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Preliminary studies on cryopreservation of Sydney rock oyster (Saccostrea glomerata) larvae.

ABSTRACT In this study several parameters critical to the success of cryopreserving Sydney rock oyster (Saccostrea glomerata) larvae were investigated. They were: (1) cryoprotectants (10% dimethyl sulfoxide and 10% propylene glycol), (2) freezing protocols (with or without the seeding step), (3) larval concentrations (1,000, 3,000, 5,000, 10,000 and 30,000 individuals [mL.sup.-1]), and (4) larval ages (6, 12, 24, 48, and 96 h old). The survival rates were determined as percentages of postthaw larvae performing active movements for the 6 and 12 h larvae or active cilia movement for the 24, 48, and 96 h larvae. Analyses showed that the difference in survival rates between different age classes was significant in all the experiments conducted, with the maximum survival rate being achieved in the 24-h-old larvae. When the freezing protocol without the seeding step was applied in 24-h-old larvae the postthaw survival tares of larvae cryopreserved with 10% dimethyl sulfoxide (93.1 [+ or -] 0.2%) were significantly higher (P < 0.001) than those with 10% propylene glycol (81.5 [+ or -] 0.4%). Differences in postthaw survival rates between different concentrations (1,000--30,000 individuals [mL.sup.-1]) were not significant within each of the three larval age classes (6-, 12-, and 24-hold) used.

KEY WORDS: cryopreservation, Saccostrea glomerata, larvae, DMSO, PG, oyster


The cryopreservation of gametes and embryos has been studied for nearly six decades since Polge et al. (1949) reported the first effective sperm cryopreservation in 1949. Now cryopreservation techniques are widely applied in hospitals, livestock breeding, and conservation of endangered species. So far researches on cryopreservation of aquatic animals have mainly focused on finfish and some shellfish species (Chao & Liao 2001).

Studies on molluscan cryopreservation started in 1970s with oysters (Lannan 1971, Hughes 1973). Since then about 30 papers have been published (Hughes 1973, Zell et al. 1979, Yankson & Moyse 1991, Gwo 1995, Naidenko 1997, Chao et al. 1997, Paniagua-Chavez et al. 1998a, Paniagua-Chavez et al. 1998b, Paniagua-Chavez & Tiersch 2001, Smith et al. 2001), with primary focuses on spermatozoa and very few reports on eggs, embryos, and larvae (Brougrier & Rabenomanana 1986, Usuki et al. 1997, Gwo 2000, Paniagua-Chavez & Tiersch 2001, Tervit et al. 2005). Oysters are the only molluscan group that has been intensively investigated. Spermatozoa from four oyster species, Crassostrea virginica, C. tulipa, C. iredalei, and C. gigas have been successfully cryopreserved with different methods. Cryopreservations on eggs, embryos, and larvae have been tried in C. virginica and C. gigas with varying degrees of success (Renard 1991, Chao et al. 1994, Chao et al. 1997, Chao & Liao 2001, Gwo 1995, Naidenko 1997, Paniagua-Chavez et al. 1998, Lin et al. 1999, Paniagua-Chavez & Tiersch 2001, Smith et al. 2001, Tervit et al. 2005) and revealed that procedures and parameters were species specific and should be optimized prior to their application in a new species (Gwo 1995, Chao et al. 1997).

The Sydney rock oyster, Saccostrea glomerata (Gould 1850) is the most important commercial molluscan species cultured in estuaries along the eastern coasts of Australia (Nell 1993). Recently, its production reduced substantially because of the outbreak of the QX disease in some areas. A selective breeding program has therefore been established to improve their resistance to this disease and growth rates at the same time. It is anticipated that the development of cryopreservation techniques would further enhance the capacity and efficiency of the program. In this study the effects of cryoprotectants, freezing protocols, larval concentrations, and larval ages on postthaw survival rates were investigated.


Larval Preparation

The Sydney rock oyster, S. glomerata was purchased from a seafood market in Adelaide, South Australia, where they were originally imported from New South Wales, Australia. The oysters were then acclimatized at 22[degrees]C in a 90-L bucket for at least three days before being used in experiments. Gametes were collected by strip-spawning method and their quality was evaluated under the microscope. Males with more than 80% active sperm and females with healthy looking eggs were chosen. Eggs from 5 individuals were pooled in a 250-mL beaker and sperm from at least 3 individuals were pooled in a 50-mL beaker containing filtered seawater. To separate debris from gametes eggs were washed through a 70-[micro]m screen and collected on a 15- [micro]m screen, whereas sperm was filtered through a 15-[micro]m screen. The fertilization was conducted in a 2-L beaker at an egg:sperm ratio of approximate 1:10 to avoid polyspermism. At 20 min postfertilization the fertilized eggs were washed on a 15-[micro]m screen and then stored in 50-L tanks at a concentration of approximate 20 individuals per milliliter. The concentration reduced to 10 individuals per milliliter after the water change at 24 h postfertilization and remained at this level until 96 h. During this period the water was maintained at 28[degrees]C [+ or -] 1[degrees]C and changed every day. The larvae were fed with an algal mixture of Isochrysis sp. and Pavlova sp. once a day after water change. The larvae used in the experiments were collected on a 20-[micro]m screen first and then washed into a 10-mL tube, where their density was counted and diluted to a level twice as high as the final concentration required in the experiment. In this study the trochophore and D-larvae stages appeared, respectively, at 12 and 24 h postfertilization.

The seawater used at the fertilization and larval rearing stages was filtered with a 1-[micro]m cartridge and was approximately 37 [per thousand] in salinity.

Cryopreservation Procedures

The dimethyl sulfoxide (DMSO) and propylene glycol (PG) used in this study as cryoprotectants were purchased from Sigma-Aldrich Pty Ltd. Their stock solutions (20%, v/v) were prepared freshly in 0.45 [micro]m filtered seawater on the day the experiment was conducted. The stock solution was then mixed with the same volume of larval suspension, resulting in a larvae-cryoprotectant mixture with 10% cryoprotectant and a larval concentration required for the experiment. The mixture was gently stirred by hand and transferred into three 0.5-mL French plastic straws with a pipette. A quantity of 0.25 mL per straw was used to minimize the potential heat effect on larvae from sealing the straw end with hot forceps. After equilibrated at the room temperature (22[degrees]C [+ or -] 1[degrees]C) for 20 min the straws were cooled in a programmable Freeze Control CL863 (Cryologic P/L, Australia) using a freezing protocol either with or without the seeding step (Table 1). Straws frozen with the seeding step were held at -12[degrees]C for 5 min. During this period, if ice formation had not occurred in a straw it would be touched with a liquid nitrogen cooled cotton bud for seeding. When the freezing steps were completed the straws were quickly plunged into liquid nitrogen (-196[degrees]C).

After at least 24 h storage in liquid nitrogen the straws were thawed in a 30[degrees]C water bath for 10-15 s. The straw content was released into a beaker containing 50 mL fresh seawater. The thawed larvae were then evaluated under the invert microscopy 1-2 h later. A 6- or 12-h-old larva was considered alive when it showed rotary motion with cilia and a larva of 24 h old or older was recorded as alive when it showed active cilia movements. For each straw at least 100 individuals were counted and the postthaw survival rate was calculated by dividing the number of alive individuals with the total individuals counted. In this study three experiments were conducted. Each experiment was replicated three rimes with three straws.

Effects of Cryoprotectants and Freezing Protocols on Postthaw Survival Rates

In this experiment the combination effects of 10% DMSO or 10% PG with a freezing protocol with or without the seeding step were investigated using 12- and 24-h-old larvae at the final concentration of 1,000 individuals [mL.sup.-1].

Effects of Larval Ages on Postthaw Survival Rates

Differences in postthaw survival rates between larvae of different ages (12, 24, 48, and 96 h old) were assessed using the freezing protocol with the seeding step and 10% DMSO or 10% PG. The final larval concentration used in this experiment was 1,000 individuals [mL.sup.-1] for all age classes.

Effects of Larval Concentrations on Postthaw Survival Rates

The concentrations assessed in this study were 1,000, 3,000, 5,000, 10,000, and 30,000 individuals [mL.sup.-1] for 6, 12, and 24 h old larvae, respectively. The freezing protocol with the seeding step and 10% DMSO were used.

Data Analysis

The postthaw survival rates (percentages) were arcsine square-root transformed and analyzed using SAS 9.1 software. Three-way ANOVA with a repeated measures option was applied to assess the effect of freezing protocols, cryoprotectants and larval ages in the experiment "Effects of cryoprotectants and freezing protocols on post-thaw survival rates." Two-way ANOVA with a repeated measures option was used to investigate the effect of larval ages and cryoprotectants in the experiment on "Effects of larval ages on post-thaw survival rates." Two-way ANOVA with a repeated measures option was also used to study the effect of larval concentrations and larval ages in the experiment on "Effects of larval concentrations on postthaw survival rates." The repeated measures option was selected because the three replicates used in each experiment were not independent from each other; they were from the larvae produced from the same batches of pooled gametes. Differences were considered statistically significant at P values less than 0.05.


Effects of Cryoprotectants and Freezing Protocols on Postthaw Survival Rates

Analyses showed that when the freezing protocol with the seeding step was used the effects of different cryoprotectants on postthaw survival rates were not significant (P = 0.567) (Fig. lA), whereas the differences between the two age classes were significant (P < 0.001), with postthaw survival rates being approximately 45% for 12-h-old larvae and 90% for 24-h-old larvae. When the freezing protocol without the seeding step was applied, the postthaw survival rates were affected significantly by larval ages and cryoprotectants used. The 24-h-old larvae resulted in significantly higher survival rates than the 12-h-old larvae (P < 0.001). The survival rates of 24-h-old-larvae cryo-preserved with 10% DMSO (93.1 [+ or -] 0.2%) were significantly higher (P< 0.001) than those with 10% PG (81.5 [+ or -] 0.4%, Fig. 1B).

Effects of Larval Ages on Postthaw Survival Rates

This experiment showed that the postthaw survival rates were affected significantly by the ages of larvae used (P < 0.001), but were not significantly affected by the types of cryoprotectants used (P = 0.762, Fig. 2). The postthaw survival rates increased with the development of larvae, from approximately 45 % for 12-h larvae to 88% for 24-h larvae. The rates decreased when larvae became older, down to approximately 65% for 96-h-old larvae.


Effects of Larval Concentrations on Postthaw Survival Rates

When the freezing protocol with the seeding step and 10% DMSO were used in the cryopreservation, the postthaw survival rates were not affected significantly (P = 0.114) by the larval concentrations within each age classes (6-, 12-, or 24-h old) used in this experiment (Fig. 3). The effects of age classes on postthaw survival rates were, however, significant (P < 0.001), with the survival rates of 24-h-old larvae being significantly higher than those in the other two age classes. Two straws with 30,000 individuals [mL.sup.-1] 12-h-old larvae were lost at the thawing stage by accident, only the survival rate from one replicate (straw) was therefore used in the analysis and provided in Figure 3.


DMSO and PG have successfully been used in cryopreservations of embryos and trochophore larvae in C. gigas and C. virginica (Naidenko 1997, Chao et al. 1997, Paniagua-Chavez et al. 1998, Paniagua-Chavez & Tiersch 2001). A postthaw survival rate of approximately 75% was obtained in C. gigas with 2M DMSO using freezing protocols with the seeding step (Chao et al. 1997, Lin et al. 1999) and as high as about 100% was reported in C. virginica (Paniagua-Chavez & Tiersch 2001) using freezing protocols without the seeding step and 10-15% PG solution. In this study freezing protocols with or without the seeding step produced similar postthaw survival rates (~48%) in 12-h-old larvae when 10% DMSO or 10% PG was used. In 24-h-old larvae, however, the larvae treated with 10% PG and frozen with the freezing protocol without the seeding step produced significantly lower postthaw survival rates (~81%, Fig. 1B) than those with other treatments (~91%, Fig. 1).


In molluscs, larvae prior to the formation of the larval shell (D-larvae) are typically selected as experimental material in cryopreservation studies (Naidenko 1997, Chao et al. 1997, Lin et al. 1999, Paniagua-Chavez et al. 1998, Paniagua-Chavez & Tiersch 2001). In C. gigas Gwo (1995) found that during this period the stage of larval development appeared to be a critical factor affecting postthaw survivals; the older larvae (trochophore stage) were more resistant to cryoprotectant exposure and produced a better survival rate after freezing than the younger ones. Choi and Chang (2003) extended the evaluation to the more advanced larval developmental stages in the pearl oyster Pinctadafucata martensii and discovered that prior to the umbonal stage, postthaw survival rates increased as the larvae developed, with the best survival rate being as high as 91% at late D-larval stage, followed by a decrease in survival with age. A similar trend was also observed in this study on Sydney rock oysters. However, the best postthaw survival rate was found at the early D-larval stage (24 h post fertilization at 28[degrees]C). The survival rate then decreased when older larvae were used. The possible reasons for this decline include that after 24 h postfertilization the larvae's capability to protect themselves from environmental changes has been enhanced substantially with the development of the second larval shell (prodissoconch II) and larval muscles. When larvae are stimulated by changes in external factors such as the exposure to cryoprotectants in cryopreservation studies, the animals withdraw their soft body into the shells immediately to prevent themselves from further exposure to these chemicals. This could result in the cryoprotectant concentration in their tissue being not high enough to protect them from the effects of freezing during cryopreservation. The other possibility for the decrease in the postthaw survival rates of older larvae could be caused by the additional differentiation of larval organs after the D-larval stage. Some organs might be more sensitive to cryo-injuries than others. Injuries of a certain organ could cause the death of the organ and result in the death of the whole organism.


In Eastern oysters C. virginica Paniagua-Chavez and Tiersch (2001) reported that the postthaw survival rate of trochophore larvae (12 h postfertilization at 25[degrees]C) was affected by the larval concentration used during cryopreservation, and decreased as the larval concentration increased, from about 100% at 10 individuals [mL.sup.-1] to less than 40% at 100 individuals [mL.sup.-1] and further decreased to less 10% when larval concentration increased to 10,000 individuals [mL.sup.-1]. A freezing protocol without the seeding step and a freezing rate of 2.5[degrees]C per min were adopted in their study. In this study, effects of larval concentrations on postthaw survival rates were assessed at 5 levels (from 1,000-30,000 individuals [mL.sup.-l]) using a freezing protocol with the seeding step. Results showed that within each age class the postthaw survival rates were not affected significantly by the larval concentrations used, being approximately 44%, 48%, and 95% for 6-, 12-, and 24-h-old larvae, respectively. In addition, the postthaw survival rates of trochophore larvae (12 h old) frozen at the concentrations of 1,000 and 10,000 individuals [mL.sup.-1] in this study (~48%) were much higher than those reported by PaniaguaChavez and Tiersch in 2001 for Eastern oysters (<30% at 1,000 individuals [mL.sup.-1] and <10% at 10,000 individual [mL.sup.-1]). The discrepancy between these two studies at these two larval concentrations might be because of differences in species and/ or freezing protocols used. The difference in cryo-tube sizes used in these two studies might be another critical contributing factor. Liquid in 0.5-mL French straws used in this study could be cooled (and thawed) more evenly than that in 5-mL macrotubes used in the Paniagua-Chavez and Tiersch's experiments. This remains to be investigated.

Sydney rock oysters are endemic to eastern costs of Australia and are found in bays, inlets and sheltered estuaries. This species does not inhabit South Australian waters. Therefore, further incubation of thawed larvae was not attempted in this study. However, the results from this study highlight the potential to develop a practical larval cryopreservation technique(s) in this species.


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(1) Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; (2) South Australian Research and Development Institute, 2 Hamra Avenue, West Beach, SA 5024, Australia

* Corresponding author:
Two freezing protocols used to cryopreserve the Sydney rock
oyster (S. glomerata) embryos and larvae.

     Freezing Protocol with            Freezing Protocol without
          Seeding Step                       Seeding Step

Initial temperature 21[degrees]C   Initial temperature 21[degrees]C
  -7[degrees]C/min [down arrow]      -7[degrees]C/min [down arrow]
    0[degrees]C                        0[degrees]C
  -1[degrees]C/min [down arrow]              [down arrow]
    -12[degrees]C                  Holding for 15 min
          [down arrow]               -2.5[degrees]C/min [down arrow]
Seeding and holding for 5 min          -36[degrees]C
  -2[degrees]C/min [down arrow]              [down arrow]
    -36[degrees]C                  Holding for 10 min
          [down arrow]                       [down arrow]
Holding for 10 min                 Liquid nitrogen
          [down arrow]
Liquid nitrogen
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Article Details
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Author:Liu, Baozhong; Li, Xiaoxu
Publication:Journal of Shellfish Research
Article Type:Abstract
Geographic Code:8AUST
Date:Dec 1, 2008
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