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. Cryopreservation containers; The Resulting larvae were fixed with 2 drops of suitability of two cryopreservation 40% formalin and the larval growth was containers (cryovials or straws) was also measured. Larval growth (ΔL) was defined studied. Our prior work was performed as the maximum dimension of the first 35 using cryovials because they offer several 484 experimental advantages, and the with ammonium formate (HCO2NH4) 0.5 possibility of storing large volumes was M to eliminate the sea salt. The dry weight more suitable for the future application. of oocytes was recorded in a precision However, since straws offer certain storing scale, after drying at 60ºC until constant advantages (a higher number of straws can weight (24 h). The net oocyte weight was be banked in a Dewar flask, allowing to divided among the number of oocytes to store easily different treatments, densities calculate the weight per oocyte (g oocyte- or family lines), an experiment was 1). conducted to compare the results of cryopreserving the same batch of embryos Histological analysis; A thin slice of in two different containers (2 ml cryovials each female gonad was fixed in and 0.25 ml straws), using the freezing Davidson’s solution, glycerine, protocol described above, and incubating formaldehyde, Ethanol 99%, filtered sea the resulting embryos until 4-arm pluteus water and acetic acid (proportions larvae were achieved. This experiment was 1:2:3:3:1) and sent to ECIMAT (Vigo replicated 3 times using 3 different pairs of University) histological services, for sea urchins. histological preparation. Gonads were embedded in paraffin and paraffin blocks Biological parameters were sectioned at 5 µm with a microtome. Condition Index; The condition index Tissue sections were deparaffinized and (C.I.) was calculated according to Byrne stained with Harris’ haematoxylin and (13) as: C.I.=(Gonad dry weight / total eosin. Finally, samples were prepared to be body dry weight) × 100. Several formulas examined by light microscopy. are used to calculate the C.I. in different marine species, the formula used in this In this work, the classification in work has been commonly used for the different stages of gonad development was study of natural sea urchin populations (13, considered following Byrne’s (13) 30, 42, 44, 46). classification, the description of the gonad cycle is divided in 6 stages: F1 - Gonadal Gonads from both male and female reconstruction: the gonad is full of were carefully separated from their nutritive phagocytes and reserve carcasses and placed in aluminum foil substances. F2 - Oocyte growth: pieces. The dry weight of cleaned gametogenesis takes place. F3 – Oocyte carcasses and gonads was recorded after pre-maturation: oocytes grow from the drying at 60ºC until constant weight (48 h) gonad wall and migrate to the interior of The dry weight of oocytes extracted for the gonad. F4 – Mature Gonad: the gonad oocytes weight measures was added to the is full of mature oocytes, without trace of gonad dry weight but not the weight of the reserve material. F5 – Partially spawned slice extracted for gonad histology, gonad: empty spaces appear in the gonad therefore the slice size was standardized left by the already spawned oocytes. F6 – and minimized to interfere in the C.I. Post-Spawning: the gonad appears mostly calculation as less as possible. empty. Reserve material starts to regenerate and there might still be some Oocytes weight; Sea urchin oocytes oocytes left which will be reabsorbed. were collected from the female gonad and concentrated into a known small volume of ASW. The total number of oocytes was calculated. Subsequently, the oocytes were filtered using a 20 μm mesh and washed

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Statistics Statistical analyses were conducted using the SPSS® version 15.0 statistical software. Differences among treatments were analyzed by one-way analysis of variance (ANOVA) followed by the Bonferroni test for multiple comparisons. To study the relationships among different factors we used the Pearson correlation.

RESULTS

Methodological parameters CPAs aqueous media; Embryos cryopreserved with CPAs solutions prepared with ASW produced larvae which grew in average 68% ± 23 of the control larvae after 96 hours incubation (Figure 1a), but when using CPAs solutions prepared with ultrapure water (17.5 ‰), the embryonic development was impaired, and most blastulae were partially destroyed or lysed once returning to sea water Figure 1. Growth/length of sea urchin 4-arm salinity post-thawing. pluteus larvae after 96 hours incubation Error bars indicate standard deviation, p < 0.05. (a) Embryo density; As it can be observed Comparison of two different CPAs aqueous in Figure 1b, no significant differences media: ASW (grey) and ultrapure water (black). were found among density treatments and (b) Effects of the embryo density in the all treatments were significantly different cryopreservation outcome. (c) Comparison of from the control. Therefore two different cryopreservation containers: 2 mL cryopreservation was not affected by vials and 0.25 mL straws. In all cases n = 35 density and the average growth was ca. larvae measured per treatment. 50% of the control larvae. Biological parameters Cryopreservation containers; No Condition Index; The reproductive significant differences were found in the season of Paracentrotus lividus in the Ría results obtained with 0.25 ml straws or 2 de Vigo extends from March to October. ml vials (Figure 1c). The average growth This sea urchin population presents one was 62% ±12 (58.9 and 66.8% of the single spawning peak along the year, control larvae, for straws and vials, which, according to the C.I. measurements, respectively). can be located at the beginning of summer (June-July), when the C.I. decreases (Figure 2). The C.I. of males and females showed a similar pattern of variation along the reproductive period, and a significant positive correlation was observed (r = 0.64, p =0.011, n = 13).

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Oocytes weight; The maximum and gonad samples into four categories (F3, F4, minimum oocyte weights recorded along F5 and F6) out of the six categories the spawning season were 5.06×10-07 (in described by Byrne (13). According to this March) and 5.97×10-10 gr (in July), analysis, oocyte prematuration (F3) occurs respectively (506 ng and 0.6 ng). The in April-May, and mature gonads (F4) are oocyte weight increased significantly with found during June-August, when natural increasing female C.I. (r=0.65, p=0.019, spawning takes place. Partially spawned n=13), R2 = 0.43). gonads (F5) were observed during August- September and the beginning of post Histological analysis; The female spawning (F6) occurs in September- histological study collected from April to November. Finally, although not covered September 2012, allowed us to classify by the sampling period described here,

Figure 2. Male and female condition index (C.I.) of sea urchins in Ría de Vigo (NW. Spain) along the reproductive season.

Figure 3. Growth of sea urchin 4-arm pluteus larvae obtained from cryopreserved/thawed blastulas incubated for 48, 72 and 96 hours at 20ºC. Error bars indicate standard deviation.

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during winter sea urchins will go through 2×106 oocytes/mL and implies that large gonadal reconstruction (F1) and oocyte amounts of embryos can be cryopreserved growth (F2). and stored in small volumes for future use. Cryopreservation of sea urchin embryos We also found that there is no difference in A progressive increase in larval growth using straws or cryovials for of cryopreserved embryos with increasing cryopreservation, despite the differences incubation periods was observed (Figure between both containers. Plastic straws are 3). The 4-arm pluteus larvae resulting from made of thin plastic and have a higher cryopreserved blastulae reach, in average, surface area to volume than 2 mL vials 19.4% ±7.8, 30.8% ±14.4, 50% ±10.8 the which are also made of thicker plastic, that size of larvae resulting from unfrozen could influence the cooling and thawing blastulae, after 48, 72 and 96 hours rates (28, 29). Also, the absence of any incubation, respectively (considering April difference between survival in cryovials and June-a values as outliers). and straws may indicate that these embryos support a wide range of cooling and Therefore, 96 hours was chosen as the warming rates, which could offer most appropriate incubation period. No flexibility in the protocol. The possibility significant correlation was found between of using straws instead of vials without the cryopreservation outcome and the C.I., differences in the cryopreservation the oocyte weight or the histological outcome, represents also an alternative in classification of the female gonads case of needing to store small amounts of embryos from a great number of parental DISCUSSION lines without using such a great amount of space as when using cryovials. This result The present study investigates the effects is in disagreement with published data for of different methodological factors and the Perna canaliculus trochophore larvae variability of several reproductive cryopreserved using 3 different containers, parameters, on the cryopreservation 0.25 mL straws, 2 and 4 mL vials (38), outcome of sea urchin (Paracentrotus where the % of normal D-larvae obtained lividus) embryos. One of the from the vials (either 2 or 4 mL vials) was methodological factors investigated is the dramatically lower than from straws, when density of embryos in the cryopreservation using similar cooling rates and higher containers. Our data indicate that the thawing rates than those described here. embryo density presented no effect on the Often, cryopreservation studies with cryopreservation outcome in the range 250 early developmental stages of marine to 5000 embryos/mL. This finding implies invertebrates use CPAs prepared in that our trials can be scaled up, and higher ultrapure water (MiliQ) (3, 4, 35). densities of embryos can be cryopreserved Regarding sea urchins, we found that the in vials with no detrimental effects. Ten use of ultrapure water to CPAs preparation thousand embryos per vial would provide a had a detrimental effect and caused the high enough number to start an lysis of all cells in the culture after experimental culture, but we have verified cryopreservation. In order to understand that this density can be scaled up further if how the use of ultrapure water affected necessary, up to a value of 50,000 embryos before and afterwards the embryos/mL, without detrimental effect cryopreservation process, we repeated the (unpublished data). This finding is in experiment of addition and removal of agreement with high densities CPA prepared with ultrapure water, but cryopreserved for other species, Mytilus skipping the freezing step. When cooling is galloprovinciallis (47) 3×1015 not involved, embryos continued to individuals/mL or Crassostrea gigas (45) at develop once transferred to ASW for 96 488 hours incubation, thereforefresh embryos substances (mostly glycogen) are resisted the stepwise addition and removal accumulated in the gonads during the of the CPAs with clean ASW. The simplest reproductive cycle and are used as source hypothesis is that the combination of both of energy for oocyte maturation previous stressing conditions as exposure to low to spawning, and as oocyte yolk reserves. salinity and the side effects of being Therefore there is a clear relationship exposed to such low temperatures, resulted between glycogen content and C.I., which in cellular death. The membrane fluidity is in agreement with the positive and integrity might be reduced due to the correlation found here between the oocyte cryopreservation process; this has been weight and C.I., Garrido and Barber (19) detected in human sperm and mouse reported that maximum mass in oocytes is oocytes (1, 22) and very likely happens to found previous to the optimum spawning other organisms during the freeze-thawing peak, whereas lower levels are detected in process. It is also well known that sea the post spawning period and late in the urchins have mechanisms to repair reproductive season. Our results are in membrane integrity disruptions (34), agreement with these findings, since we therefore the damage produced due to the report maximum and minimum oocyte osmotic change (volume changes) was weight in March and July (506 ng and 0.6 important or the repair mechanisms were ng respectively), in correspondence to the still inactive after the cryopreservation. spawning period location. The fertilization of sea urchins is The C.I. results and the histology external, occurs when gametes are study of female gonads (F4 mature gonad spawned into the sea, so male and female stage in Byrne’s classification), suggest gonad development has to be synchronized that the sea urchin natural spawning period to ensure oocytes and sperm are spawned in the Ría de Vigo occurs between June at the same time to the surrounding water and July. This result coincides with our (12). Our study confirms the previous observations in the area, which synchronization of the gonad development also point to a slight interannual variation of males and females along the of the reproductive period, depending on reproductive season, as shown by the the variation of different endogenous and correlation of the C.I. Factors affecting the exogenous factors (e.g. weather conditions, volume of the gonad are mainly food food supply, and water temperature). In availability and reproductive cycle - which general, these results are in accordance is also affected by food availability and with previous studies in the East Atlantic water temperature (19). Sea urchins were coast where several authors agree that sea collected from a population located in a urchins present a single spawning period small area subjected to homogeneous whose duration varies depending on the environmental conditions (quantity of food population, starting around May and availability, temperature, photoperiod, finishing around August (7, 15, 25, 36, 44). hydrodynamic conditions); therefore, Cryopreservation results obtained taking into account the relatively low throughout the reproductive season dispersal potential of adults, it is indicate a broad difference in the larval reasonable to assume that our samples development of cryopreserved embryos were fairly representative of the when compared to unfrozen controls. reproductive cycle of this population at this Unfrozen sea urchin embryos reach the 4- particular location. arm pluteus larvae at 48 hours when The change in gonad composition incubated at 20ºC, with an average larval during the course of the reproductive size of 400-500 µm. However, season has previously been reported (10, cryopreserved embryos development 12, 36, 48), indicating that reserve seems to be delayed, achieving after 48

489 hours incubation, in average, 20% the size exposed to a great number of sources of of controls under the same conditions. stress during cryopreservation (e.g. Either due to the slow activation of handling, CPA toxicity, exposure to low metabolism following the exposure to cold temperatures, membrane deformation, or temperatures, or to activation of some exposure to high concentration of salt restoration mechanisms (34) dealing with during cooling), causing a possible side effects of cryopreservation (1, 6, 22) increase in the production of Reactive the fact is that in the first 48 hours there is Oxygen Species (ROS) (6). Depending on a slow active growth, and most embryos previous biological conditions, organisms during that period were still in blastula or might deal differently with stress gastrula stage. When incubated for longer magnification. Finally, it is also possible periods we observed increasing that some random microescale variations development and growth, so larvae which in the freezing process may cause these survived cryopreservation will successfully fluctuations in the cryopreservation grow (though a little slower) and reach the success. Addressing the success of 4-arm pluteus stage after 96 hours of cryopreserved embryos during the larval incubation. This finding has also been rearing process in comparison to non- detected when incubating cryopreserved cryopreserved larvae will be the step and thawed embryos from other marine forward for improving sea urchin invertebrates as Perna canaliculus (38), aquaculture and study long term effects of Mytilus galloprovincialis (39, 47) or cryopreservation. Crassostrea gigas (39), where differences between frozen and unfrozen larvae were Acknowledgements: Authors want to observed not only during the embryonic acknowledge personnel from the Estación development, but also throughout larval de Ciencias Mariñas de Toralla- rearing. Universidade de Vigo for technical We found no influence of any of the assistance. E. Paredes is funded by reproductive parameters monitored (C.I., Ministerio de Educación under a FPU oocyte weight or histological fellow classification) on the cryopreservation outcome. This means that once the REFERENCES reproductive season starts, we only need to check for healthy blastulas previous to 1. Ahn, H.f.; Sohn, I.P.; Kwon, H.C.; Jo, cryopreservation to obtain 4-arm pluteus D.H.; Park, Y.D.; Min, C.K. larvae at 96 hours of incubation. However, Characteristics of the cell membrane there is still some source of variability in fluidity, Actin fibres, and mitochondrial the cryopreservation outcome that we have dysfunctions of frozen-thawed two-cell not identified yet. This variability might be mouse embryos. Mollecular due to the quality of oocyte reserves that is Reproduction and development 61, affected by food composition (18-20). 2002, Pages 466 – 476. Macroalgae availability and seasonality 2. Adams, S.L.; Smith, J.F.; Roberts, R.D.; will provide some temporal heterogeneity Janke, A.R.; King, N.G.; Tervit, R.H.; on the quality of food resources available. Webb, S.C. Application of sperm As a result of diet quality fluctuations not cryopreservation in selective breeding only membrane composition can be of the Pacific oyster, Crassostrea gigas affected and therefore influence (Thunberg). Aquaculture Research 39, cryopreservation survival but also embryos 2008, Pages 143-144. might have a different quality of reserves and therefore influence recovery and 3. Adams, S.L.; Smith, J.F.; Tervit, H.R.; growth post-thawing. Also, embryos were McGowan, L.T.; Roberts, R.D.; Janke, A.R.; King, N.G.; Gale, S.L.; Webb, 490

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