CryoLetters 35 (6), 482-494 (2014) © CryoLetters, [email protected] SEA URCHIN (PARACENTROTUS LIVIDUS) CRYOPRESERVED EMBRYOS SURVIVAL AND GROWTH: EFFECTS OF CRYOPRESERVATION PARAMETERS AND REPRODUCTIVE SEASONALITY E. Paredes1* and J. Bellas2 1Departamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo, Estrada Colexio Universitario s/n, 36310 Vigo, Galicia, Spain. 2Instituto Español de Oceanografía, Centro Oceanográfico de Vigo, Cabo Estai – Canido, 36200 Vigo, Galicia, Spain *Corresponding author email: [email protected] Abstract BACKGROUND: The cryopreservation of embryos can be a powerful biotechnological tool to supply all year-round biological material for sea urchin aquaculture production. This study investigates different methodological and biological factors that may affect the result of the cryopreservation process of sea urchin (Paracentrotus lividus) embryos. Our data indicate that neither embryo density nor the use of different cryopreservation containers presented effect on the cryopreservation outcome. Contrary to other marine invertebrates, for sea urchin embryo cryopreservation ultrapure water cannot be used as CPA solvent, yielding zero survival. After studying the reproductive parameters along the reproductive season, we found a positive correlation between both male and female Condition Index (C.I.), and between the oocyte weight and C.I. Both the histology study of female gonads and the C.I. variation, suggest that the sea urchin natural spawning period in the Ría de Vigo occurs between June and July. We found no correlation between any of the reproductive parameters monitored and the cryopreservation outcome. Keywords: Cryopreservation, sea urchin, Paracentrotus lividus, Aquaculture urchin inland or semi-inland based culture INTRODUCTION facilities would allow a more sustainable and reliable production, with the Sea urchin fishery has flourished in possibility of combined cultivation with many areas like Japan, Russia, USA, and other species, use of triploid sea urchins Canada along the last decades and many that will prevent gametogenesis and sea urchin populations are suffering from gonadal fluctuations, the use of an artificial overfishing (5), to prevent the collapse of diet would standardize and allow diet many populations the sea urchin fisheries manipulation (16, 11, 21). would need to rely less on the natural In addition to previously listed tools production and take advantage of new that can enhance sea urchin culture, biotechnological tools available. Sea 482 cryopreservation is a very powerful tool marine quality assessment (8, 37). In these for aquaculture. studies, the main steps of the sea urchin A significant amount of gametes, embryos cryopreservation protocol were embryos and larvae from different established, from the selection of suitable organisms have been successfully cryoprotecting agents (CPAs), to the cryopreserved for different purposes, such definition of protocol details, namely: CPA as endangered animal and fisheries addition/removal methodology, cooling conservation (23, 27, 29, 49), animal rates, thawing and incubation time (8, 37). reproduction and aquaculture production In the present study we investigate other (33, 2, 14, 41), , food and industry (43), or cryopreservation parameters like the effects of using ultrapure (MiliQ) for CPAs laboratory research (17, 37). preparation, embryos density effect, and In particular, cryopreservation can be suitability of different containers (2 mL used in aquaculture to provide food supply vials and 0.25 mL straws). We also for some species –like groupers or investigated the seasonal evolution of snappers- where very small zooplankton is reproductive parameters (condition index - needed as first feed (24, 32). Also, the C.I.- as an indirect measurement of the cryopreservation of marine invertebrate maturity of the gonads, the oocytes weight embryos and larvae would enable a year- as an indirect measurement of the oocytes round spat supply without the need to quality, and histological analysis of the condition broodstock (3). It can help female gonads) and their possible improve the management of selective relationship with the cryopreservation breeding programs (easy transportation, outcome. allowing crossings from different breeding seasons, meeting market requirements or resistance to illnesses) (3, 49), and MATERIALS AND METHODS provides a certainty of supply that will help plan the crossings and the Biological material implementation of new breeding designs Sea urchins, Paracentrotus lividus, were (3). collected in the outer part of the Ría de Vigo (Galicia, NW Iberian Peninsula) The test species chosen here is the during the natural reproductive season, at edible sea urchin Paracentrotus lividus least once a month. Sea urchins of 5 to 6 (Lamarck 1816), a large regular sea urchin cm diameter were selected, transported to widely distributed throughout the the laboratory in a portable icebox, Mediterranean Sea and European Atlantic maintained in aquaria with running natural coast with important ecological roles in the seawater (18°C, 35.5‰) and used within functioning, dynamics and structure of one week. Gametes were obtained directly benthic assemblages (9, 25, 26). Several from the gonads with a Pasteur pipette studies have also shown the importance of after dissection of a single pair of adults sea urchin pluteus larvae in the for each experiment. Only batches of composition and biomass of zooplankton healthy oocytes and motile sperm were communities, playing a significant role in used for fertilization (sperm was added to the pelagic foodweb (31). This species is the oocyte suspension (4:1 ratio) and exploited in some European countries for carefully stirred for one minute to allow its highly valued gonads (9, 30, 40, 44). fertilization, fertilization percentage >90%). Fertilized oocytes were incubated In previous work we developed a in Artificial Sea Water (ASW), in cryopreservation protocol for sea urchin darkness, at 20ºC for 8 hours, until embryos with potential application on reaching the blastula stage. Blastulas were aquaculture, fisheries conservation and 483 checked out under the microscope and individuals per replicate (L) (n =3 cryopreservation experiments were replicates per treatment), subtracting the performed only if those blastulas looked average diameter of fertilized eggs (L0), healthy. Oocytes from the same batch were ΔL = L - L0. Data were normalized to the weighted as described below. A thin slice larval size in the controls (ΔLc), to obtain of female gonad was also extracted for the percentage of larval growth, % growth histological analysis. Finally, the gonads = (ΔL/ ΔLc)*100. from both male and female were separated from their carcasses for Conditon Index Methodological parameters (C.I.) calculation. CPAs aqueous media; Cryopreservation trials were carried out to study the effect of Cryopreservation methodology dissolving the CPAs in different aqueous The cryopreservation methodology was matrixes artificial sea (ASW) water (final performed following methods described sample salinity of 35%), and ultrapure elsewhere (8). The CPA combination used (Milli-Q) water (final sample salinity was dimethyl sulfoxide 1.5 M (Me2SO) + 17.5%). The standard cryopreservation 0.04 M trehalose (TRE), 1 ml of solution methodology was followed as described was stepwise added to 1 ml of embryos above. The whole process was repeated suspension (blastula stage) in ASW, in 15 with 4 different sea urchin crossings. CPAs equimolar steps one minute apart (1:1 final in both cases were diluted stepwise -post dilution), at 19 ± 1ºC. The containers used thawing- with ASW and were transferred were 2 ml vials and the cooling ramp to 20 ml vials with clean ASW (post- (Cryologic programmable freezer) started thawing salinity 35%) for incubation until with a hold at 4ºC for 2 min, then cooled at 4-arm pluteus larvae were achieved. a rate of 1ºC min-1 to -12ºC. At this point Another experiment was placed following vials were seeded during a 2 min hold the above procedure of addition and followed by cooling at 1ºC min-1 to -80ºC. removal of CPAs prepared with ultrapure A final hold of 2 min was placed at -80ºC water, but skipping the freezing step, the and vials were transferred to liquid resulting embryos were transferred to nitrogen for storage. Thawing was ASW for 96 hours incubation. performed by immersion into a 17 ± 1ºC water bath until the ice was melted (± 2 Embryo density; To inquire if there was minutes). CPAs were then removed with an effect of the embryo density on the clean ASW in 12 equimolar steps one cryopreservation outcome, an experiment minute apart at room temperature 19 ± was conducted where embryos were 1ºC. Embryos were finally transferred to cryopreserved at different densities 20 ml vials with clean ASW for incubation (cryopreservation protocol previously (at 20ºC, in darkness) until 4-arm pluteus explained), namely: 10,000; 5,000; 2,500; larvae stage was achieved. 1,250 and 500 embryos/vial, in 2 ml vials. Once embryos were thawed and CPAs Whilst unfrozen P. lividus embryos diluted, the same amount of 500 embryos normally develop to 4-arm-pluteus larvae (for a final incubation density of 25 -1 in 48 hours, the development of embryos ml ) were transferred to 20 ml frozen/thawed embryos was stated to be vials with clean ASW for incubation until slower (9), so the incubation time for 4-arm-pluteus larvae were achieved. cryopreserved larvae was either recorded at 48, 72 and 96 hours post fertilization.