CryoLetters 30 (5), 373-381 (2009) © CryoLetters, [email protected]

EFFECTS OF CRYOPROTECTANT ON THE EMBRYOS OF BANDED CORAL ( HISPIDUS); PRELIMINARY STUDIES TO ESTABLISH FREEZING PROTOCOLS

S. Tsai and C. Lin*

National Museum of Marine Biology & Aquarium, 2 Houwan Road, Checheng, Pingtung, 944, Taiwan. *Correspondence author e-mail: [email protected]

Abstract The addition of cryoprotectants is a necessary step in cryopreservation procedures because they can minimize cellular injury during cryopreservation. Toxicity of cryoprotectant depends on the type, concentration, temperature and exposure period. The aim of this study was to investigate the toxicity of cryoprotectant to embryos of banded coral shrimp (Stenopus hispidus) in order to inform the development of a cryopreservation protocol. Three stages of embryonic development (eye-formation, heart beat and pre-hatch stage) embryos were selected and exposed to different concentration of cryoprotectants (0.25M-5M) for an equilibration period of 10, 20 or 30 min at room temperature. Hatching percentage indicated that the toxicity of tested cryoprotectants increased in the order of methanol, ethylene glycol (EG), dimethyl sulphoxide (DMSO), glycerol and dimethylacetamide (DMA). The No Observed Effect Concentrations (NOECs) for eye-formation stage embryos were 1M, 0.25M, 0.25M, 0.25M and 0.25M respectively after 10 min incubation whilst the NOECs for heart beat and pre-hatch stage embryos were 1M, 0.5M, 0.5M, 0.5M and 0.25M respectively. Pre-hatch stage embryos appeared to be more tolerant to cryoprotectant toxicity than eye-formation and heart beat stage embryos. Keywords: cryoprotectant, banded coral shrimp (Stenopus hispidus), embryo, methanol, dimethylacetamide (DMA)

373 INTRODUCTION

Banded coral shrimp (Stenopus hispidus) is one of the most popular species in the aquarium trade industry (20). S. hispidus is a reef-associated with a worldwide distribution. At present, most marine species marketed in the aquarium trade are collected from the wild, particularly from ecosystems, and the collection practices sometimes cause severe damage to the reefs. Availability of cryopreserved embryos would greatly help in minimizing wild collection whilst sustaining the aquarium industry and creating new employment opportunities. Research on cryopreservation of invertebrates has been attempted for a few species and techniques are still being developed. Earlier studies have reported successful cryopreservation in larvae (2, 10), crustancean embryo (9, 16) and molluscan embryos (18, 12 Optimum cryopreservation techniques for shrimp embryos require an understanding their response to cryoprotectants. The addition of cryoprotectants is a necessary step in cryopreservation procedures because they can minimize cellular injury during cryopreservation. However, cryoprotectants can cause cellular injury by osmotic trauma and many are toxic. Toxicity of cryoprotectant depends on the type, concentration,, temperature and exposure period (5, 17). Information on the relative toxicity of cryoprotectants is of key importance in protocol design. Limited studies have been done on relative toxicities of different cryoprotectants on aquatic invertebrate species (3, 9, 13, 15), and there is no any information on S. hispidus. This study set out to obtain baseline information regarding tolerance of different stages of banded coral shrimp embryos in response to cryoprotectants and to examine the effects of some commonly used cryoprotectants on survival of banded coral shrimp (S. hispidus) embryos. The commonly used cryoprotectants methanol, dimethyl sulphoxide (DMSO), ethylene glycol (EG), glycerol and dimethylacetamide (DMA) were used in this study. In the present study, late-stage embryos (eye-formation, heart beat and pre-hatch stage) were tested for their tolerance of the cryoprotectant toxicity. The prenauplius embryo stages were selected because it showed higher tolerance of handling stress and higher resistance to cryoprotectant toxicity and cooling compared with other embryonic stages (4).

MATERIALS AND METHODS

Adult male and female S. hispidus were purchased locally (collected from the wild 1-2 days prior to our purchase) and maintained in a recirculating seawater system under 14 h light : 10 h dark. Average weight and total body length of females (N = 20) were 4.5 ± 0.3 g and 45 ± 1.4 mm, respectively. One female and one male S. hispidus were kept in the same individual tanks (9 females and 9 males in total of nine tanks). They were fed twice a day, morning and evening, with frozen Antarctic krill at a rate of 5-10% body weight. Average values of water temperature, salinity and pH of the holding tanks were 22 ± 1.8°C, 35.2 ± 1.6 ppt and 8.0 ± 0.4, respectively. Females were then naturally mated and inseminated in a circular tank, spawning began within 15-25 min of copulation and lasts approximately 10 min. Females S. hispidus then deposited a blue-green egg mass on the swimmerets under the abdomen (20). Embryos were gently collected from the female

374 abdomen and kept in a 1 L beaker of seawater. Each female produced approximately 3,000 embryos

Embryo developmental stages After spawning, the embryos mass was removed from female abdomen and embryos were incubated in a plastic container. Embryos were collected at 192h (eye-formation stage), 240h (heart beat stage) and 288h (pre-hatch stage) after spawning respectively, and these three embryo development stages were used in cryoprotectant toxicity studies. Developmental stages were classified according to Zhang et al. (20).

Cryoprotectant toxicity The cryoprotectants used in the study were methanol, DMSO, EG, glycerol and DMA. All the chemicals were purchased from sigma chemicals (St. Louis, MO, USA). Solutions of cryoprotectants were made up in aged and filtered (0.4 µm) natural seawater (35 part per thousand) at a range of concentrations: 0.25M, 0.5M , 2M, 3M, 4M and 5M. 15-30 embryos were put into each well of a 6-well culture plate. The filtered natural seawater was then removed and 2 ml cryoprotectant solution was added. Embryos were incubated in cryoprotectant solutions for 10, 20 and 30 min at room temperature (22 °C). After incubation in cryoprotectants, embryos were washed twice with filtered natural seawater and allowed to hatch at room temperature in beakers (50 ml) supplied with aeration. Control embryos were allowed to hatch without the cryoprotectant treatment. Cryoprotectant toxicity was expressed as the percentage of hatched nauplii to total embryo counted.

Statistical analysis For all experiments, three replicates were used for each treatment and experiments were repeated at least three times. Statistical analysis was carried out using SPSS (Version 17). One-Sample Kolmogorov-Smirnov test was used to ensure the data was normally distributed. One-way ANOVA was then performed and homogeneous of variance was tested using Levene’s test (p > 0.05). Where differences were found Tukey’s post-hoc test was carried out in order to identify which groups differ. All data were expressed as mean ± SEM across the three replicates and p values of less than 0.05 were considered to be significant.

RESULTS

Effect of cryoprotectants and exposure time on hatching percentage of S. hispidus embryos The No Observed Effect Concentrations (NOECs) for methanol, EG, DMSO, glycerol and DMA for embryos at three different developmental stages after 10, 20 and 30 min exposure are shown in Table 1. The results showed that the toxicity of cryoprotectants to embryos generally increased with concentration and their hatching percentage decreased as duration of exposure increased. The toxicity of the tested cryoprotectants increased in the order of methanol, EG, DMSO, glycerol and DMA for eye-formation, heart beat and pre-hatch stage embryos. The NOECs for eye-formation

375 stage embryos were 1M, 0.25M, 0.25M, 0.25M and 0.25M respectively after 10 min incubation whilst the NOECs for heart beat and pre-hatch stage embryos were 1M, 0.5M, 0.5M, 0.5M and 0.25M respectively (Table 1). Methanol is the least toxic cryoprotectant to S. hispidus embryos. The hatching percentage for eye-formation, heart beat and pre-hatch stage embryos after treated in 1M methanol for 10 min at room temperature were 51.4 ± 4.9%, 53.6 ± 4.8% and 60.8 ± 4.3% respectively when compared with 56.5 ± 2.3%, 58.4 ± 2.7% and 65.4 ± 3.5% for their controls.

Table 1. No Observed Effect Concentrations (NOECs) of cryoprotectants for eye-formation, heart beat and pre-hatch stage S. hispidus embryos

Exposure time Eye-formation Cryoprotectant Heart beat stage Pre-hatch stage period (min) stage 10 1M 1M 1M Methanol 20 0.5M 0.5M 1M 30 0.25M 0.25M 0.5M

10 0.25M 0.5M 0.5M DMSO 20 0.25M 0.25M 0.5M 30 <0.25M 0.25M 0.25M

10 0.25M 0.5M 0.5M EG 20 0.25M 0.5M 0.5M 30 0.25M 0.25M 0.25M

10 0.25M 0.5M 0.5M Glycerol 20 0.25M 0.25M 0.5M 30 <0.25M <0.25M 0.25M

10 0.25M 0.25M 0.25M DMA 20 <0.25M 0.25M 0.25M 30 <0.25M <0.25M 0.25M

Hatching of pre-hatch stage embryos did not change significantly from the control with exposure time of 10-20 min to 0.5M DMSO, 0.5M EG and 0.5M glycerol with average value of 61.3 ± 2.6%. However, hatching percentage decreased significantly when exposed to 0.5M DMSO, 0.5M EG and 0.5M glycerol for 30min (average value 48.4 ± 3.3%) (p < 0.05). EG was moderately toxic. Hatching percentage for eye-formation, heart beat and pre-hatch stage embryos were significantly reduced to 27.5 ± 2.6%, 37.9 ± 3.2% and 48.0 ± 2.6% after 10 min exposure in 1M EG, when compared to the control groups with 56.5 ± 2.4%, 58.4 ± 2.7% and 65.4 ± 3.5%(p < 0.05). DMSO and glycerol showed similar toxicity to S. hispidus embryos. An increase in incubation period at each concentration resulted in a significant decline in hatching percentage

376 (Figure 1). Embryos of eye-formation, heart beat and pre-hatch stage exposed to 1M DMA for 10 min showed limited hatching percentage (13.6 ± 4.1%, 15.4 ± 4.4% and 23.0 ± 2.9%).

a

80

) Control

% a Methanol ( a a a a DMSO e a a a a a a DMA g 60 b EG a

t Glycerol

n b b e b

c b r 40 c e b p bc b g

n c i c c

h 20 c

c c

t c c

a b

H c c c 0 0.25M 0.5M 1M 2M 3M Concentration of CPAs b 80 Control ) Methanol

% a

( a a a a a DMSO a a a a a DMA e 60 a a a a

g EG

a Glycerol t n

e b c c r 40 b e

p bc

b

g b c n i b c h 20 c

c c t a

H c c c c 0 0.25M 0.5M 1M 2M 3M Concentration of CPAs c 80

) a a a a a a a Control % a a a a ( a a a a Methanol DMSO e DMA g 60 b

a b EG t Glycerol n b e b c

r 40 b b e c p b

g c c n

i c

h 20

c c t c c a c H 0 0.25M 0.5M 1M 2M 3M Concentration of CPAs Figure 1. Toxic effect of cryoprotectants on hatching percentage of embryos at different

377 stages. Eyes-formation (a), heart beat (b) and pre-hatch (c) stage embryos were exposed to up to 3M cryoprotectants for 10 min at room temperature. Hatching of eyes-forming, heart beat and pre-hatch embryos controls averaged 56.5 ± 2.3%, 58.4 ± 2.7% and 65.4 ± 3.5%. Error bars represent Standard Errors of the Mean. Different letters represent significant differences between cryoprotectants at the same concentration (p < 0.05).

Effect of embryos developmental stage on hatch rate Hatching percentage for embryos of three different embryonic stages after incubated in methanol, EG and DMSO are shown in Figure 2. There was no significant difference (p > 0.05) in hatching percentage for eye-formation, heart beat and pre-hatch stage embryos after treated in 1M methanol for 10 min at room temperature (Figure 2a). Eye-formation and heart beat stage embryos showed less resistance to cryoprotectant than pre-hatch stage embryos (Figure 2b & c). Hatching percentage for eye-formation, heart beat stage and pre-hatch stage embryos after exposure in 1M EG and 1M DMSO for 10 min at room temperature were 27.5 ± 2.6%, 37.9 ± 3.2% and 48.0 ± 2.6%, and 16.7 ± 2.5%, 21.1 ± 2.9% and 32.2 ± 3.8%, respectively. The results also showed that hatching percentage for pre-hatch stage embryos exposed to all cryoprotectants at 0.5M for 10 min were 49.2% to 61.1%, not significantly different from that of the control (65.4 ± 3.5%) (p > 0.05).

a

80 ) a Eye-forming

% a

( a

a a Heart beating a e a a a a Pre-hatching

g 60 a a a t

n a e

c a r 40 a a e a p

a g n i 20 h c t a

H 0 Control 0.25M 0.5M 1M 2M 3M Concentration of methanol

378 b

80 ) a Eye-formation %

( Heart beat a a b e a a a b Pre-hatch

g 60

a c t

n a b e c

r 40 e a b p

g ab n i 20

h a c t a a a a H 0 Control 0.25M 0.5M 1M 2M 3M Concentration of EG c

80 ) a Eye-formation % ( a a a b Heart beat e a a Pre-hatch g 60 b a t n

e a c r 40 b e p a b g a n i 20 ab h a b c t

a a a H 0 Control 0.25M 0.5M 1M 2M 3M Concentration of DMSO

Figure 2. Effect of methanol (a), EG (b) and DMSO (c) toxicity to hatching percentage of embryos at different stages. Embryos were exposed to methanol, EG and DMSO for 10 min at room temperature (22 °C). Control embryos were incubated in filtered natural seawater under the same conditions. Error bars represent Standard Errors of the Mean. Different letters represent significant differences between developmental stages at the same concentration (p < 0.05).

DISCUSSION

Cryoprotectant toxicity The results obtained in the present study showed that the toxicity of cryoprotectants increase in the order of methanol, EG, DMSO, glycerol and DMA. Methanol is the least toxic cryoprotectant to S. hispidus embryos when compared with other tested

379 cryoprotectants, because of its low osmolality and high permeability (21). The importance of developing methods to lower cryoprotectant toxicity to achieve effective levels of cryoprotectant was recognized in penaeid embryos by Robertson and Lawrence (14). Several previous studies have also shown methanol to be the least toxic cryoprotectant to (1, 7, 11). Studies showed that methanol proved to be relatively nontoxic to morula, blastocele, prenauplius, nauplii and zoea of P. japonicus, when compared to DMSO, glycerol, EG and PG (7). Preston and Coman (11) reported that methanol was the least toxic cryoprotectant to embryos of P. esculentus, as compared to EG and DMSO. Alfaro et al. (1) also found that methanol is not toxic to embryos of T. Byrdi after exposure in 2M methanol at 12 °C. In this study, EG, DMSO, glycerol and DMA were considered to be more toxic cryoprotectants to S. hispidus embryos than methanol. EG has been successfully used for the cryopreservation of embryos and larvae of marine shrimp species (9). In the present study, EG toxicity was similar to that found with embryos of P. monodon reported by Vuthiphandchai et al. (19). DMSO has been successful used as an appropriate cryoprotectant for cryopreservation of gamete and embryos in a number of vertebrate and invertebrate species. However, Gwo and Lin (7) reported that DMSO was one of most toxic cryoprotectant to embryos, nauplii and zoea of P. japonicus. Glycerol has also been reported to be a toxic cryoprotectants for embryos of Pacific oyster (Crassostrea gigas)(6), embryos of P. indicus (9, 16), embryos of Trachypenaeus byrdi (1) and embryo, nauplii and zoea of P. japonicus (7). DMA was first used for sperm by McNiven et al. (8) who demonstrated the usefulness of DMA as a cryoprotectant for rainbow trout spermatozoa. The results obtained from the present study identified DMA as the most toxic cryoprotectant for S. hispidus embryos.

Effect of embryos developmental stage on hatch rate The results obtained from present study showed tolerance of S. hispidus embryos to cryoprotectants toxicity increase with embryonic development. The resistance of late stage embryos to higher cryorotectant concentrations and longer equilibration time over early stage embryos has also been reported in P. indicus (9, 16), P. japonicus (7) and P. monodon (19). The higher tolerance of cryoprotectant toxicity at later embryonic developmental stages appeared to correspond with the limited success of the cropreservtion at these stages in penaeid shrimp (4). Dre to physiological changes associated with the development, it is expected that later stage embryos had higher tolerance to cryoprotectants than the early stages embryos. In the present study, pre-hatch stage embryos were relatively less sensitive to an increase in cryoprotectant concentration than eye-formation, heart beat stage embryos. The better tolerance to cryoprotectants of later stage embryos is probably due to a lower penetration of cryoprotantants and an increase in their complex structure affecting cryoprotectant permeation. Whether high resistance of the toxicity of cryoprotectant will lead to better results in cryopreservation remains unknown. Further studies are required to understand the permeability of shrimp embryo to overcome the limited success of cryopreservation.

380 Acknowledgments The authors wish to express their deepest appreciation to Prof. David Rawson and Prof. Tiantian Zhang, Institute of Research in the Applied Natural Sciences (LRANS), University of Bedfordshire, UK. for valuable comments and suggestions on the manuscript.

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