Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e59013, marzo 2024 (Publicado Mar. 01, 2024)

Effect of gamete aging on fertilization success of the sea urchin

Arbacia dufresnii (Arbacioida: Arbaciidae)

Jimena Pía Fernández1, 2**; https://orcid.org/0000-0002-7834-2313

Florencia Di Marco1, 2**; https://orcid.org/0009-0007-3198-956X

Tamara Rubilar1, 2; https://orcid.org/0000-0003-1728-3273

Maximiliano Cledón3; https://orcid.org/0000-0002-3811-1481

Ximena Gonzalez Pisani1, 2*; https://orcid.org/0000-0002-4163-411X

1. Laboratorio de Oceanografía Biológica, Centro para el Estudio de Sistemas Marinos, Centro Nacional Patagónico,

Consejo Nacional de Investigaciones Científicas y Técnicas, Blvd. Almte. Brown 2915, Puerto Madryn, Argentina;

jimena.pia.fernandez@gmail.com, fdimarco@cenpat-conicet.gob.ar, rubilar@cenpat-conicet.gob.ar,

xgpisani@gmail.com (*Correspondence).

2. Instituto Patagónico del Mar, National University of Patagonia San Juan Bosco, Bv. Almte. Brown 3051, Puerto

Madryn, Argentina.

3. Centro de Investigación Aplicada y Transferencia tecnológica en recursos Marinos “Almirante Storni”, Güemes 1030,

San Antonio Oeste, Argentina; mcledon@gmail.com

** Both authors contributed equally to the development of this work.

Received 02-VII-2023. Corrected 27-XII-2023. Accepted 05-I-2024.

ABSTRACT

Introduction: Short-term gametes storage is an inexpensive and simple technique that allows the use of the same

batch of eggs or sperm at different times, maximizing the application of research protocols and the use of gametes

in production. Arbacia dufresnii is a sea urchin species with proven aquaculture potential and already used in the

nutraceutical industry. Aging of its gametes is unknown and is a needed information to scale up the production.

Objective: Determine the effect of male and female gamete aging on the fertilization success of Arbacia dufresnii.

This will allow optimizing the use of gametes after collection decoupling spawning from fertilization.

Methods: A. dufresnii individuals were induced to spawn and gametes were kept at 12 ± 1 °C throughout each

bioassay. Sperm was separated into two treatments: activated sperm in seawater (AS), and dry sperm (DS). Two

bioassays were made: Bioassay 1 evaluated the effect of time on fertility by performing fertilization tests at 0 h,

24 h, 48 h, 72 h, and 96 h after spawning. Bioassay 2 evaluated the contribution of each type of aged gamete on

fertility, combining aged gametes (96 h) with fresh gametes (0 h).

Results: Bioassay 1: the fertilization success obtained by combining eggs (E) with AS or DS presented important

differences. While the fertilization success remained acceptable (greater than 50 %) for up to 72 h using ExDS,

it only remained acceptable for up to 48 h using ExAS. Bioassay 2: acceptable fertilization success was found by

combining aged E (96 h) with fresh sperm, or aged DS (96 h) with fresh E, but not using aged AS with fresh E.

Conclusions: The findings of this work show that fertilization success in A. dufresnii gametes remains relatively

unchanged for up to 48 h after spawning when combining ExAS, and for up to 72 h when combining ExDS.

However, when combining aged E or aged DS with a fresh gamete, post-collection fertilization can be extended

up to 96 h. In this work, the first steps have been taken to understand the conservation time of A. dufresnii gam-

etes with minimum intervention.

Key words: short-term storage; gamete handling; sperm; eggs; echinoderms.

https://doi.org/10.15517/rev.biol.trop..v72iS1.59013

SUPPLEMENT

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e59013, marzo 2024 (Publicado Mar. 01, 2024)

INTRODUCTION

Gamete handling standardization proce-

dures are a necessary tool for the development

of hatchery techniques and for the use of gam-

etes in laboratory research (Beirão et al., 2019;

Fabbrocini et al., 2023; Ramos-Júdez et al.,

2019). In this way, optimal gametes preserva-

tion for a short period before fertilization is a

valuable tool to the use of samples after their

collection (Contreras et al., 2020).

Short-term gametes storage is an inex-

pensive and simple technique that allows the

use of the same batch of eggs or sperm at dif-

ferent times, maximizing the application of

research protocols and the use of gametes in

production (Bokor et al., 2021; Cejko et al.,

2022; Kristan et al., 2020; Yasui et al., 2015).

Besides, using short-term storage techniques,

gametes transportation from broodstock-rear-

ing sites to other teachings, laboratory research,

or aquaculture facilities is possible (Kiyomoto,

2019). In the last decade, the welfare of natu-

ral populations of invertebrates has become a

greater concern, highlighting the importance

of minimizing animal collection for research,

education, or production (Beirão et al., 2019;

Fabbrocini et al., 2023; Rubilar & Crespi-Abril,

2017). Short-term gamete storage enables

the reduction of animal collection by shar-

ing gamete samples for laboratory research,

which is also in accordance with recommended

guidelines such as ARRIVE (Animal Research:

Reporting of In Vivo Experiments, https://

nc3rs.org.uk/arrive-guidelines).

Most procedures for gametes handling in

aquatic species have been developed for aqua-

culture purposes and mostly for fish (Bokor

et al., 2021; Kristan et al., 2020; Ramos-Júdez

et al., 2019). However, in recent years, the

development of breeding methods to repro-

duce model species on a laboratory scale has

RESUMEN

Efecto del envejecimiento de los gametos sobre el éxito en la fecundación

del erizo de mar Arbacia dufresnii (Arbacioida: Arbaciidae)

Introducción: El almacenamiento de gametos a corto plazo es una técnica económica y sencilla que permite utili-

zar el mismo lote de óvulos o espermatozoides en diferentes momentos, maximizando la aplicación de protocolos

de investigación y el uso de gametos en la producción. Arbacia dufresnii es una especie con probado potencial

acuícola como fuente de gametos para la industria nutracéutica. Sin embargo, se desconoce el envejecimiento de

sus gametos y es una información necesaria para escalar la producción.

Objetivo: Determinar el efecto del envejecimiento de los gametos masculinos y femeninos en el éxito de la fecun-

dación de Arbacia dufresnii con el fin de optimizar el aprovechamiento de los gametos después de la recolecta

desincronizando el desove de la fecundación.

Métodos: Se indujo el desove de individuos de A. dufresnii y los gametos se mantuvieron a 12 ± 1 °C durante cada

bioensayo. El esperma se separó en dos tratamientos: esperma activado en agua de mar (AS) y esperma seco (DS).

Se realizaron dos bioensayos: El Bioensayo 1 evaluó el efecto del tiempo sobre la fertilidad realizando pruebas de

fecundación a las 0 h, 24 h, 48 h, 72 h y 96 h después del desove. El bioensayo 2 evaluó la contribución de cada tipo

de gameta envejecida (96 h) sobre la fertilidad, combinando gametos envejecidas (96 h) con gametos frescas (0 h).

Resultados: Bioensayo 1: el éxito de fecundación obtenido combinando huevos (E) con AS o DS presentó diferen-

cias importantes. Si bien el éxito de la fecundación se mantuvo aceptable (más del 50 %) durante un máximo de 72

h con ExDS, solo permaneció aceptable hasta 48 h con ExAS. Bioensayo 2: se encontró un éxito de fecundación

aceptable combinando E envejecidos (96 h) con esperma fresco, o DS envejecido (96 h) con E fresco (0 h), pero

no usando AS envejecido con E fresco (0 h).

Conclusiones: Los hallazgos de este trabajo muestran que el éxito de la fecundación en los gametos de A. dufres-

nii permanece relativamente sin cambios hasta 48 h después del desove cuando se combina ExAS, y hasta 72 h

cuando se combina ExDS. Sin embargo, cuando se combina E envejecido o DS envejecido con un gameto fresco,

el tiempo entre la recolección y la fecundación puede extenderse hasta 96 h. En este trabajo se han dado los pri-

meros pasos para entender el tiempo de conservación de los gametos de A. dufresnii con mínima intervención.

Palabras clave: almacenamiento a corto plazo; manejo de gametos; esperma; óvulos; equinodermos.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e59013, marzo 2024 (Publicado Mar. 01, 2024)

increased (Cirino et al., 2017; Fabbrocini &

D’Adamo, 2011; Fabbrocini et al., 2021; Tsang

et al., 2017). Echinoderms are excellent ani-

mal models among marine organisms, it is

a Phylum of invertebrates exclusively marine

that plays important ecological roles in each

environment they inhabit (Brusca & Brusca,

2003; Chiarelli et al., 2019; Matranga & Corsi,

2012; McClay, 2011). Sea urchins and starfish

embryos have been used for embryological

studies (Boveri, 1901; Briggs & Wessel, 2006;

Dufossé, 1847; Garner et al., 2016; McClay,

2011; Williams & Anderson, 1975), ecotoxicol-

ogy analysis (Rahman et al., 2009), and more

recently for neurogenesis and molecular studies

(Garner et al., 2016; Hinman & Burke, 2018;

McClay, 2011). In addition, throughout history,

humans have been consuming both sea urchins

and sea cucumbers, being highly valued fishing

and aquaculture resources, considered a gastro-

nomic delicacy in many regions of the world

(Lawrence, 2007; Stefansonn et al., 2017; Sun &

Chiang, 2015).

The green sea urchin Arbacia dufresnii

(Blainville, 1825) is an abundant coastal spe-

cies from the coast of Buenos Aires in Argen-

tina (35⁰ S) to Puerto Montt in Chile (42⁰ S)

including some islands of the South Atlan-

tic Ocean (Bernasconi, 1947; Brogger et al.,

2013). In recent years, great advances have been

made in the aquaculture management of A.

dufresnii, optimizing its nutrition, water qual-

ity parameters, culture density in recirculating

aquaculture systems, and early developmental

culture (Chaar et al., 2021; Fernández et al.,

2021; Sepúlveda et al., 2021; Vera-Piombo et

al., 2022). Additionally, significant advance-

ments have been made in the purification of

pigment extracts from gametes, which can be

utilized in the development of nutraceutical

products and potentially beneficial for human

health (Avaro et al., 2022; Barbieri et al., 2021;

Rubilar et al., 2021). All this knowledge made it

possible that A. dufresnii is now a species with

great aquaculture potential and as a source for

the nutraceutical industry (Rubilar & Cardozo,

2021; Rubilar et al., 2016). However, there is

still a lack of information on the appropriate

handling and storage of gametes in A. dufresnii,

which is of great value to scale up its produc-

tivity. Therefore, the aim of this study was to

determine the effect of male and female gamete

aging on the fertilization success of the sea

urchin A. dufresnii in order to optimize the use

of gametes after collection decoupling spawn-

ing from fertilization.

MATERIALS AND METHODS

Collection and maintenance of experi-

mental animals: 30 A. dufresnii adults (30-35

mm test diameter) (Epherra et al., 2015) were

collected by scuba diving in Nuevo gulf, Argen-

tina (42º 46’ 44’’ S & 64º 59’ 52’’ W). Individuals

were transferred, in thermal containers with

seawater and controlled temperature, to the

Experimental Aquarium of the CCT-CONI-

CET-CENPAT in Puerto Madryn, Chubut,

Argentina. For acclimatization, the individuals

were kept for seven days in recirculating 100 L

tanks under similar to local natural conditions

of temperature and salinity (12 ⁰C ± 1, 35 ± 1

ppm) and starvation.

Spawning induction and collection of

gametes: Males and females were induced to

spawn by injection through the peristomial

membrane of 0.3 mL of 0.5 M KCl solution

(Strathmann, 1987). To account for the fertility

variability observed among individuals of A.

dufresnii (Fernández et al., 2021), standardized

fertilization was performed in each bioassay

by utilizing an egg pool from five females and

a sperm pool from five males. Eggs were col-

lected on filtered and UV sterilized seawater

and placed in a single container, generating a

pool of eggs. Then, the pool of eggs was quanti-

fied in triplicate in a Sedgewick Rafter chamber

using a Leica DM 2 500 microscope. Sperm was

collected dry in a single container and kept on

ice for further processing.

Experimental design: Two bioassays were

performed to test the effect when both types of

gametes are aged and when one type of gametes

is aged. In both bioassays, each treatment and

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e59013, marzo 2024 (Publicado Mar. 01, 2024)

the control had 3 replicas. Each replica consist-

ed of a 250 ml glass beaker, with filtered seawa-

ter conditioned through a series of mechanical

filters (10, 5, and 1 µm) and sterilized (UV fil-

ter), constant aeration, the temperature of 12 ⁰C

± 1, the salinity of 35 ± 1 ppm and photoperiod

(12:12 h). Each container was independent and

contained about 100 000 eggs.

Aging Gametes: To age the eggs, they were

placed in the glass beakers filled with seawater

and kept at a temperature of 12 ± 1 °C during

each bioassay. As for the sperm, two different

ways were used: activated sperm, which con-

sisted of a pool of sperm in seawater (1: 1 000

v/v) that was kept at a temperature of 12 ± 1

°C during each bioassay, and dry sperm, which

consisted of a pool of sperm that was kept dry

at the same temperature during each bioassay.

Before each fertilization, an aliquot of the dry

sperm was resuspended in seawater to activate

it (1: 1 000 v/v), and then it was immediately

used to fertilize the eggs.

Bioassay 1. Fertilization success with

both gametes aged over time: The impact

of gametes aging in fertilization success was

investigated by performing fertilization tests

with gametes aged for 0 h, 24 h, 48 h, 72 h, and

96 h (Table 1). At each time point, eggs were

fertilized with 1: 100 000 v/v sperm in seawater.

The sperm used for fertilization were either

activated sperm or dry sperm. Fertilization

success was daily checked by observing the

appearance of the fertilization envelope after 30

minutes of launching the assay. The fertilization

percentage was calculated using the formula F

= Fertilized eggs/total eggs x 100.

Bioassay 2. Effect of aged gametes on

fertility: To determine the contribution of aged

sperm and eggs to the decline in fertilization

success, two separate spawning and fertilization

tests were conducted. The fertilization success

of aged sperm was evaluated individually by

combining fresh eggs (0 h) with aged activated

sperm (96 h) and aged dry sperm (96 h), as

well as using fresh sperm (0 h) as the control

(Table 2). Similarly, the fertilization success of

aged eggs was assessed by conducting a fer-

tilization test with fresh sperm (0 h) and aged

eggs (96 h), as well as using fresh eggs (0 h) as

the control (Table 2).

Data analysis: For bioassay 1, a General-

ized Linear Model (GLM) was applied to ana-

lyze fertilizing success over time after spawning.

As a first step, a graphical exploration based on

scatter and residual plots of the data was per-

formed to understand the relationship between

time and fertilizing success. Different replicates

were included in the model analysis to evaluate

method error and to determine if the repli-

cates presented similar values. Starting with

a null model, different models were created,

incrementing the complexity of the models

Table 1

Assay design to study fertilization success of simultaneous aging of male and female gametes. E: Eggs, AS: Activated sperm,

DS: Dry sperm.

Fresh 24 h Aged 48 h Aged 72 h Aged 96 h Aged

E x DS 0 h (E X DS) 24 h (E X DS) 48 h (E X DS) 72 h (E X DS) 96 h (E X DS)

E x AS 0 h (E X AS) 24 h (E X AS) 48 h (E X AS) 72 h (E X AS) 96 h (E X AS)

Table 2

Bioassay design to evaluate the incidence of aged sperm or aged eggs on the fertilization success. E: Eggs, S: Sperm, AS:

Activated sperm, DS: Dry sperm.

Fresh sperm (S) Aged dry sperm (DS) Aged activated sperm (AS)

Fresh eggs (E) E (0 h) X S (0 h) E (0 h) X DS (96 h) E (0 h) X AS (96 h)

Aged eggs (E) E (96 h) X S (0 h) - -

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e59013, marzo 2024 (Publicado Mar. 01, 2024)

by combining time (h) and type of gametes

combined (Eggs x Activated sperm or Eggs x

Dry sperm) in a double interaction model. The

best model was selected based on the Akaike

criteria, taking into account residual analysis,

explained variance (deviation), and the prin-

ciple of parsimony or normality (Hurvich et

al., 1998). For bioassay 2, one-way ANOVA was

applied to analyze the contribution of each type

of aged gamete on the decline in fertilization

success. Normality was tested using the Shap-

iro-Wilks test and homoscedasticity of variance

was tested using the F-test (Zar, 1984). All GLM

analyses were performed with the free software

RStudio (version 3.5.1) (R Core Team, 2016).

The software InfoStat (InfoStat, 2016) was used

for ANOVA, Shapiro-Wilks, and F-tests.

RESULTS

Bioassay 1. Fertilizing success over time

after spawning: The fertilization success of

eggs and sperm decreased over time, being

less than 30 % by the end of the experiment at

96 h (Fig. 1). Eggs combined with dry sperm

presented high values of fertilization success

(95.1 ± 0.7 %) for up to 48 h. Afterwards, the

fertilization success fell rapidly, with only 26.5

± 1.4 % of eggs being fertilized at 96 h. On the

other hand, fertilization success of eggs com-

bined with activated sperm was high (96.1 ±

1.3 %) only for 24 h, values fell slightly at 48 h

and finally fell rapidly with only 15.5 ± 2.5 %

of eggs being fertilized at 96 h. GLM analysis

revealed that the type of gametes combined

(Eggs x Activated sperm or Eggs x Dry sperm)

in interaction with time (h) influences fertiliza-

tion success (Table 3).

Bioassay 2. Effect of aged gametes on

fertility: Fertilization success presented sig-

nificant differences when fresh eggs (0 h)

were combined with the three types of sperm

(F2,6 : 191.28, p < 0.0001) (Fig. 2). The fertiliza-

tion success of 96 h aged dry sperm decreased

to 51.1 ± 2.2 %, and the fertilization success of

96 h aged activated sperm decreased to 13.1

± 3 %, whereas fresh sperm (0 h) achieved

a success rate of 86.8 ± 2.8 % (Fig. 2). Simi-

larly, significant differences were observed in

the fertilization success when fresh sperm (0

h) was combined with the two types of eggs

(F1,42 : 3.35, P < 0.05). The fertilization suc-

cess with 96 h aged eggs decreased to 72.3 ±

4.8 % when compared to fresh eggs (0 h) that

achieved a success rate of 96.8 ± 1.7 % (Fig. 3).

DISCUSSION

Under laboratory conditions, the influence

of gamete age on fertilization success is one

of the main limitations to optimize the use of

Table 3

GLM analysis for fertilization success in A. dufresnii of both eggs and sperm over time. In bold, the model with the best fit

according to the Akaike information criteria (AIC).

Model AIC DIF

1Null 1 296.79 107.85

2Type of gametes combined 298.28 109.33

3Tiempo (h) 212.88 23.94

4Type of gametes combined + time 201.34 12.40

5Type of gametes combined * time  188.94 0.00

Fig. 1. Fertilization success of both eggs and sperm over

time calculated as percent fertilization (Mean ± SEM, n =

3) at different times after spawning in A. dufresnii. E: eggs,

DS: Dry sperm, AS: Activate sperm.

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e59013, marzo 2024 (Publicado Mar. 01, 2024)

samples after their collection (Contreras et al.,

2020). In general, a higher fertilization success

rate (over 80 %) is desirable as it increases the

efficiency of gamete utilization and reduces

the cost of producing embryos or larvae (Fab-

brocini et al., 2021; Lera & Pellegrini, 2006).

However, depending on the context and goals

of the study or application, a fertilization suc-

cess rate of 50 % may be considered acceptable.

For example, if the goal is to obtain a large

number of sea urchin embryos for education, a

fertilization success rate of 50 % may be accept-

able as long as a sufficient number of embryos

are obtained (Epel et al., 2004; Kiyomoto et

al., 2014). On the other hand, if the goal is to

produce sea urchin larvae for aquaculture pur-

poses, a higher fertilization success rate may be

necessary to meet the industrial requirements

(Contreras et al., 2020; Fabbrocini et al., 2021).

The findings of the present study show that

the fertilization success in the sea urchin A.

dufresnii gametes remains relatively unchanged

for up to 48 h after spawning. Although there is

a slight decrease in fertilization success between

24 and 48 h when using activated sperm, using

both types of gametes combinations (Eggs x

Activated sperm or Eggs x Dry sperm) results

in a fertilization success rate over 87 %, which

represents a high fertilization success. In prac-

tice, this means that both female and male

gametes of A. dufresnii can be preserved for 48

h post-spawning with a very high probability

of fertilization between them. Additionally, the

sperm can be kept dry or activated for up to

48 h. This aspect is highly variable between

different marine invertebrates and even within

the same taxon, being necessary to determine

in each species in order to standardize its

handling. For example, in the asteroid Asterias

rubens, fertilization reaches 100 % only during

the first 4 h after spawning and then falls to 0

% at 24 h (Williams & Bentley, 2002), while in

eggs of the echinoid Strongylocentrotus droe-

bachiensis fertilization success is high for 48 to

72 h (Meidel & Yund, 2001).

On the other hand, we found important

differences in fertilization success when com-

bining eggs with dry sperm or activated sperm

of A. dufresnii at 72 and 96 h of gamete aging.

Further analysis using generalized linear mod-

els (GLM) confirmed that the effect of time on

fertilization success varied depending on the

type of gamete combination used, with Eggs

x Activated sperm exhibiting lower fertiliza-

tion success rates. This difference may be due

to the fact that activated sperm is activated

at the beginning of the bioassay (0 h) and is

allowed to age, which results in the depletion

of its energy reserves and a noticeable reduc-

tion in fertilization capability after 72 h. In

contrast, dry sperm was aged and activated

immediately before each fertilization, enabling

it to retain its energy reserves and maintain a

Fig. 3. Fertilization success of fresh eggs (Fresh E) and 96

h aged eggs (Aged E) when combined with fresh sperm,

calculated as the percentage of fertilization success (Mean

± SEM, n = 3) in A. dufresnii. The asterisk (*) indicates

treatments that showed significant differences (P < 0.05).

Fig. 2. Fertilization success of fresh eggs combined with

fresh sperm (Fresh S), 96 h aged dry sperm (Aged DS),

and 96 h aged activated sperm (Aged AS), calculated as

the percentage of fertilization success (Mean ± SEM, n =

3) in A. dufresnii. S: sperm, DS: Dry sperm, AS: Activate

sperm. The asterisk (**) indicates treatments that showed

significant differences (P < 0.0001).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72(S1): e59013, marzo 2024 (Publicado Mar. 01, 2024)

higher fertilization capability. Under labora-

tory conditions, Eggs x Dry sperm showed a

fertilization success rate of over 50 % at 72 h,

making it a viable option for certain purposes.

In contrast, the use of Eggs x Activated sperm

aged 72 h, which exhibited a fertilization suc-

cess rate of 36 %, would not be advisable. At 96

h, Eggs x Dry Sperm still had a higher fertiliza-

tion success rate compared to Eggs x Activated

sperm, but the overall fertilization success rate

for both types of gametes combinations was

less than 30 %, indicating that using either dry

or activated sperm to fertilize aged eggs would

not be recommended. Therefore, this means

that if both the female and male gametes of A.

dufresnii are aged simultaneously, acceptable

fertilization rates can be achieved up to 72 h

only using dry sperm.

When analyzing the results of bioassay 2, it

becomes evident that each type of aged gamete

contributes differently to the decline in fertil-

ization success. Furthermore, it is observed that

the fertilization success after 96 h of gamete

aging differs between male and female gam-

etes. This observation is consistent with prior

research, which has indicated that the process

of sperm aging is more pronounced than that

of eggs and that the rate of sperm aging accel-

erates with increasing sperm dilution (Benzie

& Dixon, 1994). In A. dufresnii, a significant

decrease in fertilization success was observed

after 96 h of gamete aging when combining aged

sperm (96 h) with fresh eggs (0 h). Notably, the

decrease was significantly more pronounced in

the case of activated sperm (96 h) compared to

dry sperm (96 h). Therefore, under laboratory

conditions, it would be possible to use aged dry

sperm (96 h) to fertilize fresh eggs (0 h) and

obtain a fertilization success of approximately

50 %. On the contrary, it is not recommended

to use aged activated sperm (96 h) combined

with fresh eggs, since fertilization success was

under 30 %. When evaluating the fertiliza-

tion success of the aged eggs (96 h) combined

with fresh sperm (0 h), a decrease of less than

25 % is observed. Therefore, under laboratory

conditions, it could be feasible to use eggs for

up to 96 h and obtain acceptable fertilization

percentages when combining them with fresh

sperm. In practice, this implies that the exten-

sion of fertilization capability of aged eggs and

dry sperm up to 96 h can be achieved by com-

bining fresh gametes. These findings could have

significant implications for the management of

A. dufresnii gametes, including the develop-

ment of hatchery techniques and their use in

education and laboratory research (Beirão et

al., 2019; Fabbrocini et al., 2023; Ramos-Júdez

et al., 2019). However, in order to ensure viable

offspring, a more extended study would be nec-

essary, analyzing the normal development of A.

dufresnii progenies from aged gametes. None-

theless, these results provide valuable insights

into the aging process of A. dufresnii gametes

and offer a foundation for future studies on the

management and conservation of this species.

Taken together, the results of this work

suggest that the fertilization success of aged

gametes in A. dufresnii is influenced by several

factors, such as the type of aged gamete, the

duration of aging, the dilution of sperm, and

the gamete combination. In addition, in some

studies, it has been possible to obtain a longer

duration of the gametes by avoiding bacterial

proliferation, since it is the main cause of egg

deterioration. Epel et al. (2004), described for

the first time that it is possible to satisfactorily

increase the post-spawning time by keeping

gametes in antibiotic-modified seawater. In

another study, Kiyomoto et al. (2014) evaluated

the egg storage conditions of five sea urchin

species in antibiotic-modified seawater and

found that depending on the species there are

differences in the time and temperature range

used in storage. This evidence indicates that

there are multiple factors to take into account

for the conservation of sea urchin gametes,

such as temperature, the application of antibi-

otics, and the species of sea urchin analyzed.

Knowledge of the proper management of each

of these variables could even improve the sur-

vival time described for A. dufresnii gametes

and their effectiveness in fertilization. In this

context, in the present work the first steps

have been taken to understand the conserva-

tion of A. dufresnii gametes with the minimum

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72(S1): e59013, marzo 2024 (Publicado Mar. 01, 2024)

possible intervention, that is, in filtered and

sterilizer seawater, temperature similar to the

environment and without the addition of anti-

biotics or other substances. Based on these

results, further research can be conducted to

understand the survival of A. dufresnii gam-

etes and to explore modifications to seawater

conditions, and the use of antibiotics, that can

potentially increase their viability and fertiliza-

tion success.

Ethical statement: the authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

ACKNOWLEDGMENTS

We are grateful to the diver Ricardo Bebo

Vera for the collection of the sea urchins, to

Mariano Moris for helping with the experi-

ments, and to Norberto Garin for assisting with

a microscope. This work was financially sup-

ported by PICT 2018-01729-(2020-2023). The

sea urchins were collected by the Provincial

Permit N° 386/20.

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