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Development of a programmable freezing technique on larval cryopreservation in the Pacific oyster Crassostrea gigas
Aquaculture 523 (2020) 735199
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aquaculture
Short communication
Development of a programmable freezing technique on larval cryopreservation in the Pacific oyster Crassostrea gigas
T ⁎
Yibing Liua, Mark Gluisb, Penny Miller-Ezzyb, Jiabo Hana, Jianguang Qinc, Xin Zhand, , ⁎ Xiaoxu Lib, a
Dalian Key Laboratory of Conservation Biology for Endangered Marine Mammals and Dalian Key Laboratory of Genetic Resources for Marine Shellfish, Liaoning Ocean and Fisheries Science Research Institute, Dalian 116023, China b South Australian Research and Development Institute, Aquatic Sciences Centre, Adelaide 5024, Australia c College of Science and Engineering, Flinders University, Adelaide 5042, Australia d Department of Aquaculture, College of Marine Sciences, Hainan University, Haikou 570228, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Crassostrea gigas Larval cryopreservation Ficoll Polyvinylpyrrolidone
This study investigated three vital factors to develop a programmable cryopreservation technique for the larvae of Pacific oyster Crassostrea gigas, namely (1) larval developmental stages; (2) cryoprotectant agents (CPAs) and (3) larval density. The D-larval rate was used as the post-thaw survival indicator, which was calculated as the percentage of larvae that develop into D-larvae after 24 h post-fertilization. The results showed that the postthaw D-larval rate above 80% was achieved when the 18 h post-fertilization (PF) larvae were cryopreserved with the CPA consisting of 10% ethylene glycol +5% Ficoll +0.2% polyvinylpyrrolidone. No significant difference in the rate of post-thaw D-larvae (P > .05) was found between initial larval density of 4 × 105 mL−1 and 1 × 106 mL−1. The shell length of progenies produced from fresh and cryopreserved larvae had no significant difference at all developmental stages investigated. In addition, similar relative mortality rates were found from umbo larvae (day 8 PF) to spat (day 27 PF) between progenies produced from fresh and cryopreserved larvae. Therefore, the larval cryopreservation technique developed in this study could be used to facilitate breeding programs and hatchery management in Pacific oyster aquaculture industry.
1. Introduction The Pacific oyster, Crassostrea gigas, is one of the most important economic aquaculture species in the world due to its volume of production and market demand (Paredes et al., 2013; de Melo et al., 2016). To support the long-term sustainable development of this species, genetic improvement programs, such as selective breeding, have been integrated into its hatchery management (de Melo et al., 2016; Ugalde et al., 2018). However, the lack of a reliable technique to protect the superior genetic resource from loss could put the breeding programs at high risk. For example, the outbreak of oyster herpesvirus in Tasmania, Australia has caused mortalities up to 87% in all infected growing areas for C. gigas (Ugalde et al., 2018). This unpredictable risk has almost destroyed many years' efforts on the selective breeding programs in Tasmania (Ugalde et al., 2018). Therefore, development of a reliable technique that could preserve the superior genetic resources is urgent and important.
Up to date, larval cryopreservation has been widely acknowledged as an effective and reliable technique to preserve germplasm (Paredes et al., 2013; Labbé et al., 2018). However, the application of this technique is very challenging in marine bivalves as a low percentage of cryopreserved larvae could further develop into competent pediveliger larvae in oysters (Paredes et al., 2013; Labbé et al., 2018), clams (Simon and Yang, 2018) and mussels (Wang et al., 2011; Paredes et al., 2012, 2013). The programmable freezing technique is the dominant method having been used in the larval cryopreservation of marine bivalve species. One of the key parameters investigated in the literature is to optimize the cryoprotectant agents (CPA). Usually, CPA can be divided into permeable and non-permeable types depending on their ability to penetrate the cell membrane, and the usage of these two types of CPA together is also common in the larval cryopreservation (Youngs, 2011). In Pacific oysters, ethylene glycol (EG) is commonly used as a permeable CPA, but little research has focused on the evaluation of nonpermeable CPAs (Paredes et al., 2013; Labbé et al., 2018).
⁎
Corresponding authors. E-mail addresses: [email protected] (Y. Liu), [email protected] (M. Gluis), [email protected] (P. Miller-Ezzy), [email protected] (J. Han), jian.qin@flinders.edu.au (J. Qin), [email protected] (X. Zhan), [email protected] (X. Li). https://doi.org/10.1016/j.aquaculture.2020.735199 Received 16 December 2019; Received in revised form 28 February 2020; Accepted 3 March 2020 Available online 04 March 2020 0044-8486/ © 2020 Published by Elsevier B.V.
Aquaculture 523 (2020) 735199
Y. Liu, et al.
results in the larval cryopreservation in M. galloprovincialis (Liu et al., 2020). The larvae were collected at 10, 15, 18 and 21 h PF and were mixed with CPA for 10 min on ice. The mixtures were then transferred into 0.25 mL straws and maintained at 0 °C for 5 min in the programmable freezer. The straws were cooled at a rate of −1 °C/min from 0 to −10 °C and at −0.3 °C/min from −10 to −34 °C before being plunged into liquid nitrogen (LN). After at least 12 h storage in LN, the straws were thawed individually in a 28 °C water bath until ice melted and recovered in an 18 °C seawater bath. The content in each straw was then expelled into a 4 mL tube and diluted for 10 min using 0.25 mL medium consisting of 9% sucrose. The CPA concentration was further diluted twice by adding a same volume of FSW as that in the tube at 10 min intervals. The larvae were cultured in 500 mL containers until reaching D-larval stage (24 h PF). The D-larval rate was calculated as the percentage of larvae that develop into D-larvae. Controls were established using the same batch of broodstock. Each experiment was replicated three times.
Our recent study on larval cryopreservation in Mytilus galloprovincialis has demonstrated that the non-permeable CPA Ficoll (FIC) or polyvinylpyrrolidone (PVP) could improve the resultant post-thaw Dlarval rate and the application of FIC and PVP together could further improve the rate to exceed 80% (Liu et al., 2020). In addition, progenies produced from the larvae that cryopreserved with the combination of FIC and PVP experienced similar relative mortality rates in comparison with those in the control (fresh larvae) after 8 days of postfertilization. In this study, the suitability of this technique will be evaluated for the development of a programmable larval cryopreservation technique in C. gigas. 2. Materials and methods 2.1. Larval collection Mature broodstock were supplied by the Zippel Enterprises Pty Ltd. in Smoky Bay, South Australia (SA) and transported in a refrigerated container overnight to Aquatic Sciences Center, South Australian Research and Development Institute (SARDI). Upon arrival, the animals were washed with 1 μm filtered seawater (FSW) before being opened for gamete collection. A sample from gonad was extracted and examined under a light microscope to determine the sex. The eggs were collected by a pipette in terms of lacerating the gonad wall and gently scraping and washing contents into 1 L container filled with 1 μm FSW at 23 °C. The eggs were left undisturbed for 1 h prior to being poured into a 90 μm sieve to remove large debris and retained on a 15 μm sieve. They were then gently rinsed by 1 μm FSW, and washed into a settlement beaker. The eggs were collected from at least 5 individuals. The sperm were extracted from the gonad using the same method and filtered through a 25 μm sieve to remove the debris. The sperm quality was examined under a light microscope and those with motility above 80% were pooled from at least 5 individuals. The fertilization was conducted at a sperm to egg ratio of 20:1. At 15 min post-fertilization (PF), the fertilized eggs were gently washed on a 15 μm sieve and then incubated in 50 L tanks at a density of approximate 20 individuals mL−1. When the fertilized eggs were developed for the pre-determined hours PF, the larvae were collected on a 25 μm sieve from the top of tanks. The larvae were transferred into 10 mL tubes and stored on ice. The density of larvae was counted and diluted to 4 × 105 mL−1 for experiments unless otherwise described.
2.3.2. Effects of different FIC concentrations on post-thaw D-larval rate The highest post-thaw D-larval rate was achieved when the larvae were cryopreserved at 18 h PF in the previous experiment. Therefore, this stage of larvae was used for this and subsequent experiments. In this experiment, 10% EG combined with 1, 3, 5 or 7.5% FIC were evaluated on post-thaw D-larval rate. The other procedures were the same as Experiment 2.3.1. 2.3.3. Comparison of different CPAs on post-thaw D-larval rate The highest post-thaw D-larval rate was achieved when larvae were cryopreserved with 10% EG + 5% FIC in the previous experiment. Therefore, this CPA was used for this experiment. Recent study on larval cryopreservation in M. galloprovincialis has shown that the application of FIC and PVP together can significantly improve the postthaw survival rate (Liu et al., 2020). Therefore, different CPAs, 10% EG + 5% FIC, 10% EG + 0.2% PVP and 10% EG + 5% FIC + 0.2% PVP were evaluated on post-thaw D-larval rate in this experiment. The other procedures were the same as Experiment 2.3.2. 2.3.4. Effects of different larval densities on post-thaw D-larval rate The highest post-thaw D-larval rate was achieved when the larvae were cryopreserved with 10% EG + 5% FIC + 0.2% PVP in the previous experiment. Therefore, this CPA was used in this experiment to evaluate two larval densities (4 × 105 mL−1 and 1 × 106 mL−1) on post-thaw D-larval rate. The other procedures were the same as Experiment 2.3.3.
2.2. Chemicals and equipment Sucrose, EG, FIC and PVP were purchased from Sigma-Aldrich Pty Ltd. (St. Louis, MO, USA). The cryoprotective stock solution was prepared in Milli-Q water at a concentration two times as high as that required in the experiments. Therefore, when a same volume of stock solution and larval suspension were mixed, the required final chemical concentration was produced. A programmable controller (Cryologic, Mulgrave, Victoria, Australia) was used in this study. Straws at volume of 0.25 mL (Minitube, Germany) were placed into a cryochamber (model: CC23F) with a lid on top and cooled by LN. The temperature was regulated by an electronic device inside the cryochamber in accordance with the protocol set and monitored with the CryoGenesis software (V5) from the Cryologic. A thawing seawater bath was used with the required temperature was produced by mixing the ambient and boiled seawater. The temperature required in a recovery bath (18 °C) was produced by mixing the ambient and cold seawater.
2.3.5. Performance comparison between progenies produced between fresh and cryopreserved larvae The cryopreservation protocol (larvae: 18 h PF; larval density 1 × 106 mL−1, CPA: 10% EG + 5% FIC + 0.2% PVP; post-thaw CPA removal medium: 9% sucrose; thawing temperature: 28 °C; straw volume: 0.25 mL) was applied to compare the performance between progenies produced from fresh and cryopreserved larvae. After the assessment of D-larval rate, the larvae were transferred into 30 L tanks and stocked at a density of ~10 individuals mL−1 for both cryopreserved (3 tanks) and fresh (3 tanks) larvae. Methods for larval culture and settlement were the same as those used by Li (2009). The survival and relative mortality rates and shell length were compared at D-larvae (24 h PF), umbo larvae (8 d PF), eyed larvae (22 d PF) and spat (27 d PF). The survival rate (%) was calculated by dividing the number of alive larvae or spat at sampling (d PF) by the number of larvae stocked at the start of the experiment. The relative mortality rate (%) was calculated by dividing the difference in survival rate between adjacent sampling collection dates with the survival rate at the start of this period. For the larvae shell length measurement, 30 individuals from each tank were randomly collected.
2.3. Experiments 2.3.1. Effects of different larval developmental stages on post-thaw D-larval rate In this study, 10% EG + 7.5% FIC was used as CPA according to the 2
Aquaculture 523 (2020) 735199
Y. Liu, et al.
100.0 D -larval rate (%)
2.4. Statistical analysis The D-larval rates were normalized to the control mean percentage of larval abnormality using Abbot's formula: P = (Pe-Pc)/(100-Pc) x 100, where Pc and Pe are the control and experimental percentages of response, respectively (Paredes et al., 2013). The data were presented as mean ± standard deviation (SD) and arcsine transformed for statistical analyses using SPSS 26. One-way analysis of variance (ANOVA) was applied to analyze the data on the effects of cryopreservation on larval developmental stages, different FIC concentrations, CPA combinations. The Least-Significant Difference (LSD) comparison test was used when significance was observed. A t-test was applied to compare the larval density on post-thaw D-larval rate and a paired sample t-test was applied to compare the performances (survival rate, relative mortality rate and larval shell length) between progenies produced from fresh and cryopreserved larvae. Differences were considered statistically significant at P < .05.
80.0
A
A
60.0 B
B
40.0 20.0 0.0 10% EG + 1% 10% EG + 3% 10% EG + 5% 10% EG + 7.5% FIC FIC FIC FIC FIC concentrations
Fig. 2. Post-thaw D-larval rates (%) when larvae were cryopreserved in 10% EG with different FIC concentrations, n = 3. Different letter indicates significant difference (P < .05). All the data have been normalized to the controls.
D-larval rate (%)
100.0
3. Results 3.1. Effects of different larval developmental stages on post-thaw D-larval rate The highest post-thaw D-larval rate of 69.5 ± 8.5% was achieved when 18 h PF larvae were cryopreserved, which was significantly higher than 10 h and 21 h PF larvae (P < .05), but not in comparison with 15 h PF larvae (P > .05; Fig. 1).
80.0
A B
B
60.0 40.0 20.0 0.0 10% EG + 5% FIC
10% EG + 0.2% 10% EG + 5% FIC PVP + 0.2% PVP CPA combinations
Fig. 3. Post-thaw D-larval rates (%) when larvae were cryopreserved in different CPA combinations, n = 3. Different letter indicates significant difference (P < .05). All the data have been normalized to the controls.
3.2. Effects of different FIC concentrations on post-thaw D-larval rate No significant difference (P > .05) on post-thaw D-larval rate was found when larvae were cryopreserved with 10% EG + 5% FIC and 10% EG + 7.5% FIC (Fig. 2). However, these two treatments achieved significantly higher post-thaw D-larval rate than 10% EG + 1% FIC and 10% EG + 3% FIC treatments (P < .05; Fig. 2).
3.5. Performance comparison between progenies produced between fresh and cryopreserved larvae
The highest post-thaw D-larval rate was achieved when larvae were cryopreserved with 10% EG + 5% FIC + 0.2% PVP, which was significantly higher than other groups (P < .05; Fig. 3).
Table 1 showed that cryopreserved larvae produced significantly lower rate of D larvae, umbo larvae, eyed larvae and spat in comparison with fresh larvae (P < .05). However, no significant difference was found on the larval shell length at the developmental stages evaluated (P > .05; Fig. 4). Moreover, from day 8 PF and onward no significant difference in relative mortality rate between progenies resulted from fresh and cryopreserved larvae (P > .05; Table 1).
3.4. Effects of different larval densities on post-thaw embryo quality
4. Discussion
No significant difference was found between larvae cryopreserved at the initial density of 4 × 105 mL−1 and 1 × 106 mL−1 (P > .05), resulting in post-thaw D-larval rates of 77.5 ± 10.0% and 80.7 ± 10.3, respectively.
This study has developed a programmable larval cryopreservation technique for C. gigas. The post-thaw survival rate has been improved to > 80% when 18 h PF larvae were cryopreserved with 10% EG + 5% FIC + 0.2% PVP. Larvae at different developmental stages have various ability for cryoresistance, therefore, selection of suitable developmental stage is important for the success of cryopreservation (Paredes et al., 2012, 2013; Liu et al., 2020). In this study, larvae collected at 18 h PF had the highest resistance to cryoinjury, with about 70% post-thaw D-larval rate achieved. This collection time was later than the 14 h PF reported by Paredes et al. (2013) on the same species. Prior to natural feeding, bivalve embryos and early stage larvae normally have a high level of lipid as an energy reserve for development (Goslling, 2015). Larvae with high lipid content would be more sensitive to cryopreservation and this phenomenon has been shown in pigs (Dobrinsky, 2002), cattle (Massip, 2001; Pryor et al., 2011), sheep (Massip, 2001) and mussels (Liu et al., 2020). The cryoprotective medium containing both permeable and nonpermeable CPAs is common for larval cryopreservation (Saragusty and Arav, 2011; Paredes et al., 2012, 2013). The most dominant permeable CPA used in C. gigas is EG (Paredes et al., 2013; Labbé et al., 2018). On
3.3. Comparison of different CPAs on post-thaw D-larval rate
D-larval rate (%)
100.0 A
80.0 A
60.0 B
40.0 B
20.0 0.0 10 h
15 h 18 h Post fertilization hours
21 h
Fig. 1. Post-thaw D-larval rates (%) when larvae were cryopreserved at different larval developmental stages, n = 3. Different letter indicates significant difference (P < .05). All the data have been normalized to the controls. 3
Aquaculture 523 (2020) 735199
Y. Liu, et al.
Table 1 Comparison of survival rates and relative mortality rate between progenies produced from fresh and cryopreserved larvae in C. gigas, n = 3. Different letter indicates significant difference (P < .05). Days post fertilization
Survival rate (%)
Relative mortality rate (%)
Fresh larvae Day Day Day Day
1 (D larvae) 8 (umbo larvae) 22 (eyed larvae) 27 (spat)
82.0 46.7 33.7 18.3
± ± ± ±
2.2 7.5 4.5 4.0
A A′ A″ A‴
Cryopreserved larvae
Fresh larvae
Cryopreserved larvae
60.9 ± 7.1 B 14.9 ± 3.0 B′ 7.5 ± 2.2 B″ 4.9 ± 1.3 B‴
43.0 ± 9.5 b 26.4 ± 16.3 a′ 45.0 ± 12.8 a″
75.6 ± 2.7 a 46.9 ± 26.3 a′ 33.5 ± 2.4 a″
Acknowledgments
600
Shell length (µm)
500
This research was funded by the Talent Project of Revitalizing Liaoning (Project No. XLYC1807087), Department of Ocean and Fisheries of Liaoning (Project No. 201829), China Scholarship Council and South Australian Research and Development Institute. We thank Mr. Gary Zippel of Zippel Enterprises Pty Ltd. for the provision of Pacific oyster broodstock.
Fresh larvae
400
Cryopreserved larvae
300 200 100 0 D larvae
umbo larvae
eyed larvae
Declaration of Competing Interest
spat
Larval developmental stages
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Fig. 4. Shell length (μm) of D-larvae, umbo larvae, eyed larvae and spats produced from fresh and cryopreserved larvae, n = 90.
References
the contrary, only a few non-permeable CPAs, such as polyvinylpyrrolidone (PVP) and trehalose, have been evaluated on their suitability in oyster larval cryopreservation (Paredes et al., 2013). In our recent study in M. galloprovincialis, the application of non-permeable CPAs, FIC and PVP, has improved the post-thaw larvae survival rates, which could be further enhanced by applying FIC and PVP together (Liu et al., 2020). A similar trend has been reported in livestock species (Saha et al., 1996; Titterington and Robinson, 1996). This was also confirmed in the current study, where the post-thaw D-larval rate was improved to > 80% when both PIC and PVP were applied together (Fig. 3). This result indicates that the combined effects of these two CPAs could play a key role in protecting larvae from cryoinjury in C. gigas. Increase in initial larval density would increase the efficiency of application of cryopreservation technique in breeding programs and hatchery production if a similar post-thaw survival rate can be achieved (Tervit et al., 2005). In this study, the initial larval density of 1 × 106 mL−1 achieved the similar post-thaw D-larval rate as the density of 4 × 105. The former is close to the highest larval density that could be achieved in this study. No significant difference between fresh and cryopreserved larvae was found in their shell length at any ages evaluated (Fig. 4). Although the survival rates were significantly lower in the cryopreserved than those in the fresh larvae (Table 2), the relative mortality rates after day 8 PF remained similar (P > .05) between them (Table 2). These results indicate that cryopreservation may mainly affect the larval performances at the early developmental stage in C. gigas. Similar phenomenon has also been observed by Labbé et al. (2018) in the same species and our study on oocyte and larval cryopreservation in M. galloprovincialis (Liu and Li, 2015; Liu et al., 2020). In conclusion, the larval cryopreservation technique developed for C. gigas has achieved the post-thaw D-larval rate > 80%. The cryodamage was mainly expressed at early developmental stages, as relative mortality rates remained similar after day 8 PF between progenies produced from fresh and cryopreserved larvae. Thus, this technique could be applied for all year round supply of larvae for hatchery productions and assist in superior genetic preservation for breeding programs in oyster industry.
De Melo, C.M.R., Durland, E., Langdon, C., 2016. Improvements in desirable traits of the Pacific oyster, Crassostrea gigas, as a result of five generations of selection on the West Coast, USA. Aquaculture 460, 105–115. Dobrinsky, J.R., 2002. Advancements in cryopreservation of domestic animal embryos. Theriogenology 57, 285–302. Goslling, E., 2015. Marine Bivalve Molluscs, second ed. Blackwell Publishing, UK. Labbé, C., Haffray, P., Mingant, C., Quittet, B., Diss, B., Tervit, H.R., Adams, S.L., Rimond, F., Suquet, M., 2018. Cryopreservation of Pacific oyster (Crassostrea gigas) larvae: revisiting the practical limitations and scaling up the procedure for application to hatchery. Aquaculture 488, 227–234. Li, 2009. Development of Techniques for Production of Homozygous Pacific Oysters. FRDC Final Report. Liu, Y., Li, X., 2015. Successful oocyte cryopreservation in the blue mussel Mytilus galloprovincialis. Aquaculture 438, 55–58. Liu, Y., Gluis, M., Miller-Ezzy, P., Qin, J., Zhan, X., Li, X., 2020. Development of a programmable freezing technique on larval cryopreservation in Mytilus galloprovincialis. Aquaculture 516, 734554. Massip, A., 2001. Cryopreservation of embryos of farm animals. Reprod. Domest. Anim. 36, 49–55. Paredes, E., Adams, S.L., Tervit, H.R., Smith, J.F., McGowan, L.T., Gale, S.L., Morrish, J.R., Watts, E., 2012. Cryopreservation of GreenshellTM mussel (Perna canaliculus) trochophore larvae. Cryobiology 65, 256–262. Paredes, E., Bellas, J., Adams, S.L., 2013. Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and blue mussel (Mytilus galloprovincialis). Cryobiology 67, 274–279. Pryor, J.H., Looney, C.R., Romo, S., Kraemer, D.C., Long, C.R., 2011. Cryopreservation of in vitro produced bovine embryos: effects of lipid segregation and post-thaw laser assisted hatching. Theriogenology 75, 24–33. Saha, S., Otoi, T., Takagi, M., Boediono, A., Sumantri, C., Suzuki, T., 1996. Normal calves obtained after direct transfer of vitrified bovine embryos using ethylene glycol, trehalose, and polyvinylpyrrolidone. Cryobiology 33, 291–299. Saragusty, J., Arav, A., 2011. Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction 141, 1–19. Simon, N.A., Yang, H., 2018. Cryopreservation of trochophore larvae from the hard clam Mercenaria mercenaria: evaluation of the cryoprotecant toxicity, cooling rate and thawing temperature. Aquac. Res. 49, 2869–2880. Tervit, H.R., Adams, S.L., Roberts, R.D., McGowan, L.T., Pugh, P.A., Smith, J.F., Janke, A.R., 2005. Successful cryopreservation of Pacific oyster (Crassostrea gigas) oocytes. Cryobiology 51, 142–151. Titterington, J.L., Robinson, J., 1996. The protective action of polyvinylpyrrolidonePercoll during the cryopreservation of mouse 2-cell embryos and its effect on subsequent developmental potential post-thaw in vitro and in vivo. Hum. Reprod. 11, 2697–2702. Ugalde, S.C., Preston, J., Ogier, E., Crawford, C., 2018. Analysis of farm management strategies following herpesvirus (OsHV-1) disease outbreaks in Pacific oysters in Tasmania, Australia. Aquaculture 495, 179–186. Wang, H., Li, X., Wang, M., Clarke, S., Gluis, M., Zhang, Z., 2011. Effects of larval cryopreservation on subsequent development of the blue mussel, Mytilus galloprovincialis Lamarck. Aquac. Res. 42, 1816–1823. Youngs, C.R., 2011. Cryopreservation of preimplantation embryos of cattle, sheep, and goats. J. Vis. Exp. 54, e2764.
4
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aquaculture
Short communication
Development of a programmable freezing technique on larval cryopreservation in the Pacific oyster Crassostrea gigas
T ⁎
Yibing Liua, Mark Gluisb, Penny Miller-Ezzyb, Jiabo Hana, Jianguang Qinc, Xin Zhand, , ⁎ Xiaoxu Lib, a
Dalian Key Laboratory of Conservation Biology for Endangered Marine Mammals and Dalian Key Laboratory of Genetic Resources for Marine Shellfish, Liaoning Ocean and Fisheries Science Research Institute, Dalian 116023, China b South Australian Research and Development Institute, Aquatic Sciences Centre, Adelaide 5024, Australia c College of Science and Engineering, Flinders University, Adelaide 5042, Australia d Department of Aquaculture, College of Marine Sciences, Hainan University, Haikou 570228, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Crassostrea gigas Larval cryopreservation Ficoll Polyvinylpyrrolidone
This study investigated three vital factors to develop a programmable cryopreservation technique for the larvae of Pacific oyster Crassostrea gigas, namely (1) larval developmental stages; (2) cryoprotectant agents (CPAs) and (3) larval density. The D-larval rate was used as the post-thaw survival indicator, which was calculated as the percentage of larvae that develop into D-larvae after 24 h post-fertilization. The results showed that the postthaw D-larval rate above 80% was achieved when the 18 h post-fertilization (PF) larvae were cryopreserved with the CPA consisting of 10% ethylene glycol +5% Ficoll +0.2% polyvinylpyrrolidone. No significant difference in the rate of post-thaw D-larvae (P > .05) was found between initial larval density of 4 × 105 mL−1 and 1 × 106 mL−1. The shell length of progenies produced from fresh and cryopreserved larvae had no significant difference at all developmental stages investigated. In addition, similar relative mortality rates were found from umbo larvae (day 8 PF) to spat (day 27 PF) between progenies produced from fresh and cryopreserved larvae. Therefore, the larval cryopreservation technique developed in this study could be used to facilitate breeding programs and hatchery management in Pacific oyster aquaculture industry.
1. Introduction The Pacific oyster, Crassostrea gigas, is one of the most important economic aquaculture species in the world due to its volume of production and market demand (Paredes et al., 2013; de Melo et al., 2016). To support the long-term sustainable development of this species, genetic improvement programs, such as selective breeding, have been integrated into its hatchery management (de Melo et al., 2016; Ugalde et al., 2018). However, the lack of a reliable technique to protect the superior genetic resource from loss could put the breeding programs at high risk. For example, the outbreak of oyster herpesvirus in Tasmania, Australia has caused mortalities up to 87% in all infected growing areas for C. gigas (Ugalde et al., 2018). This unpredictable risk has almost destroyed many years' efforts on the selective breeding programs in Tasmania (Ugalde et al., 2018). Therefore, development of a reliable technique that could preserve the superior genetic resources is urgent and important.
Up to date, larval cryopreservation has been widely acknowledged as an effective and reliable technique to preserve germplasm (Paredes et al., 2013; Labbé et al., 2018). However, the application of this technique is very challenging in marine bivalves as a low percentage of cryopreserved larvae could further develop into competent pediveliger larvae in oysters (Paredes et al., 2013; Labbé et al., 2018), clams (Simon and Yang, 2018) and mussels (Wang et al., 2011; Paredes et al., 2012, 2013). The programmable freezing technique is the dominant method having been used in the larval cryopreservation of marine bivalve species. One of the key parameters investigated in the literature is to optimize the cryoprotectant agents (CPA). Usually, CPA can be divided into permeable and non-permeable types depending on their ability to penetrate the cell membrane, and the usage of these two types of CPA together is also common in the larval cryopreservation (Youngs, 2011). In Pacific oysters, ethylene glycol (EG) is commonly used as a permeable CPA, but little research has focused on the evaluation of nonpermeable CPAs (Paredes et al., 2013; Labbé et al., 2018).
⁎
Corresponding authors. E-mail addresses: [email protected] (Y. Liu), [email protected] (M. Gluis), [email protected] (P. Miller-Ezzy), [email protected] (J. Han), jian.qin@flinders.edu.au (J. Qin), [email protected] (X. Zhan), [email protected] (X. Li). https://doi.org/10.1016/j.aquaculture.2020.735199 Received 16 December 2019; Received in revised form 28 February 2020; Accepted 3 March 2020 Available online 04 March 2020 0044-8486/ © 2020 Published by Elsevier B.V.
Aquaculture 523 (2020) 735199
Y. Liu, et al.
results in the larval cryopreservation in M. galloprovincialis (Liu et al., 2020). The larvae were collected at 10, 15, 18 and 21 h PF and were mixed with CPA for 10 min on ice. The mixtures were then transferred into 0.25 mL straws and maintained at 0 °C for 5 min in the programmable freezer. The straws were cooled at a rate of −1 °C/min from 0 to −10 °C and at −0.3 °C/min from −10 to −34 °C before being plunged into liquid nitrogen (LN). After at least 12 h storage in LN, the straws were thawed individually in a 28 °C water bath until ice melted and recovered in an 18 °C seawater bath. The content in each straw was then expelled into a 4 mL tube and diluted for 10 min using 0.25 mL medium consisting of 9% sucrose. The CPA concentration was further diluted twice by adding a same volume of FSW as that in the tube at 10 min intervals. The larvae were cultured in 500 mL containers until reaching D-larval stage (24 h PF). The D-larval rate was calculated as the percentage of larvae that develop into D-larvae. Controls were established using the same batch of broodstock. Each experiment was replicated three times.
Our recent study on larval cryopreservation in Mytilus galloprovincialis has demonstrated that the non-permeable CPA Ficoll (FIC) or polyvinylpyrrolidone (PVP) could improve the resultant post-thaw Dlarval rate and the application of FIC and PVP together could further improve the rate to exceed 80% (Liu et al., 2020). In addition, progenies produced from the larvae that cryopreserved with the combination of FIC and PVP experienced similar relative mortality rates in comparison with those in the control (fresh larvae) after 8 days of postfertilization. In this study, the suitability of this technique will be evaluated for the development of a programmable larval cryopreservation technique in C. gigas. 2. Materials and methods 2.1. Larval collection Mature broodstock were supplied by the Zippel Enterprises Pty Ltd. in Smoky Bay, South Australia (SA) and transported in a refrigerated container overnight to Aquatic Sciences Center, South Australian Research and Development Institute (SARDI). Upon arrival, the animals were washed with 1 μm filtered seawater (FSW) before being opened for gamete collection. A sample from gonad was extracted and examined under a light microscope to determine the sex. The eggs were collected by a pipette in terms of lacerating the gonad wall and gently scraping and washing contents into 1 L container filled with 1 μm FSW at 23 °C. The eggs were left undisturbed for 1 h prior to being poured into a 90 μm sieve to remove large debris and retained on a 15 μm sieve. They were then gently rinsed by 1 μm FSW, and washed into a settlement beaker. The eggs were collected from at least 5 individuals. The sperm were extracted from the gonad using the same method and filtered through a 25 μm sieve to remove the debris. The sperm quality was examined under a light microscope and those with motility above 80% were pooled from at least 5 individuals. The fertilization was conducted at a sperm to egg ratio of 20:1. At 15 min post-fertilization (PF), the fertilized eggs were gently washed on a 15 μm sieve and then incubated in 50 L tanks at a density of approximate 20 individuals mL−1. When the fertilized eggs were developed for the pre-determined hours PF, the larvae were collected on a 25 μm sieve from the top of tanks. The larvae were transferred into 10 mL tubes and stored on ice. The density of larvae was counted and diluted to 4 × 105 mL−1 for experiments unless otherwise described.
2.3.2. Effects of different FIC concentrations on post-thaw D-larval rate The highest post-thaw D-larval rate was achieved when the larvae were cryopreserved at 18 h PF in the previous experiment. Therefore, this stage of larvae was used for this and subsequent experiments. In this experiment, 10% EG combined with 1, 3, 5 or 7.5% FIC were evaluated on post-thaw D-larval rate. The other procedures were the same as Experiment 2.3.1. 2.3.3. Comparison of different CPAs on post-thaw D-larval rate The highest post-thaw D-larval rate was achieved when larvae were cryopreserved with 10% EG + 5% FIC in the previous experiment. Therefore, this CPA was used for this experiment. Recent study on larval cryopreservation in M. galloprovincialis has shown that the application of FIC and PVP together can significantly improve the postthaw survival rate (Liu et al., 2020). Therefore, different CPAs, 10% EG + 5% FIC, 10% EG + 0.2% PVP and 10% EG + 5% FIC + 0.2% PVP were evaluated on post-thaw D-larval rate in this experiment. The other procedures were the same as Experiment 2.3.2. 2.3.4. Effects of different larval densities on post-thaw D-larval rate The highest post-thaw D-larval rate was achieved when the larvae were cryopreserved with 10% EG + 5% FIC + 0.2% PVP in the previous experiment. Therefore, this CPA was used in this experiment to evaluate two larval densities (4 × 105 mL−1 and 1 × 106 mL−1) on post-thaw D-larval rate. The other procedures were the same as Experiment 2.3.3.
2.2. Chemicals and equipment Sucrose, EG, FIC and PVP were purchased from Sigma-Aldrich Pty Ltd. (St. Louis, MO, USA). The cryoprotective stock solution was prepared in Milli-Q water at a concentration two times as high as that required in the experiments. Therefore, when a same volume of stock solution and larval suspension were mixed, the required final chemical concentration was produced. A programmable controller (Cryologic, Mulgrave, Victoria, Australia) was used in this study. Straws at volume of 0.25 mL (Minitube, Germany) were placed into a cryochamber (model: CC23F) with a lid on top and cooled by LN. The temperature was regulated by an electronic device inside the cryochamber in accordance with the protocol set and monitored with the CryoGenesis software (V5) from the Cryologic. A thawing seawater bath was used with the required temperature was produced by mixing the ambient and boiled seawater. The temperature required in a recovery bath (18 °C) was produced by mixing the ambient and cold seawater.
2.3.5. Performance comparison between progenies produced between fresh and cryopreserved larvae The cryopreservation protocol (larvae: 18 h PF; larval density 1 × 106 mL−1, CPA: 10% EG + 5% FIC + 0.2% PVP; post-thaw CPA removal medium: 9% sucrose; thawing temperature: 28 °C; straw volume: 0.25 mL) was applied to compare the performance between progenies produced from fresh and cryopreserved larvae. After the assessment of D-larval rate, the larvae were transferred into 30 L tanks and stocked at a density of ~10 individuals mL−1 for both cryopreserved (3 tanks) and fresh (3 tanks) larvae. Methods for larval culture and settlement were the same as those used by Li (2009). The survival and relative mortality rates and shell length were compared at D-larvae (24 h PF), umbo larvae (8 d PF), eyed larvae (22 d PF) and spat (27 d PF). The survival rate (%) was calculated by dividing the number of alive larvae or spat at sampling (d PF) by the number of larvae stocked at the start of the experiment. The relative mortality rate (%) was calculated by dividing the difference in survival rate between adjacent sampling collection dates with the survival rate at the start of this period. For the larvae shell length measurement, 30 individuals from each tank were randomly collected.
2.3. Experiments 2.3.1. Effects of different larval developmental stages on post-thaw D-larval rate In this study, 10% EG + 7.5% FIC was used as CPA according to the 2
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100.0 D -larval rate (%)
2.4. Statistical analysis The D-larval rates were normalized to the control mean percentage of larval abnormality using Abbot's formula: P = (Pe-Pc)/(100-Pc) x 100, where Pc and Pe are the control and experimental percentages of response, respectively (Paredes et al., 2013). The data were presented as mean ± standard deviation (SD) and arcsine transformed for statistical analyses using SPSS 26. One-way analysis of variance (ANOVA) was applied to analyze the data on the effects of cryopreservation on larval developmental stages, different FIC concentrations, CPA combinations. The Least-Significant Difference (LSD) comparison test was used when significance was observed. A t-test was applied to compare the larval density on post-thaw D-larval rate and a paired sample t-test was applied to compare the performances (survival rate, relative mortality rate and larval shell length) between progenies produced from fresh and cryopreserved larvae. Differences were considered statistically significant at P < .05.
80.0
A
A
60.0 B
B
40.0 20.0 0.0 10% EG + 1% 10% EG + 3% 10% EG + 5% 10% EG + 7.5% FIC FIC FIC FIC FIC concentrations
Fig. 2. Post-thaw D-larval rates (%) when larvae were cryopreserved in 10% EG with different FIC concentrations, n = 3. Different letter indicates significant difference (P < .05). All the data have been normalized to the controls.
D-larval rate (%)
100.0
3. Results 3.1. Effects of different larval developmental stages on post-thaw D-larval rate The highest post-thaw D-larval rate of 69.5 ± 8.5% was achieved when 18 h PF larvae were cryopreserved, which was significantly higher than 10 h and 21 h PF larvae (P < .05), but not in comparison with 15 h PF larvae (P > .05; Fig. 1).
80.0
A B
B
60.0 40.0 20.0 0.0 10% EG + 5% FIC
10% EG + 0.2% 10% EG + 5% FIC PVP + 0.2% PVP CPA combinations
Fig. 3. Post-thaw D-larval rates (%) when larvae were cryopreserved in different CPA combinations, n = 3. Different letter indicates significant difference (P < .05). All the data have been normalized to the controls.
3.2. Effects of different FIC concentrations on post-thaw D-larval rate No significant difference (P > .05) on post-thaw D-larval rate was found when larvae were cryopreserved with 10% EG + 5% FIC and 10% EG + 7.5% FIC (Fig. 2). However, these two treatments achieved significantly higher post-thaw D-larval rate than 10% EG + 1% FIC and 10% EG + 3% FIC treatments (P < .05; Fig. 2).
3.5. Performance comparison between progenies produced between fresh and cryopreserved larvae
The highest post-thaw D-larval rate was achieved when larvae were cryopreserved with 10% EG + 5% FIC + 0.2% PVP, which was significantly higher than other groups (P < .05; Fig. 3).
Table 1 showed that cryopreserved larvae produced significantly lower rate of D larvae, umbo larvae, eyed larvae and spat in comparison with fresh larvae (P < .05). However, no significant difference was found on the larval shell length at the developmental stages evaluated (P > .05; Fig. 4). Moreover, from day 8 PF and onward no significant difference in relative mortality rate between progenies resulted from fresh and cryopreserved larvae (P > .05; Table 1).
3.4. Effects of different larval densities on post-thaw embryo quality
4. Discussion
No significant difference was found between larvae cryopreserved at the initial density of 4 × 105 mL−1 and 1 × 106 mL−1 (P > .05), resulting in post-thaw D-larval rates of 77.5 ± 10.0% and 80.7 ± 10.3, respectively.
This study has developed a programmable larval cryopreservation technique for C. gigas. The post-thaw survival rate has been improved to > 80% when 18 h PF larvae were cryopreserved with 10% EG + 5% FIC + 0.2% PVP. Larvae at different developmental stages have various ability for cryoresistance, therefore, selection of suitable developmental stage is important for the success of cryopreservation (Paredes et al., 2012, 2013; Liu et al., 2020). In this study, larvae collected at 18 h PF had the highest resistance to cryoinjury, with about 70% post-thaw D-larval rate achieved. This collection time was later than the 14 h PF reported by Paredes et al. (2013) on the same species. Prior to natural feeding, bivalve embryos and early stage larvae normally have a high level of lipid as an energy reserve for development (Goslling, 2015). Larvae with high lipid content would be more sensitive to cryopreservation and this phenomenon has been shown in pigs (Dobrinsky, 2002), cattle (Massip, 2001; Pryor et al., 2011), sheep (Massip, 2001) and mussels (Liu et al., 2020). The cryoprotective medium containing both permeable and nonpermeable CPAs is common for larval cryopreservation (Saragusty and Arav, 2011; Paredes et al., 2012, 2013). The most dominant permeable CPA used in C. gigas is EG (Paredes et al., 2013; Labbé et al., 2018). On
3.3. Comparison of different CPAs on post-thaw D-larval rate
D-larval rate (%)
100.0 A
80.0 A
60.0 B
40.0 B
20.0 0.0 10 h
15 h 18 h Post fertilization hours
21 h
Fig. 1. Post-thaw D-larval rates (%) when larvae were cryopreserved at different larval developmental stages, n = 3. Different letter indicates significant difference (P < .05). All the data have been normalized to the controls. 3
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Table 1 Comparison of survival rates and relative mortality rate between progenies produced from fresh and cryopreserved larvae in C. gigas, n = 3. Different letter indicates significant difference (P < .05). Days post fertilization
Survival rate (%)
Relative mortality rate (%)
Fresh larvae Day Day Day Day
1 (D larvae) 8 (umbo larvae) 22 (eyed larvae) 27 (spat)
82.0 46.7 33.7 18.3
± ± ± ±
2.2 7.5 4.5 4.0
A A′ A″ A‴
Cryopreserved larvae
Fresh larvae
Cryopreserved larvae
60.9 ± 7.1 B 14.9 ± 3.0 B′ 7.5 ± 2.2 B″ 4.9 ± 1.3 B‴
43.0 ± 9.5 b 26.4 ± 16.3 a′ 45.0 ± 12.8 a″
75.6 ± 2.7 a 46.9 ± 26.3 a′ 33.5 ± 2.4 a″
Acknowledgments
600
Shell length (µm)
500
This research was funded by the Talent Project of Revitalizing Liaoning (Project No. XLYC1807087), Department of Ocean and Fisheries of Liaoning (Project No. 201829), China Scholarship Council and South Australian Research and Development Institute. We thank Mr. Gary Zippel of Zippel Enterprises Pty Ltd. for the provision of Pacific oyster broodstock.
Fresh larvae
400
Cryopreserved larvae
300 200 100 0 D larvae
umbo larvae
eyed larvae
Declaration of Competing Interest
spat
Larval developmental stages
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Fig. 4. Shell length (μm) of D-larvae, umbo larvae, eyed larvae and spats produced from fresh and cryopreserved larvae, n = 90.
References
the contrary, only a few non-permeable CPAs, such as polyvinylpyrrolidone (PVP) and trehalose, have been evaluated on their suitability in oyster larval cryopreservation (Paredes et al., 2013). In our recent study in M. galloprovincialis, the application of non-permeable CPAs, FIC and PVP, has improved the post-thaw larvae survival rates, which could be further enhanced by applying FIC and PVP together (Liu et al., 2020). A similar trend has been reported in livestock species (Saha et al., 1996; Titterington and Robinson, 1996). This was also confirmed in the current study, where the post-thaw D-larval rate was improved to > 80% when both PIC and PVP were applied together (Fig. 3). This result indicates that the combined effects of these two CPAs could play a key role in protecting larvae from cryoinjury in C. gigas. Increase in initial larval density would increase the efficiency of application of cryopreservation technique in breeding programs and hatchery production if a similar post-thaw survival rate can be achieved (Tervit et al., 2005). In this study, the initial larval density of 1 × 106 mL−1 achieved the similar post-thaw D-larval rate as the density of 4 × 105. The former is close to the highest larval density that could be achieved in this study. No significant difference between fresh and cryopreserved larvae was found in their shell length at any ages evaluated (Fig. 4). Although the survival rates were significantly lower in the cryopreserved than those in the fresh larvae (Table 2), the relative mortality rates after day 8 PF remained similar (P > .05) between them (Table 2). These results indicate that cryopreservation may mainly affect the larval performances at the early developmental stage in C. gigas. Similar phenomenon has also been observed by Labbé et al. (2018) in the same species and our study on oocyte and larval cryopreservation in M. galloprovincialis (Liu and Li, 2015; Liu et al., 2020). In conclusion, the larval cryopreservation technique developed for C. gigas has achieved the post-thaw D-larval rate > 80%. The cryodamage was mainly expressed at early developmental stages, as relative mortality rates remained similar after day 8 PF between progenies produced from fresh and cryopreserved larvae. Thus, this technique could be applied for all year round supply of larvae for hatchery productions and assist in superior genetic preservation for breeding programs in oyster industry.
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