissue biochemical composition and feeding ac-

tivity, will be necessary to determine whether O.

argentinensis follows a predominantly capital, in-

come, or mixed breeding strategy.

The digestive physiology of O. argentinensis has

received limited attention despite its importance

for understanding the species’ biology and feeding

ecology. In the present study, the characterization

of digestive enzymes revealed measurable alkaline

peptidase and lipase activities in wild adult females

(Table 3). These baseline enzyme activities provide

fundamental insights into the digestive capacity of

the species. Silversides are agastric sh; therefore,

they lack gastric digestion and rely on an elongat-

ed intestine with an increased microvillar surface

Marine and Fishery sciences 40 (1): xxx-xxx (2027)

area to maximize nutrient digestion and absorp-

tion (Horn et al. 2006; Toledo-Cuevas et al. 2024).

Early development in related Odontesthes species

(O. bonariensis and O. hatcheri) is characterized

by the rapid onset of high cytosolic peptidase ac-

tivity, indicating early functional maturation of the

digestive tract (Toledo-Cuevas et al. 2024). This

adaptation compensates for the absence of a stom-

ach and supports efcient nutrient digestion despite

the simplied gastrointestinal anatomy.

It has been reported that other silverside species

possess a broad range of digestive enzymes, includ-

ing amylase, maltase, sucrase, lipase, trypsin, and

aminopeptidase-N, enabling the efcient hydroly-

sis of carbohydrates, proteins, and lipids (Horn et

al. 2006; Logothetis et al. 2001). Digestive enzyme

proles in atheriniform shes are associated with

ecological niche and feeding strategy, with estu-

arine herbivores exhibiting longer intestines and

higher carbohydrase activity, whereas carnivorous

species have shorter intestines and higher pepti-

dase activity (Logothetis et al. 2001). Building on

the intermediate, omnivorous enzyme prole es-

tablished for Odontesthes argentinensis (Albanesi

et al. 2026), comparisons between estuarine and

marine populations revealed stable trypsin activity

alongside variable carbohydrase levels and energy

reserves. This suggests that proteolytic capacity

remains conservative across habitats, whereas car-

bohydrate digestion and lipid storage respond dy-

namically to environmental and dietary variations

(Albanesi et al. 2025). In this work, the observed

alkaline peptidase and lipase activities (Table 3)

align with prior ndings, supporting an omnivo-

rous digestive prole in O. argentinensis.

Diet quality has received increasing attention

in aquaculture because broodstock nutrition di-

rectly inuences egg quality, larval survival, and

reproductive performance. Protein source selection

is particularly important for maintaining energy

reserves and ensuring offspring tness. In recent

decades, soybean meal has become one of the main

protein sources used to partially replace shmeal in

aquafeeds owing to its consistent availability and

favorable nutritional properties. Soybean meal pro-

vides a high protein content, a balanced amino acid

prole, and bioactive compounds with antioxidant

properties, making it a valuable ingredient in aqua-

feed formulations (Alarcón et al. 2001; Coscueta

et al. 2016). However, its use is often limited by

the presence of antinutritional factors (ANFs), in-

cluding protease inhibitors, which negatively affect

digestive processes (Alarcón et al. 2001).

Trypsin is an endogenous digestive enzyme that

plays a key role in protein hydrolysis. Protease

inhibitors interfere with trypsin activity, thereby

reducing protein digestibility and exerting antinu-

tritional effects. Soybean meal contains two major

classes of protease inhibitors: Kunitz trypsin in-

hibitors, which primarily inhibit trypsin, and Bow-

man-Birk inhibitors, which inhibit both trypsin and

chymotrypsin (Erdaw and Beyene 2018). There-

fore, selecting an appropriate processing method

is essential when incorporating soybean meal into

aquafeeds (Vagadia et al. 2017). Enzymatic treat-

ment has been extensively investigated as an effec-

tive approach to improve the functional properties

and nutritional quality of soybean meal (Wu and

Muir 2010), thereby enhancing its value as a func-

tional feed ingredient.

Table 3. Peptidase and lipase activities in the digestive tract of female Odontesthes argentinensis. Values are means ± SE (n = 20).

Total activity (U ml-1) Specic activity (U mg protein-1)

Peptidase 0.86 ± 0.10 0.33 ± 0.04

Lipase 33.50 ± 6.91 12.67 ± 2.36

RodRiguez et al.: FRom waste to Feed oF OdOntesthes argentinensis 11

In the present study, digestive enzymes recov-

ered from marine shery by-products were charac-

terized and evaluated for their potential to improve

soybean meal as a protein source. Digestive ex-

tracts obtained from P. brasiliensis (Pb), U. bra-

siliensis (Ub), and O. trimaculatus (Ot) were se-

lected because these by-products contain enzymes

with favorable technological properties, including

stability over a broad pH range and at moderate

temperatures, while being readily inactivated at

relatively low temperatures (Bonadero et al. 2022;

Friedman et al. 2022). Their enzymatic activity was

conrmed prior to soybean meal treatment (supple-

mentary material, Table S2).

Incubation of O. argentinensis digestive extracts

with untreated commercial soybean meal signi-

cantly reduced alkaline peptidase activity, indicat-

ing the presence of residual antinutritional factors

despite industrial heat processing (Figure 1). This

inhibition is likely associated with the presence

of thermostable Bowman-Birk inhibitors, which

can persist after conventional heat treatment (Chen

et al. 2014). In contrast, incubation with soybean

meal treated using marine enzymes (Pb2, Ub2, and

Ub8) resulted in signicantly higher peptidase ac-

tivity than that observed with untreated soybean

meal (supplementary material, Table S3; Figure

1). These ndings indicate that enzymatic treat-

ment with marine enzymes effectively reduces the

antinutritional effects of soybean meal, most likely

through the hydrolysis of protease inhibitors such

as Bowman-Birk inhibitors. Nevertheless, addi-

tional in vivo studies are required to conrm the

nutritional benets of this approach.

Proteins from soybean meal have been shown

to provide health benets, either as intact proteins

or in partially digested forms, known as bioactive

peptides. Previous studies have demonstrated that

enzymatic treatment of soybean meal can increase

the production of soy-derived peptides with anti-

oxidant properties (Sila and Bougatef 2016). In the

present study, DPPH radical scavenging activity

was used to evaluate the antioxidant capacity of

untreated and enzyme-treated soybean meal. Both

commercial soybean meal and soybean meal treat-

ed with marine enzymes recovered from shery

waste exhibited DPPH radical scavenging activi-

ty, although signicant differences were observed

among treatments (supplementary material, Table

S3; Figure 2). Commercial soybean meal (control)

showed the highest antioxidant activity. Similarly,

soybean meal treated with U. brasiliensis enzymes

under acidic conditions (Ub2) and with O. trimac-

ulatus enzymes under alkaline conditions (Ot8)

Figure 1. Peptidase activity of Odontesthes argentinensis with commercial (SM) and enzyme-treated soybean meal. P. brasiliensis

at pH 2 and 8 (Pb2, Pb8), U. brasiliensis at pH 2 and 8 (Ub2, Ub8), and O. trimaculatus at pH 8 (Ot8). Bars represent

means ± SE. Different letters indicate signicant differences.

Marine and Fishery sciences 40 (1): xxx-xxx (2027)

Figure 3. Total activity of peptidase (A and B) and lipase activity (C and D) of Odontesthes argentinensis (Oa) mixed with exoge-

nous enzymes of sheries waste (P. muelleri (Pm) or O. trimaculatus (Ot)). Vertical bars represent the mean and standard

error. Different letters indicate signicant differences.

Figure 2. Antioxidant activity of commercial (SM) and enzyme-treated soybean meal: P. brasiliensis at pH 2 and 8 (Pb2, Pb8),

U. brasiliensis at pH 2 and 8 (Ub2, Ub8), and O. trimaculatus at pH 8 (Ot8). Bars represent means ± SE. Different letters

indicate signicant differences.

RodRiguez et al.: FRom waste to Feed oF OdOntesthes argentinensis 13

exhibited antioxidant activities comparable to the

control (Figure 2). In contrast, antioxidant activity

decreased in soybean meal treated with P. brasil-

iensis enzymes under acidic conditions (Pb2) and

with P. brasiliensis and U. brasiliensis enzymes

under alkaline conditions (Pb8 and Ub8) compared

with the control (Figure 2).

Oxidative stress contributes to the development

of several diseases and accelerates the aging pro-

cess; therefore, antioxidants play an important role

in maintaining health. In aquaculture, the use of

antioxidant-enriched diets has been associated with

several benecial effects. Chen et al. (1995) hy-

drolyzed soybean meal using different peptidases

and observed a signicant increase in antioxidant

activity due to the production of bioactive peptides.

Ma et al. (2023) proposed that the bioactivity of

soybean peptides depends mainly on peptide chain

length, amino acid composition, and amino acid se-

quence. Amino acids such as tryptophan, tyrosine,

glycine, histidine, alanine, phenylalanine, methio-

nine, leucine, and valine have been identied as

key constituents of antioxidant peptides (Sun et al.

2022). Recent studies on marine sh have shown

that fermentation or enzymatic hydrolysis of soy-

bean meal can enhance in vitro antioxidant capacity

(Zhang et al. 2023). In the present study, however,

the DPPH results did not consistently follow this

pattern. The control (SM) exhibited the highest

antioxidant activity, with only the Ot8 and Ub2

treatments reaching values that were statistically

comparable to those of the control. In contrast, the

remaining enzyme-treated meals exhibited signif-

icantly lower antioxidant activity.

In recent years, the application of exogenous

enzymes in aquaculture has increased because of

their ability to promote growth, enhance immune

function, and improve the overall health of cultured

sh. Furthermore, the use of exogenous enzymes

as feed additives has been proposed as a strategy

for enhancing nutrient digestibility in aquaculture.

Previous studies have demonstrated the effective-

ness of exogenous enzymes in improving the in

vitro digestibility of dietary proteins (Rodriguez

et al. 2017, 2021; del Valle et al. 2022). Conse-

quently, these enzymes have become valuable feed

additives, attracting considerable interest from both

the aquafeed industry and the research community

(Zheng et al. 2020).

In the present study, the compatibility between

the endogenous digestive enzymes of O. argen-

tinensis and exogenous enzymes derived from the

crustaceans P. muelleri (Pm) and O. trimaculatus

(Ot) was evaluated to assess potential synergistic

effects. When O. argentinensis digestive extracts

(Oa) were combined with either Pm or Ot enzyme

extracts, alkaline peptidase activity increased by

more than 25% compared with Oa alone (supple-

mentary material, Table S3; Figure 3 A and 3 B).

This result provides the rst experimental evidence

that digestive enzymes recovered from crustacean

processing by-products are functionally compatible

with the endogenous proteases of O. argentinensis,

under in vitro conditions tested, providing support

for further evaluation of their potential as exoge-

nous feed enzymes in vivo trials with this species.

In contrast, lipase activity did not differ signi-

cantly after combining Oa with either crustacean

enzyme extract (supplementary material, Table

S3; Figure 3 C and 3 D). Collectively, these in

vitro results demonstrate that crustacean-derived

enzyme extracts are compatible with the digestive

physiology of O. argentinensis and can increase

proteolytic activity. This represents a promising

strategy for improving dietary protein utilization

while simultaneously promoting the valorization

of crustacean processing by-products within a cir-

cular bioeconomy framework. However, testing is

required in future feeding trials.

CONCLUSIONS

This study provides the biological characteriza-

tion of wild female O. argentinensis, contributing

fundamental knowledge to the biology of atherin-

iform shes. Energy reserve analysis revealed dis-

Marine and Fishery sciences 40 (1): xxx-xxx (2027)

tinct allocation patterns, suggesting that the liver is

a key site of lipid concentration in autumn, whereas

muscle showed the highest protein concentration

among the tissues analyzed. The characterization

of digestive enzymes provides baseline values for

alkaline peptidase and lipase activities that may

help ll an important gap in the understanding of

the digestive physiology of this species and could

provide comparative data for other members of the

Atherinopsidae family. These ndings may also

contribute to a better understanding of the digestive

adaptations of agastric shes.

Additionally, this study explores potential strate-

gies for the future development of highly digestible

feeds through a circular economy approach based

on the valorization of shery waste. Enzymatic

treatment of soybean meal with U. brasiliensis

enzymes at pH 2 reduced antinutritional factors,

and the treated meal retained DPPH antioxidant

activity comparable to that of the untreated control.

Furthermore, cross-species enzyme compatibility

assays showed that mixtures of exogenous marine

enzymes with the endogenous digestive extract of

O. argentinensis maintained or increased proteolyt-

ic activity relative to the endogenous extract alone,

indicating the biotechnological potential of shery

by-products as feed additives. Likewise, exoge-

nous enzymes recovered from crustacean process-

ing waste resulted in greater activity in mixtures

than with the endogenous extract alone.

Overall, these ndings provide a valuable phys-

iological and biochemical baseline for O. argen-

tinensis and establish a foundation for future stud-

ies aimed at developing nutritional strategies for

broodstock management, and could support the

diversication of marine aquaculture in South

America through the culture of a native sh spe-

cies. Moreover, this study provides in vitro evi-

dence that enzymatic treatment of soybean meal

and cross-species enzyme mixtures can reduce

antinutritional factors and increase proteolytic ac-

tivity relative to the endogenous digestive extract

of O. argentinensis, indicating the biotechnologi-

cal potential of shery by-products as candidate

feed additives. However, these conclusions are

based exclusively on in vitro assays; before these

enzymatic strategies could be considered for in-

corporation into feeding programs for this species,

their stability under feed processing and storage

conditions, safety, digestibility and efcacy in vivo,

production costs, and environmental impact would

need to be specically evaluated.

ACKNOWLEDGEMENTS

The authors thank Dr. Luis I. Granone for as-

sistance with EPR spectra determinations and Lic.

Cecilia Bonadero for assistance with laboratory

assays. This work was supported by the Technolog-

ical Development and Innovation Promotion Na-

tional Agency from Argentina [PICT 2020, 01851;

AVFG], CONICET Multiyear Research Projects

[PIP 2021/2023, 353 11220200101093CO; AVFG]

and Mar del Plata National University [EXA

1194/24; AVFG]. The funders had no role in study

design, data collection and analysis, decision to

publish, or preparation of the manuscript. This is

INIDEP contribution no 2544.

Conict of interest

The authors declare that they have no known

competing nancial or non-nancial, professional,

or personal conicts that could have appeared to

inuence the work reported in this paper.

Author contributions

Yamila E. Rodriguez: conceptualization; meth-

odology; formal analysis and investigation; writ-

ing-original draft preparation. Juana C. del Valle:

conceptualization; methodology; writing-original

draft preparation; supervision. Ivana S. Friedman:

methodology; formal analysis and investigation;

writing, review and editing. María Victoria Laitano:

RodRiguez et al.: FRom waste to Feed oF OdOntesthes argentinensis 15

methodology; formal analysis and investigation;

writing, review and editing. Clara Liébana: meth-

odology; formal analysis and investigation; writing,

review and editing. Aldo N. Zanazzi: methodology.

Analia V. Fernández-Gimenez: conceptualization;

methodology; writing-original draft preparation;

writing, review and editing; funding acquisition;

resources; supervision.

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