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Factors Affecting Survival of Cryopreserved Oyster (Crassostrea gigas) Embryos
Cryobiology 39, 192–196 (1999) Article ID cryo.1999.2196, available online at http://www.idealibrary.com on
BRIEF COMMUNICATION Factors Affecting Survival of Cryopreserved Oyster (Crassostrea gigas) Embryos Ta-Te Lin,* Nai-Hsien Chao,† and Hsiao-Tsuei Tung* *Department of Agricultural Machinery Engineering, National Taiwan University, Taipei, Taiwan, Republic of China; and †Department of Aquaculture, Taiwan Fisheries Research Institute, Keelung, Taiwan, Republic of China A conventional two-step freezing procedure was developed and optimized in order to cryopreserve oyster embryos. The effects of cooling rate, choice of cryoprotectant, and seeding temperature on the survival of late-stage oyster embryos were examined. When these factors were optimized, improved survival rates of 78 6 8 and 83 6 7% were achieved using 2 M Me 2SO or glycerol, respectively, as the cryoprotectant. The experimental results indicate that oyster embryos survive after freezing over a broad range of cooling rates ranging from 20.5 to 216°C/min. Me 2SO, glycerol, propylene glycol, and ethylene glycol may be used as cryoprotectants for the cryopreservation of oyster embryos. © 1999 Academic Press Key Words: oyster embryo; cryoprotectant; cryopreservation; cooling rate; bivalve.
Recent progress in the cryopreservation of shellfish embryos has shed light on the feasibility of this technique for establishing gene banks and manipulating spawning programs (2, 3, 5, 7–9, 12). However, in order to improve cryopreservation protocols to obtain higher survival rates for oyster embryos following freezing, a systematic study of the factors affecting the survival of cryopreserved embryos is required. In the pursuit of optimal cryopreservation protocols for oyster embryos, we have previously investigated the toxicity tolerance of selected cryoprotectants to oyster embryos (1). The apparent toxicity of cryoprotectants is dependent on the type and concentration of cryoprotectant, the equilibration time, and the temperature during loading. The stage of embryonic development is also a critical factor and it was found that later stages of oyster embryos were more tolerant. Intracellular ice formation (IIF) was also examined in oyster embryos and eggs (6). The IIF process in oyster eggs and embryos was found to be time and temperature dependent. Received May 10, 1999; accepted August 2, 1999. This work was supported by the National Science Council, R.O.C. under Grant No. NSC 83-0409-B-002-096 and No. NSC 84-2312-B-002-083.
0011-2240/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
The probability of IIF increased directly with time and inversely with temperature. This information is useful in designing practical cryopreservation procedures and saves effort in optimizing procedures experimentally. We have previously reported a successful cryopreservation protocol for oyster embryos using Me 2SO as the cryoprotectant (5); a survival rate of 68 6 18% of cryopreserved embryos was obtained. This study is focused mainly on a comparison of procedural factors affecting the survival of cryopreserved oyster embryos in order to improve the cryopreservation procedure. The cryopreservation protocol was examined stepwise and optimized by modifying cooling rates and seeding temperatures. The feasibility of using different cryoprotectants was also explored. Oyster (Crassostrea gigas) sperm and eggs were collected from male and female oysters and incubated in natural sea water (34 6 2‰ salinity). The sex and maturity of individual oysters was first determined by observing the dissected gonad material under a light microscope. Gametes from four or more oysters of the same sex were then pooled in separate beakers. Eggs were artificially inseminated 1 h after their mechanical removal from the ovary. When
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newly fertilized eggs were examined under a microscope, the suggested number of sperm surrounding the egg to assure ideal fertilization was approximately 5 6 1. The 1-h incubating period in sea water was found to significantly reduce the percentage of polyspermic eggs (11). The fertilized eggs were washed and filtered to remove excess spermatozoa. The concentration of fertilized eggs was adjusted to about 1000/ml for further incubation. Embryos were obtained from the inseminated oocytes 4 h after fertilization in sea water at 28°C, when the ciliated blastula embryos start to exhibit rotary motion. It is generally observed that with good quality oyster gametes, more than 90% of the resulting embryos are normal using this procedure. Experiments were designed to compare the effects of seeding temperature, cooling rates, and the use of different cryoprotectants on the survival of cryopreserved embryos. Conventional two-step freezing experiments were conducted using a programmable freezer (KRYO 10 Series III; Planer Biomed, UK). Ciliated blastula embryos were equilibrated with 2 M Me 2SO (or other cryoprotectants) in sea water for 10 min at 25°C and were then cooled at 21°C/min from 25 to 212°C (or different seeding temperatures). Next, 0.5-ml straws containing thousands of embryos were held at 212°C for 5 min to allow equilibration after seeding with precooled tweezers. Embryos were then cooled slowly at 22.0°C/min (or at different cooling rates) to 235°C and equilibrated for 5 min before quenching in liquid nitrogen. For most experiments in this study, embryos were held in liquid nitrogen for 1 h before thawing. Thawing was carried out in a water bath at 28°C. Immediately after thawing, embryos were transferred to sea water to unload the cryoprotectant. Figure 1 is a flow chart that illustrates the sequence of events in the optimized twostep freezing protocol for oyster embryos using Me 2SO as a cryoprotectant. For all freezing experiments, at least three straws were used as replicates for each treatment. The surviving embryos were assayed by counting the embryos showing active rotary
193
FIG. 1. Flow chart of the optimized two-step cryopreservation protocol for oyster embryos using 2 M Me 2SO as a cryoprotectant.
motion under a light microscope. The presence of surviving embryos was determined 1–2 h after thawing. The survival rate for each treatment was normalized by comparing the survival rate of the thawed embryos to that of the control. With the cryopreservation protocol described in Fig. 1, a survival rate of 78 6 8% was obtained for oyster embryos quenched in liquid nitrogen. The reduction of survival was minimal during the cryoprotectant loading step. There was a decrease in survival rate (from 94 6 4 to 86 6 4%) during the freezing process from 212 to 235°C and further during the quenching process (from 86 6 4 to 78 6 8%). In a conventional two-step freezing method, samples are initially cooled to a temperature equal to or lower than the freezing point of the suspension solution before freezing at a con-
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FIG. 2. Effect of seeding temperature on the survival of cryopreserved oyster embryos. Me 2SO was used as a cryoprotectant. The cooling rate was 22°C/min.
stant cooling rate. Supercooling of the solution usually occurs and manual seeding is necessary. This procedure can be accomplished by gripping the straw with a pair of tweezers previously quenched in liquid nitrogen. The low temperature induces the nucleation of the supercooled solution in the straw and the ice propagates until the solution is solidified. In this study, the seeding temperature was also examined with experiments in the protocol optimization process. Figure 2 shows the effect of seeding temperature on the survival of cryopreserved oyster embryos when Me 2SO was used as the cryoprotectant. The cooling rate in these experiments was 22°C/min. In the temperature range from 210 to 216°C, seeding at 212°C yielded the best result. The survival rate of embryos frozen to 235°C was compared with those quenched in liquid nitrogen. Warmer seeding temperatures resulted in the melting of the ice induced by the seeding procedure and the solution in the straw was found to remain supercooled. In effect this defers the nucleation of the solution to a lower temperature during constant-rate freezing. In contrast, seeding at lower temperatures, such as 216°C, resulted in rapid propagation of the ice front in the solution during the solidification process. Following seeding or at the end of the constant-rate freezing (at 235°C), it is beneficial to maintain isothermal conditions for a certain length of time to allow for intracellular and extracellular osmotic
equilibration. However, some of our experiments (unpublished data) indicated that prolonging the holding time more than necessary increased the probability of intracellular ice formation and thus decreased the overall survival rate (10). The most critical factor to be considered when optimizing the cryopreservation protocol is cooling rate. Figure 3 shows the effect of cooling rate on the survival of cryopreserved oyster embryos loaded with 2 M Me 2SO or 2 M glycerol. Cooling rates ranging from 20.5 to 216°C/min were examined. The optimum cooling rate, which gave the highest survival rate in this series of experiments, differed in experiments that used Me 2SO or glycerol as cryoprotectants. The optimum cooling rate was 22°C/ min for embryos treated with Me 2SO and 24°C/min for glycerol. It is worth mentioning that with 2 M glycerol as a cryoprotectant, a high survival rate of 83 6 7% was obtained at a cooling rate of 24°C/min. This experiment also showed that various levels of survival rate were obtained over a relatively broad range of cooling rates. This result also agrees with the broad range of cooling rates (20.5 to 215°C/ min) previously reported from various research groups in cryopreservation procedures used for oyster embryos (2, 3, 8, 9). To compare the feasibility of using different cryoprotectants for oyster embryo cryopreservation, separate experiments were performed at
FIG. 3. Effect of cooling rate on the survival of cryopreserved oyster embryos. Me 2SO and glycerol were used as cryoprotectants.
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FIG. 4. Comparison of the effect of four different cryoprotectants on the survival of cryopreserved oyster embryos. The cooling rate was 22°C/min.
a cooling rate of 22°C/min. Figure 4 compares the effect of using 2 M Me 2SO, glycerol, ethylene glycol (EG), or propylene glycol (PG) as the cryoprotectant. For the cryoprotectant loading step, there were no significant differences between the selected cryoprotectants. For all the cryoprotectants, direct loading of the cryoprotectant at a concentration of 2 M caused minimal loss of oyster embryos. The survival rates of oyster embryos subjected to constant-rate freezing and quenching in liquid nitrogen were similar whether Me 2SO, EG, or glycerol were used as cryoprotectants. However, the survival rate of cryopreserved oyster embryos using PG as the cryoprotectant was significantly (P , 0.01) lower than those for Me 2SO, EG, and glycerol. The viability of oyster embryos after freezing is a complex function of embryonic characteristics and the operating parameters of the cryopreservation protocol. The aim of the present study was to examine the effect of major factors affecting the survival of cryopreserved embryos and thereafter to apply this information to optimize the cryopreservation protocol. Our results show that high survival rates (78 6 8% with Me 2SO at 22°C/min; 83 6 7% with glycerol at 24°C/min) of cryopreserved oyster embryos can be achieved using the optimized protocol. Our results also reveal that cryopreserved oyster embryos can survive a relatively broad range of
cryopreservation parameters, such as cooling rate and the selection of cryoprotectants. It should be noted that, similar to mammalian embryos, the survival rate of cryopreserved oyster embryos is highly dependent on the quality of gametes (4). Although a rate of more than 90% of embryos being normal can be regularly obtained from artificial fertilization procedures in our laboratory, a low fertilization rate occasionally occurred due to poor quality of oyster gametes. As a general trend, the normalized survival rate of cryopreserved embryos is usually low for experiments using batches of embryos with a low fertilization rate. Therefore, in this study the cryopreservation experiments were performed only when the fertilization rate of oyster embryos was above 90% in experimental controls. In this study, we did not attempt to incubate the thawed embryos continuously throughout the settlement. The survival of cryopreserved embryos was assayed 1–2 h after thawing and was based on the motility criterion. However, in a separate set of experiments using glycerol as a cryoprotectant and a cooling rate of 24°C/min, 32 6 12% of thawed embryos and 55 6 12% of untreated embryos developed to D-larvae stage. For hatchery and aquacultural applications, development of larvae through settlement is of considerable importance. Paniagua-Chavez et al. (9) first reported a successful production of seed oysters from cryopreserved oyster larvae. Their results confirmed the feasibility of practical applications of oyster embryo cryopreservation. In combination with the results from this study, the future development of optimized cryopreservation protocols for the complete life cycle of the oyster for use in hatcheries and other practical purposes can be anticipated. REFERENCES 1. Chao, N. H., Chiang, C. P., Hsu, H. W., Tsai, C. T., and Lin, T. T. Toxicity tolerance of oyster embryos to selected cryoprotectants. Aquatic Living Resources 7, 99 –104 (1994). 2. Chao, N. H., Lin, T. T., Chen, Y. L., Hsu, H. W., and Liao, I. C. Cryopreservation of early larvae and embryos in oyster and hard clam. Aquaculture 155, 31– 44 (1997).
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3. Gwo, J. C. Cryopreservation of oyster (Crassostrea gigas) embryos. Theriogenology 43, 1163–1174 (1995). 4. Han, Y. M., Yamashina, H., Koyama, N., Lee, K. K., and Fukui, Y. Effects of quality and developmental stage on the survival of IVF-derived bovine blastocysts cultured in vitro after freezing and thawing. Theriogenology 42, 645– 654 (1994). 5. Lin, T. T., Tung, H. T., and Chao, N. H. Cryopreservation of oyster embryos with conventional freezing procedure and vitrification. Cryobiology 30, 614 (1993). [Abstract] 6. Lin, T. T., and Lung, K. IIF characteristics of oyster embryos and eggs determined by a feedback controlled directional cryomicroscope. Cryobiology 32, 566 (1995). [Abstract] 7. McFadzen, I. R. B. Growth and survival of cryopreserved oyster and clam larvae along a pollution gradient in the German Bight. Mar. Ecol. Prog. Ser. 91, 215–220 (1992).
8. Naidenko, T. Cryopreservation of Crassostrea gigas oocytes, embryos and larvae using antioxidant echinochrome A and antifreeze protein AFP 1. CryoLett. 18, 375–382 (1997). 9. Paniagua-Chavez, C. G., Buchman, J. T., Supan, J. E., and Tiersch, T. R. Settlement and growth of Eastern oysters produced from cryopreserved larvae. CryoLett. 19, 283–292 (1998). 10. Pitt, R. E., Myers, S. P., Lin, T. T., and Steponkus, P. L. Subfreezing volumetric behavior and stochastic modeling of intracellular ice formation in Drosophila melanogaster embryos. Cryobiology 28, 72– 86 (1991). 11. Stephano, J. L., and Gould, M. Avoiding polyspermy in the oyster (Crassostrea gigas). Aquaculture 73, 295–307 (1988). 12. Toledo, J. D., Kurokura, H., and Kasahara, S. Preliminary studies on the cryopreservation of blue mussel embryos. Nippon Suisan Gakkaishi 55, 1661 (1989).
BRIEF COMMUNICATION Factors Affecting Survival of Cryopreserved Oyster (Crassostrea gigas) Embryos Ta-Te Lin,* Nai-Hsien Chao,† and Hsiao-Tsuei Tung* *Department of Agricultural Machinery Engineering, National Taiwan University, Taipei, Taiwan, Republic of China; and †Department of Aquaculture, Taiwan Fisheries Research Institute, Keelung, Taiwan, Republic of China A conventional two-step freezing procedure was developed and optimized in order to cryopreserve oyster embryos. The effects of cooling rate, choice of cryoprotectant, and seeding temperature on the survival of late-stage oyster embryos were examined. When these factors were optimized, improved survival rates of 78 6 8 and 83 6 7% were achieved using 2 M Me 2SO or glycerol, respectively, as the cryoprotectant. The experimental results indicate that oyster embryos survive after freezing over a broad range of cooling rates ranging from 20.5 to 216°C/min. Me 2SO, glycerol, propylene glycol, and ethylene glycol may be used as cryoprotectants for the cryopreservation of oyster embryos. © 1999 Academic Press Key Words: oyster embryo; cryoprotectant; cryopreservation; cooling rate; bivalve.
Recent progress in the cryopreservation of shellfish embryos has shed light on the feasibility of this technique for establishing gene banks and manipulating spawning programs (2, 3, 5, 7–9, 12). However, in order to improve cryopreservation protocols to obtain higher survival rates for oyster embryos following freezing, a systematic study of the factors affecting the survival of cryopreserved embryos is required. In the pursuit of optimal cryopreservation protocols for oyster embryos, we have previously investigated the toxicity tolerance of selected cryoprotectants to oyster embryos (1). The apparent toxicity of cryoprotectants is dependent on the type and concentration of cryoprotectant, the equilibration time, and the temperature during loading. The stage of embryonic development is also a critical factor and it was found that later stages of oyster embryos were more tolerant. Intracellular ice formation (IIF) was also examined in oyster embryos and eggs (6). The IIF process in oyster eggs and embryos was found to be time and temperature dependent. Received May 10, 1999; accepted August 2, 1999. This work was supported by the National Science Council, R.O.C. under Grant No. NSC 83-0409-B-002-096 and No. NSC 84-2312-B-002-083.
0011-2240/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
The probability of IIF increased directly with time and inversely with temperature. This information is useful in designing practical cryopreservation procedures and saves effort in optimizing procedures experimentally. We have previously reported a successful cryopreservation protocol for oyster embryos using Me 2SO as the cryoprotectant (5); a survival rate of 68 6 18% of cryopreserved embryos was obtained. This study is focused mainly on a comparison of procedural factors affecting the survival of cryopreserved oyster embryos in order to improve the cryopreservation procedure. The cryopreservation protocol was examined stepwise and optimized by modifying cooling rates and seeding temperatures. The feasibility of using different cryoprotectants was also explored. Oyster (Crassostrea gigas) sperm and eggs were collected from male and female oysters and incubated in natural sea water (34 6 2‰ salinity). The sex and maturity of individual oysters was first determined by observing the dissected gonad material under a light microscope. Gametes from four or more oysters of the same sex were then pooled in separate beakers. Eggs were artificially inseminated 1 h after their mechanical removal from the ovary. When
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newly fertilized eggs were examined under a microscope, the suggested number of sperm surrounding the egg to assure ideal fertilization was approximately 5 6 1. The 1-h incubating period in sea water was found to significantly reduce the percentage of polyspermic eggs (11). The fertilized eggs were washed and filtered to remove excess spermatozoa. The concentration of fertilized eggs was adjusted to about 1000/ml for further incubation. Embryos were obtained from the inseminated oocytes 4 h after fertilization in sea water at 28°C, when the ciliated blastula embryos start to exhibit rotary motion. It is generally observed that with good quality oyster gametes, more than 90% of the resulting embryos are normal using this procedure. Experiments were designed to compare the effects of seeding temperature, cooling rates, and the use of different cryoprotectants on the survival of cryopreserved embryos. Conventional two-step freezing experiments were conducted using a programmable freezer (KRYO 10 Series III; Planer Biomed, UK). Ciliated blastula embryos were equilibrated with 2 M Me 2SO (or other cryoprotectants) in sea water for 10 min at 25°C and were then cooled at 21°C/min from 25 to 212°C (or different seeding temperatures). Next, 0.5-ml straws containing thousands of embryos were held at 212°C for 5 min to allow equilibration after seeding with precooled tweezers. Embryos were then cooled slowly at 22.0°C/min (or at different cooling rates) to 235°C and equilibrated for 5 min before quenching in liquid nitrogen. For most experiments in this study, embryos were held in liquid nitrogen for 1 h before thawing. Thawing was carried out in a water bath at 28°C. Immediately after thawing, embryos were transferred to sea water to unload the cryoprotectant. Figure 1 is a flow chart that illustrates the sequence of events in the optimized twostep freezing protocol for oyster embryos using Me 2SO as a cryoprotectant. For all freezing experiments, at least three straws were used as replicates for each treatment. The surviving embryos were assayed by counting the embryos showing active rotary
193
FIG. 1. Flow chart of the optimized two-step cryopreservation protocol for oyster embryos using 2 M Me 2SO as a cryoprotectant.
motion under a light microscope. The presence of surviving embryos was determined 1–2 h after thawing. The survival rate for each treatment was normalized by comparing the survival rate of the thawed embryos to that of the control. With the cryopreservation protocol described in Fig. 1, a survival rate of 78 6 8% was obtained for oyster embryos quenched in liquid nitrogen. The reduction of survival was minimal during the cryoprotectant loading step. There was a decrease in survival rate (from 94 6 4 to 86 6 4%) during the freezing process from 212 to 235°C and further during the quenching process (from 86 6 4 to 78 6 8%). In a conventional two-step freezing method, samples are initially cooled to a temperature equal to or lower than the freezing point of the suspension solution before freezing at a con-
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FIG. 2. Effect of seeding temperature on the survival of cryopreserved oyster embryos. Me 2SO was used as a cryoprotectant. The cooling rate was 22°C/min.
stant cooling rate. Supercooling of the solution usually occurs and manual seeding is necessary. This procedure can be accomplished by gripping the straw with a pair of tweezers previously quenched in liquid nitrogen. The low temperature induces the nucleation of the supercooled solution in the straw and the ice propagates until the solution is solidified. In this study, the seeding temperature was also examined with experiments in the protocol optimization process. Figure 2 shows the effect of seeding temperature on the survival of cryopreserved oyster embryos when Me 2SO was used as the cryoprotectant. The cooling rate in these experiments was 22°C/min. In the temperature range from 210 to 216°C, seeding at 212°C yielded the best result. The survival rate of embryos frozen to 235°C was compared with those quenched in liquid nitrogen. Warmer seeding temperatures resulted in the melting of the ice induced by the seeding procedure and the solution in the straw was found to remain supercooled. In effect this defers the nucleation of the solution to a lower temperature during constant-rate freezing. In contrast, seeding at lower temperatures, such as 216°C, resulted in rapid propagation of the ice front in the solution during the solidification process. Following seeding or at the end of the constant-rate freezing (at 235°C), it is beneficial to maintain isothermal conditions for a certain length of time to allow for intracellular and extracellular osmotic
equilibration. However, some of our experiments (unpublished data) indicated that prolonging the holding time more than necessary increased the probability of intracellular ice formation and thus decreased the overall survival rate (10). The most critical factor to be considered when optimizing the cryopreservation protocol is cooling rate. Figure 3 shows the effect of cooling rate on the survival of cryopreserved oyster embryos loaded with 2 M Me 2SO or 2 M glycerol. Cooling rates ranging from 20.5 to 216°C/min were examined. The optimum cooling rate, which gave the highest survival rate in this series of experiments, differed in experiments that used Me 2SO or glycerol as cryoprotectants. The optimum cooling rate was 22°C/ min for embryos treated with Me 2SO and 24°C/min for glycerol. It is worth mentioning that with 2 M glycerol as a cryoprotectant, a high survival rate of 83 6 7% was obtained at a cooling rate of 24°C/min. This experiment also showed that various levels of survival rate were obtained over a relatively broad range of cooling rates. This result also agrees with the broad range of cooling rates (20.5 to 215°C/ min) previously reported from various research groups in cryopreservation procedures used for oyster embryos (2, 3, 8, 9). To compare the feasibility of using different cryoprotectants for oyster embryo cryopreservation, separate experiments were performed at
FIG. 3. Effect of cooling rate on the survival of cryopreserved oyster embryos. Me 2SO and glycerol were used as cryoprotectants.
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FIG. 4. Comparison of the effect of four different cryoprotectants on the survival of cryopreserved oyster embryos. The cooling rate was 22°C/min.
a cooling rate of 22°C/min. Figure 4 compares the effect of using 2 M Me 2SO, glycerol, ethylene glycol (EG), or propylene glycol (PG) as the cryoprotectant. For the cryoprotectant loading step, there were no significant differences between the selected cryoprotectants. For all the cryoprotectants, direct loading of the cryoprotectant at a concentration of 2 M caused minimal loss of oyster embryos. The survival rates of oyster embryos subjected to constant-rate freezing and quenching in liquid nitrogen were similar whether Me 2SO, EG, or glycerol were used as cryoprotectants. However, the survival rate of cryopreserved oyster embryos using PG as the cryoprotectant was significantly (P , 0.01) lower than those for Me 2SO, EG, and glycerol. The viability of oyster embryos after freezing is a complex function of embryonic characteristics and the operating parameters of the cryopreservation protocol. The aim of the present study was to examine the effect of major factors affecting the survival of cryopreserved embryos and thereafter to apply this information to optimize the cryopreservation protocol. Our results show that high survival rates (78 6 8% with Me 2SO at 22°C/min; 83 6 7% with glycerol at 24°C/min) of cryopreserved oyster embryos can be achieved using the optimized protocol. Our results also reveal that cryopreserved oyster embryos can survive a relatively broad range of
cryopreservation parameters, such as cooling rate and the selection of cryoprotectants. It should be noted that, similar to mammalian embryos, the survival rate of cryopreserved oyster embryos is highly dependent on the quality of gametes (4). Although a rate of more than 90% of embryos being normal can be regularly obtained from artificial fertilization procedures in our laboratory, a low fertilization rate occasionally occurred due to poor quality of oyster gametes. As a general trend, the normalized survival rate of cryopreserved embryos is usually low for experiments using batches of embryos with a low fertilization rate. Therefore, in this study the cryopreservation experiments were performed only when the fertilization rate of oyster embryos was above 90% in experimental controls. In this study, we did not attempt to incubate the thawed embryos continuously throughout the settlement. The survival of cryopreserved embryos was assayed 1–2 h after thawing and was based on the motility criterion. However, in a separate set of experiments using glycerol as a cryoprotectant and a cooling rate of 24°C/min, 32 6 12% of thawed embryos and 55 6 12% of untreated embryos developed to D-larvae stage. For hatchery and aquacultural applications, development of larvae through settlement is of considerable importance. Paniagua-Chavez et al. (9) first reported a successful production of seed oysters from cryopreserved oyster larvae. Their results confirmed the feasibility of practical applications of oyster embryo cryopreservation. In combination with the results from this study, the future development of optimized cryopreservation protocols for the complete life cycle of the oyster for use in hatcheries and other practical purposes can be anticipated. REFERENCES 1. Chao, N. H., Chiang, C. P., Hsu, H. W., Tsai, C. T., and Lin, T. T. Toxicity tolerance of oyster embryos to selected cryoprotectants. Aquatic Living Resources 7, 99 –104 (1994). 2. Chao, N. H., Lin, T. T., Chen, Y. L., Hsu, H. W., and Liao, I. C. Cryopreservation of early larvae and embryos in oyster and hard clam. Aquaculture 155, 31– 44 (1997).
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3. Gwo, J. C. Cryopreservation of oyster (Crassostrea gigas) embryos. Theriogenology 43, 1163–1174 (1995). 4. Han, Y. M., Yamashina, H., Koyama, N., Lee, K. K., and Fukui, Y. Effects of quality and developmental stage on the survival of IVF-derived bovine blastocysts cultured in vitro after freezing and thawing. Theriogenology 42, 645– 654 (1994). 5. Lin, T. T., Tung, H. T., and Chao, N. H. Cryopreservation of oyster embryos with conventional freezing procedure and vitrification. Cryobiology 30, 614 (1993). [Abstract] 6. Lin, T. T., and Lung, K. IIF characteristics of oyster embryos and eggs determined by a feedback controlled directional cryomicroscope. Cryobiology 32, 566 (1995). [Abstract] 7. McFadzen, I. R. B. Growth and survival of cryopreserved oyster and clam larvae along a pollution gradient in the German Bight. Mar. Ecol. Prog. Ser. 91, 215–220 (1992).
8. Naidenko, T. Cryopreservation of Crassostrea gigas oocytes, embryos and larvae using antioxidant echinochrome A and antifreeze protein AFP 1. CryoLett. 18, 375–382 (1997). 9. Paniagua-Chavez, C. G., Buchman, J. T., Supan, J. E., and Tiersch, T. R. Settlement and growth of Eastern oysters produced from cryopreserved larvae. CryoLett. 19, 283–292 (1998). 10. Pitt, R. E., Myers, S. P., Lin, T. T., and Steponkus, P. L. Subfreezing volumetric behavior and stochastic modeling of intracellular ice formation in Drosophila melanogaster embryos. Cryobiology 28, 72– 86 (1991). 11. Stephano, J. L., and Gould, M. Avoiding polyspermy in the oyster (Crassostrea gigas). Aquaculture 73, 295–307 (1988). 12. Toledo, J. D., Kurokura, H., and Kasahara, S. Preliminary studies on the cryopreservation of blue mussel embryos. Nippon Suisan Gakkaishi 55, 1661 (1989).