Fish Sci (2014) 80:1257–1267 DOI 10.1007/s12562-014-0796-9
ORIGINAL ARTICLE
Aquaculture
The development of oocyte cryopreservation techniques in blue mussels Mytilus galloprovincialis Hanru Wang • Xiaoxu Li • Meiqing Wang Steven Clarke • Mark Gluis
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Received: 22 December 2013 / Accepted: 29 March 2014 / Published online: 28 August 2014 Ó Japanese Society of Fisheries Science 2014
Abstract Reliable techniques for the cryopreservation of both sperm and oocytes of the blue mussel Mytilus galloprovincialis Lamarck would increase the availability of seed supplies out-of-season and enhance efficiency in selective breeding. We have investigated the optimal cryotechnique for blue mussel oocytes. The toxicity of three cryoprotective agents (CPAs) [dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG)] at different concentrations (1–5 M) and exposure times (0.25–30 min) were investigated for mussel oocytes at room temperature (20 °C) or on ice. The same CPAs (1, 1.5 and 2 M) as well as three different cryoprotectant mixtures [1.5 M EG ? 0.2 M trehalose ? 100 % Milli-Q water (EGTM); 1.5 M EG ? 0.2 M trehalose ? 75 % Milli-Q water ? 25 % seawater; 1.5 M EG ? 0.2 M sucrose ? 100 % Milli-Q water] were tested by comparing the postthaw oocyte fertilization rate after using the slow-cooling method. Vitrification was also examined; however, this method failed to produce any post-thaw surviving oocytes. Among the tested CPAs, EG was the least toxic to oocytes. There was a tendency for the equilibration of CPAs on ice to achieve a higher oocyte fertilization rate compared with that at room temperature, and this difference was
H. Wang M. Wang Dalian Ocean University, Dalian, China H. Wang School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, Roseworthy, SA, Australia X. Li (&) S. Clarke M. Gluis South Australia Research and Development Institute, West Beach, SA 5024, Australia e-mail:
[email protected]
significant at concentrations of 3 and 4 M (P \ 0.01). The DMSO, EG and PG treatments all resulted in post-thaw fertilization, with EGTM achieving the highest number of surviving oocytes (32 %). At the optimal seeding temperature (-7 °C), the addition of 0.2 M trehalose to EG resulted in a better fertilization rate of post-thawed oocytes than the addition of 0.2 M sucrose. All of the treatments evaluated produced D-larvae from post-thawed oocytes, although the rates were low. Keywords Slow-cooling method Vitrification Cryoprotectant Oocytes Cryopreservation
Introduction The application of gamete/embryo cryopreservation technologies has been demonstrated for some aquatic species to play a major role in seed production, genetic improvement programs and the conservation of natural resources. Whittingham et al. [1] were the first to successfully cryopreserve embryos of any animal species. Over the years, there have been several published papers on fish embryo cryopreservation [2, 3] and on a range of studies on the cryopreservation of bivalve molluscs embryos/larvae [4– 10]. To date, however, only three studies have been published on oocyte cryopreservation in marine organisms [11–13]. The first successful cryopreservation of oocytes of aquatic species was that of Pacific oyster Crassostrea gigas oocytes in 2005 using the slow-cooling method[11]. In that same year, Hamaratoglu et al. [12] used another method, denoted vitrification, to cryopreserve starfish oocytes. In 2009, Adams et al. [13] applied the slow-cooling method to cryopreserve greenshell mussel (Perna canaliculus)
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oocytes. Unfortunately, there was a very low production of early stage progenies in these last two studies. Slow-cooling cryopreservation is a commonly recognized method that uses a lower concentration of cryoprotectant agent (CPA; 1–2 M) and gradually decreases the temperature to a predetermined subzero temperature prior to plunge the treated oocyte into -196 °C liquid nitrogen (LN2). The vitrification method, in comparison, uses a higher concentration of CPA (3–5 M) and a very high cooling speed, which is achieved by placing the cryopreservation materials directly into LN2 [9, 14]. Several studies have demonstrated that the application of the slow-cooling technique to bivalve oocytes is species specific, while the vitrification method has yet to be evaluated [11–13]. In comparison with other biological materials, such as sperm, embryos, etc., oocytes are more difficult to cryopreserve because of their large size, low surface area to volume ratio, high water content and low hydraulic conductivity [15]. The survival of cryopreserved oocytes could also be affected by other factors, such as CPAs, cryopreservation procedures and their combinations. Studies of the toxicity of CPAs on embryos/oocytes have been published for some marine species (fish, starfish, and bivalves) [16–22]. Commonly used CPAs for marine species include glycerol, 1,2-propanediol, ethylene glycol (EG), propylene glycol (PG), dimethyl sulfoxide (DMSO), and methanol, with one or more supplements (i.e. trehalose, fructose, glucose, galactose, sucrose, polyvinylpyrrolidone and polyethylene glycol) [19–23]. The toxicity of CPAs to oocytes/embryos is dependent on type and concentration, as well as the temperature and duration of exposure [9]. The blue mussel Mytilus galloprovincialis Lamarck is one of the most important commercial aquaculture bivalve species globally and also a common bio-indicator used in environmental monitoring programs [24–27]. The development of reliable cryopreservation techniques for this species would therefore provide opportunities for out-of-season seed supplies and enhance the capacity and efficiency of genetic improvement and coastal environmental monitoring programs. In this study the key factors and procedures critical to the establishment of oocyte cryopreservation were studied, including: (1) CPA toxicity investigations at two temperatures [20 °C (room temperature) and on ice]; (2) the effectiveness of stepwise increases in CPA concentrations in reducing CPA toxicity to mussel oocytes; (3) optimization of seeding temperature; (4) evaluation of key steps affecting oocytes cryopreservation with the slow-cooling method; (5) cryopreservation using the vitrification method.
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Materials and methods Gamete supply The mussel broodstock used in this study were originally from a commercial aquaculture venture in Port Lincoln, South Australia and were transported in a refrigerated container overnight to the South Australian Research and Development Institute, West Beach, South Australia. On arrival they were washed with filtered seawater (pore size 25 lm) prior to being used for spawning. The mussels were spawned individually in 1-l beakers at a water temperature of 18–20 °C and salinity of 37 %. The gamete quality was evaluated under a light microscope at 4009 magnification. Males with active sperm of [80 % and females with healthy looking oocytes were chosen for the subsequent experiments. In each experiment, oocytes from at least five individuals were pooled and sperm from at least three individuals were pooled. To separate debris from the gametes, we washed oocytes through a 90-lm screen and collected them on a 25-lm screen, whereas sperm were filtered through a 15-lm screen and stored in a refrigerator. The density of oocytes was then calculated and standardized at 1 9 106 oocytes/ml. Cryoprotective agents All CPA stock solutions used in this study (DMSO, EG, PG—with or without sugar) were prepared in Milli-Q water (or 75 % Milli-Q water with 25 % filtered seawater in some treatments in Experiment D) at a concentration double the final requirement for 1 and 2 M treatments. CPA solutions were then mixed with oocyte suspensions at a 1:1 ratio (v/v). To achieve final concentrations of C3 M, the original concentration of the CPA was made up to the respective target concentration (3, 4 or 5 M) ? 11 % (single-step loading) or 5 % (two-step loading). CPA solutions were then mixed with oocyte suspensions at a 1:10 ratio (v/v) to achieve the required experimental chemical concentrations. A mixing ratio of 1:10 instead of 1:1 to achieve final concentrations of C3 M could also potentially avoid the osmotic pressure from the CPA. All chemicals used in this study were purchased from Sigma Aldrich Pty Ltd (Castle Hill, NSW, Australia). Experiments This study consisted of three sections in which we examined different aspects of the cryopreservation of blue mussel oocytes: Section 1, the toxicity of CPAs on blue mussel oocytes; Section 2, cryopreservation of blue mussel oocytes using the slow-cooling method; Section 3,
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cryopreservation of mussel oocytes with the vitrification method. Section 1: toxicity of CPAs on blue mussel oocytes Two experiments (A, B) were conducted to evaluate the toxicity effects of one-step loading of a single CPA and two-step loading of CPAs, respectively, at increasing concentrations on the fertilization rate of blue mussel oocytes after various exposure periods at room temperature (20 °C) or on ice. After being exposed to a CPA for a predetermined period, oocytes were diluted 10-fold with filtered seawater for 20 min and then poured onto a 25-lm screen and rinsed with filtered seawater. The oocytes were then fertilized with freshly collected sperm at a ratio of 1:10 (oocyte:sperm) and left standing for 20 min, following which the fertilized oocytes were rinsed with filtered seawater and transferred into a petri dish. At 1.5 h post-fertilization, a subsample of the fertilized oocytes was viewed under the microscope, and at least 100 randomly selected oocytes were used for assessing survival. Oocytes that had developed into at least two cells were assessed as being fertilized and alive. The survival rate in each replicate was then calculated as the proportion of fertilized oocytes in all the oocytes assessed. In these experiments, each treatment was replicated three times. The controls were established using oocytes collected from the same batch of mussels in the same experiment but not exposed to CPA treatments. They were fertilized using the same techniques as those in the respective CPA treatment groups. Experiment A: effects of single CPAs on oocyte fertilization rates In this experiment, the following parameters were investigated in petri dishes: three CPAs (DMSO, EG, and PG), five final concentrations (1, 2, 3, 4 and 5 M), two temperatures [room temperature (20 °C) or on ice] and nine CPA exposure periods (0.25, 0.5, 0.75, 1, 5, 10, 15, 20 and 30 min), at a standard oocyte concentration of 0.5 9 106 oocytes/ml. In the treatments where the final CPA concentrations were B2 M, the exposure duration was 1, 5, 10, 15, 20 or 30 min; in those treatments where the final CPA concentrations were C3 M, the duration was 0.25, 0.5, 0.75, 1, 5, 10, 15, 20 or 30 min. Experiment B: effect of two-step loading of a high concentration of EG on the fertilization rate of post-thawed oocytes The two-step loading procedures evaluated in this experiment were all carried out on ice. The required low (2 M) and high (3 or 4 M) final CPA concentrations were achieved following the methods described in section ‘‘Cryoprotective agents’’. To facilitate description, we developed abbreviations for each treatment in which DMSO, EG and PG were further shortened to D, E and P,
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respectively, and a number indicated the concentration (in moles). For example, 2D ? 3E represents 2 M DMSO used in the first step and 3 M EG used in the second step. Exposure period for the first step was 5 min, and up to 1-min exposure periods (0.25, 0.5, 0.75, or 1 min) were used at the second step. Section 2: Cryopreservation of mussel oocytes with the slow-cooling method The effects of different CPA solutions and seeding temperatures on the fertilization rate of post-thawed oocytes and the development of resultant progenies were evaluated in this section using the slow-cooling oocyte cryopreservation method published for the Pacific oyster and greenshell mussel [11, 13]. After being mixed with a CPA solution, the oocytes were placed on ice for 10 min, during which time the oocyte suspension was loaded into 0.25 ml French straws (IMV Technologies, Paris, France), which were then put into a CL863 programmable freezer (Cryologic P/L, Mulgrave, VIC, Australia) and held at 0 °C for another 10 min. The freezer was programmed to cool at a rate of -1 °C min-1 to a predetermined seeding temperature (-5, -7, -10 or -12 °C) and then to hold the target temperature for 10 min during which time the straws were touched with a LN2cooled cotton bud (seeding stage). The straws were then further cooled at a rate of -0.4 °C min-1 to the target temperature of -33 °C, held for 5 min and then plunged into LN2 and stored for at least 2 h. After being thawed in water at 26 °C for 5 s the content of one straw was emptied into a petri dish and diluted with 6 ml filtered seawater [12]. The thawed oocytes were left in the petri plate for a further 30 min before being fertilized at a oocyte:sperm ratio of 1:10. The fertilization rate was calculated as previously described in Section 1. The fertilized oocytes were transferred to larval rearing beakers containing 500 ml filtered seawater and incubated for 36–48 h at room temperature (approx. 20 °C). Control replicates were established from oocytes that were also loaded and unloaded from straws, and handled and reared as per the methods used for the treatment groups, except that they had not been through the freezing process. Experiment C: effects of CPA type and concentration on the fertilization rate of post-thawed oocytes DSMO, EG and PG were evaluated at the final concentrations of 1, 1.5, 2 M with or without 0.2 M trehalose to compare their cryoprotective effects on mussel oocytes. The seeding temperature was standardized at -7 °C. The CPA concentrations were determined based on the results of Experiment A and of published studies on bivalve oocytes, embryos and larvae [4–11, 13, 28].
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Experiment D: effects of seeding temperature and different CPA solutions on the fertilization rate of postthawed oocytes The CPA solution (1.5 M EG with 0.2 M trehalose) resulting in the highest fertilization rate of postthawed oocytes in Experiment C was modified to investigate whether further improvement in the fertilization rate of post-thawed oocytes could be achieved by adding 25 % seawater or replacing trehalose with sucrose. The CPA solutions used in this experiment included: (1) 1.5 M EG ? 0.2 M trehalose ? 100 % Milli-Q water (EGTM); (2) 1.5 M EG ? 0.2 M trehalose ? 75 % Milli-Q water ? 25 % seawater (EGTMS); (3) 1.5 M EG ? 0.2 M sucrose ? 100 % Milli-Q water (EGSM). At the same time the effects of different seeding temperatures on the fertilization rate of post-thawed oocytes were also evaluated by seeding straws at -5, -7, -10 and -12 °C, respectively. Experiment E: effects of key freezing steps (after seeding at -7 °C, at -33 °C and in LN2) on the fertilization rates and subsequent D-larval yields Based on the results of Experiment D, the seeding temperature of -7 °C was selected in this experiment. The effect of the three CPA solutions (EGTM, EGTMS, EGSM) used in Experiment D were further evaluated at each key freezing step (after seeding at -7 °C, at -33 °C, and in LN2) to determine the steps that caused the most damage in terms of D-larval yields. The D-larval yield was determined as the percentage of fertilized oocytes developing into D-larvae. Section 3: Cryopreservation of mussel oocytes with vitrification method In this section only one experiment (Experiment F) was conducted and two methods were used to load high concentrations of CPAs. In the first method, the oocytes were directly exposed to 3 M EG for \1 min or 4 M for \0.75 min on ice. In the second method, the oocytes were exposed to a 2 M CPA (2 M DMSO, 2 M EG or 2 M PG) for 5 min and then to 3 M EG or 4 M EG for 1 or 0.75 min. Within this period each suspension of CPA oocytes was loaded into a pulled straw (0.25 mm in diameter) and plunged into LN2 directly. The experimental procedures from thawing and subsequent fertilization were the same as previously described for the slow-cooling method in Section 2. Statistics The percentage data were arcsine-transformed, and analyses undertaken using SPSS version 17.0 software (SPSS Inc., Chicago, IL). Untransformed data were used in figures and expressed as the mean ± standard deviation (SD). Experiment A was analyzed using a four-way analysis of variance (ANOVA; CPA loading type, exposure duration,
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CPA concentration and temperature); Experiment B, using a three-way ANOVA [CPA loading type, exposure duration and CPA concentration (3 or 4 M)]; Experiment C, using a three-way ANOVA [CPA loading type (with or without 0.2 M trehalose), CPA concentration]; Experiment D, using a two-way ANOVA (CPA solution and seeding temperature); Experiment E, using a one-way ANOVA. When significant differences were found, the Student– Newman–Keuls multiple range test (SNK) was used to compare means. The significant level was set at P \ 0.05.
Results Experiment A: Effects of single CPAs on oocyte fertilization rates The overall toxicity of the three CPAs was EG \ DMSO \ PG at the same concentration evaluated in this study. The toxicity increased with an increase in either exposure period from 0.25 to 30 min at the same CPA concentration, or with increasing concentration from 1 to 5 M in the same CPA. Mussel oocytes could not survive in 5 M concentration for all three CPAs. The four-way ANOVA showed that the overall trend of fertilization rates at the low temperature (on ice) were higher than, but not significantly different from, those at room temperature (20 °C) (P [ 0.05) in 1 and 2 M CPAs (Fig. 1a, b). However, when the CPA concentrations increased to 3 and 4 M, fertilization rates were significantly higher at low temperature (on ice) than at room temperature (20 °C) (P \ 0.05; Fig. 1c, d). Therefore, only on-ice data are presented in the following sections, although data from both on-ice and room temperature treatments are used in the relevant figures. The analyses showed that the fertilization rates of oocytes exposed to 1 M of CPA (DMSO, EG or PG) for 1 min were not significantly different from those of the control (90.1 ± 2.7 %). When the period of exposure was extended to 30 min, the fertilization rates decreased significantly to about 73, 58 and 47 % in 1 M EG, DMSO and PG, respectively (Fig. 1a). At a CPA concentration of 2 M there was a significant difference in fertilization rates between the control oocytes and those exposed to DMSO and EG for [5 min and to PG for [1 min (P \ 0.05; Fig. 1b). However, the oocytes exposed to 2 M DMSO and EG for 20 min still yielded fertilization rates of about 75 and 65 %, respectively; the fertilization rate was 45 % when the exposure period was further extended to 30 min. Mussel oocytes were relatively more sensitive to 2 M PG, with fertilization rates dropping from 83 % after 1 min of exposure to 18 % after 30 min.
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Fig. 1 Fertilization rates (%) of mussel oocytes exposed to different cryoprotective agents [CPAs: dimethyl sulfoxide (DMSO), ethylene glycol (EG) propylene glycol (PG)] at concentrations of 1 M (a), 2 M (b), 3 M (c) or 4 M (d), respectively, for 1, 5, 10, 15, 20 or 30 min at either room temperature (20 °C; temperature not indicated in treatment codes) or on ice (as indicated). In all figure parts, the bars represent the mean ± standard deviation (SD) of n = 3 measurements. Bars sharing the same small or capital letter within each exposure time do not differ significantly from each other in the treatments on ice or at room temperature, respectively, at P \ 0.05
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At 3 and 4 M CPA concentrations, the fertilization rates were all significantly lower than those of the controls (90.1 ± 2.7 %) (P \ 0.01; Fig. 1c, d), even when the exposure period was only 0.25 min. The highest tolerance
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fertilization rate at 0.75 min which then dropped quickly to 39 % after 1 min, and those exposed to 3 M PG had a 47 % fertilization rate after 0.25 min, but this had dropped to only about 20 % by 1 min. When oocytes were exposed to 4 M of the respective CPA, their fertilization rates were very low; at an exposure period of 0.25 min, the fertilization rate was 23 % in EG and \5 % in the other two CPAs. Experiment B: The effect of two-step loading of EG at a high concentration on oocyte fertilization rates The three-way ANOVA showed that when 3 M EG was used at the second step (Fig. 2a), similar fertilization rates were obtained for all treatments, although the 2P ? 3E treatment yielded numerically the lowest fertilization rates for exposure periods of up to 0.75 min in duration. Both the 2D ? 3E and 2E ? 3E treatments yielded 70–80 % fertilization rates within 0.5 min, which dropped to around 50 % at 1 min. When oocytes were exposed to 4 M EG at the second step (Fig. 2b), all two-step treatments showed significantly higher fertilization rates than following exposure to the equivalent CPA in the one-step treatments. In addition, the fertilization rates in treatment groups were all significantly lower than those of the control (P \ 0.05). The 2E ? 4E and 2P ? 4E treatments yielded about 40–50 % fertilization rates within a 0.5-min exposure time, rapidly dropping thereafter to around 10 % within 1 min. Experiment C: Effect of CPA type and concentration on fertilization rate of post-thawed oocytes The three-way ANOVA on post-thawed fertilization rate showed that the use of 0.2 M trehalose did not improve the cryoprotective effects of DMSO and EG at the three CPA concentrations tested. For PG, however, the results were different depending on the concentration of PG: at 1 M PG,
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the addition of 0.2 M trehalose to the treatment resulted in a higher fertilization rate of post-thawed oocytes than without, while at 1.5 M PG, the addition of 0.2 M trehalose resulted in a significantly lower fertilization rate of postthawed oocytes than without (Fig. 3). In this experiment the oocytes could not survive all 2 M CPAs tested. Of the three concentrations evaluated, 1.5 M resulted in significantly better protection than other two concentrations (1 and 2 M). The 1.5 M EG with or without 0.2 M trehalose and 1.5 M PG without trehalose resulted in about a 30 % fertilization rate of post-thawed oocytes. Experiment D: Effect of seeding temperature and different CPA solutions on the fertilization rate of post-thawed oocytes The two-way ANOVA revealed that the interaction between seeding temperature (-5, -7, -10 or -12 °C) and CPA solutions (EGTM, EGTMS or EGSM) significantly affected the fertilization rate of post-thawed oocytes (P \ 0.05; Fig. 4a). Seeding at -7 °C resulted in a slightly higher fertilization rate than seeding at -12 °C, which in turn was better than seeding at -5 and -10 °C. The fertilization rates of post-thawed oocytes in all treatments were significantly lower than those of the control (89.0 ± 5.6 %). At -7 °C seeding temperature, the EGTM solution achieved the highest fertilization rate of post-thawed oocytes (32 %; Fig. 4a). Experiment E: Effects of key freezing steps (seeding after -7 °C, at -33 °C and in LN2) on fertilization and D-larval yield rates The one-way ANOVA demonstrated that the fertilization rate of post-thawed oocytes after experiencing freezing at -7 °C and then -33 °C and finally emersion in LN2 were all significantly lower than that the control group
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Fig. 2 a Fertilization rates (%) of mussel oocytes on ice (ice exposure treatment) to 2 M of the respective CPA(s) [EG (E), DMSO (D) and/or PG (P)] for 5 min, following by exposure to 3 M EG for 0.25, 0.5 or 1 min, or to 3 M EG directly for 0.25, 0.5 or 1 min. b Fertilization rates (%) of mussel oocytes on ice to 2 M of the respective CPA(s) (EG, DMSO and/or PG) for 5 min, followed by exposure to 4 M EG for 0.25, 0.5 or 1 min, or to 4 M EG directly for 0.25, 0.5 or 1 min. Bars represent the mean ± SD (n = 3). Bars sharing the same letter within each exposure period do not differ significantly from each other at P \ 0.05
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Fig. 3 Fertilization rate of postthawed oocytes (%) of mussel oocytes cryopreserved with the slow-cooling method in different CPA combinations: EG, PG and DMSO at 1, 1.5 or 2 M; with (?) or without (-) 0.2 M trehalose. Bars represent the mean ± SD (n = 3). Bars sharing the same letter within each CPA concentration do not differ significantly from each other at P \ 0.05
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(P \ 0.05; Fig. 4b). Furthermore, freezing to -7 °C resulted in significantly higher fertilization rates (67 %) than freezing to -33 °C (37.9 ± 4.0 %) and emersion in LN2 (32.0 ± 5.6 %), with the latter two treatments significantly different from each other (P \ 0.05; Fig. 4b).
The one-way ANOVA indicated that the D-larval yields from oocytes frozen with EGTM to -7 and -33 °C and in LN2 were all significantly lower than those of the control (48.8 ± 7.0 %) (P \ 0.05; Fig. 4c). D-larval yields at -7 °C (about 20 %) were significantly higher than those at
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Fig. 4 a Fertilization rate of post-thawed oocytes (%) of mussel oocytes cryopreserved in different CPA solutions (EGTM, EGTMS and EGSM) at different seeding temperatures (-5, -7, -10 and -12 °C). Bars sharing the same letter within each seeding temperature do not differ significantly from each other at P \ 0.05. b Fertilization rate of postthawed oocytes (%) of mussel oocytes after oocytes were frozen in EGTM to -7 °C (after seeding), then to -33 °C and finally plunged into liquid nitrogen (LN2). Bars sharing different letters differ significantly from each other at P \ 0.05. c Post-thaw D-larval yield rates (%) after oocytes were frozen to -7 °C (just after seeding), then to -33 °C (before being plunged into LN2) and storage in LN2 (EGTM). Bars sharing different letters differ significantly from each other at P \ 0.05. EGTM 1.5 M EG ? 0.2 M trehalose ? 100 % Milli-Q water, EGTMS 1.5 M EG ? 0.2 M trehalose ? 75 % Milli-Q water ? 25 % seawater, EGSM 1.5 M EG ? 0.2 M sucrose ? 100 % Milli-Q water. In all figure parts, the bars represent the mean ± SD of n = 3 measurements.
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-33 °C (about 10 %), which in turn were significantly higher than when LN2 was used (\1 %). Experiment F: Cryopreservation of mussel oocytes with the vitrification method No fertilization occurred in oocytes cryopreserved using the vitrification method.
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Discussion The aim of the CPA toxicity experiments (Experiments A and B) was to select CPAs that would be suitable to cryopreserve blue mussel oocytes using either the slowcooling or vitrification methods. The fertilization rate of blue mussel oocytes treated with the CPAs evaluated in this study decreased with increases in CPA concentration and
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duration of exposure (Fig. 1). These results are similar to those reported in previous studies showing that CPA toxicity was concentration- and exposure period-dependent— the higher the CPA concentration and the longer the exposure period, the lower the oocyte survival rate [16– 20]. The CPA exposure treatments performed on ice tended to result in higher fertilization rates than their counterparts carried out at room temperature (20 °C). This was particularly evident when high CPA concentrations (3 or 4 M) were applied. This trend was consistent with that observed in a previous study, where the authors found that an exposure temperature of 4 °C produced higher viability than one at 26 °C in the copepod Acartia tonsa (Dana) oocytes challenged with different CPAs [22]. In our study, low CPA concentrations of 1 and 2 M CPA were evaluated at long exposure times, a standard procedure used by others who have applied the slow-cooling method [5, 9–11, 13]. Alternatively, the higher CPA concentrations and relatively short duration times used in our study have been successfully applied for the vitrification of livestock embryos at \1 min exposure periods [29–31]. Our experiments revealed that at the same concentration and duration of exposure, EG was the least toxic of the three CPAs tested, followed by DMSO and PG. Importantly, at a concentration of 1 M and 2 M, all three of these CPAs achieved a 75 % (Fig. 1a) and 65 % (Fig. 1b) fertilization rate, respectively, in the 20-min exposure treatment; these rates are suitable for optimizing subsequent cryopreservation steps. In Experiment C, these concentrations were further refined to 1.5 M, which is consistent with other slow-cooling studies [5, 10, 11, 13]. The CPAs at 3 and 4 M concentrations all resulted in a significantly lower fertilization rate than the controls. Among these, EG showed the least toxicity to oocytes, followed by DMSO. Mussel oocytes were unable to tolerate exposure to 5 M CPAs. It was anticipated that oocyte tolerance to higher CPA concentrations could be improved by two-step loading methods as this method has been used in a number of vitrification studies in oocytes and embryos [29–31]. Our results show that loading 2 M DMSO or EG as the first step, followed by 3 M EG for 0.5 min achieved a higher fertilization rate, 80–90 %, in comparison with one-step direct 3 M EG loading (approx. 64 %). However, when the exposure duration was extended to 1 min, differences in the fertilization rate, which dropped to about 50 %, were insignificant between the one-and two-step loading methods (Fig. 2a). The advantage with two-step loading was further demonstrated when 4 M EG was applied in the second step, which resulted in a 45 % fertilization rate after a 0.5-min exposure, whereas only a 20 % fertilization rate was achieved with one-step loading in 4 M EG for 0.25 min (Fig. 2b).
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The slow-cooling protocol used to optimize CPAs in blue mussel oocytes was initially based on results from our previous investigation on the cryopreservation of blue mussel D-larvae [5] and from another published study on Pacific oysters [11], including a -7 °C seeding temperature, -1 and -0.4 °C min-1 cooling speeds for pre- and after seeding temperature, respectively, and the use of 0.2 M trehalose. Of the CPAs tested, treatment with 1.5 M EG ? 0.2 M trehalose achieved by far the highest fertilization rate of post-thawed oocytes (32.0 ± 6.9 %), followed by that with 1.5 M PG without sugar, with a fertilization rate of postthawed oocytes of 30.9 ± 3.7 %. The addition of sugar to the EG treatment at a 1.5 M concentration did not significantly affect the fertilization rate of post-thawed oocytes, when added to PG it did significantly reduced the postthaw fertilization. These results suggest that the addition of trehalose impacts the cryoprotective functions of various CPAs differently. With respect to the effect of trehalose, our results are similar to those of a cryopreservation study by Adams et al. [13] on greenshell mussel oocytes, but differ from those reported by Tervit et al. [11] in their study of Pacific oyster oocytes where the addition of trehalose was found to be unfavorable. The use of the DMSO, EG and PG as CPAs in our study resulted in fertilization rates of post-thawed oocytes that differ from those reported by Adams et al. [13]. Using the slow-cooling method, the latter authors found that DMSO and PG were detrimental to greenshell mussel oocytes. Our results are, however, similar to those reported by Wang et al. [5] on the cryopreservation of blue mussel D-larvae. These authors found that DMSO, EG and PG protected blue mussel D-larvae from cryo-injury, with 5 % DMSO with or without 0.2 M trehalose being the optimal treatment. Taken together, these results reveal that the protection effects of these three CPAs are species- and development stage-dependent. Detailed investigation of seeding temperature, sugar type and salinity on the fertilization rate of post-thawed oocytes revealed that -7 °C was the best seeding temperature for oocyte cryopreservation in blue mussels (Fig. 4a). This result is consistent with the results of larval cryopreservation in the same species [13]. However, in Pacific oysters, -12 and -10 °C are considered to be the optimal seeding temperature for embryos and oocytes, respectively [11, 32]. In our study, the highest fertilization rate of post-thawed oocytes was achieved with the CPA prepared in fresh water (1.5 M EG ? 0.2 M trehalose ? 100 % Milli-Q water), which agrees with the results of Stachecki et al. [33] and Tervit et al. [11]. Stachecki et al. [33] reported the detrimental effects of sodium during mouse oocyte cryopreservation, and Tervit et al. [11] reported the use of Milli-Q
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water rather than seawater as a base medium significantly improved the fertilization rate of post-thawed oocytes in Pacific oysters. We observed that the addition of 0.2 M trehalose to EG at the optimal seeding temperature (-7 °C) resulted in a better fertilization rate of post-thawed oocytes than that of 0.2 M sucrose. However, this result should be interpreted cautiously as no difference was found between sugar types at other seeding temperatures. Our detailed cryo-damage investigation at each cryopreservation step revealed that the fertilization rate of the post-thawed oocytes decreased with a decrease in cooling temperature, dropping from 70 % at -7 °C to 40 % at -33 °C to 32 % following emersion in LN2 (Fig. 4b). Clearly, the LN2 treatment had little additional effect on oocyte survival when compared to the -33 °C treatment. D-larval yields followed a similar trend as the fertilization rate of post-thawed oocytes, decreasing from 25 % after seeding at -7 °C to 10 % at -33 °C to \1 % following emersion LN2 (Fig. 4c). To the contrary, D-larval yield rate for greenshell mussels dropped to \1 % after seeding at -10 °C in the study of Adams et al. [13], suggesting that greenshell mussel oocytes are more sensitive to subzero temperature decreases. To date, only Pacific oyster oocytes have been successfully cryopreserved, and attempts to cryopreserve mussel oocytes have resulted in low D-larval yields, although the cryopreservation protocols optimized in this study and that by Adams et al. [13] were similar to those used in oysters [11]. Reasons for the low production of post-thaw D-larvae in mussels remain largely unexplored. In terms of oocyte volumes, mussel oocytes are much larger than those of Pacific oysters, and it is known that the volume of water in oocytes plays a pivotal role in cryopreservation [34]. The formation of ice from water during freezing is believed to cause damage to both internal oocyte organelles and membrane systems. Water exosmosis could avoid ice formation inside of the oocytes, thus preventing damage from ¨ zkavukcu and Erdemli [34] calculated the rate of ice. O exosmosis of water during freezing using four equations, summarizing the optimum conditions of cryopreservation to avoid the potential intracellular ice damage to embryos, which have a low surface area to volume ratio, low water permeability and slow-cooling rates. The diameter of blue mussel oocytes has been reported to be about 68 lm [35], which is consistent with our observations, and much larger than the 46 lm reported for Pacific oysters [36]. As such, the ratio of surface area to volume of mussel oocytes would be smaller than that for oysters, resulting in the water permeability of mussel oocytes being lower than that of oyster oocytes. Therefore, mussel oocytes should be subjected to a slower cooling rate than oyster oocytes. However, in the current study, mussel oocytes were programmed to cool at a rate of -1 °C min-1 to a
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predetermined seeding temperature (-5, -7, -10 or -12 °C) followed by the rate of -0.4 °C min-1 to -33 °C. These freezing rates are not much different from those reported as optimal for Pacific oyster oocytes (-1 °C min-1 to -10 °C followed by -0.4 °C min-1 post-seeding [11]). Therefore, the low production of D-larvae in our study might be the result of an inappropriate freezing rate that led to the damage caused by ice during cryopreservation. Future research is required to investigate the effect of freezing rate on mussel post-thaw fertilization and D-larval production. Vitrification is the rapid transformation of a substance into a glass-like solid without the formation of ice crystals during the cryopreservation process. This method normally requires plunging prepared biological samples into LN2 directly and may thereby offer some practical benefits in cryopreservation, including saving time and eliminating the need for an expensive programmable freezing machine. This method has been applied routinely in the cryopreserving of mammal oocytes from a variety of domestic and laboratory species [30, 37]. However, when this method was applied in our study, no mussel oocytes survived the vitrification treatment, which is in agreement with previously reported findings in fish species [3]. Mussel oocytes are highly sensitive to high concentrations of the CPA. A 0.5-min exposure to C4 M CPA concentrations resulted in very low survival rates (\5 %); exposure to 4 M is the lowest concentration used to cryopreserve oocytes in livestock and laboratory animals [37, 38]. In addition, a 0.5-min exposure period might not be enough for the CPA to equilibrate, which could therefore reduce its protective capacity during vitrification. Vitrification with the open pulled-straw technique was used in our study as it enabled a slightly larger volume of solution to be used in comparison to the nylon cryoloop [30] and metal mesh [29] techniques. However, the open pulled-straw technique might compromise the freezing speed more than the other two methods. Therefore, further research should focus on those methods that will increase both the freezing speed and CPA tolerance, including CPA loading methods to reduce the cryo-damage by vitrification and the toxicity of CPAs on mussel oocytes. Acknowledgments The authors would like to thank Assoc. Prof. David Stone and Dr. Ron Smernik for their assistance in proofreading this manuscript.
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