Aquaculture 437 (2015) 366–369

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Aquaculture
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Short communication

Sea urchin (Paracentrotus lividus) larval rearing — Culture from

cryopreserved embryos
E. Paredes a,⁎, J. Bellas b, D. Costas c
a
Departamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo, Estrada Colexio Universitario, 36310 Vigo, Galicia, Spain
b
Instituto Español de Oceanografía, Centro Oceanográfico de Vigo, Cabo Estai - Canido, 36200 Vigo, Galicia, Spain
c
Estación de Ciencias Mariñas de Toralla (ECIMAT), Universidade de Vigo, Illa de Toralla - Coruxo, 36331 Vigo, Galicia, Spain

a r t i c l e i n f o a b s t r a c t

Article history: This is the first attempt to cryopreserve Paracentrotus lividus embryos for the aquaculture industry. The cryopres-
Received 18 June 2014 ervation protocol used for sea urchin embryos has been proven not only to be able to produce viable outcome, but
Received in revised form 10 December 2014 also embryos that can grow into competent larvae, metamorphose and settle. Cryopreserved blastulas yielded an
Accepted 14 December 2014
initial survival of 50% at day 3 and 29% by the end of larval rearing (day 20), which represents the 71% survival of
Available online 20 December 2014
control, non-cryopreserved blastulas. Cryopreserved blastulas suffered a delay in growth and development,
Keywords:
achieving the prism or 4-arm pluteus stage at 96 h after fertilization. Larval development showed differences
Cryopreservation with the control blastulas after 10 days of the larval rearing (staying at 4-arm pluteus until day 8, early 6-arm
Sea urchin embryos pluteus by day 10). By day 13 cryopreserved blastulas achieved the 8-arm pluteus stage and, from that point
Paracentrotus lividus on, developed the rudiment and larvae achieved competence to metamorphose almost at the same time than
Larval rearing fresh controls. The settlement percentage of cryopreserved larvae was 7%, which represents the 25% of control
larvae settlement.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
The sea urchin Paracentrotus lividus (Lamarck 1816) is a large regular
sea urchin widely distributed throughout the Mediterranean Sea and
European Atlantic coast. This species is exploited in some European
countries for its highly valued gonads (Boudouresque and Verlaque,
2001). The regions with more active P. lividus fisheries in Europe are
in Ireland, Scotland, Italy and Spain (Andrew et al., 2002; Kelly and
Chamberlain, 2010; Norman et al., 2010).
Sea urchin fishery has flourished in many areas along the last de-
cades due to the increasing value of sea urchin roe, which has led to
overfishing of many natural populations (Andrew et al., 2002). In addi-
tion, production remains very traditional, relying on the natural envi-
ronment supply of juveniles with capture numbers at the mercy of
climatic conditions and natural or human-provoked disasters. Due to
the concern about the collapse of natural populations (Andrew et al.,
2002) there has been active research into: land-based culture (James,
2006), larval and juvenile feeding (George et al., 2001, 2004), artificial
feed (Fernandez, 1997), larval rearing systems (Christiansen and
Siikavuopio, 2007), gonad enhancement (Akiyama et al., 2001;
⁎ Corresponding author. Tel.: +34 986 818780.
E-mail address: eparedes@uvigo.es (E. Paredes).
Fernandez and Boudouresque, 2000; Shpigel et al., 2005), and wild
stock enhancement (Gonzalez-Henriquez et al., 2009).
Cryopreservation can be a very powerful biotechnological tool for
aquaculture production, i.e. for fisheries conservation and wild stock en-
hancement (Zhang, 2004), for the production of all year-round spat
supply without the need to condition broodstock (Adams et al.,
2011a) and for the improvement of selective breeding program man-
agement (Adams et al., 2011a; Zhang, 2004).
Although cryopreservation protocols have been successfully devel-
oped for aquaculture of many valuable species as sturgeons (Mims
et al., 2011), salmonid fishes (Lahnsteiner, 2011), or marine invertebrates
as oysters, mussels or abalone (Adams et al., 2011a; Lin and Chao, 2011;
Paniagua-Chavez et al., 2011), few research has been carried out
regarding sea urchin cryopreservation (gametes, embryos or larvae).
Fragmentary information was published on the species Hemicentrotus
pulcherrimus, Strongylocentrotus nudus and Stongilocentrotus intermedius
by Asahina and Takahashi (1978, 1979), Naidenko et al. (1991) and
Naidenko and Koltsova (1998), Tetrapigus niger by Barros et al. (1996,
1997), or Evechinus Chloroticus by Adams and Hessian (2004) and
Adams et al. (2006), and long term development studies from embryo
to metamorphosis and settlement have only been reported in one study
(Naidenko et al., 1991). Recently, we have developed a cryopreservation
protocol (Bellas and Paredes, 2011) producing high survival and healthy
larvae for P. lividus, but a complete larval rearing of cryopreserved and

http://dx.doi.org/10.1016/j.aquaculture.2014.12.022
0044-8486/© 2014 Elsevier B.V. All rights reserved.
 E. Paredes et al. / Aquaculture 437 (2015) 366–369
thawed early embryos to settlement has never been carried out before for
this species.
The aim of this work was to test the viability of cryopreserved sea ur-
chin embryos and examine their ability to survive and develop from
embryo through larval rearing to juvenile stage.
2. Materials and methods
Mature sea urchins, P. lividus, were collected in the outer part of the
Ría de Vigo (Galicia, NW Iberian Peninsula) during the natural repro-
ductive season, maintained in aquaria with running natural filtered sea-
water (18 ± 2 °C, 35‰) and were fed at demand with fresh green algae
Ulva lactuca. Gametes were obtained directly from the gonads with a
Pasteur pipette after dissection of a single pair of adults for each exper-
iment. Oocyte quality and sperm motility were checked prior to fertili-
zation, a few microliters of undiluted sperm were added to a 100 mL
cylinder with oocytes and stirred for 2 min to allow fertilization accord-
ing to Beiras and Saco-Álvarez (2006). The percentage of fertilization
was then calculated checking the formation of the fertilization mem-
brane and only batches with a fertilization rate N90% were used for
obtaining the embryos; batches not meeting this quality criteria were
discarded. Blastulas were obtained by incubation in Artificial Sea
Water (ASW, prepared following Lorenzo et al., 2002), in darkness, at
20 °C for 8 h and were checked out under the microscope previous to
cryopreservation experiments. Oocytes from the same batch were fertil-
ized and immediately introduced in two larval rearing tanks for each
batch control. Microalgae cultures of Tetraselmis suecica (Provasoli–
Guillard National Center for Marine Algae and Microbia, CCMP904),
Isocrysis aff. galbana clon T-ISO (Provasoli–Guillard National Center for
Marine Algae and Microbia, CCMP1324), Chaetoceros gracilis (ECIMAT
collection), Phaeodactilum tricornutum (ECIMAT collection) and
Cylindrotheca closterium (ECIMAT collection) used during the experi-
ments were provided by ECIMAT (Universidade de Vigo).
The cryopreservation methodology was performed following the
methods described in Bellas and Paredes (2011). Sea urchin embryos
were cryopreserved at the blastula stage (8 h incubation at 20 °C), the
cryoprotecting agent (CPA) combination used was 1.5 M dimethyl sulf-
oxide (Me2SO) + 0.04 M trehalose (TRE). One mL of CPA solution was
added to 1 mL of embryo suspension in ASW, in 15 equimolar steps
1 min apart (1:1 final dilution), at 19 ± 1 °C. We used a programmable
controlled freezer (Cryologic Lty. Ltd, Australia). The cooling ramp
started with a hold at 4 °C for 2 min, and then cooled at a rate of
1 °C min − 1 to − 12 °C. At this point vials were seeded during a
2 min hold, followed by cooling at 1 °C min − 1 to − 80 °C. A final
hold of 2 min was placed at − 80 °C and vials were transferred to
liquid nitrogen for storage. Thawing was performed by immersion
into a 17 ± 1 °C water bath until the ice was melted. CPAs were then re-
moved with clean ASW in 12 equimolar steps 1 min apart at room tem-
perature 19 ± 1 °C and embryos were finally rinsed with clean ASW.
Gametes from two male and female pairs were used to make indi-
vidual crossings: the first fertilizations were reared as fresh controls,
the second fertilizations using the same parental line, were incubated
for 8 h until the blastula stage, cryopreserved and conserved for 24 h
in liquid nitrogen, blastulas were thawed and embryos were finally
ready to be introduced in the larval rearing tanks. This study was carried
out by duplicate for each cross and treatment.
Larvae were reared for 20 days, until they were competent to meta-
morphose, in 100 L polypropylene cylindroconical tanks in closed circuit
of filtered sea water (FSW) (0.5 μm + Ultraviolet), with three complete
water changes per week (larvae were collected with a 40 μm mesh). The
initial amount of 105 larvae per tank were incubated at 19 ± 1 °C with a
24 h light schedule (7.1 μE m−2 s−1), and were fed with 40 to 80 equiv-
alents (a diet based on T. suecica, I. aff. galvana clon T-iso, C. gracilis and
P. tricornutum, in a number of algal cells equivalent in dry weight to
I. galbana, from day 0–7 with 40 equivalents, 60 equivalents until day
367
14 and 80 until day 20) as described in Casal et al. (2011) and Muller-
Feuga et al. (2003).
During larval rearing information was compiled to measure the
larval performance: survival counts (n = 100) and length measure-
ments (maximum dimension of the first 35 individuals, embryos
included).
Seven days before reaching competence, 10 Petri dishes per treat-
ment were inoculated with 15 mL of FSW (0.22 μm + Ultraviolet)
with f/2 medium and 5 mL of a benthonic diatom C. closterium
forming a biofilm to use as settlement attractant for competent lar-
vae. The dishes were incubated at 18 °C and 54.2 μE m − 2 s − 1 and
were rinsed with FSW to eliminate dead cells from the surface previ-
ous to the settlement experiment start point. After 20 days of larval
rearing 60 competent larvae per dish were introduced, which were
maintained in semidarkness (0.5 μE m− 2 s− 1) at 18 °C, with a single
food dose of 50 equivalents and were checked for settlement every
8–10 days. One-way analysis of variance (ANOVA) followed by the
Bonferroni test for multiple comparisons were conducted to assess
the differences among treatments using the SPSS® version 15.0 sta-
tistical software.
3. Results
Cryopreserved blastulas yielded an initial survival of 50% at day 3
while fresh controls sustained a 100% survival (Fig. 1a). In both treat-
ments larval survival decreased slowly, achieving 40.5% survival in
fresh controls and 29% survival in cryopreserved treatments at the end
of the rearing period. There was a significant variability in survival in
culture among batches of sea urchins (results shown are the average ±
SD of two different batches), both among fresh and cryopreserved
treatments.
During larval rearing samples were taken every two days to measure
larval size (Fig. 1b) and to study larval development differences (Fig. 2).
At day 4 fresh control larvae had an average size of 647.8 μm while the
3-day larvae from the cryopreserved treatments presented a size of
388.56 μm, representing 60% of the fresh control size. Larval size was
significantly lower in cryopreserved treatments (n = 35, p b 0.05)
than in fresh controls until 15 days in culture (Fig. 1b). During larval de-
velopment, echinoplutei reach their maximum size at day 15, then lar-
vae increase in volume as their arms shorten (inverted U shape curve
in Fig. 1b). The same pattern is observed in larvae from cryopreserved
treatments. After day 16 no significant size differences were observed
between control larvae and larvae which developed from cryopreserved
blastulae (n = 35, p b 0.05).
Sea urchin larvae reached the 4-arm pluteus stage at 48 h, the early
6-arm pluteus stage by day 7 (Fig. 2), the mature 6-arm pluteus stage by
day 11, the 8-arm pluteus stage by day 14, and from that point on, the
rudiment developed during days 16 to 20, when larvae achieved com-
petence to metamorphose. Despite the fact that cryopreserved larvae
suffered a delay in growth and development achieving the prism or 4-
arm pluteus stages at 96 h, differences in development stage with the
fresh controls were only observed during the first 10 days (4-arm
pluteus until day 8, early 6-arm pluteus by day 10). At day 13 larvae
achieved the 8-arm pluteus stage (although still smaller than 8-arm
pluteus larvae from controls at this point) and from that point on the ru-
diment developed and larvae achieved competence to metamorphose
at the same time compared with fresh controls.
After 18 days of incubation in the Petri dishes the control average
settlement was 28.21% and the cryopreserved larval settlement 7.09%
(25.14% of control settlement).
Larval settlement varied importantly among batches of sea urchins
and incubation time (not only varied among treatments but also varied
among Petri dishes along the whole experiment). For both batches
settlement increased from day 10 to 18, this difference was only
statistically significant among controls.
368 E. Paredes et al. / Aquaculture 437 (2015) 366–369

Fig. 1. (a) Percentage of larval survival (n = 100) for fresh fertilized oocytes (filled symbols) and for cryopreserved blastulas (open symbols), and (b) larvae size during larval rearing, for
fresh fertilized oocytes (black) and for cryopreserved blastulas (grey) (n = 35). Error bars indicate standard deviation.

4. Discussion by De la Uz et al. (2009) (75–85%), by Casal et al. (2011) (53–65%), or by

The aim of this work was to test the viability of cryopreserved sea ur-
chin (P. lividus) embryos during larval development, metamorphosis
and settlement. Survival of embryos immediately after cryopreservation
is very complicated to measure (it can indirectly be measured as mem-
brane integrity or motility). Moreover, adequate embryonic and larval
development is not warranted merely by the embryo survival within
few hours post-thawing. Therefore, our first survival checkpoint was
done at 96 h of incubation. At this point cryopreserved embryos have al-
ready developed to the 4-arm pluteus larval stage, and yielded a surviv-
al of 50% in comparison to the fresh control culture. From this point
forward (from early embryonic stage to settlement), survival, growth
and development were checked every two days.
It must be mentioned that there was a noticeable variability in per-
formance among batches of sea urchins, both in the fresh and cryopre-
served treatments, being this variability higher in the latter. Similar
variability in the survival of non-cryopreserved larvae throughout larval
rearing (44–85.5% for different batches) has been reported for P. lividus
by De la Uz et al. (2009) and Ojea et al. (2009). Furthermore, this
has also been noticed among different batches of sea urchins for
P. lividus cryopreserved embryos (Bellas and Paredes, 2011), and for
E. chloroticus cryopreserved pluteus larvae (Adams et al., 2006). At the
end of 20 days of larval rearing the fresh culture yielded an average sur-
vival of 40.5%, which is lower than that previously reported for P. lividus
Ojea et al. (2009) (47–67%), while the cryopreserved treatments
yielded an average survival of 29%.
The study of larval growth from cryopreserved embryo treatments,
showed a significant delay in development. After 96 h, the size of larvae
which developed from cryopreserved embryos was only 60% of the size
of fresh control larvae, but the differences in growth disappeared at the
end of the incubation time (unnoticeable from day 16 onwards). Also,
despite the fact that differences in the developmental stage were signif-
icant during the first 10 days of rearing and were reduced during devel-
opment and by the end of the larval rearing period, when larvae were
ready to settle, most of control larvae and larvae which resulted from
cryopreserved embryos were competent echinopluteus (8-arm larvae
with developed rudiment).
Once larvae were competent to metamorphose it is important to
study their ability to metamorphose, settle, and give rise to a juvenile.
Results from this work indicate that after 18 days the percentage of con-
trol larvae settled was 28.21%. These results indicate lower settlement
that was previously reported with this attractant (70%) for P. lividus
(Casal et al., 2011). On the other side, cryopreserved larvae achieved a
7.09% settlement by day 18.
Only one previous work reared sea urchin (Strongylocentrotus
intermedius) cryopreserved embryos until metamorphosis (Naidenko
et al., 1991), reporting 0.1–0.2% survival. As for later stages of develop-
ment, Adams et al. (2006) successfully developed a cryopreservation

Fig. 2. Larval morphology and development during 20 days of larval rearing, (a) are fresh controls; and (b) cryopreserved embryos.
 E. Paredes et al. / Aquaculture 437 (2015) 366–369
protocol that yielded a 6% settlement for cryopreserved 4-arm pluteus
larvae of Evechinus choloticus. In both studies, as well as in the present
work, the cryopreservation protocol included similar CPA concentra-
tions, using Me2SO from 1 to 1.5 M, but apart from this, the other param-
eters, including cooling or thawing rates, were different.
Although survival and settlement of fresh larvae were lower than
that previously published for P. lividus (our incubation volumes used
for settlement were scaled down in comparison to previous published
work), performance of cryopreserved larvae compared with fresh con-
trols (71% survival and 25% settlement in comparison to fresh controls),
was better than in previous studies. It must be said that, standard cul-
ture methodologies and timing for invertebrate larvae must be slightly
readjusted when working with cryopreserved embryos. Specific charac-
teristics such as the slower development (and therefore smaller size
than controls at a given time), the variability in survival to cryopreserva-
tion and the variability in survival during larval rearing and settlement
process, should be taken into account.
This first attempt to cryopreserve P. lividus embryos for the aquacul-
ture industry is encouraging. The cryopreservation protocol used for sea
urchin embryos has been proven not only to be able to produce viable
outcome, but also to result in acceptable numbers of embryos that can
develop into larvae, metamorphose, and settle into juveniles.
Acknowledgments
Authors want to acknowledge personnel from the Estación de
Ciencias Mariñas de Toralla-Universidade de Vigo, especially Arantxa
Martinez for technical assistance. E. Paredes is funded by Ministerio de
Educación under a FPU fellow (AP2009-2751).
References
Adams, S.L., Hessian, P.A.V., 2004. Cryopreservation of sea urchin (Evechinus chloroticus)
sperm. Cryoletters 25 (4), 287–299.
Adams, S.L., Hessian, P.A., Mladenov, P.V., 2006. The potential for cryopreserving larvae of
the sea urchin, Evechinus chloroticus. Cryobiology 52, 139–145.
Adams, S.L., Tervit, H.R., Salinas-Flores, L., Smith, J.F., McGowan, L.T., Roberts, R.D., Janke,
A., King, N., Webb, S.C., Gale, S.L., 2011. Cryopreservation of Pacific oyster oocytes.
In: Tiersch, T.R., Green, C.C. (Eds.), Cryopreservation in Aquatic Species, 2nd edition
World Aquaculture Society, Baton Rouge, Louisiana, pp. 616–623.
Akiyama, T., Unuma, T., Yamamoto, T., 2001. Optimum protein level in a purified diet for
young red sea urchin Pseudocentrotus depressus. Fish. Sci. 67, 361–363.
Andrew, N.L., Agatsuma, Y., Ballesteros, E., Bazhin, A.G., Creaser, E.P., Barnes, D.K.A.,
Botsford, L.W., Bradbury, A., Campbell, A., Dixon, J.D., Eirnarsson, S., Gerring, P.K.,
Hebet, K., Hunter, M., Hur, S.B., Johnson, C.R., Juino-Meñez, M.A., Kalvass, P., Miller,
R.J., Moreno, C.A., Palleiro, J.S., Rivas, D., Robinson, S.M.L., Schroeter, S.C., Steneck,
R.S., Vadas, R.L., Woodby, D.A., Xiaoqi, Z., 2002. Status and management of world
sea urchin fisheries. In: Barnes, H., Gibson, R.N., Barnes, M., Atkingson, R.J.A. (Eds.),
Oceanography and Marine Biology, Annual Review. vol. 40. Taylor and Francis,
pp. 343–426.
Asahina, E., Takahashi, T., 1978. Freezing tolerance in embryos and spermatozoa of the sea
urchin. Cryobiology 15, 122–127.
Asahina, E., Takahashi, T., 1979. Cryopreservation of sea urchins embryos and sperm. Dev.
Growth Differ. 21, 423–430.
Barros, C., Muller, A., Wood, M.J., Whittingham, D.G., 1996. High survival of sea urchin
semen (Tetrapigus niger) pluteus larvae (Loxechinus albus) frozen in 1.0 M Me2SO.
Cryobiology 33, 646.
Barros, C., Muller, A., Wood, M.J., 1997. High survival of spermatozoa and pluteus larvae of
sea urchins frozen in Me2SO. Cryobiology 35, 341.
Beiras, R., Saco-Álvarez, L., 2006. Toxicity of seawater and sand affected by the Prestige
fuel-oil spill using bivalve and sea urchin embryogenesis bioassays. Water Air Soil
Pollut. 117, 457–466.
369
Bellas, J., Paredes, E., 2011. Advances in the cryopreservation of sea-urchin embryos: po-
tential application in marine water quality assessment. Cryobiology 62, 174–180.
Boudouresque, C.F., Verlaque, M., 2001. Ecology of Paracentrotus lividus. In: Lawrence, J.M.
(Ed.), Edible Sea Urchins: Biology and Ecology, 1st edition Elsevier, Amsterdam,
pp. 247–286 (2007 2nd edition).
Casal, A., Costas, D., Rodiguez, R., Costoya, N., Rivera, L., Rial, D., 2011. Asentamiento de
larvas competentes de erizo de mar, Paracentrotus lividus Lamarck 1816, expuestas
a diferentes inductores algales. Abstracts Congreso Nacional de Acuicultura
(Barcelona).
Christiansen, J.S., Siikavuopio, S.I., 2007. The relationship between feed intake and gonad
growth of single and stocked green sea urchin (Strongylocentrotus droebachiensis) in
raceway culture. Aquaculture 262, 163–167.
De la Uz, S., Carrasco, J.F., Rodriguez, C., 2009. Cultivo larvario de erizo de mar
(Paracentrotus lividus) Resultados obtenidos 2006–2009. Abstracts Congreso
Nacional de Acuicultura, pp. 546–547 (Madrid).
Fernandez, C., 1997. Effect of diet on the biochemical composition of Paracentrotus lividus
(Echinodermata: Echinoidea) under natural and rearing condition (effect of diet on
biochemical composition of urchins). Comp. Biochem. Physiol. 118A (4), 1377–1384.
Fernandez, C., Boudouresque, C.F., 2000. Nutrition of the sea urchin Paracentrotus lividus
(Echinodermata: Echinoidea) fed different artificial food. Mar. Ecol. Prog. Ser. 204,
131–141.
George, S.B., Lawrence, J.M., Lawrence, A.L., Smiley, J., Plank, L., 2001. Carotenoids in the
adult diet enhance egg and juvenile production in the sea urchin Lytechinus
variegatus. Aquaculture 199, 353–369.
George, S.B., Lawrence, J.M., Lawrence, A.L., 2004. Complete larval development of the sea
urchin Lytechinus variegatus fed artificial feed. Aquaculture 242, 217–228.
Gonzalez-Henriquez, N., Grimon, M., Catoira, J.L., 2009. Cultivo exterior para la
repoblacion de Paracentotus lividus en las Islas Canarias. Abstracts Congreso Nacional
de Acuicultura, pp. 44–45 (Madrid).
James, P.J., 2006. A comparison of roe enhancement of the sea urchin Evechinus
Chloroticus in sea-based and land-based cages. Aquaculture 253, 290–300.
Kelly, M.S., Chamberlain, J., 2010. Recent advances in sea-urchin aquaculture and en-
hancement in Scotland and Ireland bull. Aquac. Assoc. Can. 108–1, 23–24.
Lahnsteiner, F., 2011. Cryopreservation protocols for sperm of salmonid fishes. In: Tierch,
T.R., Green, C.C. (Eds.), Cryopreservation of Aquatic Species, 2nd edition World Aqua-
culture Society, pp. 409–420.
Lin, T.-T., Chao, N.-H., 2011. Cryopreservation of eggs and embryos of shellfish. In: Tierch,
T.R., Green, C.C. (Eds.), Cryopreservation of Aquatic Species, 2nd edition World Aqua-
culture Society, pp. 604–615.
Lorenzo, J.I., Nieto, O., Beiras, R., 2002. Effect of humic acids on speciation and toxicity of
copper to Paracentrotuslividus larvae in seawater. Aquatic Toxicology 58 (2002),
27–41.
Mims, S.D., Tsvetkova, L.I., Wayman, W.R., Horváth, A., Urbányi, B., Gomelsky, B., 2011.
Cryopreservation of sturgeon and paddlefish sperm. In: Tierch, T.R., Green, C.C.
(Eds.), Cryopreservation of Aquatic Species, 2nd edition World Aquaculture Society,
pp. 366–380.
Muller-Feuga, A., Robert, R., Calu, C., Robin, J., Divanach, P., 2003. Uses of microalgae in
aquaculture. In: Stǿttrup, J.G., McEvoy, L.A. (Eds.), Live Feeds in Marine Aquaculture.
Blackwell publishing, pp. 253–318.
Naidenko, T.K., Koltsova, E.A., 1998. The use of antioxidant echinochrome-A in cryopres-
ervation of sea urchin embryos and larvae. Russ. J. Mar. Biol. 24, 203–206.
Naidenko, T.K., Gakhova, E.N., Naidenko, V.P., Veprintsev, B.N., 1991. Evaluation of viabil-
ity of sea urchin larvae after cryopreservation of embryos. In: Yanagisawa, T.,
Yasumasu, I., Oguro, C., Suzuki, N., Motokawa, T. (Eds.), Biology of Echinodermata.
A.A. Balkema Publishers, pp. 261–269.
Norman, J.C., Guerrero, D., Ortega, C., Alvarez, P.A., Ibañez, A.J., Lopez, C., 2010. Erizo de
Mar: gestión, cultivo y utilización. Consejeria de Agricultura y pesca de Andalucía.
Ojea, J., Martinez-Patiño, D., Novoa, S., Catoira, J.L., 2009. Cultivo de erizo de mar en
criadero: desoves y desarrollos larvarios. Abstracts Congreso Nacional de Acuicultura,
pp. 282–283 (Madrid).
Paniagua-Chavez, C.G., Buchanan, J.T., Supan, J.E., Tierch, T.R., 2011. cryopreservation of
sperm and larvae of the eastern oyster. In: Tierch, T.R., Green, C.C. (Eds.), Cryopreser-
vation of Aquatic Species, 2nd edition World Aquaculture Society, pp. 595–603.
Shpigel, M., McBride, S.C., Marciano, S., Ron, S., Ben-Amotz, A., 2005. Improving gonad
color and somatic index in the European sea urchin Paracentrotus lividus. Aquaculture
245, 101–109.
Zhang, T., 2004. Cryopreservation of gametes and embryos of aquatic species. In: Fuller,
B.J., Lane, N., Benson, E.E. (Eds.), Life in the Frozen State. CRC Press (Pages 415-136).