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Cryopreservation of sperm of the Pacific oyster (Crassostrea gigas): development of a practical method for commercial spat production
Aquaculture 242 (2004) 271 – 282 www.elsevier.com/locate/aqua-online
Cryopreservation of sperm of the Pacific oyster (Crassostrea gigas): development of a practical method for commercial spat production Serean L. Adamsa, John F. Smithb, Rodney D. Robertsc,*, Achim R. Jankec, Heinrich F. Kaspar c, H. Robin Tervitb, P. Anne Pughb, Steven C. Webbc, Nick G. Kingc a
Department of Marine Science, University of Otago, P. O. Box 56, Dunedin, New Zealand b AgResearch Ltd, Private Bag 3123, Hamilton, New Zealand c Cawthron Institute, Private Bag 2, Nelson, New Zealand
Received 1 March 2004; received in revised form 20 August 2004; accepted 24 August 2004
Abstract This paper describes a simple method for cryopreserving sperm of the Pacific oyster (Crassostrea gigas Thunberg) in quantities suitable for commercial spat production. Experiments to refine the cryoprotectant mixtures demonstrated the key role of trehalose. Trehalose alone (at 0.45 M final concentration) was an effective cryoprotectant. The addition of 2.5–15% dimethyl sulphoxide (DMSO) in combination with 0.45 M trehalose gave only modest improvement in fertility over trehalose alone ( p=0.056). There was no significant difference in fertility among DMSO concentrations ( p=0.611). Seawater (SW) without cryoprotectant gave very poor results, but yielded some fertilization at very high sperm concentrations (7F1% at 107 sperm mL 1, 21F2% at 3.2107 sperm mL 1, meanFS.E., n=3). The fertility of unfrozen sperm was 30- to 100-fold higher than that of sperm cryopreserved with DMSO and/or trehalose. For sperm cryopreserved in 4.5-mL cryovials, two simplified freezing methods gave fertilization rates equivalent to sperm cryopreserved by controlled rate freezing ( p=0.386). These methods involved securing the cryovials to aluminium canes and then either placing them into a bath of methanol chilled with dry ice, or holding them on a floating rack 3 cm above liquid nitrogen. A third technique of plunging the cryovials directly into liquid nitrogen gave reduced and variable fertility relative to the methanol/dry ice bath method
* Corresponding author. Tel.: +64 3 548 2319; fax: +64 3 546 9464. E-mail address: [email protected] (R.D. Roberts). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.08.034
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( p=0.032). The commercial applicability of the protocols was demonstrated on a batch of 30 million eggs. Fertilization with cryopreserved sperm yielded 81% fertilization, and larval rearing by normal commercial practises yielded 3.7 million settled spat, which was comparable to the 2.5 million spat from a parallel batch fertilized with unfrozen sperm. D 2004 Elsevier B.V. All rights reserved. Keywords: Pacific oyster; Sperm; Cryopreservation; Hatchery; Commercial application
1. Introduction The Pacific oyster (Crassostrea gigas Thunberg) is farmed in many countries and is one of the few shellfish species for which culture is based substantially on hatchery-reared juveniles. The ability to routinely cryopreserve sperm of this species would enable hatcheries to store sperm from individual males for selective breeding programmes (McFadzen, 1995; Smith et al., 2001). Adult oysters conditioned with ample food are often predominantly female (Thompson et al., 1998), so cryopreservation could also provide a guaranteed supply of sperm for spat production outside the natural breeding season, reducing the costs associated with conditioning broodstock. Cryopreservation of oyster sperm was achieved decades ago (e.g., Lannan, 1971; Staeger, 1974) but is still not used by oyster hatcheries. Most past work has been conducted at small scale, often with controlled rate freezers, and has not verified development beyond early larval stages. Recent work using cryopreserved sperm of the eastern oyster, Crassostrea virginica, addressed some of these shortcomings and led to successful rearing of tens of thousands of larvae to pediveliger stage, and hundreds of oysters to juvenile stage (Paniagua-Chavez, 1999). The development of affordable, reliable, commercial-scale techniques for sperm cryopreservation is still required for hatcheries to begin to use cryopreserved sperm routinely. Previous work on cryopreserving Pacific oyster sperm has found that dimethyl sulphoxide (DMSO) is more effective than other cryoprotective agents (CPAs) in maintaining postthaw fertility (Hwang and Chen, 1973; Bougrier and Rabenomanana, 1986; Iwata et al., 1989; Yankson and Moyse, 1991; Smith et al., 2001). However, the concentration of DMSO considered to be optimal has varied among studies, and different base diluents have been used making comparisons difficult. The addition of trehalose to the diluent has been more effective than complex salt solutions or seawater (SW) alone (Smith et al., 2001) and trehalose was shown to reduce the toxicity of DMSO to Pacific oyster embryos (Chao et al., 1994). The cooling rate used to freeze Pacific oyster sperm has ranged from 4.7 8C min 1 (Yankson and Moyse, 1991) to immediate plunging in liquid nitrogen (Hwang and Chen, 1973). This wide variation in cooling rate suggests that there should be scope to use simple freezing methods in place of controlled rate freezers, making cryopreservation simpler and more affordable for hatcheries. Simplified freezing methods have been tested with low-volume ampoules or straws (e.g., Staeger, 1974; Usuki et al., 1999) but for commercial requirements, large-volume straws or cryovials, which will cool more slowly, need to be evaluated.
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Past research on Pacific oyster sperm cryopreservation has not delivered protocols that can be reliably applied at commercial scale. The present study aimed to (1) refine the concentration of CPAs used in cryopreservation of Pacific oyster sperm; (2) develop simple methods for freezing sperm in commercial quantities; and (3) demonstrate the applicability of the protocols in commercial-scale spat production.
2. Methods 2.1. Collection of oysters and collection and care of oyster gametes Sexually mature Pacific oysters were obtained during the natural spawning season (November–December) from marine farms in the Marlborough Sounds, New Zealand. Sperm and eggs were obtained by bstrip spawningQ. The oysters were opened and a small sample of gonad tissue was examined microscopically to determine sex. Eggs were collected by lacerating the gonad wall with the tip of a transfer pipette (Samco Scientific 222, San Fernando, CA, USA) and gently scraping and washing the gonad contents into 70-mL plastic jars (Labserv LBS32003N, Auckland, New Zealand) containing ~50 mL of 1-Am cartridge-filtered seawater (FSW). Eggs were maintained at ~4 8C to minimise any loss of viability associated with aging. Sperm were collected into jars in the same fashion but without the addition of any FSW, then held on ice prior to experiments to minimise aging effects. Eggs or sperm from each individual were maintained separately. Eggs were examined for maturity based on egg shape and distinctness of its nucleus, and sperm were visually assessed for vigorous motility after activation with FSW. Batches of gametes that were considered inadequate were not used. Equivalent volumes of the gametes from at least three individuals were pooled for each run of each experiment, to reduce variability (Palumbi, 1999; Boudry et al., 2002). 2.2. Experiments 2.2.1. Experiment 1: effect of CPAs on postthaw fertility of cryopreserved sperm In this experiment, the addition of 0.45 M trehalose (Sigma T 5251, St. Louis, MO) alone, or in combination with 0%, 2.5%, 5%, 7.5%, 10%, 12.5% or 15% DMSO (Sigma D 5879) was evaluated to determine the effect of DMSO concentration on postthaw fertility of sperm. Cryoprotectant solutions were prepared in distilled water and were combined with sperm at the rate of 10 parts CPA solution to 1 part sperm (Bougrier and Rabenomanana, 1986; Smith et al., 2001) to give the above final (in-straw) concentrations. All solutions were cooled on ice before being added to sperm in 10 equal volume steps, 10 to 20 s apart, to avoid osmotic injury. Diluted sperm were aspirated into 0.25 mL plastic straws and sealed with coloured PVC powder (Instruments de Me´dicine Ve´te´rinaire, l’Aigle, France). The straws were then loaded into a controlled rate freezer (Kryo-10 Series II, Planer Products, Sunbury-on-Thames, England) programmed to cool from 0 to 80 8C at 50 8C min 1, held at 80 8C for 10 min, then plunged directly into and stored in liquid nitrogen. The interval between the dilution of sperm and the start of cooling was approximately 45 min. For thawing, straws were removed from liquid nitrogen and placed
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immediately in a FSW bath at ~20 8C for 15 to 20 s, by which time they were fully thawed. The experiment was replicated three times. 2.2.2. Experiment 2: comparison of three freezing methods Two simple methods for freezing oyster sperm in commercial quantities (4.5-mL cryovials) were evaluated and compared to the controlled rate freezing described above in Experiment 1. The first method involved mounting cryovials onto aluminium canes (Instruments de Me´dicine Ve´te´rinaire, l’Aigle, France) for ease of handling, and placing the canes directly into a styrofoam box containing several litres of methanol cooled to ~ 75 8C with dry ice pellets. A lid was added and the cryovials remained in the bath for 10 min before being plunged into liquid nitrogen. The second method involved gently placing the canes with cryovials on a styrofoam rectangular frame (brackQ) that floated on liquid nitrogen and held the vials 3 cm above several litres of liquid nitrogen in a styrofoam box. A lid was added and the cryovials remained on the rack for 10 min before being plunged into liquid nitrogen. The controlled rate freezer programme was as in Experiment 1, but with cryovials standing in the freezing chamber attached to canes. Sperm were divided into 4 mL aliquots, diluted 1:10 with DMSO in 0.6 M trehalose (in distilled water) to give a final concentration of 5% DMSO and 0.55 M trehalose. The solution was added in 10 fixed volume steps, 10 to 20 s apart. Diluted sperm were loaded into 4.5-mL cryovials (Nalgene Nunc International, Denmark), and frozen using the three different freezing methods. The temperature profile of sperm frozen by each method was measured by sealing the sensor of a thermocouple connected to a microprocessor (Model HH21, Omega, Stamford, CT, USA) into a cryovial containing diluted sperm and by recording the temperature change during cooling. Cryopreserved sperm were held in liquid nitrogen for at least 60 min before being thawed in a FSW bath at ~20 8C until they were completely thawed (5 to 8 min). The experiment was replicated three times. 2.2.3. Experiment 3: plunging vials of sperm directly into liquid nitrogen Based on the success of Experiment 2, Experiment 3 was designed to ascertain whether the freezing method could be further simplified by plunging cryovials of sperm on canes directly into liquid nitrogen (several litres in a styrofoam box). This method was compared to freezing with the methanol/dry ice bath technique (methodology as in Experiment 2). Experiment 3 was replicated four times. 2.2.4. Experiment 4: demonstration of commercial applicability Sperm from several males were combined, frozen using the methanol/dry ice bath method, and thawed, all as described for Experiment 2. Sixty million eggs were collected from several females and were fertilized with cryopreserved or unfrozen sperm from the same pool of males. Cryopreserved sperm (31.5 mL at 1.7109 mL 1) were added to 30 million eggs (30 min poststripping) in 300 mL of FSW. The sperm/egg mix was diluted to 2 L with FSW after 15 min and transferred after a further 30 min to a tank with 150 L of FSW containing 1 mg L 1 EDTA. After 2 days, these tanks were drained through a 45-Am mesh and the retained d-larvae were transferred to a continuous exchange larval rearing system (Janke et al., 2004) and reared through to settlement on a mixed diet of
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Chaetoceros calcitrans and Isochrisis galbana. Tanks were cleaned and the larvae were screened every second day to remove dead or slow-growing larvae and other debris. Competent larvae were induced to metamorphose by bath application of 10 4 M epinephrine (Coon et al., 1985) for 2 to 3 h, and spat were reared in down-wellers. 2.3. Fertilization assays Fertilization assays were used to evaluate the fertility of sperm before and after cryopreservation. Preliminary experiments showed no effect of gamete age up until at least 4 h for dry stripped sperm held on ice (data not presented). This enabled the same pools of eggs to be used in assays with unfrozen sperm and cryopreserved sperm. In situations where cryopreserved sperm were held for longer periods, two control fertilization assays were carried out. Control 1 assessed the fertility of the pooled sperm before it was frozen. Control 2 used unfrozen sperm from a different pool of males to assess the fertility of eggs that were used in assays with cryopreserved sperm at the time that the sperm were thawed. Pooled batches of eggs and sperm were used for fertilizations in all experiments. Egg concentrations were determined by counting 50 AL aliquots from an even egg suspension. Sperm concentrations were determined using a Neubauer haemocytometer. Eggs were diluted in FSW to a concentration of ~2,700 eggs mL 1 in a 70-mL plastic jar (Labserv) and held in at ~4 8C until used in fertilization assays. Each well of a 12-well tissue culture plate (Falcon 353043, Becton Dickinson and Company, Franklin Lakes, NJ, USA) received 2750 AL of FSW, 225 AL of egg suspension containing ~600 eggs and 30 AL of sperm solution at 100 times the target concentration. Total assay volume was 3 mL and egg concentration was ~200 eggs mL 1. Cryopreserved sperm were diluted serially in FSW and added to each well to give final concentrations of between 104 and 107 sperm mL 1. Controls with unfrozen sperm were assayed at concentrations from 102 to 105 sperm mL 1. Duplicate wells were run at all sperm concentrations for accuracy and at least two wells containing eggs but no sperm acted as negative controls. When embryos had reached the 4-cell stage or beyond, 300 AL of 4% borax-buffered formalin was added to each well to arrest further development. Approximately 100 eggs in each well were examined microscopically to determine the percentage fertilized. Eggs were considered fertilized if they had undergone cleavage. Eggs with polar bodies that had not divided were not scored as fertilized. bFertilityQ was measured as the sperm density corresponding to 50% fertilisation in dose–response curves constructed for the standard fertilisation assay. 2.4. Statistical analysis Statistical analysis for all experiments was performed using Minitab 13.31 software (Minitab, State College, PA, USA). To compare between treatments, the trapezoidal rule formula was used to estimate the total area under the fertilization curve (Conte, 1965). Only treatments that were measured over the same sperm concentrations could be compared using this method. For analysis of the effect of CPAs, treatment means were compared using two-way ANOVA. Dimethyl sulphoxide concentration was
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analysed as a brepeated measures factorQ because treatment levels were not independent (i.e., the same batches of pooled sperm were used for each DMSO concentration that was evaluated). The analysis was done with and without 0.45 M trehalose alone (i.e., 0% DMSO) included as a level. Treatment means were also compared using two-way ANOVA for the experiment comparing different freezing methods and freezing method was analysed as a repeated measures factor. When appropriate, Tukey’s test was used to identify significant differences between treatment groups. For all ANOVA tests, residuals were checked to confirm that the assumptions of normality and homogeneity of variance were not violated. For Experiment 3, a paired sample t-test was used.
3. Results 3.1. Experiment 1: effect of CPAs on postthaw fertility of cryopreserved sperm Sperm that were frozen in FSW without CPA were agglutinated upon thawing and their postthaw fertility was near zero (Fig. 1), rising to only 7F1% at 107 sperm mL 1 and 21F2% at 3.2107 sperm mL 1 (meanFS.E., n=3). The addition of 0.45 M trehalose as a cryoprotectant yielded sperm with high postthaw fertility, 80F5 % at 3.2106 sperm mL 1 (meanFS.E., n=3; Fig. 1). Addition of DMSO to the trehalose yielded modest improvement over trehalose alone ( F=2.89, p=0.056, df=6, 12). There was no significant difference between DMSO concentrations ( F=0.74, p=0.611, df=5, 10), but concen-
Fig. 1. Fertilization rates (meanFS.E., n=3) of Pacific oyster sperm frozen in various CPA concentrations. Control 1 assesses the fertility of the pooled sperm before they were frozen. Control 2 represents a second batch of unfrozen sperm used to assess the fertility of the eggs used in assays with thawed sperm. 0% (SW) samples were diluted with seawater and frozen without any cryoprotectant. 0% (TRE) samples were frozen with 0.45 M trehalose in distilled water. Other treatments represent samples frozen with 2.5–15% DMSO and 0.45 M trehalose. Data represent three batches of pooled sperm, collected, cryopreserved and assayed independently.
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trations of 5% to 12.5% DMSO yielded slightly higher average fertility than 2.5% or 15% DMSO (Fig. 1). Postthaw fertilization differed significantly between the three replicate runs of the experiment ( F=22.63, pb0.001, df=2, 12). Cryopreservation with DMSO and/or trehalose reduced the fertility of sperm by ~30- to 100-fold relative to unfrozen controls. In the assay system used, the sperm concentration required to produce 50% fertilization was ~104 sperm mL 1 for unfrozen sperm (50 sperm per egg) and between 3.2105 sperm mL 1 and 1106 sperm mL 1 (1600 to 5000 sperm per egg) for cryopreserved sperm (Fig. 1). 3.2. Experiment 2: alternative freezing methods Postthaw fertility did not vary significantly among the three methods tested ( F=1.22, p=0.386, df=2, 4). Sperm that were frozen using the methanol/dry ice bath or rack over liquid nitrogen had postthaw fertility comparable to that of sperm frozen in the controlled rate freezer (Fig. 2). Postthaw fertility again differed significantly among the three replicate runs of the experiment ( F=72.58, p=0.001, df=2, 4). The fertility of fresh sperm in Experiment 2 (50% fertilization at ~3103 sperm mL 1, Fig. 2) was about threefold higher than in Experiment 1 (Fig. 1). 3.3. Experiment 3: plunging vials of sperm directly into liquid nitrogen Sperm frozen by directly plunging cryovials on canes into liquid nitrogen had consistently lower postthaw fertilization than sperm frozen using the methanol/dry ice bath method (t=3.81, p=0.032, df=3; Fig. 3).
Fig. 2. Fertilization rates (meanFS.E., n=3) of Pacific oyster sperm frozen by different methods. Data represent three batches of pooled sperm that were collected, cryopreserved and assayed independently. The control assesses the fertility of the pooled sperm before it was cryopreserved. The same batches of eggs were used with both the unfrozen and cryopreserved sperm.
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Fig. 3. Comparison of postthaw fertility of sperm frozen by methanol/dry ice bath method or by direct plunging of cryovials into liquid nitrogen. Data shown are for fertilization assays carried out at 106 sperm mL 1 in four replicates of the experiment.
3.4. Cooling and warming profiles The cooling rates that sperm were subjected to during freezing varied widely among the four freezing methods evaluated in Experiments 2 and 3 (Fig. 4). The controlled rate freezer was programmed to cool at 50 8C min 1 to 80 8C, but sperm samples actually cooled at rates between 1 and 22 8C min 1, reaching 80 8C only at the end of the 10min hold period, immediately prior to plunging into liquid nitrogen. Samples in the methanol/dry ice bath stabilised at 76 8C before being plunged into liquid nitrogen, while samples on the rack reached 140 8C before plunging (Fig. 4). Of the simplified freezing methods, the rack over liquid nitrogen most closely mimicked the cooling rate of the controlled rate freezer (Fig. 4). The average rate of cooling from 0 to 60 8C was 9.5
Fig. 4. Representative profiles of cooling or warming of Pacific oyster sperm in 4.5-mL cryovials. Vials containing diluted sperm were frozen by four different methods, and thawed in a 20 8C water bath. The rapid cooling near the end of profiles occurred when the cryovials were plunged into liquid nitrogen.
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8C min 1 for the controlled rate freezer, 13.5 8C min 1 for the rack, 26.8 8C min 1 for the methanol/dry ice bath and 106.8 8C min 1 for the liquid nitrogen bath. Cooling profiles were more erratic in those freezing methods that relied on liquid nitrogen vapour, namely the controlled rate freezer and rack over liquid nitrogen. The same thawing method was used for all freezing methods. Initial warming was very rapid, averaging 460 8C min 1 over the first 15 s. Warming rate then dropped rapidly, with the samples taking about 5 min to reach 0 8C. Above 0 8C, there was an increase in warming rate, once all the material had thawed (Fig. 4). 3.5. Experiment 4: demonstration of commercial applicability Cryopreserved sperm fertilized 81% of ~30 million eggs. After larval rearing and metamorphosis, 3.7 million metamorphosed spat were obtained. A parallel batch of 30 million eggs fertilized with unfrozen sperm gave a slightly lower yield of 2.5 million spat.
4. Discussion This study presents a simple, affordable and effective cryopreservation method for Pacific oyster sperm, and demonstrates its commercial applicability. Sperm frozen either by plunging 4.5-mL cryovials into a methanol/dry ice bath or by placing them on a rack over liquid nitrogen had postthaw fertility comparable to sperm frozen with a controlled rate freezer. Either of these methods could therefore be used in hatcheries to carry out routine cryopreservation of Pacific oyster sperm. The commercial applicability of the methanol/dry ice bath method was demonstrated by producing 3.7 million spat from 30 million eggs. This represents a very good rate of survival from egg to spat using commercial rearing practises (Holliday, 1986; Utting and Spencer, 1991; Guo et al., 1996). A single large male oyster can yield ~10 mL of bdryQ stripped sperm at a concentration of ~21010 sperm mL 1. If cryopreserved sperm gives 80% fertilization at 2000 sperm per egg (Experiment 4) and 12% survival from egg to spat (Experiment 4), then the cryopreserved sperm from one male could fertilize 80 million eggs and produce 12 million spat. Hence, the cryopreservation technique is commercially relevant, even where production is based on selected individual males. Cryopreserved sperm are 30- to 100fold less fertile than fresh sperm (Figs. 1 and 2), so hatcheries may use fresh sperm for an initial production run, then cryopreserve excess sperm for later use. Sperm that were frozen using the simplified freezing methods cooled much faster than sperm frozen using the controlled rate freezer. The programmed freezing rate of 50 8C min 1 was established using 0.25- and 0.5-mL plastic straws (Smith et al., 2001). However, this freezing rate was not attained when 4.5-mL cryovials were used in the same controlled rate freezer during this study. The actual cooling rate in the 4.5-mL cryovials lagged behind that recorded in the chamber of the freezer, averaging only 9.5 8C min 1 from 0 to 60 8C. Fertility of cryopreserved sperm was equally good at cooling rates of 9.5 (controlled rate freezer), 13.5 (rack) and 27 8C min 1 (methanol/dry ice bath), but reduced at 107 8C min 1 (direct liquid nitrogen plunge). Other studies on Pacific
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oyster sperm have reported successful fertilization using sperm cooled at rates of 4.7 to N 100 8C min 1 (Hwang and Chen, 1973; Yankson and Moyse, 1991; McFadzen, 1995; Smith et al., 2001). It appears that Pacific oyster sperm are able to tolerate a relatively wide range of cooling rates without any appreciable change in postthaw fertility. The effect of DMSO concentration was investigated in the presence of the nonpermeating CPA, trehalose, which was previously shown to reduce the toxicity of DMSO to Pacific oyster embryos (Chao et al., 1994). The main purpose was to determine an optimal DMSO concentration for cryopreserving Pacific oyster sperm, as the concentration suggested in previous studies ranged from 5% to 17.7% (Staeger, 1974; Bougrier and Rabenomanana, 1986; Iwata et al., 1989; Yankson and Moyse, 1991; Smith et al., 2001). Sperm were also frozen in seawater without DMSO or trehalose because sea urchin sperm show modest postthaw fertility and survival following cryopreservation without CPA (Adams et al., 2004). The postthaw fertility of oyster sperm frozen in seawater was very poor, but not zero. DMSO concentration had no significant effect on postthaw fertility of sperm in the presence of trehalose, and no concentration of DMSO gave significantly higher postthaw fertility than trehalose alone. These results may, in part, explain why such a large range has been suggested to be optimal by other studies on Pacific oyster sperm. They also highlight the beneficial role of trehalose in protecting Pacific oyster sperm during cryopreservation. Trehalose is thought to protect cells by several mechanisms. One is by stabilizing the phospholipids in the cell membrane, preventing damage caused by dehydration (Strauss et al., 1986; Anchordoguy et al., 1987). Another is by changing the properties of the extracellular solution. Trehalose may alter the pattern of ice crystallisation, thereby preventing mechanical damage to cells (Woelders et al., 1997). It may also trap salts in a viscous phase, stopping eutectic freezing from occurring (Nicolajsen and Hvidt, 1994). Finally, trehalose protects cells by reducing the salt concentration in the unfrozen fraction at a given temperature (Holt, 2000). In this study, trehalose was diluted in distilled water rather than seawater, further reducing the likelihood of sperm being exposed to high salt concentrations. We diluted 1 part stripped sperm with 10 parts CPA solution because previous work showed that this returned sperm with ~2.5-fold higher fertility than a 1:1 dilution (Smith et al., 2001). However, if storage space were the main constraint, then the 1:1 dilution offers the best compromise because the higher number of sperm in a given volume more than offsets the lower fertility of 1:1 diluted sperm. Thus, about twice as many eggs could be fertilized from a given volume of frozen sperm. Combining gametes to reduce variation between experiments has been a feature of bioassays for water quality testing and for developmental biology and embryology research. In the work reported here, sperm and eggs used in each run were pooled from at least three males and three females. The quality of sperm and eggs was checked visually before the gametes from a particular individual were added to a pool. However, significant variation in postthaw fertility among replicate runs of experiments occurred. It is likely that this variation is at least partly attributable to variation in the fertility of different pools of sperm and eggs. Where pools of gametes are used in future studies, this variation should be reduced by pooling gametes from a larger number of animals. However, for selective breeding programmes using cryopreserved sperm for pairwise matings an understanding
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of the factors contributing to this variation amongst individuals will be important in minimising its impact on the breeding programme. It has been suggested that DNA damage to oyster sperm during cryopreservation may result in increased embryonic mortality of eggs fertilized with cryopreserved sperm (Gwo et al. 2003). We did not observe any increase in embryonic mortality in this study, although DNA damage may account, at least in part, for the reduction in fertility observed. Indeed, the number of spat obtained with cryopreserved sperm in this study was comparable to that obtained when unfrozen sperm were used (3.7 million spat with cryopreserved sperm and 2.5 million with unfrozen sperm).
5. Commercial cryopreservation protocol We have used the methods outlined in this paper on numerous occasions with consistent success, and believe they will be useful to staff of Pacific oyster hatcheries wishing to use sperm cryopreservation. For ease of application, a simple summary of the protocol used is given below. Sperm are stripped bdryQ and stored in a water bath at ~4 8C until diluted with 10 parts of CPA solution in 10 equal volume steps, 10 to 20 s apart (CPA solution: Solution A=15.2 g of trehalose+40 mL distilled water. Combine 30 mL of solution A, 7.8 mL of distilled water and 2.2 mL of DMSO, mix and cool in a 4 8C water bath.). The diluted sperm solution is transferred to labelled 4.5-mL cryovials, attached to aluminium canes and then cooled for 10 min in a lidded styrofoam box by either 1) placing in a methanol bath chilled to ~ 75 8C with dry ice pellets or 2) gently placing on a rack floating 3 cm above liquid nitrogen. At the end of 10 min, the samples are plunged into liquid nitrogen for at least several minutes, and then transferred to a liquid nitrogen storage container where they remain viable indefinitely. For thawing, cryovials are transferred from liquid nitrogen to a lidded FSW bath at ~20 8C until thawed (5–8 min). Thawed sperm are combined with freshly spawned or stripped eggs at a ratio of ~2000 sperm per egg, and an egg density of ~100,000 eggs mL 1. After 10 min, the eggs are diluted into larval hatching tanks and reared following normal practises. Acknowledgements We thank the staff at the Cawthron Institute for technical assistance and Dr. Paul Hessian at the University of Otago for helpful comments on this manuscript. This research was supported by the New Zealand Foundation for Research Science and Technology (contracts CAW801, CAWX0004 and CAWX0304). References Adams, S.L., Hessian, P.H., Mladenov, P.V., 2004. Cryopreservation of sea urchin (Evechinus chloroticus) sperm. Cryo. Lett. 25, 287 – 299. Anchordoguy, T.J., Rudolph, A.S., Carpenter, J.F., Crowe, J.H., 1987. Modes of interaction of cryoprotectants with membrane phospholipids during freezing. Cryobiology 24, 324 – 331.
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Boudry, P., Collet, B., Cornette, F., Hervouet, V., Bonhomme, F., 2002. High variance in reproductive success of the Pacific oyster (Crassostrea gigas, Thunberg) revealed by microsatellite-based parentage analysis of multifactorial crosses. Aquaculture 204, 283 – 296. Bougrier, S., Rabenomanana, L.D., 1986. Cryopreservation of spermatozoa of the Japanese oyster, Crassostrea gigas. Aquaculture 58, 277 – 280. Chao, N.H., Chiang, C.P., Hsu, H.W., Tsai, C.T., Lin, T.T., 1994. Toxicity tolerance of oyster embryos to selected cryoprotectants. Aquat. Living Resour. 7, 99 – 104. Conte, S.D., 1965. Elementary Numerical Analysis: An Algorithmic Approach. McGraw-Hill, New York. Coon, S.L., Bonar, D.B., Weiner, R.M., 1985. Induction of settlement and metamorphosis of the Pacific oyster Crassostrea gigas (Thunberg) by l-DOPA and catecholamines. J. Exp. Mar. Biol. Ecol. 94, 211 – 221. Guo, X., DeBrosse, G.A., Allen, S.K., 1996. All-triploid Pacific oysters (Crassostrea gigas Thunberg) produced by mating tetraploids and diploids. Aquaculture 142, 149 – 161. Gwo, J.-C., Wu, C.-Y., Chang, W.-S.P., Cheng, H.-Y., 2003. Evaluation of damage in Pacific oyster (Crassostrea gigas) spermatozoa before and after cryopreservation using comet assay. Cryo. Lett. 24, 171 – 180. Holliday, J.E. 1986. International developments in oyster hatchery technology, 1983–1985. Division of Fisheries. Department of Agriculture. New South Wales. Australia. Holt, W.V., 2000. Basic aspects of frozen storage of semen. Anim. Reprod. Sci. 62, 3 – 22. Hwang, S.W., Chen, H.P., 1973. Fertility of male oyster gametes after freezing and thawing. Chin.-Am. Jt. Comm. Rural Reconstr. Fish. Ser. 15, 1 – 5. Iwata, N., Kurokura, H., Hirano, R., 1989. Cryopreservation of Pacific oyster, Crassostrea gigas, sperm. Suisan Zoshoku 37, 163 – 166. Janke, A., Kaspar, H., King, N., Foster, S., 2004. Cawthron’s ultra density larval system (CUDLS) for rearing larvae of the Pacific oyster, Crassostrea gigas. Abstracts of the World Aquaculture Society Conference, 1st – 4th March 2004, Hawaii. World Aquaculture Society, Louisiana State University, Baton Rouge, Louisiana, pp. 312. Lannan, J.E., 1971. Experimental self-fertilisation of the pacific oyster Crassostrea gigas, utilising cryopreserved sperm. Genetics 6, 599 – 601. McFadzen, I.R.B., 1995. Cryopreservation of sperm of the Pacific oyster Crassostrea gigas. In: Day, J.G., McLellan, M.R. (Eds.), Cryopreservation and Freeze-Drying Protocols. Humana Press, Totowa, pp. 145 – 149. Nicolajsen, H., Hvidt, A., 1994. Phase behavior of the system trehalose–NaCl–water. Cryobiology 31, 199 – 205. Palumbi, S.R., 1999. All males are not created equal: fertility differences depend on gamete recognition polymorphisms in sea urchins. Evolution 53, 12632 – 12637. Paniagua-Chavez, C.G., 1999. Cryopreservation of gametes and larvae of the Eastern oyster Crassostrea virginica. PhD thesis. Louisiana State University. Smith, J.F., Pugh, P.A., Tervit, H.R., Roberts, R.D., Janke, A.R., Kaspar, H.F., Adams, S.L., 2001. Cryopreservation of shellfish sperm, eggs and embryos. Proc. N. Z. Soc. Anim. Prod. 61, 31 – 34. Staeger, W.H., 1974. Cryobiological Investigations of the Gametes of the Pacific Oyster, Crassostrea gigas, Department of Fisheries and Wildlife. Oregon State University, Corvallis. Strauss, G., Schurtenberger, P., Hauser, H., 1986. The interaction of saccharides with lipid bilayer vesicles: stabilization during freeze-thawing and freeze-drying. Biochim. Biophys. Acta 858, 169 – 180. Thompson, R.J., Newell, R.I.E., Kennedy, V.S., Mann, R., 1998. Reproductive processes and early development. In: Kennedy, V.S., Newell, R.I.E., Eble, A.F. (Eds.), The Eastern Oyster, Crassostrea virginica. Maryland Sea Grant College, Maryland. Usuki, H., Hamaguchi, M., Ishioka, H., 1999. Cryopreservation of Pacific Oyster sperm and larvae. Bull. Natl. Res. Inst. Aquacult. (Suppl. 1), 3 – 6. Utting, S.D., Spencer, B.E., 1991. The hatchery culture of bivalve mollusc larvae and juveniles. Lab. Leafl., MAFF Fish. Res. 68 (Lowestoft) 32 pp. Woelders, H., Matthijs, A., Engel, B., 1997. Effects of trehalose and sucrose, osmolalilty of the freezing medium and cooling rate on viability and intactness of bull sperm after freezing and thawing. Cryobiology 35, 93 – 105. Yankson, K., Moyse, J., 1991. Cryopreservation of the spermatozoa of Crassostrea tulipa and three other oysters. Aquaculture 97, 259 – 267.
Cryopreservation of sperm of the Pacific oyster (Crassostrea gigas): development of a practical method for commercial spat production Serean L. Adamsa, John F. Smithb, Rodney D. Robertsc,*, Achim R. Jankec, Heinrich F. Kaspar c, H. Robin Tervitb, P. Anne Pughb, Steven C. Webbc, Nick G. Kingc a
Department of Marine Science, University of Otago, P. O. Box 56, Dunedin, New Zealand b AgResearch Ltd, Private Bag 3123, Hamilton, New Zealand c Cawthron Institute, Private Bag 2, Nelson, New Zealand
Received 1 March 2004; received in revised form 20 August 2004; accepted 24 August 2004
Abstract This paper describes a simple method for cryopreserving sperm of the Pacific oyster (Crassostrea gigas Thunberg) in quantities suitable for commercial spat production. Experiments to refine the cryoprotectant mixtures demonstrated the key role of trehalose. Trehalose alone (at 0.45 M final concentration) was an effective cryoprotectant. The addition of 2.5–15% dimethyl sulphoxide (DMSO) in combination with 0.45 M trehalose gave only modest improvement in fertility over trehalose alone ( p=0.056). There was no significant difference in fertility among DMSO concentrations ( p=0.611). Seawater (SW) without cryoprotectant gave very poor results, but yielded some fertilization at very high sperm concentrations (7F1% at 107 sperm mL 1, 21F2% at 3.2107 sperm mL 1, meanFS.E., n=3). The fertility of unfrozen sperm was 30- to 100-fold higher than that of sperm cryopreserved with DMSO and/or trehalose. For sperm cryopreserved in 4.5-mL cryovials, two simplified freezing methods gave fertilization rates equivalent to sperm cryopreserved by controlled rate freezing ( p=0.386). These methods involved securing the cryovials to aluminium canes and then either placing them into a bath of methanol chilled with dry ice, or holding them on a floating rack 3 cm above liquid nitrogen. A third technique of plunging the cryovials directly into liquid nitrogen gave reduced and variable fertility relative to the methanol/dry ice bath method
* Corresponding author. Tel.: +64 3 548 2319; fax: +64 3 546 9464. E-mail address: [email protected] (R.D. Roberts). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.08.034
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( p=0.032). The commercial applicability of the protocols was demonstrated on a batch of 30 million eggs. Fertilization with cryopreserved sperm yielded 81% fertilization, and larval rearing by normal commercial practises yielded 3.7 million settled spat, which was comparable to the 2.5 million spat from a parallel batch fertilized with unfrozen sperm. D 2004 Elsevier B.V. All rights reserved. Keywords: Pacific oyster; Sperm; Cryopreservation; Hatchery; Commercial application
1. Introduction The Pacific oyster (Crassostrea gigas Thunberg) is farmed in many countries and is one of the few shellfish species for which culture is based substantially on hatchery-reared juveniles. The ability to routinely cryopreserve sperm of this species would enable hatcheries to store sperm from individual males for selective breeding programmes (McFadzen, 1995; Smith et al., 2001). Adult oysters conditioned with ample food are often predominantly female (Thompson et al., 1998), so cryopreservation could also provide a guaranteed supply of sperm for spat production outside the natural breeding season, reducing the costs associated with conditioning broodstock. Cryopreservation of oyster sperm was achieved decades ago (e.g., Lannan, 1971; Staeger, 1974) but is still not used by oyster hatcheries. Most past work has been conducted at small scale, often with controlled rate freezers, and has not verified development beyond early larval stages. Recent work using cryopreserved sperm of the eastern oyster, Crassostrea virginica, addressed some of these shortcomings and led to successful rearing of tens of thousands of larvae to pediveliger stage, and hundreds of oysters to juvenile stage (Paniagua-Chavez, 1999). The development of affordable, reliable, commercial-scale techniques for sperm cryopreservation is still required for hatcheries to begin to use cryopreserved sperm routinely. Previous work on cryopreserving Pacific oyster sperm has found that dimethyl sulphoxide (DMSO) is more effective than other cryoprotective agents (CPAs) in maintaining postthaw fertility (Hwang and Chen, 1973; Bougrier and Rabenomanana, 1986; Iwata et al., 1989; Yankson and Moyse, 1991; Smith et al., 2001). However, the concentration of DMSO considered to be optimal has varied among studies, and different base diluents have been used making comparisons difficult. The addition of trehalose to the diluent has been more effective than complex salt solutions or seawater (SW) alone (Smith et al., 2001) and trehalose was shown to reduce the toxicity of DMSO to Pacific oyster embryos (Chao et al., 1994). The cooling rate used to freeze Pacific oyster sperm has ranged from 4.7 8C min 1 (Yankson and Moyse, 1991) to immediate plunging in liquid nitrogen (Hwang and Chen, 1973). This wide variation in cooling rate suggests that there should be scope to use simple freezing methods in place of controlled rate freezers, making cryopreservation simpler and more affordable for hatcheries. Simplified freezing methods have been tested with low-volume ampoules or straws (e.g., Staeger, 1974; Usuki et al., 1999) but for commercial requirements, large-volume straws or cryovials, which will cool more slowly, need to be evaluated.
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Past research on Pacific oyster sperm cryopreservation has not delivered protocols that can be reliably applied at commercial scale. The present study aimed to (1) refine the concentration of CPAs used in cryopreservation of Pacific oyster sperm; (2) develop simple methods for freezing sperm in commercial quantities; and (3) demonstrate the applicability of the protocols in commercial-scale spat production.
2. Methods 2.1. Collection of oysters and collection and care of oyster gametes Sexually mature Pacific oysters were obtained during the natural spawning season (November–December) from marine farms in the Marlborough Sounds, New Zealand. Sperm and eggs were obtained by bstrip spawningQ. The oysters were opened and a small sample of gonad tissue was examined microscopically to determine sex. Eggs were collected by lacerating the gonad wall with the tip of a transfer pipette (Samco Scientific 222, San Fernando, CA, USA) and gently scraping and washing the gonad contents into 70-mL plastic jars (Labserv LBS32003N, Auckland, New Zealand) containing ~50 mL of 1-Am cartridge-filtered seawater (FSW). Eggs were maintained at ~4 8C to minimise any loss of viability associated with aging. Sperm were collected into jars in the same fashion but without the addition of any FSW, then held on ice prior to experiments to minimise aging effects. Eggs or sperm from each individual were maintained separately. Eggs were examined for maturity based on egg shape and distinctness of its nucleus, and sperm were visually assessed for vigorous motility after activation with FSW. Batches of gametes that were considered inadequate were not used. Equivalent volumes of the gametes from at least three individuals were pooled for each run of each experiment, to reduce variability (Palumbi, 1999; Boudry et al., 2002). 2.2. Experiments 2.2.1. Experiment 1: effect of CPAs on postthaw fertility of cryopreserved sperm In this experiment, the addition of 0.45 M trehalose (Sigma T 5251, St. Louis, MO) alone, or in combination with 0%, 2.5%, 5%, 7.5%, 10%, 12.5% or 15% DMSO (Sigma D 5879) was evaluated to determine the effect of DMSO concentration on postthaw fertility of sperm. Cryoprotectant solutions were prepared in distilled water and were combined with sperm at the rate of 10 parts CPA solution to 1 part sperm (Bougrier and Rabenomanana, 1986; Smith et al., 2001) to give the above final (in-straw) concentrations. All solutions were cooled on ice before being added to sperm in 10 equal volume steps, 10 to 20 s apart, to avoid osmotic injury. Diluted sperm were aspirated into 0.25 mL plastic straws and sealed with coloured PVC powder (Instruments de Me´dicine Ve´te´rinaire, l’Aigle, France). The straws were then loaded into a controlled rate freezer (Kryo-10 Series II, Planer Products, Sunbury-on-Thames, England) programmed to cool from 0 to 80 8C at 50 8C min 1, held at 80 8C for 10 min, then plunged directly into and stored in liquid nitrogen. The interval between the dilution of sperm and the start of cooling was approximately 45 min. For thawing, straws were removed from liquid nitrogen and placed
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immediately in a FSW bath at ~20 8C for 15 to 20 s, by which time they were fully thawed. The experiment was replicated three times. 2.2.2. Experiment 2: comparison of three freezing methods Two simple methods for freezing oyster sperm in commercial quantities (4.5-mL cryovials) were evaluated and compared to the controlled rate freezing described above in Experiment 1. The first method involved mounting cryovials onto aluminium canes (Instruments de Me´dicine Ve´te´rinaire, l’Aigle, France) for ease of handling, and placing the canes directly into a styrofoam box containing several litres of methanol cooled to ~ 75 8C with dry ice pellets. A lid was added and the cryovials remained in the bath for 10 min before being plunged into liquid nitrogen. The second method involved gently placing the canes with cryovials on a styrofoam rectangular frame (brackQ) that floated on liquid nitrogen and held the vials 3 cm above several litres of liquid nitrogen in a styrofoam box. A lid was added and the cryovials remained on the rack for 10 min before being plunged into liquid nitrogen. The controlled rate freezer programme was as in Experiment 1, but with cryovials standing in the freezing chamber attached to canes. Sperm were divided into 4 mL aliquots, diluted 1:10 with DMSO in 0.6 M trehalose (in distilled water) to give a final concentration of 5% DMSO and 0.55 M trehalose. The solution was added in 10 fixed volume steps, 10 to 20 s apart. Diluted sperm were loaded into 4.5-mL cryovials (Nalgene Nunc International, Denmark), and frozen using the three different freezing methods. The temperature profile of sperm frozen by each method was measured by sealing the sensor of a thermocouple connected to a microprocessor (Model HH21, Omega, Stamford, CT, USA) into a cryovial containing diluted sperm and by recording the temperature change during cooling. Cryopreserved sperm were held in liquid nitrogen for at least 60 min before being thawed in a FSW bath at ~20 8C until they were completely thawed (5 to 8 min). The experiment was replicated three times. 2.2.3. Experiment 3: plunging vials of sperm directly into liquid nitrogen Based on the success of Experiment 2, Experiment 3 was designed to ascertain whether the freezing method could be further simplified by plunging cryovials of sperm on canes directly into liquid nitrogen (several litres in a styrofoam box). This method was compared to freezing with the methanol/dry ice bath technique (methodology as in Experiment 2). Experiment 3 was replicated four times. 2.2.4. Experiment 4: demonstration of commercial applicability Sperm from several males were combined, frozen using the methanol/dry ice bath method, and thawed, all as described for Experiment 2. Sixty million eggs were collected from several females and were fertilized with cryopreserved or unfrozen sperm from the same pool of males. Cryopreserved sperm (31.5 mL at 1.7109 mL 1) were added to 30 million eggs (30 min poststripping) in 300 mL of FSW. The sperm/egg mix was diluted to 2 L with FSW after 15 min and transferred after a further 30 min to a tank with 150 L of FSW containing 1 mg L 1 EDTA. After 2 days, these tanks were drained through a 45-Am mesh and the retained d-larvae were transferred to a continuous exchange larval rearing system (Janke et al., 2004) and reared through to settlement on a mixed diet of
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Chaetoceros calcitrans and Isochrisis galbana. Tanks were cleaned and the larvae were screened every second day to remove dead or slow-growing larvae and other debris. Competent larvae were induced to metamorphose by bath application of 10 4 M epinephrine (Coon et al., 1985) for 2 to 3 h, and spat were reared in down-wellers. 2.3. Fertilization assays Fertilization assays were used to evaluate the fertility of sperm before and after cryopreservation. Preliminary experiments showed no effect of gamete age up until at least 4 h for dry stripped sperm held on ice (data not presented). This enabled the same pools of eggs to be used in assays with unfrozen sperm and cryopreserved sperm. In situations where cryopreserved sperm were held for longer periods, two control fertilization assays were carried out. Control 1 assessed the fertility of the pooled sperm before it was frozen. Control 2 used unfrozen sperm from a different pool of males to assess the fertility of eggs that were used in assays with cryopreserved sperm at the time that the sperm were thawed. Pooled batches of eggs and sperm were used for fertilizations in all experiments. Egg concentrations were determined by counting 50 AL aliquots from an even egg suspension. Sperm concentrations were determined using a Neubauer haemocytometer. Eggs were diluted in FSW to a concentration of ~2,700 eggs mL 1 in a 70-mL plastic jar (Labserv) and held in at ~4 8C until used in fertilization assays. Each well of a 12-well tissue culture plate (Falcon 353043, Becton Dickinson and Company, Franklin Lakes, NJ, USA) received 2750 AL of FSW, 225 AL of egg suspension containing ~600 eggs and 30 AL of sperm solution at 100 times the target concentration. Total assay volume was 3 mL and egg concentration was ~200 eggs mL 1. Cryopreserved sperm were diluted serially in FSW and added to each well to give final concentrations of between 104 and 107 sperm mL 1. Controls with unfrozen sperm were assayed at concentrations from 102 to 105 sperm mL 1. Duplicate wells were run at all sperm concentrations for accuracy and at least two wells containing eggs but no sperm acted as negative controls. When embryos had reached the 4-cell stage or beyond, 300 AL of 4% borax-buffered formalin was added to each well to arrest further development. Approximately 100 eggs in each well were examined microscopically to determine the percentage fertilized. Eggs were considered fertilized if they had undergone cleavage. Eggs with polar bodies that had not divided were not scored as fertilized. bFertilityQ was measured as the sperm density corresponding to 50% fertilisation in dose–response curves constructed for the standard fertilisation assay. 2.4. Statistical analysis Statistical analysis for all experiments was performed using Minitab 13.31 software (Minitab, State College, PA, USA). To compare between treatments, the trapezoidal rule formula was used to estimate the total area under the fertilization curve (Conte, 1965). Only treatments that were measured over the same sperm concentrations could be compared using this method. For analysis of the effect of CPAs, treatment means were compared using two-way ANOVA. Dimethyl sulphoxide concentration was
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analysed as a brepeated measures factorQ because treatment levels were not independent (i.e., the same batches of pooled sperm were used for each DMSO concentration that was evaluated). The analysis was done with and without 0.45 M trehalose alone (i.e., 0% DMSO) included as a level. Treatment means were also compared using two-way ANOVA for the experiment comparing different freezing methods and freezing method was analysed as a repeated measures factor. When appropriate, Tukey’s test was used to identify significant differences between treatment groups. For all ANOVA tests, residuals were checked to confirm that the assumptions of normality and homogeneity of variance were not violated. For Experiment 3, a paired sample t-test was used.
3. Results 3.1. Experiment 1: effect of CPAs on postthaw fertility of cryopreserved sperm Sperm that were frozen in FSW without CPA were agglutinated upon thawing and their postthaw fertility was near zero (Fig. 1), rising to only 7F1% at 107 sperm mL 1 and 21F2% at 3.2107 sperm mL 1 (meanFS.E., n=3). The addition of 0.45 M trehalose as a cryoprotectant yielded sperm with high postthaw fertility, 80F5 % at 3.2106 sperm mL 1 (meanFS.E., n=3; Fig. 1). Addition of DMSO to the trehalose yielded modest improvement over trehalose alone ( F=2.89, p=0.056, df=6, 12). There was no significant difference between DMSO concentrations ( F=0.74, p=0.611, df=5, 10), but concen-
Fig. 1. Fertilization rates (meanFS.E., n=3) of Pacific oyster sperm frozen in various CPA concentrations. Control 1 assesses the fertility of the pooled sperm before they were frozen. Control 2 represents a second batch of unfrozen sperm used to assess the fertility of the eggs used in assays with thawed sperm. 0% (SW) samples were diluted with seawater and frozen without any cryoprotectant. 0% (TRE) samples were frozen with 0.45 M trehalose in distilled water. Other treatments represent samples frozen with 2.5–15% DMSO and 0.45 M trehalose. Data represent three batches of pooled sperm, collected, cryopreserved and assayed independently.
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trations of 5% to 12.5% DMSO yielded slightly higher average fertility than 2.5% or 15% DMSO (Fig. 1). Postthaw fertilization differed significantly between the three replicate runs of the experiment ( F=22.63, pb0.001, df=2, 12). Cryopreservation with DMSO and/or trehalose reduced the fertility of sperm by ~30- to 100-fold relative to unfrozen controls. In the assay system used, the sperm concentration required to produce 50% fertilization was ~104 sperm mL 1 for unfrozen sperm (50 sperm per egg) and between 3.2105 sperm mL 1 and 1106 sperm mL 1 (1600 to 5000 sperm per egg) for cryopreserved sperm (Fig. 1). 3.2. Experiment 2: alternative freezing methods Postthaw fertility did not vary significantly among the three methods tested ( F=1.22, p=0.386, df=2, 4). Sperm that were frozen using the methanol/dry ice bath or rack over liquid nitrogen had postthaw fertility comparable to that of sperm frozen in the controlled rate freezer (Fig. 2). Postthaw fertility again differed significantly among the three replicate runs of the experiment ( F=72.58, p=0.001, df=2, 4). The fertility of fresh sperm in Experiment 2 (50% fertilization at ~3103 sperm mL 1, Fig. 2) was about threefold higher than in Experiment 1 (Fig. 1). 3.3. Experiment 3: plunging vials of sperm directly into liquid nitrogen Sperm frozen by directly plunging cryovials on canes into liquid nitrogen had consistently lower postthaw fertilization than sperm frozen using the methanol/dry ice bath method (t=3.81, p=0.032, df=3; Fig. 3).
Fig. 2. Fertilization rates (meanFS.E., n=3) of Pacific oyster sperm frozen by different methods. Data represent three batches of pooled sperm that were collected, cryopreserved and assayed independently. The control assesses the fertility of the pooled sperm before it was cryopreserved. The same batches of eggs were used with both the unfrozen and cryopreserved sperm.
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Fig. 3. Comparison of postthaw fertility of sperm frozen by methanol/dry ice bath method or by direct plunging of cryovials into liquid nitrogen. Data shown are for fertilization assays carried out at 106 sperm mL 1 in four replicates of the experiment.
3.4. Cooling and warming profiles The cooling rates that sperm were subjected to during freezing varied widely among the four freezing methods evaluated in Experiments 2 and 3 (Fig. 4). The controlled rate freezer was programmed to cool at 50 8C min 1 to 80 8C, but sperm samples actually cooled at rates between 1 and 22 8C min 1, reaching 80 8C only at the end of the 10min hold period, immediately prior to plunging into liquid nitrogen. Samples in the methanol/dry ice bath stabilised at 76 8C before being plunged into liquid nitrogen, while samples on the rack reached 140 8C before plunging (Fig. 4). Of the simplified freezing methods, the rack over liquid nitrogen most closely mimicked the cooling rate of the controlled rate freezer (Fig. 4). The average rate of cooling from 0 to 60 8C was 9.5
Fig. 4. Representative profiles of cooling or warming of Pacific oyster sperm in 4.5-mL cryovials. Vials containing diluted sperm were frozen by four different methods, and thawed in a 20 8C water bath. The rapid cooling near the end of profiles occurred when the cryovials were plunged into liquid nitrogen.
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8C min 1 for the controlled rate freezer, 13.5 8C min 1 for the rack, 26.8 8C min 1 for the methanol/dry ice bath and 106.8 8C min 1 for the liquid nitrogen bath. Cooling profiles were more erratic in those freezing methods that relied on liquid nitrogen vapour, namely the controlled rate freezer and rack over liquid nitrogen. The same thawing method was used for all freezing methods. Initial warming was very rapid, averaging 460 8C min 1 over the first 15 s. Warming rate then dropped rapidly, with the samples taking about 5 min to reach 0 8C. Above 0 8C, there was an increase in warming rate, once all the material had thawed (Fig. 4). 3.5. Experiment 4: demonstration of commercial applicability Cryopreserved sperm fertilized 81% of ~30 million eggs. After larval rearing and metamorphosis, 3.7 million metamorphosed spat were obtained. A parallel batch of 30 million eggs fertilized with unfrozen sperm gave a slightly lower yield of 2.5 million spat.
4. Discussion This study presents a simple, affordable and effective cryopreservation method for Pacific oyster sperm, and demonstrates its commercial applicability. Sperm frozen either by plunging 4.5-mL cryovials into a methanol/dry ice bath or by placing them on a rack over liquid nitrogen had postthaw fertility comparable to sperm frozen with a controlled rate freezer. Either of these methods could therefore be used in hatcheries to carry out routine cryopreservation of Pacific oyster sperm. The commercial applicability of the methanol/dry ice bath method was demonstrated by producing 3.7 million spat from 30 million eggs. This represents a very good rate of survival from egg to spat using commercial rearing practises (Holliday, 1986; Utting and Spencer, 1991; Guo et al., 1996). A single large male oyster can yield ~10 mL of bdryQ stripped sperm at a concentration of ~21010 sperm mL 1. If cryopreserved sperm gives 80% fertilization at 2000 sperm per egg (Experiment 4) and 12% survival from egg to spat (Experiment 4), then the cryopreserved sperm from one male could fertilize 80 million eggs and produce 12 million spat. Hence, the cryopreservation technique is commercially relevant, even where production is based on selected individual males. Cryopreserved sperm are 30- to 100fold less fertile than fresh sperm (Figs. 1 and 2), so hatcheries may use fresh sperm for an initial production run, then cryopreserve excess sperm for later use. Sperm that were frozen using the simplified freezing methods cooled much faster than sperm frozen using the controlled rate freezer. The programmed freezing rate of 50 8C min 1 was established using 0.25- and 0.5-mL plastic straws (Smith et al., 2001). However, this freezing rate was not attained when 4.5-mL cryovials were used in the same controlled rate freezer during this study. The actual cooling rate in the 4.5-mL cryovials lagged behind that recorded in the chamber of the freezer, averaging only 9.5 8C min 1 from 0 to 60 8C. Fertility of cryopreserved sperm was equally good at cooling rates of 9.5 (controlled rate freezer), 13.5 (rack) and 27 8C min 1 (methanol/dry ice bath), but reduced at 107 8C min 1 (direct liquid nitrogen plunge). Other studies on Pacific
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oyster sperm have reported successful fertilization using sperm cooled at rates of 4.7 to N 100 8C min 1 (Hwang and Chen, 1973; Yankson and Moyse, 1991; McFadzen, 1995; Smith et al., 2001). It appears that Pacific oyster sperm are able to tolerate a relatively wide range of cooling rates without any appreciable change in postthaw fertility. The effect of DMSO concentration was investigated in the presence of the nonpermeating CPA, trehalose, which was previously shown to reduce the toxicity of DMSO to Pacific oyster embryos (Chao et al., 1994). The main purpose was to determine an optimal DMSO concentration for cryopreserving Pacific oyster sperm, as the concentration suggested in previous studies ranged from 5% to 17.7% (Staeger, 1974; Bougrier and Rabenomanana, 1986; Iwata et al., 1989; Yankson and Moyse, 1991; Smith et al., 2001). Sperm were also frozen in seawater without DMSO or trehalose because sea urchin sperm show modest postthaw fertility and survival following cryopreservation without CPA (Adams et al., 2004). The postthaw fertility of oyster sperm frozen in seawater was very poor, but not zero. DMSO concentration had no significant effect on postthaw fertility of sperm in the presence of trehalose, and no concentration of DMSO gave significantly higher postthaw fertility than trehalose alone. These results may, in part, explain why such a large range has been suggested to be optimal by other studies on Pacific oyster sperm. They also highlight the beneficial role of trehalose in protecting Pacific oyster sperm during cryopreservation. Trehalose is thought to protect cells by several mechanisms. One is by stabilizing the phospholipids in the cell membrane, preventing damage caused by dehydration (Strauss et al., 1986; Anchordoguy et al., 1987). Another is by changing the properties of the extracellular solution. Trehalose may alter the pattern of ice crystallisation, thereby preventing mechanical damage to cells (Woelders et al., 1997). It may also trap salts in a viscous phase, stopping eutectic freezing from occurring (Nicolajsen and Hvidt, 1994). Finally, trehalose protects cells by reducing the salt concentration in the unfrozen fraction at a given temperature (Holt, 2000). In this study, trehalose was diluted in distilled water rather than seawater, further reducing the likelihood of sperm being exposed to high salt concentrations. We diluted 1 part stripped sperm with 10 parts CPA solution because previous work showed that this returned sperm with ~2.5-fold higher fertility than a 1:1 dilution (Smith et al., 2001). However, if storage space were the main constraint, then the 1:1 dilution offers the best compromise because the higher number of sperm in a given volume more than offsets the lower fertility of 1:1 diluted sperm. Thus, about twice as many eggs could be fertilized from a given volume of frozen sperm. Combining gametes to reduce variation between experiments has been a feature of bioassays for water quality testing and for developmental biology and embryology research. In the work reported here, sperm and eggs used in each run were pooled from at least three males and three females. The quality of sperm and eggs was checked visually before the gametes from a particular individual were added to a pool. However, significant variation in postthaw fertility among replicate runs of experiments occurred. It is likely that this variation is at least partly attributable to variation in the fertility of different pools of sperm and eggs. Where pools of gametes are used in future studies, this variation should be reduced by pooling gametes from a larger number of animals. However, for selective breeding programmes using cryopreserved sperm for pairwise matings an understanding
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of the factors contributing to this variation amongst individuals will be important in minimising its impact on the breeding programme. It has been suggested that DNA damage to oyster sperm during cryopreservation may result in increased embryonic mortality of eggs fertilized with cryopreserved sperm (Gwo et al. 2003). We did not observe any increase in embryonic mortality in this study, although DNA damage may account, at least in part, for the reduction in fertility observed. Indeed, the number of spat obtained with cryopreserved sperm in this study was comparable to that obtained when unfrozen sperm were used (3.7 million spat with cryopreserved sperm and 2.5 million with unfrozen sperm).
5. Commercial cryopreservation protocol We have used the methods outlined in this paper on numerous occasions with consistent success, and believe they will be useful to staff of Pacific oyster hatcheries wishing to use sperm cryopreservation. For ease of application, a simple summary of the protocol used is given below. Sperm are stripped bdryQ and stored in a water bath at ~4 8C until diluted with 10 parts of CPA solution in 10 equal volume steps, 10 to 20 s apart (CPA solution: Solution A=15.2 g of trehalose+40 mL distilled water. Combine 30 mL of solution A, 7.8 mL of distilled water and 2.2 mL of DMSO, mix and cool in a 4 8C water bath.). The diluted sperm solution is transferred to labelled 4.5-mL cryovials, attached to aluminium canes and then cooled for 10 min in a lidded styrofoam box by either 1) placing in a methanol bath chilled to ~ 75 8C with dry ice pellets or 2) gently placing on a rack floating 3 cm above liquid nitrogen. At the end of 10 min, the samples are plunged into liquid nitrogen for at least several minutes, and then transferred to a liquid nitrogen storage container where they remain viable indefinitely. For thawing, cryovials are transferred from liquid nitrogen to a lidded FSW bath at ~20 8C until thawed (5–8 min). Thawed sperm are combined with freshly spawned or stripped eggs at a ratio of ~2000 sperm per egg, and an egg density of ~100,000 eggs mL 1. After 10 min, the eggs are diluted into larval hatching tanks and reared following normal practises. Acknowledgements We thank the staff at the Cawthron Institute for technical assistance and Dr. Paul Hessian at the University of Otago for helpful comments on this manuscript. This research was supported by the New Zealand Foundation for Research Science and Technology (contracts CAW801, CAWX0004 and CAWX0304). References Adams, S.L., Hessian, P.H., Mladenov, P.V., 2004. Cryopreservation of sea urchin (Evechinus chloroticus) sperm. Cryo. Lett. 25, 287 – 299. Anchordoguy, T.J., Rudolph, A.S., Carpenter, J.F., Crowe, J.H., 1987. Modes of interaction of cryoprotectants with membrane phospholipids during freezing. Cryobiology 24, 324 – 331.
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